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Sample records for acetylated lysine residues

  1. Acetyl-Phosphate Is a Critical Determinant of Lysine Acetylation in E. coli

    DEFF Research Database (Denmark)

    Weinert, Brian T; Iesmantavicius, Vytautas; Wagner, Sebastian A

    2013-01-01

    Lysine acetylation is a frequently occurring posttranslational modification in bacteria; however, little is known about its origin and regulation. Using the model bacterium Escherichia coli (E. coli), we found that most acetylation occurred at a low level and accumulated in growth-arrested cells...... acetylate lysine residues in vitro and that AcP levels are correlated with acetylation levels in vivo, suggesting that AcP may acetylate proteins nonenzymatically in cells. These results uncover a critical role for AcP in bacterial acetylation and indicate that most acetylation in E. coli occurs at a low...

  2. SPOTing Acetyl-Lysine Dependent Interactions

    Directory of Open Access Journals (Sweden)

    Sarah Picaud

    2015-08-01

    Full Text Available Post translational modifications have been recognized as chemical signals that create docking sites for evolutionary conserved effector modules, allowing for signal integration within large networks of interactions. Lysine acetylation in particular has attracted attention as a regulatory modification, affecting chromatin structure and linking to transcriptional activation. Advances in peptide array technologies have facilitated the study of acetyl-lysine-containing linear motifs interacting with the evolutionary conserved bromodomain module, which specifically recognizes and binds to acetylated sequences in histones and other proteins. Here we summarize recent work employing SPOT peptide technology to identify acetyl-lysine dependent interactions and document the protocols adapted in our lab, as well as our efforts to characterize such bromodomain-histone interactions. Our results highlight the versatility of SPOT methods and establish an affordable tool for rapid access to potential protein/modified-peptide interactions involving lysine acetylation.

  3. Acetylation and glycation of fibrinogen in vitro occur at specific lysine residues in a concentration dependent manner: A mass spectrometric and isotope labeling study

    Energy Technology Data Exchange (ETDEWEB)

    Svensson, Jan, E-mail: jan.svensson@ki.se [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden); Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm (Sweden); Bergman, Ann-Charlotte [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden); Adamson, Ulf [Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm (Sweden); Blombaeck, Margareta [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden); Wallen, Hakan; Joerneskog, Gun [Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm (Sweden)

    2012-05-04

    Highlights: Black-Right-Pointing-Pointer Fibrinogen was incubated in vitro with glucose or aspirin. Black-Right-Pointing-Pointer Acetylations and glycations were found at twelve lysine sites by mass spectrometry. Black-Right-Pointing-Pointer The labeling by aspirin and glucose occurred dose-dependently. Black-Right-Pointing-Pointer No competition between glucose and aspirin for binding to fibrinogen was found. -- Abstract: Aspirin may exert part of its antithrombotic effects through platelet-independent mechanisms. Diabetes is a condition in which the beneficial effects of aspirin are less prominent or absent - a phenomenon called 'aspirin resistance'. We investigated whether acetylation and glycation occur at specific sites in fibrinogen and if competition between glucose and aspirin in binding to fibrinogen occurs. Our hypothesis was that such competition might be one explanation to 'aspirin resistance' in diabetes. After incubation of fibrinogen in vitro with aspirin (0.8 mM, 24 h) or glucose (100 mM, 5-10 days), we found 12 modified sites with mass spectrometric techniques. Acetylations in the {alpha}-chain: {alpha}K191, {alpha}K208, {alpha}K224, {alpha}K429, {alpha}K457, {alpha}K539, {alpha}K562, in the {beta}-chain: {beta}K233, and in the {gamma}-chain: {gamma}K170 and {gamma}K273. Glycations were found at {beta}K133 and {gamma}K75, alternatively {gamma}K85. Notably, the lysine 539 is a site involved in FXIII-mediated cross-linking of fibrin. With isotope labeling in vitro, using [{sup 14}C-acetyl]salicylic acid and [{sup 14}C]glucose, a labeling of 0.013-0.084 and 0.12-0.5 mol of acetylated and glycated adduct/mol fibrinogen, respectively, was found for clinically (12.9-100 {mu}M aspirin) and physiologically (2-8 mM glucose) relevant plasma concentrations. No competition between acetylation and glycation could be demonstrated. Thus, fibrinogen is acetylated at several lysine residues, some of which are involved in the cross-linking of

  4. New lysine-acetylated proteins screened by immunoaffinity and liquid chromatography-mass spectrometry

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The lack of selective extraction specific for lysine-acetylated proteins has been a major problem in the field of acetylation biology,though acetylation plays a key role in many biological processes.In this paper,we report for the first time the proteomic screening of lysine-acetylated proteins from a mouse liver tissue,by a new approach of immunoaffinity purification of lysine-acetylated peptides combined with nano-HPLC/MS/MS analysis.We have found 20 lysine-acetylated proteins with 21 lysine-acetylated sites,among which 12 lysine-acetylated proteins and 16 lysine-acetylated sites have never been reported before.Notably,three acetyltransferases harboring in mitochondrion are newly discovered acetyltransferases responsible for the acetylation of nonhistone proteins.We have explored the significant patterns of residue preference by the hierarchical clustering analysis of amino acid residues surrounding acetylation sites,which could be helpful to the prediction of new sites of lysine acetylation.Our findings provide more candidates for studying the important roles played by acetylation in diverse cellular pathways and related human diseases.

  5. Site-specific methylation and acetylation of lysine residues in the C-terminal domain (CTD) of RNA polymerase II

    Science.gov (United States)

    Voss, Kirsten; Forné, Ignasi; Descostes, Nicolas; Hintermair, Corinna; Schüller, Roland; Maqbool, Muhammad Ahmad; Heidemann, Martin; Flatley, Andrew; Imhof, Axel; Gut, Marta; Gut, Ivo; Kremmer, Elisabeth; Andrau, Jean-Christophe; Eick, Dirk

    2015-01-01

    Dynamic modification of heptad-repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 of RNA polymerase II (RNAPII) C-terminal domain (CTD) regulates transcription-coupled processes. Mass spectrometry analysis revealed that K7-residues in non-consensus repeats of human RNAPII are modified by acetylation, or mono-, di-, and tri-methylation. K7ac, K7me2, and K7me3 were found exclusively associated with phosphorylated CTD peptides, while K7me1 occurred also in non-phosphorylated CTD. The monoclonal antibody 1F5 recognizes K7me1/2 residues in CTD and reacts with RNAPIIA. Treatment of cellular extracts with phosphatase or of cells with the kinase inhibitor flavopiridol unmasked the K7me1/2 epitope in RNAPII0, consistent with the association of K7me1/2 marks with phosphorylated CTD peptides. Genome-wide profiling revealed high levels of K7me1/2 marks at the transcriptional start site of genes for sense and antisense transcribing RNAPII. The new K7 modifications further expand the mammalian CTD code to allow regulation of differential gene expression. PMID:26566685

  6. Site-specific methylation and acetylation of lysine residues in the C-terminal domain (CTD) of RNA polymerase II.

    Science.gov (United States)

    Voss, Kirsten; Forné, Ignasi; Descostes, Nicolas; Hintermair, Corinna; Schüller, Roland; Maqbool, Muhammad Ahmad; Heidemann, Martin; Flatley, Andrew; Imhof, Axel; Gut, Marta; Gut, Ivo; Kremmer, Elisabeth; Andrau, Jean-Christophe; Eick, Dirk

    2015-01-01

    Dynamic modification of heptad-repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 of RNA polymerase II (RNAPII) C-terminal domain (CTD) regulates transcription-coupled processes. Mass spectrometry analysis revealed that K7-residues in non-consensus repeats of human RNAPII are modified by acetylation, or mono-, di-, and tri-methylation. K7ac, K7me2, and K7me3 were found exclusively associated with phosphorylated CTD peptides, while K7me1 occurred also in non-phosphorylated CTD. The monoclonal antibody 1F5 recognizes K7me1/2 residues in CTD and reacts with RNAPIIA. Treatment of cellular extracts with phosphatase or of cells with the kinase inhibitor flavopiridol unmasked the K7me1/2 epitope in RNAPII0, consistent with the association of K7me1/2 marks with phosphorylated CTD peptides. Genome-wide profiling revealed high levels of K7me1/2 marks at the transcriptional start site of genes for sense and antisense transcribing RNAPII. The new K7 modifications further expand the mammalian CTD code to allow regulation of differential gene expression.

  7. DNA Damage-Induced Acetylation of Lysine 3016 of ATM Activates ATM Kinase Activity▿ †

    OpenAIRE

    Sun, Yingli; Xu, Ye; Roy, Kanaklata; Price, Brendan D.

    2007-01-01

    The ATM protein kinase is essential for cells to repair and survive genotoxic events. The activation of ATM's kinase activity involves acetylation of ATM by the Tip60 histone acetyltransferase. In this study, systematic mutagenesis of lysine residues was used to identify regulatory ATM acetylation sites. The results identify a single acetylation site at lysine 3016, which is located in the highly conserved C-terminal FATC domain adjacent to the kinase domain. Antibodies specific for acetyl-ly...

  8. Global analysis of lysine acetylation in strawberry leaves

    Directory of Open Access Journals (Sweden)

    Xianping eFang

    2015-09-01

    Full Text Available Protein lysine acetylation is a reversible and dynamic post-translational modification. It plays an important role in regulating diverse cellular processes including chromatin dynamic, metabolic pathways and transcription in both prokaryotes and eukaryotes. Although studies of lysine acetylome in plants have been reported, the throughput was not high enough, hindering the deep understanding of lysine acetylation in plant physiology and pathology. In this study, taking advantages of anti-acetyllysine-based enrichment and high-sensitive-mass spectrometer, we applied an integrated proteomic approach to comprehensively investigate lysine acetylome in strawberry. In total, we identified 1392 acetylation sites in 684 proteins, representing the largest dataset of acetylome in plants to date. To reveal the functional impacts of lysine acetylation in strawberry, intensive bioinformatic analysis was performed. The results significantly expanded our current understanding of plant acetylome and demonstrated that lysine acetylation is involved in multiple cellular metabolism and cellular processes. More interestingly, nearly 50% of all acetylated proteins identified in this work were localized in chloroplast and the vital role of lysine acetylation in photosynthesis was also revealed. Taken together, this study not only established the most extensive lysine acetylome in plants to date, but also systematically suggests the significant and unique roles of lysine acetylation in plants.

  9. Lysine Ubiquitination and Acetylation of Human Cardiac 20S Proteasomes

    Science.gov (United States)

    Lau, Edward; Choi, Howard JH; Ng, Dominic CM; Meyer, David; Fang, Caiyun; Li, Haomin; Wang, Ding; Zelaya, Ivette M; Yates, John R; Lam, Maggie PY

    2016-01-01

    Purpose Altered proteasome functions are associated with multiple cardiomyopathies. While the proteasome targets poly-ubiquitinated proteins for destruction, it itself is modifiable by ubiquitination. We aim to identify the exact ubiquitination sites on cardiac proteasomes and examine whether they are also subject to acetylations. Experimental design Assembled cardiac 20S proteasome complexes were purified from five human hearts with ischemic cardiomyopathy, then analyzed by high-resolution MS to identify ubiquitination and acetylation sites. We developed a library search strategy that may be used to complement database search in identifying PTM in different samples. Results We identified 63 ubiquitinated lysines from intact human cardiac 20S proteasomes. In parallel, 65 acetylated residues were also discovered, 39 of which shared with ubiquitination sites. Conclusion and clinical relevance This is the most comprehensive characterization of cardiac proteasome ubiquitination to-date. There are significant overlaps between the discovered ubiquitination and acetylation sites, permitting potential crosstalk in regulating proteasome functions. The information presented here will aid future therapeutic strategies aimed at regulating the functions of cardiac proteasomes. PMID:24957502

  10. The Tale of Protein Lysine Acetylation in the Cytoplasm

    Directory of Open Access Journals (Sweden)

    Karin Sadoul

    2011-01-01

    Full Text Available Reversible posttranslational modification of internal lysines in many cellular or viral proteins is now emerging as part of critical signalling processes controlling a variety of cellular functions beyond chromatin and transcription. This paper aims at demonstrating the role of lysine acetylation in the cytoplasm driving and coordinating key events such as cytoskeleton dynamics, intracellular trafficking, vesicle fusion, metabolism, and stress response.

  11. Lysine Acetylation and Deacetylation in Brain Development and Neuropathies

    Directory of Open Access Journals (Sweden)

    Alicia Tapias

    2017-02-01

    Full Text Available Embryonic development is critical for the final functionality and maintenance of the adult brain. Brain development is tightly regulated by intracellular and extracellular signaling. Lysine acetylation and deacetylation are posttranslational modifications that are able to link extracellular signals to intracellular responses. A wealth of evidence indicates that lysine acetylation and deacetylation are critical for brain development and functionality. Indeed, mutations of the enzymes and cofactors responsible for these processes are often associated with neurodevelopmental and psychiatric disorders. Lysine acetylation and deacetylation are involved in all levels of brain development, starting from neuroprogenitor survival and proliferation, cell fate decisions, neuronal maturation, migration, and synaptogenesis, as well as differentiation and maturation of astrocytes and oligodendrocytes, to the establishment of neuronal circuits. Hence, fluctuations in the balance between lysine acetylation and deacetylation contribute to the final shape and performance of the brain. In this review, we summarize the current basic knowledge on the specific roles of lysine acetyltransferase (KAT and lysine deacetylase (KDAC complexes in brain development and the different neurodevelopmental disorders that are associated with dysfunctional lysine (deacetylation machineries.

  12. The biology of lysine acetylation integrates transcriptional programming and metabolism

    Directory of Open Access Journals (Sweden)

    Mujtaba Shiraz

    2011-03-01

    Full Text Available Abstract The biochemical landscape of lysine acetylation has expanded from a small number of proteins in the nucleus to a multitude of proteins in the cytoplasm. Since the first report confirming acetylation of the tumor suppressor protein p53 by a lysine acetyltransferase (KAT, there has been a surge in the identification of new, non-histone targets of KATs. Added to the known substrates of KATs are metabolic enzymes, cytoskeletal proteins, molecular chaperones, ribosomal proteins and nuclear import factors. Emerging studies demonstrate that no fewer than 2000 proteins in any particular cell type may undergo lysine acetylation. As described in this review, our analyses of cellular acetylated proteins using DAVID 6.7 bioinformatics resources have facilitated organization of acetylated proteins into functional clusters integral to cell signaling, the stress response, proteolysis, apoptosis, metabolism, and neuronal development. In addition, these clusters also depict association of acetylated proteins with human diseases. These findings not only support lysine acetylation as a widespread cellular phenomenon, but also impel questions to clarify the underlying molecular and cellular mechanisms governing target selectivity by KATs. Present challenges are to understand the molecular basis for the overlapping roles of KAT-containing co-activators, to differentiate between global versus dynamic acetylation marks, and to elucidate the physiological roles of acetylated proteins in biochemical pathways. In addition to discussing the cellular 'acetylome', a focus of this work is to present the widespread and dynamic nature of lysine acetylation and highlight the nexus that exists between epigenetic-directed transcriptional regulation and metabolism.

  13. Expression and purification of histone H3 proteins containing multiple sites of lysine acetylation using nonsense suppression.

    Science.gov (United States)

    Young, Isaac A; Mittal, Chitvan; Shogren-Knaak, Michael A

    2016-02-01

    Lysine acetylation is a common post-translational modification, which is especially prevalent in histone proteins in chromatin. A number of strategies exist for generating histone proteins containing lysine acetylation, but an especially attractive approach is to genetically encode acetyl-lysine residues using nonsense suppression. This strategy has been successfully applied to single sites of histone acetylation. However, because histone acetylation can often occur at multiple sites simultaneously, we were interested in determining whether this approach could be extended. Here we show that we can express histone H3 proteins that incorporate up to four sites of lysine acetylation on the histone tail. Because the amount of expressed multi-acetylated histone is reduced relative to the wild type, a purification strategy involving affinity purification and ion exchange chromatography was optimized. This expression and purification strategy ultimately generates H3 histone uniformly acetylated at the desired position at levels and purity sufficient to assemble histone octamers. Histone octamers containing four sites of lysine acetylation were assembled into mononucleosomes and enzymatic assays confirmed that this acetylation largely blocks further acetylation by the yeast SAGA acetyltransferase complex.

  14. Systematic Analysis of the Functions of Lysine Acetylation in the Regulation of Tat Activity.

    Directory of Open Access Journals (Sweden)

    Minghao He

    Full Text Available The Tat protein of HIV-1 has several well-known properties, such as nucleocytoplasmic trafficking, transactivation of transcription, interaction with tubulin, regulation of mitotic progression, and induction of apoptosis. Previous studies have identified a couple of lysine residues in Tat that are essential for its functions. In order to analyze the functions of all the lysine residues in Tat, we mutated them individually to alanine, glutamine, and arginine. Through systematic analysis of the lysine mutants, we discovered several previously unidentified characteristics of Tat. We found that lysine acetylation could modulate the subcellular localization of Tat, in addition to the regulation of its transactivation activity. Our data also revealed that lysine mutations had distinct effects on microtubule assembly and Tat binding to bromodomain proteins. By correlation analysis, we further found that the effects of Tat on apoptosis and mitotic progression were not entirely attributed to its effect on microtubule assembly. Our findings suggest that Tat may regulate diverse cellular activities through binding to different proteins and that the acetylation of distinct lysine residues in Tat may modulate its interaction with various partners.

  15. Predicting post-translational lysine acetylation using support vector machines

    DEFF Research Database (Denmark)

    Gnad, Florian; Ren, Shubin; Choudhary, Chunaram

    2010-01-01

    spectrometry to identify 3600 lysine acetylation sites on 1750 human proteins covering most of the previously annotated sites and providing the most comprehensive acetylome so far. This dataset should provide an excellent source to train support vector machines (SVMs) allowing the high accuracy in silico...

  16. File list: His.Dig.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: His.Pan.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: His.Unc.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: His.Pan.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: His.Neu.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: His.Epd.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. File list: His.Brs.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: His.Dig.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: His.Prs.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: His.Liv.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: His.Myo.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: His.ALL.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: His.Emb.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: His.Pan.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: His.Unc.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: His.Adp.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  12. File list: His.Neu.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: His.Utr.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: His.ALL.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  15. File list: His.Bld.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. File list: His.CDV.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: His.Bld.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: His.Bld.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: His.Dig.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: His.Oth.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: His.Prs.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. File list: His.Epd.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: His.Neu.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: His.Unc.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: His.Brs.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: His.Lng.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: His.Kid.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: His.Adp.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: His.Oth.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: His.Bon.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: His.Bld.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  12. File list: His.Adp.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: His.Adp.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: His.CDV.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  15. File list: His.Neu.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. File list: His.Lng.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: His.PSC.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: His.Plc.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: His.Oth.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: His.Oth.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: His.Lng.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. File list: His.Prs.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: His.Myo.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: His.Unc.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: His.Epd.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: His.Prs.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: His.Bld.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: His.ALL.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: His.Neu.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: His.PSC.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: His.Pan.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  12. File list: His.ALL.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: His.Liv.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: His.Gon.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Gon.50.Pan_lysine_acetylation.AllCell hg19 Histone Pan lysine acetylation Gonad... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Gon.50.Pan_lysine_acetylation.AllCell.bed ...

  15. File list: His.ALL.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. File list: His.PSC.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: His.Prs.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: His.CDV.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.CDV.05.Pan_lysine_acetylation.AllCell hg19 Histone Pan lysine acetylation Cardi...ovascular http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.CDV.05.Pan_lysine_acetylation.AllCell.bed ...

  19. File list: His.Gon.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: His.Bon.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: His.Dig.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. File list: His.Bld.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: His.Unc.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: His.Utr.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: His.Liv.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: His.Lng.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: His.Liv.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: His.Bld.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: His.Epd.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: His.Pan.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: His.PSC.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  12. File list: His.Plc.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: His.Plc.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Plc.10.Pan_lysine_acetylation.AllCell hg19 Histone Pan lysine acetylation Place...nta http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Plc.10.Pan_lysine_acetylation.AllCell.bed ...

  14. File list: His.Prs.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  15. File list: His.Bon.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Bon.50.Pan_lysine_acetylation.AllCell mm9 Histone Pan lysine acetylation Bone h...ttp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Bon.50.Pan_lysine_acetylation.AllCell.bed ...

  16. File list: His.Lng.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: His.Unc.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: His.Utr.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: His.Adp.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: His.Gon.50.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: His.Dig.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. File list: His.Dig.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: His.Brs.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: His.Myo.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: His.CDV.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: His.Bon.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: His.Epd.05.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: His.Oth.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. Nε-lysine acetylation of a bacterial transcription factor inhibits Its DNA-binding activity.

    Directory of Open Access Journals (Sweden)

    Sandy Thao

    Full Text Available Evidence suggesting that eukaryotes and archaea use reversible N(ε-lysine (N(ε-Lys acetylation to modulate gene expression has been reported, but evidence for bacterial use of N(ε-Lys acetylation for this purpose is lacking. Here, we report data in support of the notion that bacteria can control gene expression by modulating the acetylation state of transcription factors (TFs. We screened the E. coli proteome for substrates of the bacterial Gcn5-like protein acetyltransferase (Pat. Pat acetylated four TFs, including the RcsB global regulatory protein, which controls cell division, and capsule and flagellum biosynthesis in many bacteria. Pat acetylated residue Lys180 of RcsB, and the NAD(+-dependent Sir2 (sirtuin-like protein deacetylase (CobB deacetylated acetylated RcsB (RcsB(Ac, demonstrating that N(ε-Lys acetylation of RcsB is reversible. Analysis of RcsB(Ac and variant RcsB proteins carrying substitutions at Lys180 provided biochemical and physiological evidence implicating Lys180 as a critical residue for RcsB DNA-binding activity. These findings further the likelihood that reversible N(ε-Lys acetylation of transcription factors is a mode of regulation of gene expression used by all cells.

  10. Global analysis of lysine acetylation suggests the involvement of protein acetylation in diverse biological processes in rice (Oryza sativa.

    Directory of Open Access Journals (Sweden)

    Babi Ramesh Reddy Nallamilli

    Full Text Available Lysine acetylation is a reversible, dynamic protein modification regulated by lysine acetyltransferases and deacetylases. Recent advances in high-throughput proteomics have greatly contributed to the success of global analysis of lysine acetylation. A large number of proteins of diverse biological functions have been shown to be acetylated in several reports in human cells, E.coli, and dicot plants. However, the extent of lysine acetylation in non-histone proteins remains largely unknown in monocots, particularly in the cereal crops. Here we report the mass spectrometric examination of lysine acetylation in rice (Oryza sativa. We identified 60 lysine acetylated sites on 44 proteins of diverse biological functions. Immunoblot studies further validated the presence of a large number of acetylated non-histone proteins. Examination of the amino acid composition revealed substantial amino acid bias around the acetylation sites and the amino acid preference is conserved among different organisms. Gene ontology analysis demonstrates that lysine acetylation occurs in diverse cytoplasmic, chloroplast and mitochondrial proteins in addition to the histone modifications. Our results suggest that lysine acetylation might constitute a regulatory mechanism for many proteins, including both histones and non-histone proteins of diverse biological functions.

  11. Chromosomal protein HMGN1 enhances the acetylation of lysine 14 in histone H3

    OpenAIRE

    Lim, Jae-Hwan; West, Katherine L.; Rubinstein, Yaffa; Bergel, Michael; Postnikov, Yuri V.; Bustin, Michael

    2005-01-01

    The acetylation levels of lysine residues in nucleosomes, which are determined by the opposing activities of histone acetyltransferases (HATs) and deacetylases, play an important role in regulating chromatin-related processes, including transcription. We report that HMGN1, a nucleosomal binding protein that reduces the compaction of the chromatin fiber, increases the levels of acetylation of K14 in H3. The levels of H3K14ac in Hmgn1−/− cells are lower than in Hmgn1+/+ cells. Induced expressio...

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

    Directory of Open Access Journals (Sweden)

    Michael N. Davies

    2016-01-01

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

  13. Lysine Acetylation Facilitates Spontaneous DNA Dynamics in the Nucleosome.

    Science.gov (United States)

    Kim, Jongseong; Lee, Jaehyoun; Lee, Tae-Hee

    2015-12-01

    The nucleosome, comprising a histone protein core wrapped around by DNA, is the fundamental packing unit of DNA in cells. Lysine acetylation at the histone core elevates DNA accessibility in the nucleosome, the mechanism of which remains largely unknown. By employing our recently developed hybrid single molecule approach, here we report how the structural dynamics of DNA in the nucleosome is altered upon acetylation at histone H3 lysine 56 (H3K56) that is critical for elevated DNA accessibility. Our results indicate that H3K56 acetylation facilitates the structural dynamics of the DNA at the nucleosome termini that spontaneously and repeatedly open and close on a ms time scale. The results support a molecular mechanism of histone acetylation in catalyzing DNA unpacking whose efficiency is ultimately limited by the spontaneous DNA dynamics at the nucleosome temini. This study provides the first and unique experimental evidence revealing a role of protein chemical modification in directly regulating the kinetic stability of the DNA packing unit.

  14. Induction by fructose force-feeding of histone H3 and H4 acetylation at their lysine residues around the Slc2a5 gene and its expression in mice.

    Science.gov (United States)

    Honma, Kazue; Mochizuki, Kazuki; Goda, Toshinao

    2013-01-01

    It has been reported that fructose force-feeding rapidly induced jejunal Slc2a5 gene expression in rodents. We demonstrate in this study that acetylation at lysine (K) 9 of histone H3 and acetylation at K5 and K16 of histone H4 were more enhanced in the promoter/enhancer to transcribed regions of the Slc2a5 gene in fructose force-fed mice than in glucose force-fed mice. However, fructose force-feeding did not induce acetylation at K14 of histone H3, or at K8 and K12 of histone H4 around the Slc2a5 gene. These results suggest that fructose force-feeding induced selective histone acetylation, particularly of H3 and H4, around the jejunal Slc2a5 gene in mice.

  15. Lysine acetylation stoichiometry and proteomics analyses reveal pathways regulated by sirtuin 1 in human cells.

    Science.gov (United States)

    Gil, Jeovanis; Ramírez-Torres, Alberto; Chiappe, Diego; Luna-Peñaloza, Juan; Fernandez-Reyes, Francis C; Arcos-Encarnación, Bolivar; Contreras, Sandra; Encarnación-Guevara, Sergio

    2017-09-11

    Lysine acetylation is a widespread posttranslational modification (PTM) affecting many biological pathways. Recent studies indicate that acetylated lysine residues mainly exhibit low acetylation occupancy, but challenges in sample preparation and analysis make it difficult to confidently assign these numbers, limiting understanding of their biological significance. Here, we tested three common sample preparation methods to determine their suitability for assessing acetylation stoichiometry in three human cell lines, identifying the acetylation occupancy in more than 1,300 proteins from each cell line. The stoichiometric analysis in combination with quantitative proteomics also enabled us to explore their functional roles. We found that higher abundance of the deacetylase sirtuin 1 (SIRT1) correlated with lower acetylation occupancy and lower levels of ribosomal proteins including those involved in ribosome biogenesis and rRNA processing. Treatment with the SIRT1 inhibitor EX-527 confirmed SIRT1's role in the regulation of pre-rRNA synthesis and processing. Specifically, proteins involved in pre-rRNA transcription, including subunits of the Pol 1 and SL1 complexes and the RNA polymerase I specific transcription initiation factor RRN3 were up-regulated after SIRT1 inhibition. Moreover, many protein effectors and regulators of pre-rRNA processing needed for rRNA maturation were also up-regulated after EX-527 treatment, with the outcome that pre-rRNA and 28S rRNA levels also increased. More generally, we found that SIRT1 inhibition down-regulates metabolic pathways including glycolysis and pyruvate metabolism. Together, these results provide the largest dataset thus far of lysine acetylation stoichiometry (available via ProteomeXchange with identifier PXD005903) and set the stage for further biological investigations of this central PTM. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  16. Lysine acetylation targets protein complexes and co-regulates major cellular functions

    DEFF Research Database (Denmark)

    Choudhary, Chuna Ram; Kumar, Chanchal; Gnad, Florian;

    2009-01-01

    Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600...

  17. Lysine acetylation is a common post-translational modification of key metabolic pathway enzymes of the anaerobe Porphyromonas gingivalis.

    Science.gov (United States)

    Butler, Catherine A; Veith, Paul D; Nieto, Matthew F; Dashper, Stuart G; Reynolds, Eric C

    2015-10-14

    production in this bacterium were acetylated on certain lysine residues. These enzymes were often found catalysing sequential reactions within the same catabolic pathway. The results suggest that lysine acetylation is an important mechanism of metabolic regulation in P. gingivalis vital for its adaptation and proliferation to produce disease.

  18. File list: His.NoD.20.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

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  4. File list: His.NoD.10.Pan_lysine_acetylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH).

    Science.gov (United States)

    Meyer, Jesse G; D'Souza, Alexandria K; Sorensen, Dylan J; Rardin, Matthew J; Wolfe, Alan J; Gibson, Bradford W; Schilling, Birgit

    2016-11-01

    Post-translational modification of lysine residues by NƐ-acylation is an important regulator of protein function. Many large-scale protein acylation studies have assessed relative changes of lysine acylation sites after antibody enrichment using mass spectrometry-based proteomics. Although relative acylation fold-changes are important, this does not reveal site occupancy, or stoichiometry, of individual modification sites, which is critical to understand functional consequences. Recently, methods for determining lysine acetylation stoichiometry have been proposed based on ratiometric analysis of endogenous levels to those introduced after quantitative per-acetylation of proteins using stable isotope-labeled acetic anhydride. However, in our hands, we find that these methods can overestimate acetylation stoichiometries because of signal interferences when endogenous levels of acylation are very low, which is especially problematic when using MS1 scans for quantification. In this study, we sought to improve the accuracy of determining acylation stoichiometry using data-independent acquisition (DIA). Specifically, we use SWATH acquisition to comprehensively collect both precursor and fragment ion intensity data. The use of fragment ions for stoichiometry quantification not only reduces interferences but also allows for determination of site-level stoichiometry from peptides with multiple lysine residues. We also demonstrate the novel extension of this method to measurements of succinylation stoichiometry using deuterium-labeled succinic anhydride. Proof of principle SWATH acquisition studies were first performed using bovine serum albumin for both acetylation and succinylation occupancy measurements, followed by the analysis of more complex samples of E. coli cell lysates. Although overall site occupancy was low (<1%), some proteins contained lysines with relatively high acetylation occupancy. Graphical Abstract ᅟ.

  6. Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH)

    Science.gov (United States)

    Meyer, Jesse G.; D'Souza, Alexandria K.; Sorensen, Dylan J.; Rardin, Matthew J.; Wolfe, Alan J.; Gibson, Bradford W.; Schilling, Birgit

    2016-09-01

    Post-translational modification of lysine residues by NƐ-acylation is an important regulator of protein function. Many large-scale protein acylation studies have assessed relative changes of lysine acylation sites after antibody enrichment using mass spectrometry-based proteomics. Although relative acylation fold-changes are important, this does not reveal site occupancy, or stoichiometry, of individual modification sites, which is critical to understand functional consequences. Recently, methods for determining lysine acetylation stoichiometry have been proposed based on ratiometric analysis of endogenous levels to those introduced after quantitative per-acetylation of proteins using stable isotope-labeled acetic anhydride. However, in our hands, we find that these methods can overestimate acetylation stoichiometries because of signal interferences when endogenous levels of acylation are very low, which is especially problematic when using MS1 scans for quantification. In this study, we sought to improve the accuracy of determining acylation stoichiometry using data-independent acquisition (DIA). Specifically, we use SWATH acquisition to comprehensively collect both precursor and fragment ion intensity data. The use of fragment ions for stoichiometry quantification not only reduces interferences but also allows for determination of site-level stoichiometry from peptides with multiple lysine residues. We also demonstrate the novel extension of this method to measurements of succinylation stoichiometry using deuterium-labeled succinic anhydride. Proof of principle SWATH acquisition studies were first performed using bovine serum albumin for both acetylation and succinylation occupancy measurements, followed by the analysis of more complex samples of E. coli cell lysates. Although overall site occupancy was low (<1%), some proteins contained lysines with relatively high acetylation occupancy.

  7. Proteomic investigations of lysine acetylation identify diverse substrates of mitochondrial deacetylase sirt3

    DEFF Research Database (Denmark)

    Sol, E-ri Maria; Wagner, Sebastian A; Weinert, Brian T

    2012-01-01

    Lysine acetylation is a posttranslational modification that is dynamically regulated by the activity of acetyltransferases and deacetylases. The human and mouse genomes encode 18 different lysine deacetylases (KDACs) which are key regulators of many cellular processes. Identifying substrates...... of KDACs and pinpointing the regulated acetylation sites on target proteins may provide important information about the molecular basis of their functions. Here we apply quantitative proteomics to identify endogenous substrates of the mitochondrial deacetylase Sirtuin 3 (Sirt3) by comparing site...... by modulating acetylation on diverse substrates. The experimental strategy described here is generic and can be applied to identify endogenous substrates of other lysine deacetylases....

  8. Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns

    DEFF Research Database (Denmark)

    Lundby, Alicia; Hansen, Kasper Lage; Weinert, Brian Tate;

    2012-01-01

    ,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals...... that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle...

  9. H3 lysine 4 is acetylated at active gene promoters and is regulated by H3 lysine 4 methylation.

    Directory of Open Access Journals (Sweden)

    Benoit Guillemette

    2011-03-01

    Full Text Available Methylation of histone H3 lysine 4 (H3K4me is an evolutionarily conserved modification whose role in the regulation of gene expression has been extensively studied. In contrast, the function of H3K4 acetylation (H3K4ac has received little attention because of a lack of tools to separate its function from that of H3K4me. Here we show that, in addition to being methylated, H3K4 is also acetylated in budding yeast. Genetic studies reveal that the histone acetyltransferases (HATs Gcn5 and Rtt109 contribute to H3K4 acetylation in vivo. Whilst removal of H3K4ac from euchromatin mainly requires the histone deacetylase (HDAC Hst1, Sir2 is needed for H3K4 deacetylation in heterochomatin. Using genome-wide chromatin immunoprecipitation (ChIP, we show that H3K4ac is enriched at promoters of actively transcribed genes and located just upstream of H3K4 tri-methylation (H3K4me3, a pattern that has been conserved in human cells. We find that the Set1-containing complex (COMPASS, which promotes H3K4me2 and -me3, also serves to limit the abundance of H3K4ac at gene promoters. In addition, we identify a group of genes that have high levels of H3K4ac in their promoters and are inadequately expressed in H3-K4R, but not in set1Δ mutant strains, suggesting that H3K4ac plays a positive role in transcription. Our results reveal a novel regulatory feature of promoter-proximal chromatin, involving mutually exclusive histone modifications of the same histone residue (H3K4ac and H3K4me.

  10. Differential lysine acetylation profiles of Erwinia amylovora strains revealed by proteomics

    Science.gov (United States)

    Protein lysine acetylation (LysAc) in bacteria has recently been demonstrated to be widespread in E. coli and Salmonella and to broadly regulate bacterial physiology and metabolism. However, LysAc in plant pathogenic bacteria is largely unknown. Here we report the lysine acetylome of Erwinia amylovo...

  11. Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificity and Subcellular Patterns

    Directory of Open Access Journals (Sweden)

    Alicia Lundby

    2012-08-01

    Full Text Available Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle contraction. Furthermore, we illustrate that acetylation of fructose-bisphosphate aldolase and glycerol-3-phosphate dehydrogenase serves as a cellular mechanism to switch off enzymatic activity.

  12. Proteome-wide analysis of lysine acetylation suggests its broad regulatory scope in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Henriksen, Peter; Wagner, Sebastian Alexander; Weinert, Brian Tate;

    2012-01-01

    Post-translational modification of proteins by lysine acetylation plays important regulatory roles in living cells. The budding yeast Saccharomyces cerevisiae is a widely used unicellular eukaryotic model organism in biomedical research. S. cerevisiae contains several evolutionary conserved lysin...

  13. Mitochondrial protein acetylation as a cell-intrinsic, evolutionary driver of fat storage: chemical and metabolic logic of acetyl-lysine modifications.

    Science.gov (United States)

    Ghanta, Sirisha; Grossmann, Ruth E; Brenner, Charles

    2013-01-01

    Hormone systems evolved over 500 million years of animal natural history to motivate feeding behavior and convert excess calories to fat. These systems produced vertebrates, including humans, who are famine-resistant but sensitive to obesity in environments of persistent overnutrition. We looked for cell-intrinsic metabolic features, which might have been subject to an evolutionary drive favoring lipogenesis. Mitochondrial protein acetylation appears to be such a system. Because mitochondrial acetyl-coA is the central mediator of fuel oxidation and is saturable, this metabolite is postulated to be the fundamental indicator of energy excess, which imprints a memory of nutritional imbalances by covalent modification. Fungal and invertebrate mitochondria have highly acetylated mitochondrial proteomes without an apparent mitochondrially targeted protein lysine acetyltransferase. Thus, mitochondrial acetylation is hypothesized to have evolved as a nonenzymatic phenomenon. Because the pKa of a nonperturbed Lys is 10.4 and linkage of a carbonyl carbon to an ε amino group cannot be formed with a protonated Lys, we hypothesize that acetylation occurs on residues with depressed pKa values, accounting for the propensity of acetylation to hit active sites and suggesting that regulatory Lys residues may have been under selective pressure to avoid or attract acetylation throughout animal evolution. In addition, a shortage of mitochondrial oxaloacetate under ketotic conditions can explain why macronutrient insufficiency also produces mitochondrial hyperacetylation. Reduced mitochondrial activity during times of overnutrition and undernutrition would improve fitness by virtue of resource conservation. Micronutrient insufficiency is predicted to exacerbate mitochondrial hyperacetylation. Nicotinamide riboside and Sirt3 activity are predicted to relieve mitochondrial inhibition.

  14. Proteomic investigations of lysine acetylation identify diverse substrates of mitochondrial deacetylase sirt3.

    Directory of Open Access Journals (Sweden)

    Eri Maria Sol

    Full Text Available Lysine acetylation is a posttranslational modification that is dynamically regulated by the activity of acetyltransferases and deacetylases. The human and mouse genomes encode 18 different lysine deacetylases (KDACs which are key regulators of many cellular processes. Identifying substrates of KDACs and pinpointing the regulated acetylation sites on target proteins may provide important information about the molecular basis of their functions. Here we apply quantitative proteomics to identify endogenous substrates of the mitochondrial deacetylase Sirtuin 3 (Sirt3 by comparing site-specific acetylation in wild-type murine embryonic fibroblasts to Sirt3 knockout cells. We confirm Sirt3-regulated acetylation of several mitochondrial proteins in human cells by comparing acetylation in U2OS cells overexpressing Sirt3 to U2OS cells in which Sirt3 expression was reduced by shRNA. Our data demonstrate that ablation of Sirt3 significantly increases acetylation at dozens of sites on mitochondrial proteins. Substrates of Sirt3 are implicated in various metabolic pathways, including fatty acid metabolism and the tricarboxylic acid cycle. These results imply broader regulatory roles of Sirt3 in the mitochondria by modulating acetylation on diverse substrates. The experimental strategy described here is generic and can be applied to identify endogenous substrates of other lysine deacetylases.

  15. Dietary, Metabolic, and Potentially Environmental Modulation of the Lysine Acetylation Machinery

    Directory of Open Access Journals (Sweden)

    Go-Woon Kim

    2010-01-01

    Full Text Available Healthy lifestyles and environment produce a good state of health. A number of scientific studies support the notion that external stimuli regulate an individual's epigenomic profile. Epigenetic changes play a key role in defining gene expression patterns under both normal and pathological conditions. As a major posttranslational modification, lysine (K acetylation has received much attention, owing largely to its significant effects on chromatin dynamics and other cellular processes across species. Lysine acetyltransferases and deacetylases, two opposing families of enzymes governing K-acetylation, have been intimately linked to cancer and other diseases. These enzymes have been pursued by vigorous efforts for therapeutic development in the past 15 years or so. Interestingly, certain dietary components have been found to modulate acetylation levels in vivo. Here we review dietary, metabolic, and environmental modulators of the K-acetylation machinery and discuss how they may be of potential value in the context of disease prevention.

  16. Stoichiometry of Site-specific Lysine Acetylation in an Entire Proteome*♦

    Science.gov (United States)

    Baeza, Josue; Dowell, James A.; Smallegan, Michael J.; Fan, Jing; Amador-Noguez, Daniel; Khan, Zia; Denu, John M.

    2014-01-01

    Acetylation of lysine ϵ-amino groups influences many cellular processes and has been mapped to thousands of sites across many organisms. Stoichiometric information of acetylation is essential to accurately interpret biological significance. Here, we developed and employed a novel method for directly quantifying stoichiometry of site-specific acetylation in the entire proteome of Escherichia coli. By coupling isotopic labeling and a novel pairing algorithm, our approach performs an in silico enrichment of acetyl peptides, circumventing the need for immunoenrichment. We investigated the function of the sole NAD+-dependent protein deacetylase, CobB, on both site-specific and global acetylation. We quantified 2206 peptides from 899 proteins and observed a wide distribution of acetyl stoichiometry, ranging from less than 1% up to 98%. Bioinformatic analysis revealed that metabolic enzymes, which either utilize or generate acetyl-CoA, and proteins involved in transcriptional and translational processes displayed the highest degree of acetylation. Loss of CobB led to increased global acetylation at low stoichiometry sites and induced site-specific changes at high stoichiometry sites, and biochemical analysis revealed altered acetyl-CoA metabolism. Thus, this study demonstrates that sirtuin deacetylase deficiency leads to both site-specific and global changes in protein acetylation stoichiometry, affecting central metabolism. PMID:24917678

  17. Stoichiometry of site-specific lysine acetylation in an entire proteome.

    Science.gov (United States)

    Baeza, Josue; Dowell, James A; Smallegan, Michael J; Fan, Jing; Amador-Noguez, Daniel; Khan, Zia; Denu, John M

    2014-08-01

    Acetylation of lysine ϵ-amino groups influences many cellular processes and has been mapped to thousands of sites across many organisms. Stoichiometric information of acetylation is essential to accurately interpret biological significance. Here, we developed and employed a novel method for directly quantifying stoichiometry of site-specific acetylation in the entire proteome of Escherichia coli. By coupling isotopic labeling and a novel pairing algorithm, our approach performs an in silico enrichment of acetyl peptides, circumventing the need for immunoenrichment. We investigated the function of the sole NAD(+)-dependent protein deacetylase, CobB, on both site-specific and global acetylation. We quantified 2206 peptides from 899 proteins and observed a wide distribution of acetyl stoichiometry, ranging from less than 1% up to 98%. Bioinformatic analysis revealed that metabolic enzymes, which either utilize or generate acetyl-CoA, and proteins involved in transcriptional and translational processes displayed the highest degree of acetylation. Loss of CobB led to increased global acetylation at low stoichiometry sites and induced site-specific changes at high stoichiometry sites, and biochemical analysis revealed altered acetyl-CoA metabolism. Thus, this study demonstrates that sirtuin deacetylase deficiency leads to both site-specific and global changes in protein acetylation stoichiometry, affecting central metabolism.

  18. Dichotomy in the Epigenetic Mark Lysine Acetylation is Critical for the Proliferation of Prostate Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Pathak, Ravi [Department of Structural and Chemical Biology, Mount Sinai School of Medicine, 1425 Madison Ave, New York, NY 10029 (United States); Philizaire, Marc [Medgar Evers College, City University of New York, 1638 Bedford Ave, 403D, Brooklyn, NY 11225 (United States); Mujtaba, Shiraz, E-mail: smujtaba@mec.cuny.edu [Department of Structural and Chemical Biology, Mount Sinai School of Medicine, 1425 Madison Ave, New York, NY 10029 (United States); Medgar Evers College, City University of New York, 1638 Bedford Ave, 403D, Brooklyn, NY 11225 (United States)

    2015-08-19

    The dynamics of lysine acetylation serve as a major epigenetic mark, which regulates cellular response to inflammation, DNA damage and hormonal changes. Microarray assays reveal changes in gene expression, but cannot predict regulation of a protein function by epigenetic modifications. The present study employs computational tools to inclusively analyze microarray data to understand the potential role of acetylation during development of androgen-independent PCa. The data revealed that the androgen receptor interacts with 333 proteins, out of which at least 92 proteins were acetylated. Notably, the number of cellular proteins undergoing acetylation in the androgen-dependent PCa was more as compared to the androgen-independent PCa. Specifically, the 32 lysine-acetylated proteins in the cellular models of androgen-dependent PCa were mainly involved in regulating stability as well as pre- and post-processing of mRNA. Collectively, the data demonstrate that protein lysine acetylation plays a crucial role during the transition of androgen-dependent to -independent PCa, which importantly, could also serve as a functional axis to unravel new therapeutic targets.

  19. Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xia [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Fifth People' s Hospital of Shanghai, School of Medicine, Fudan University, Shanghai, 200240 (China); Zhao, Libo [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Third People' s Hospital of Chongqing, 400014 (China); Yang, Yongtao [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Bode, Liv [Bornavirus Research Group affiliated to the Free University of Berlin, Berlin (Germany); Huang, Hua [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Liu, Chengyu [Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Huang, Rongzhong [Department of Rehabilitative Medicine, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010 (China); Zhang, Liang [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); and others

    2014-09-15

    Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs.

  20. The structural feature surrounding glycated lysine residues in human hemoglobin.

    Science.gov (United States)

    Ito, Shigenori; Nakahari, Takashi; Yamamoto, Daisuke

    2011-06-01

    Complications derived from diabetes mellitus are caused by nonenzymatic protein glycation at the specific sites. LC/MS/MS was performed for the identification of the tryptic peptides of glycated hemoglobins using glyceraldehyde. After the identification of the glycation or non-glycation site, computer analysis of the structure surrounding the sites was carried out using PDB data (1BZ0). Five glycated lysine residues (Lys-16(α), -56(α), -8(β), -82(β), and -144(β)) and four non-glycated lysine residues (Lys-7(α), -40(α), -99(α), and -132(β)) were identified. The non-glycated lysine residues, Lys-7(α), -40(α), and -132(β), are most likely to form electrostatic interactions with the β carboxyl group of Asp-74(α), C-terminal His-146(β), and Glu-7(β) by virtue of their proximity, which is 2.67-2.91 Å (N-O). Additionally, there are histidine residues within 4.55-7.38 Å (N-N) around eight sites except for Lys-7(α). We conclude that the following factors seem to be necessary for glycation of lysine residues: (i) the apparent absence of aspartate or glutamate residues to inhibit the glycation reaction by forming an electrostatic interaction, (ii) the presence of histidine residues for acid-base catalysis of the Amadori rearrangement, and (iii) the presence of an amino acid residue capable of stabilizing a phosphate during proton transfer.

  1. Differential lysine acetylation profiles of Erwinia amylovora strains revealed by proteomics.

    Science.gov (United States)

    Wu, Xia; Vellaichamy, Adaikkalam; Wang, Dongping; Zamdborg, Leonid; Kelleher, Neil L; Huber, Steven C; Zhao, Youfu

    2013-02-21

    Protein lysine acetylation (LysAc) has recently been demonstrated to be widespread in E. coli and Salmonella, and to broadly regulate bacterial physiology and metabolism. However, LysAc in plant pathogenic bacteria is largely unknown. Here we first report the lysine acetylome of Erwinia amylovora, an enterobacterium causing serious fire blight disease of apples and pears. Immunoblots using generic anti-lysine acetylation antibodies demonstrated that growth conditions strongly affected the LysAc profiles in E. amylovora. Differential LysAc profiles were also observed for two E. amylovora strains, known to have differential virulence in plants, indicating translational modification of proteins may be important in determining virulence of bacterial strains. Proteomic analysis of LysAc in two E. amylovora strains identified 141 LysAc sites in 96 proteins that function in a wide range of biological pathways. Consistent with previous reports, 44% of the proteins are involved in metabolic processes, including central metabolism, lipopolysaccharide, nucleotide and amino acid metabolism. Interestingly, for the first time, several proteins involved in E. amylovora virulence, including exopolysaccharide amylovoran biosynthesis- and type III secretion-associated proteins, were found to be lysine acetylated, suggesting that LysAc may play a major role in bacterial virulence. Comparative analysis of LysAc sites in E. amylovora and E. coli further revealed the sequence and structural commonality for LysAc in the two organisms. Collectively, these results reinforce the notion that LysAc of proteins is widespread in bacterial metabolism and virulence.

  2. Histone H4 lysine 20 acetylation is associated with gene repression in human cells

    Science.gov (United States)

    Kaimori, Jun-Ya; Maehara, Kazumitsu; Hayashi-Takanaka, Yoko; Harada, Akihito; Fukuda, Masafumi; Yamamoto, Satoko; Ichimaru, Naotsugu; Umehara, Takashi; Yokoyama, Shigeyuki; Matsuda, Ryo; Ikura, Tsuyoshi; Nagao, Koji; Obuse, Chikashi; Nozaki, Naohito; Takahara, Shiro; Takao, Toshifumi; Ohkawa, Yasuyuki; Kimura, Hiroshi; Isaka, Yoshitaka

    2016-01-01

    Histone acetylation is generally associated with gene activation and chromatin decondensation. Recent mass spectrometry analysis has revealed that histone H4 lysine 20, a major methylation site, can also be acetylated. To understand the function of H4 lysine 20 acetylation (H4K20ac), we have developed a specific monoclonal antibody and performed ChIP-seq analysis using HeLa-S3 cells. H4K20ac was enriched around the transcription start sites (TSSs) of minimally expressed genes and in the gene body of expressed genes, in contrast to most histone acetylation being enriched around the TSSs of expressed genes. The distribution of H4K20ac showed little correlation with known histone modifications, including histone H3 methylations. A motif search in H4K20ac-enriched sequences, together with transcription factor binding profiles based on ENCODE ChIP-seq data, revealed that most transcription activators are excluded from H4K20ac-enriched genes and a transcription repressor NRSF/REST co-localized with H4K20ac. These results suggest that H4K20ac is a unique acetylation mark associated with gene repression. PMID:27064113

  3. Expansion of the Lysine Acylation Landscape

    DEFF Research Database (Denmark)

    Olsen, Christian A.

    2012-01-01

    Leaving marks: The number of known posttranslational modifications for lysine has been expanded considerably. In addition to acetylation of side-chain amino functionalities of lysine residues in proteins, crotonylation, succinylation, and malonylation have now been identified as posttranslational...

  4. Proteome-wide mapping of the Drosophila acetylome demonstrates a high degree of conservation of lysine acetylation

    DEFF Research Database (Denmark)

    Weinert, Brian T; Wagner, Sebastian A; Horn, Heiko

    2011-01-01

    were significantly more conserved than were nonacetylated lysines. Bioinformatics analysis using Gene Ontology terms suggested that the proteins with conserved acetylation control cellular processes such as protein translation, protein folding, DNA packaging, and mitochondrial metabolism. We found...

  5. PSG gene expression is up-regulated by lysine acetylation involving histone and nonhistone proteins.

    Directory of Open Access Journals (Sweden)

    Soledad A Camolotto

    Full Text Available BACKGROUND: Lysine acetylation is an important post-translational modification that plays a central role in eukaryotic transcriptional activation by modifying chromatin and transcription-related factors. Human pregnancy-specific glycoproteins (PSG are the major secreted placental proteins expressed by the syncytiotrophoblast at the end of pregnancy and represent early markers of cytotrophoblast differentiation. Low PSG levels are associated with complicated pregnancies, thus highlighting the importance of studying the mechanisms that control their expression. Despite several transcription factors having been implicated as key regulators of PSG gene family expression; the role of protein acetylation has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: Here, we explored the role of acetylation on PSG gene expression in the human placental-derived JEG-3 cell line. Pharmacological inhibition of histone deacetylases (HDACs up-regulated PSG protein and mRNA expression levels, and augmented the amount of acetylated histone H3 associated with PSG 5'regulatory regions. Moreover, PSG5 promoter activation mediated by Sp1 and KLF6, via the core promoter element motif (CPE, -147/-140, was markedly enhanced in the presence of the HDAC inhibitor trichostatin A (TSA. This effect correlated with an increase in Sp1 acetylation and KLF6 nuclear localization as revealed by immunoprecipitation and subcellular fractionation assays. The co-activators PCAF, p300, and CBP enhanced Sp1-dependent PSG5 promoter activation through their histone acetylase (HAT function. Instead, p300 and CBP acetyltransferase domain was dispensable for sustaining co-activation of PSG5 promoter by KLF6. CONCLUSIONS/SIGNIFICANCE: Results are consistent with a regulatory role of lysine acetylation on PSG expression through a relaxed chromatin state and an increase in the transcriptional activity of Sp1 and KLF6 following an augmented Sp1 acetylation and KLF6 nuclear localization.

  6. Data for global lysine-acetylation analysis in rice (Oryza sativa

    Directory of Open Access Journals (Sweden)

    Yehui Xiong

    2016-06-01

    Full Text Available Rice is one of the most important crops for human consumption and is a staple food for over half of the world׳s population (Yu et al., 2002 [1]. A systematic identification of the lysine acetylome was performed by our research (Xiong et al., 2016 [2]. Rice plant samples were collected from 5 weeks old seedlings (Oryza sativa, Nipponbare. After the trypsin digestion and immunoaffinity precipitation, LC–MS/MS approach was used to identify acetylated peptides. After the collected MS/MS data procession and GO annotation, the InterProScan was used to annotate protein domain. Subcellular localization of the identified acetylated proteins was predicted by WoLF PSORT. The KEGG pathway database was used to annotate identified acetylated protein interactions, reactions, and relations. The data, supplied in this article, are related to “A comprehensive catalog of the lysine-acetylation targets in rice (O. sativa based on proteomic analyses” by Xiong et al. (2016 [2].

  7. Characterization of lysine acetylation of a phosphoenolpyruvate carboxylase involved in glutamate overproduction in Corynebacterium glutamicum.

    Science.gov (United States)

    Nagano-Shoji, Megumi; Hamamoto, Yuma; Mizuno, Yuta; Yamada, Ayuka; Kikuchi, Masaki; Shirouzu, Mikako; Umehara, Takashi; Yoshida, Minoru; Nishiyama, Makoto; Kosono, Saori

    2017-03-03

    Protein Nε-acylation is emerging as a ubiquitous post-translational modification. In Corynebacterium glutamicum, which is utilized for industrial production of L-glutamate, the levels of protein acetylation and succinylation change drastically under the conditions that induce glutamate overproduction. Here, we characterized the acylation of phosphoenolpyruvate carboxylase (PEPC), an anaplerotic enzyme that supplies oxaloacetate for glutamate overproduction. We showed that acetylation of PEPC at lysine 653 decreased enzymatic activity, leading to reduced glutamate production. An acetylation-mimic (KQ) mutant of K653 showed severely reduced glutamate production, while the corresponding KR mutant showed normal production levels. Using an acetyllysine-incorporated PEPC protein, we verified that K653-acetylation negatively regulates PEPC activity. In addition, NCgl0616, a sirtuin-type deacetylase, deacetylated K653-acetylated PEPC in vitro. Interestingly, the specific activity of PEPC was increased during glutamate overproduction, which was blocked by the K653R mutation or deletion of sirtuin-type deacetylase homologues. These findings suggested that deacetylation of K653 by NCgl0616 likely plays a role in the activation of PEPC, which maintains carbon flux under glutamate-producing conditions. PEPC deletion increased protein acetylation levels in cells under glutamate-producing conditions, supporting our hypothesis that PEPC is responsible for a large carbon flux change under glutamate-producing conditions. This article is protected by copyright. All rights reserved.

  8. Human histone acetyltransferase 1 (Hat1) acetylates lysine 5 of histone H2A in vivo.

    Science.gov (United States)

    Tafrova, Juliana I; Tafrov, Stefan T

    2014-07-01

    The primary structure of Histone Acetyltransferase 1 (Hat1) has been conserved throughout evolution; however, despite its ubiquity, its cellular function is not well characterized. To study its in vivo acetylation pattern and function, we utilized shRNAmir against Hat1 expressed in the well-substantiated HeLa (human cervical cancer) cell line. To reduce the interference by enzymes with similar HAT specificity, we used HeLa cells expressing histone acetyltransferase Tip60 with mutated acetyl-CoA binding site that abrogates its enzyme activity (mutant HeLa-tip60). Two shRNAmir were identified that reduced the expression of the cytoplasmic and nuclear forms of Hat1. Cytosolic protein preparations from these two clones showed decreased levels of acetylation of lysine 5 (K5) and K12 on histone H4, with the concomitant loss of the acetylation of histone H2A at K5. This pattern of decreased acetylation of H2AK5 was well defined in preparations of histone protein and insoluble nuclear-protein (INP) fractions as well. Abrogating the Hat1 expression caused a 74% decrease in colony-forming efficiency of mutant HeLa-tip60 cells, reduced the size of the colonies by 50%, and decreased the amounts of proteins with molecular weights below 35 kDa in the INP fractions.

  9. Post-translational serine/threonine phosphorylation and lysine acetylation: a novel regulatory aspect of the global nitrogen response regulator GlnR in S. coelicolor M145.

    Directory of Open Access Journals (Sweden)

    Rafat Amin

    2016-08-01

    Full Text Available Soil-dwelling Streptomyces bacteria such as S. coelicolor have to constantly adapt to the nitrogen (N availability in their habitat. Thus, strict transcriptional and post-translational control of the N-assimilation is fundamental for survival of this species. GlnR is a global response regulator that controls transcription of the genes related to the N-assimilation in S. coelicolor and other members of the Actinomycetales. GlnR represents an atypical orphan response regulator that is not activated by the phosphorylation of the conserved aspartate residue (Asp 50. We have applied transcriptional analysis, LC-MS/MS analysis and electrophoretic mobility shift assays (EMSAs to understand the regulation of GlnR in S. coelicolor M145. The expression of glnR and GlnR-target genes was revisited under four different N-defined conditions and a complex N-rich condition. Although, the expression of selected GlnR-target genes was strongly responsive to changing N-concentrations, the glnR expression itself was independent of the N-availability. Using LC-MS/MSanalysis we demonstrated that GlnR was post-translationally modified. The post-translational modifications of GlnR comprise phosphorylation of the serine/threonine residues and acetylation of lysine residues. In the complex N-rich medium GlnR was phosphorylated on six serine/ threonine residues and acetylated on one lysine residue. Under defined N-excess conditions only two phosphorylated residues were detected whereas under defined N-limiting conditions no phosphorylation was observed. GlnR phosphorylation is thus clearly correlated with N-rich conditions. Furthermore, GlnR was acetylated on four lysine residues independently of the N-concentration in the defined media and on only one lysine residue in the complex N-rich medium. Using EMSAs we demonstrated that phosphorylation inhibited the binding of GlnR to its targets genes, whereas acetylation had little influence on the formation of GlnR-DNA complex

  10. Exploring the possible role of lysine acetylation on Entamoeba histolytica virulence: a focus on the dynamics of the actin cytoskeleton.

    Science.gov (United States)

    López-Contreras, L; Hernández-Ramírez, V I; Lagunes-Guillén, A E; Montaño, Sarita; Chávez-Munguía, B; Sánchez-Ramírez, B; Talamás-Rohana, P

    2013-01-01

    Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

  11. Exploring the Possible Role of Lysine Acetylation on Entamoeba histolytica Virulence: A Focus on the Dynamics of the Actin Cytoskeleton

    Directory of Open Access Journals (Sweden)

    L. López-Contreras

    2013-01-01

    Full Text Available Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

  12. Histone H3 lysine 56 acetylation and the response to DNA replication fork damage

    DEFF Research Database (Denmark)

    Wurtele, Hugo; Kaiser, Gitte Schalck; Bacal, Julien;

    2012-01-01

    but are only mildly affected by hydroxyurea. We demonstrate that, after exposure to MMS, H3K56ac-deficient cells cannot complete DNA replication and eventually segregate chromosomes with intranuclear foci containing the recombination protein Rad52. In addition, we provide evidence that these phenotypes......In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) occurs in newly synthesized histones that are deposited throughout the genome during DNA replication. Defects in H3K56ac sensitize cells to genotoxic agents, suggesting that this modification plays an important role in the DNA...... damage response. However, the links between histone acetylation, the nascent chromatin structure, and the DNA damage response are poorly understood. Here we report that cells devoid of H3K56ac are sensitive to DNA damage sustained during transient exposure to methyl methanesulfonate (MMS) or camptothecin...

  13. Qualitative and quantitative analysis of lysine acetylation and methylation in yeast histone H3

    Science.gov (United States)

    Zhang, Kangling

    2008-01-01

    Histone post-translational modifications play important roles in cell functions and the modification patterns vary significantly among different organisms. It is important that histone modification patterns be identified. Flowing our previous work-identification of acetylation and methylation sites of histone H3 in a typical transcription most inactive chromatin isolated from chicken erythrocytes, here, we report using mass spectrometry to qualitatively and quantitatively analyze histone modification pattern of H3 in a typical transcription most active chromatin isolated from Saccharomyces cerevisiae. We compared the modification patterns of histone H3 between these two functionally opposite chromatins and observed that acetylation level at K9, K14, K27, K56 and methylation level at K4 and K79 are significantly higher in S. cerevisiae than in chicken erythrocytes, methylation at K9 is higher in chicken erythrocytes than in S. cerevisiae and methylation level at K36 is unchanged in these two chromatins. Contrary to other sites, acetylation levels at K18 and K23 are higher in chicken erythrocytes than in S. cerevisiae. Our data revealed the difference of acetylation and methylation pattern of individual H3 lysine between two distinct chromatins, one with more inactive form versus the other with more active form.

  14. HIF1α protein stability is increased by acetylation at lysine 709.

    Science.gov (United States)

    Geng, Hao; Liu, Qiong; Xue, Changhui; David, Larry L; Beer, Tomasz M; Thomas, George V; Dai, Mu-Shui; Qian, David Z

    2012-10-12

    Lysine acetylation regulates protein stability and function. p300 is a component of the HIF-1 transcriptional complex and positively regulates the transactivation of HIF-1. Here, we show a novel molecular mechanism by which p300 facilitates HIF-1 activity. p300 increases HIF-1α (HIF1α) protein acetylation and stability. The regulation can be opposed by HDAC1, but not by HDAC3, and is abrogated by disrupting HIF1α-p300 interaction. Mechanistically, p300 specifically acetylates HIF1α at Lys-709, which increases the protein stability and decreases polyubiquitination in both normoxia and hypoxia. Compared with the wild-type protein, a HIF1α K709A mutant protein is more stable, less polyubiquitinated, and less dependent on p300. Overexpression of the HIF1α wild-type or K709A mutant in cancer cells lacking the endogenous HIF1α shows that the K709A mutant is transcriptionally more active toward the HIF-1 reporter and some endogenous target genes. Cancer cells containing the K709A mutant are less sensitive to hypoxia-induced growth arrest than the cells containing the HIF1α wild-type. Taken together, these data demonstrate a novel biological consequence upon HIF1α-p300 interaction, in which HIF1α can be stabilized by p300 via Lys-709 acetylation.

  15. Inhibition of the acetyl lysine-binding pocket of bromodomain and extraterminal domain proteins interferes with adipogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Goupille, Olivier [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Penglong, Tipparat [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Thalassemia Research Center, Mahidol University (Thailand); Kadri, Zahra; Granger-Locatelli, Marine [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Fucharoen, Suthat [Thalassemia Research Center, Mahidol University (Thailand); Maouche-Chrétien, Leila [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); INSERM, Paris (France); Prost, Stéphane [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Leboulch, Philippe [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Thalassemia Research Center, Mahidol University (Thailand); Chrétien, Stany, E-mail: stany.chretien@cea.fr [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); INSERM, Paris (France)

    2016-04-15

    The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors. - Highlights: • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into white adipocytes. • JQ1 affected clonal cell expansion and abolished lipid accumulation. • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into brown adipocytes. • JQ1 treatment did not lead to the conversion of white adipose tissue into brown adipose tissue. • JQ1 decreased STAT5 expression, but STAT5B{sup ca} expression did not restore adipogenesis.

  16. Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription

    Science.gov (United States)

    Di Cerbo, Vincenzo; Mohn, Fabio; Ryan, Daniel P; Montellier, Emilie; Kacem, Salim; Tropberger, Philipp; Kallis, Eleni; Holzner, Monika; Hoerner, Leslie; Feldmann, Angelika; Richter, Florian Martin; Bannister, Andrew J; Mittler, Gerhard; Michaelis, Jens; Khochbin, Saadi; Feil, Robert; Schuebeler, Dirk; Owen-Hughes, Tom; Daujat, Sylvain; Schneider, Robert

    2014-01-01

    Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demonstrate that lateral surface modifications can define functionally opposing chromatin states. DOI: http://dx.doi.org/10.7554/eLife.01632.001 PMID:24668167

  17. Lysine Succinylation Is a Frequently Occurring Modification in Prokaryotes and Eukaryotes and Extensively Overlaps with Acetylation

    Directory of Open Access Journals (Sweden)

    Brian T. Weinert

    2013-08-01

    Full Text Available Recent studies have shown that lysines can be posttranslationally modified by various types of acylations. However, except for acetylation, very little is known about their scope and cellular distribution. We mapped thousands of succinylation sites in bacteria (E. coli, yeast (S. cerevisiae, human (HeLa cells, and mouse liver tissue, demonstrating widespread succinylation in diverse organisms. A majority of succinylation sites in bacteria, yeast, and mouse liver were acetylated at the same position. Quantitative analysis of succinylation in yeast showed that succinylation was globally altered by growth conditions and mutations that affected succinyl-coenzyme A (succinyl-CoA metabolism in the tricarboxylic acid cycle, indicating that succinylation levels are globally affected by succinyl-CoA concentration. We preferentially detected succinylation on abundant proteins, suggesting that succinylation occurs at a low level and that many succinylation sites remain unidentified. These data provide a systems-wide view of succinylation and its dynamic regulation and show its extensive overlap with acetylation.

  18. Mitochondrial localization of fission yeast manganese superoxide dismutase is required for its lysine acetylation and for cellular stress resistance and respiratory growth

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Hidekazu, E-mail: hidetakahashi@riken.jp [Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Suzuki, Takehiro [Biomolecular Characterization Team, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); CREST Research Project, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012 (Japan); Shirai, Atsuko; Matsuyama, Akihisa [Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Dohmae, Naoshi [Biomolecular Characterization Team, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); CREST Research Project, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012 (Japan); Yoshida, Minoru, E-mail: yoshidam@riken.jp [Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); CREST Research Project, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012 (Japan)

    2011-03-04

    Research highlights: {yields} Fission yeast manganese superoxide dismutase (MnSOD) is acetylated. {yields} The mitochondrial targeting sequence (MTS) is required for the acetylation of MnSOD. {yields} The MTS is not crucial for MnSOD activity, but is important for respiratory growth. {yields} Posttranslational regulation of MnSOD differs between budding and fission yeast. -- Abstract: Manganese-dependent superoxide dismutase (MnSOD) is localized in the mitochondria and is important for oxidative stress resistance. Although transcriptional regulation of MnSOD has been relatively well studied, much less is known about the protein's posttranslational regulation. In budding yeast, MnSOD is activated after mitochondrial import by manganese ion incorporation. Here we characterize posttranslational modification of MnSOD in the fission yeast Schizosaccharomyces pombe. Fission yeast MnSOD is acetylated at the 25th lysine residue. This acetylation was diminished by deletion of N-terminal mitochondrial targeting sequence, suggesting that MnSOD is acetylated after import into mitochondria. Mitochondrial localization of MnSOD is not essential for the enzyme activity, but is crucial for oxidative stress resistance and growth under respiratory conditions of fission yeast. These results suggest that, unlike the situation in budding yeast, S. pombe MnSOD is already active even before mitochondrial localization; nonetheless, mitochondrial localization is critical to allow the cell to cope with reactive oxygen species generated inside or outside of mitochondria.

  19. Immobilization of Antibodies on Magnetic Carbonaceous Microspheres for Selective Enrichment of Lysine-acetylated Proteins and Peptides

    Institute of Scientific and Technical Information of China (English)

    王莹寅; 姚望; 杨芃原; 邓春晖; 樊惠芝

    2012-01-01

    Lysine acetylation is a dynamic and reversible modification, which has been proved to be a key posttransla- tional modification in cellular regulation. However, the low amounts of the acetylated proteins could hardly be de- tected before enrichment. In this study, for the first time, antibody-immobilized magnetic carbonaceous micro- spheres were developed for selective enrichment of acetylated proteins and peptides. At first, standard proteins composed of acetylated bovine serum albumin, myoglobin, a-casein and ovalbumin were used as model proteins to verify the enrichment efficiency. Then, the synthesized peptide was employed to confirm the selectivity of the method. Besides, the antibody-immobilized magnetic particles were successfully applied to analyze mouse mito- chondrial proteins. After database search, 29 acetylated sites in 26 proteins were identified.

  20. SOD1 Lysine 123 Acetylation in the Adult Central Nervous System

    Science.gov (United States)

    Kaliszewski, Michael; Kennedy, Austin K.; Blaes, Shelby L.; Shaffer, Robert S.; Knott, Andrew B.; Song, Wenjun; Hauser, Henry A.; Bossy, Blaise; Huang, Ting-Ting; Bossy-Wetzel, Ella

    2016-01-01

    Superoxide dismutase 1 (SOD1) knockout (Sod1−/−) mice exhibit an accelerated aging phenotype. In humans, SOD1 mutations are linked to familial amyotrophic lateral sclerosis (ALS), and post-translational modification (PTM) of wild-type SOD1 has been associated with sporadic ALS. Reversible acetylation regulates many enzymes and proteomic studies have identified SOD1 acetylation at lysine 123 (K123). The function and distribution of K123-acetylated SOD1 (Ac-K123 SOD1) in the nervous system is unknown. Here, we generated polyclonal rabbit antibodies against Ac-K123 SOD1. Sod1 deletion in Sod1−/− mice, K123 mutation or preabsorption with Ac-K123 peptide all abolished antibody binding. Using immunohistochemistry, we assessed Ac-K123 SOD1 distribution in the normal adult mouse nervous system. In the cerebellum, Ac-K123 SOD1 staining was prominent in cell bodies of the granular cell layer (GCL) and Purkinje cell dendrites and interneurons of the molecular cell layer. In the hippocampus, Ac-K123 SOD1 staining was strong in the fimbria, subiculum, pyramidal cells and Schaffer collateral fibers of the cornus ammonis field 1 (CA1) region and granule and neuronal progenitor cells of the dentate gyrus. In addition, labeling was observed in the choroid plexus (CP) and the ependyma of the brain ventricles and central canal of the spinal cord. In the olfactory bulb, Ac-K123 SOD1 staining was prominent in axons of sensory neurons, in cell bodies of interneurons and neurites of the mitral and tufted cells. In the retina, labeling was strong in the retinal ganglion cell layer (RGCL) and axons of retinal ganglion cells (RGCs), the inner nuclear layer (INL) and cone photoreceptors of the outer nuclear layer (ONL). In summary, our findings describe Ac-K123 SOD1 distribution to distinct regions and cell types of the normal nervous system. PMID:28066183

  1. Lysine acetylation is a widespread protein modification for diverse proteins in Arabidopsis.

    Science.gov (United States)

    Wu, Xia; Oh, Man-Ho; Schwarz, Eliezer M; Larue, Clayton T; Sivaguru, Mayandi; Imai, Brian S; Yau, Peter M; Ort, Donald R; Huber, Steven C

    2011-04-01

    Lysine acetylation (LysAc), a form of reversible protein posttranslational modification previously known only for histone regulation in plants, is shown to be widespread in Arabidopsis (Arabidopsis thaliana). Sixty-four Lys modification sites were identified on 57 proteins, which operate in a wide variety of pathways/processes and are located in various cellular compartments. A number of photosynthesis-related proteins are among this group of LysAc proteins, including photosystem II (PSII) subunits, light-harvesting chlorophyll a/b-binding proteins (LHCb), Rubisco large and small subunits, and chloroplastic ATP synthase (β-subunit). Using two-dimensional native green/sodium dodecyl sulfate gels, the loosely PSII-bound LHCb was separated from the LHCb that is tightly bound to PSII and shown to have substantially higher level of LysAc, implying that LysAc may play a role in distributing the LHCb complexes. Several potential LysAc sites were identified on eukaryotic elongation factor-1A (eEF-1A) by liquid chromatography/mass spectrometry and using sequence- and modification-specific antibodies the acetylation of Lys-227 and Lys-306 was established. Lys-306 is contained within a predicted calmodulin-binding sequence and acetylation of Lys-306 strongly inhibited the interactions of eEF-1A synthetic peptides with calmodulin recombinant proteins in vitro. These results suggest that LysAc of eEF-1A may directly affect regulatory properties and localization of the protein within the cell. Overall, these findings reveal the possibility that reversible LysAc may be an important and previously unknown regulatory mechanism of a large number of nonhistone proteins affecting a wide range of pathways and processes in Arabidopsis and likely in all plants.

  2. SOD1 Lysine 123 Acetylation in the Adult Central Nervous System

    Directory of Open Access Journals (Sweden)

    Michael Kaliszewski

    2016-12-01

    Full Text Available Superoxide dismutase 1 (SOD1 knockout (Sod1-/- mice exhibit an accelerated aging phenotype. In humans, SOD1 mutations are linked to familial amyotrophic lateral sclerosis (ALS, and post-translational modification (PTM of wild-type SOD1 has been associated with sporadic ALS. Reversible acetylation regulates many enzymes and proteomic studies have identified SOD1 acetylation at lysine 123 (K123. The function and distribution of K123-acetylated SOD1 (Ac-K123 SOD1 in the nervous system is unknown. Here, we generated polyclonal rabbit antibodies against Ac-K123 SOD1. Sod1 deletion in Sod1-/- mice, K123 mutation, or preabsorption with Ac-K123 peptide all abolished antibody binding. Using immunohistochemistry, we assessed Ac-K123 SOD1 distribution in the normal adult mouse nervous system. In the cerebellum, Ac-K123 SOD1 staining was prominent in cell bodies of the granular cell layer and Purkinje cell dendrites and interneurons of the molecular cell layer. In the hippocampus, Ac-K123 SOD1 staining was strong in the fimbria, subiculum, pyramidal cells, and Schaffer collateral fibers of the cornus ammonis (CA1 region and granule and neuronal progenitor cells of the dentate gyrus. In addition, labeling was observed in the choroid plexus and the ependyma of the brain ventricles and central canal of the spinal cord. In the olfactory bulb, Ac-K123 SOD1 staining was prominent in axons of sensory neurons, in cell bodies of interneurons, and neurites of the mitral and tufted cells. In the retina, labeling was strong in the retinal ganglion cell layer and axons of retinal ganglion cells, the inner nuclear layer, and cone photoreceptors of the outer nuclear layer. In summary, our findings describe Ac-K123 SOD1 distribution to distinct regions and cell types of the normal nervous system.

  3. Lysine residue 185 of Rad1 is a topological but not a functional counterpart of lysine residue 164 of PCNA.

    Directory of Open Access Journals (Sweden)

    Niek Wit

    Full Text Available Monoubiquitylation of the homotrimeric DNA sliding clamp PCNA at lysine residue 164 (PCNA(K164 is a highly conserved, DNA damage-inducible process that is mediated by the E2/E3 complex Rad6/Rad18. This ubiquitylation event recruits translesion synthesis (TLS polymerases capable of replicating across damaged DNA templates. Besides PCNA, the Rad6/Rad18 complex was recently shown in yeast to ubiquitylate also 9-1-1, a heterotrimeric DNA sliding clamp composed of Rad9, Rad1, and Hus1 in a DNA damage-inducible manner. Based on the highly similar crystal structures of PCNA and 9-1-1, K185 of Rad1 (Rad1(K185 was identified as the only topological equivalent of PCNA(K164. To investigate a potential role of posttranslational modifications of Rad1(K185 in DNA damage management, we here generated a mouse model with a conditional deletable Rad1(K185R allele. The Rad1(K185 residue was found to be dispensable for Chk1 activation, DNA damage survival, and class switch recombination of immunoglobulin genes as well as recruitment of TLS polymerases during somatic hypermutation of immunoglobulin genes. Our data indicate that Rad1(K185 is not a functional counterpart of PCNA(K164.

  4. Histone acetylation dependent energy landscapes in tri-nucleosome revealed by residue-resolved molecular simulations

    Science.gov (United States)

    Chang, Le; Takada, Shoji

    2016-01-01

    Histone tail acetylation is a key epigenetic marker that tends to open chromatin folding and activate transcription. Despite intensive studies, precise roles of individual lysine acetylation in chromatin folding have only been poorly understood. Here, we revealed structural dynamics of tri-nucleosomes with several histone tail acetylation states and analyzed histone tail interactions with DNA by performing molecular simulations at an unprecedentedly high resolution. We found versatile acetylation-dependent landscapes of tri-nucleosome. The H4 and H2A tail acetylation reduced the contact between the first and third nucleosomes mediated by the histone tails. The H3 tail acetylation reduced its interaction with neighboring linker DNAs resulting in increase of the distance between consecutive nucleosomes. Notably, two copies of the same histone in a single nucleosome have markedly asymmetric interactions with DNAs, suggesting specific pattern of nucleosome docking albeit high inherent flexibility. Estimated transcription factor accessibility was significantly high for the H4 tail acetylated structures. PMID:27698366

  5. Acetylome analysis reveals the involvement of lysine acetylation in photosynthesis and carbon metabolism in the model cyanobacterium Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Mo, Ran; Yang, Mingkun; Chen, Zhuo; Cheng, Zhongyi; Yi, Xingling; Li, Chongyang; He, Chenliu; Xiong, Qian; Chen, Hui; Wang, Qiang; Ge, Feng

    2015-02-06

    Cyanobacteria are the oldest known life form inhabiting Earth and the only prokaryotes capable of performing oxygenic photosynthesis. Synechocystis sp. PCC 6803 (Synechocystis) is a model cyanobacterium used extensively in research on photosynthesis and environmental adaptation. Posttranslational protein modification by lysine acetylation plays a critical regulatory role in both eukaryotes and prokaryotes; however, its extent and function in cyanobacteria remain unexplored. Herein, we performed a global acetylome analysis on Synechocystis through peptide prefractionation, antibody enrichment, and high accuracy LC-MS/MS analysis; identified 776 acetylation sites on 513 acetylated proteins; and functionally categorized them into an interaction map showing their involvement in various biological processes. Consistent with previous reports, a large fraction of the acetylation sites are present on proteins involved in cellular metabolism. Interestingly, for the first time, many proteins involved in photosynthesis, including the subunits of phycocyanin (CpcA, CpcB, CpcC, and CpcG) and allophycocyanin (ApcA, ApcB, ApcD, ApcE, and ApcF), were found to be lysine acetylated, suggesting that lysine acetylation may play regulatory roles in the photosynthesis process. Six identified acetylated proteins associated with photosynthesis and carbon metabolism were further validated by immunoprecipitation and Western blotting. Our data provide the first global survey of lysine acetylation in cyanobacteria and reveal previously unappreciated roles of lysine acetylation in the regulation of photosynthesis. The provided data set may serve as an important resource for the functional analysis of lysine acetylation in cyanobacteria and facilitate the elucidation of the entire metabolic networks and photosynthesis process in this model cyanobacterium.

  6. 赖氨酸乙酰化作用:更为广泛的蛋白调控方式%Lysine acetylation, a more prevalent posttranslational regulation of protein function

    Institute of Scientific and Technical Information of China (English)

    黄的; 张华凤

    2012-01-01

    Acetylation of proteins on lysine residues,including non-histones lysine acetylation, is a prevalent and reversible posttranslational modification. Technological limitations in this field have long impeded the progress in analysis of lysine acetylation's cellular roles. In the past several years,however,quite a number of non-histones lysine acetylation have been brought to light,largely due to the maturation of detection technologies such as high-resolution mass spectrometry and label-free quantification (LFQ). Although the molecular mechanisms underlying cellular regulation of lysine acetylation remain elusive and the detection of highly dynamic lysine acetylation is still a challenge,mounting documented evidence has demonstrated that lysine acetylation is widely involved in such cellular biological activities as cell growth,apoptosis,cytokinetics and cell metabolisms. In this review,we present the progression in this field as well as our current understanding of this modification,starting with the developing detection technologies of lysine acetylation. We also highlight the function of lysine acetylation in the regulation of gene transcription,energy metabolism,cancer development as well as its therapeutic implications.%蛋白质赖氨酸残基上的乙酰化修饰,包括非组蛋白赖氨酸的乙酰化修饰,是一种普遍存在的可逆性翻译后修饰作用,然而检测技术上的限制一直阻碍着赖氨酸乙酰化修饰在细胞中的功能解析和研究.随着赖氨酸乙酰化检测技术的不断成熟,现已发现大量的非组蛋白存在赖氨酸乙酰化修饰的现象.目前,调控细胞内赖氨酸乙酰化的分子机制还不十分清楚,对于活体内高度动态的赖氨酸乙酰化修饰的捕捉尚存困难,但已有越来越多的证据表明,赖氨酸乙酰化修饰广泛地参与细胞的生长、凋亡、动力学、能量代谢等生理活动过程.本文以不断发展的赖氨酸检测技术为出发点,介绍非组蛋白

  7. Dynamic acetylation of all lysine 4-methylated histone H3 in the mouse nucleus: analysis at c-fos and c-jun.

    Directory of Open Access Journals (Sweden)

    Catherine A Hazzalin

    2005-12-01

    Full Text Available A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4-methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9-methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9-methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.

  8. Dynamic acetylation of all lysine 4-methylated histone H3 in the mouse nucleus: analysis at c-fos and c-jun.

    Directory of Open Access Journals (Sweden)

    2005-12-01

    Full Text Available A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4-methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9-methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9-methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.

  9. Progressive mitochondrial protein lysine acetylation and heart failure in a model of Friedreich's ataxia cardiomyopathy.

    Science.gov (United States)

    Stram, Amanda R; Wagner, Gregory R; Fogler, Brian D; Pride, P Melanie; Hirschey, Matthew D; Payne, R Mark

    2017-01-01

    The childhood heart disease of Friedreich's Ataxia (FRDA) is characterized by hypertrophy and failure. It is caused by loss of frataxin (FXN), a mitochondrial protein involved in energy homeostasis. FRDA model hearts have increased mitochondrial protein acetylation and impaired sirtuin 3 (SIRT3) deacetylase activity. Protein acetylation is an important regulator of cardiac metabolism and loss of SIRT3 increases susceptibility of the heart to stress-induced cardiac hypertrophy and ischemic injury. The underlying pathophysiology of heart failure in FRDA is unclear. The purpose of this study was to examine in detail the physiologic and acetylation changes of the heart that occur over time in a model of FRDA heart failure. We predicted that increased mitochondrial protein acetylation would be associated with a decrease in heart function in a model of FRDA. A conditional mouse model of FRDA cardiomyopathy with ablation of FXN (FXN KO) in the heart was compared to healthy controls at postnatal days 30, 45 and 65. We evaluated hearts using echocardiography, cardiac catheterization, histology, protein acetylation and expression. Acetylation was temporally progressive and paralleled evolution of heart failure in the FXN KO model. Increased acetylation preceded detectable abnormalities in cardiac function and progressed rapidly with age in the FXN KO mouse. Acetylation was also associated with cardiac fibrosis, mitochondrial damage, impaired fat metabolism, and diastolic and systolic dysfunction leading to heart failure. There was a strong inverse correlation between level of protein acetylation and heart function. These results demonstrate a close relationship between mitochondrial protein acetylation, physiologic dysfunction and metabolic disruption in FRDA hypertrophic cardiomyopathy and suggest that abnormal acetylation contributes to the pathophysiology of heart disease in FRDA. Mitochondrial protein acetylation may represent a therapeutic target for early intervention.

  10. In Bacillus subtilis, the SatA (formerly YyaR) acetyltransferase detoxifies streptothricin via lysine acetylation.

    Science.gov (United States)

    Burckhardt, Rachel M; Escalante-Semerena, Jorge C

    2017-08-25

    Soil is a complex niche, where survival of microorganisms is at risk due to the presence of antimicrobial agents. Many microbes chemically modify cytotoxic compounds to block their deleterious effects. Streptothricin is a broad-spectrum antibiotic produced by streptomycetes that affects Gram-positive and Gram-negative bacteria alike. Here we identify the SatA (for streptothricin acetyltransferase A, formerly YyaR) enzyme of Bacillus subtilis as the mechanism used by this soil bacterium to detoxify streptothricin. B. subtilis strains lacking satA were susceptible to streptothricin. Ectopic expression of satA(+) restored streptothricin resistance to B. subtilis satA strains. Purified BsSatA acetylated streptothricin in vitro at the expense of acetyl-CoA. A single acetyl moiety transferred onto streptothricin by SatA blocked the toxic effects of the antibiotic. SatA bound streptothricin with high affinity (Kd = 1 μM), and did not bind acetyl-CoA in the absence of streptothricin. Expression of B. subtilis satA(+) in Salmonella enterica conferred streptothricin resistance, indicating that SatA was necessary and sufficient to detoxify streptothricin. Using this heterologous system, we showed that the SatA homologue from Bacillus anthracis also had streptothricin acetyltransferase activity. Our data highlight the physiological relevance of lysine acetylation for the survival of B. subtilis in the soil.Importance Experimental support is provided for the functional assignment of gene products of the soil-dwelling bacilli Bacillus subtilis and Bacillus anthracis This study focuses on one enzyme that is necessary and sufficient to block the cytotoxic effects of a common soil antibiotic. The alluded enzyme is a member of a family of proteins that is broadly distributed in all domains of life, but poorly studied in B. subtilis and B. anthracis. The initial characterization of the enzyme provides insights into its mechanism of catalysis. Copyright © 2017 American Society for

  11. [Nourseothricin (streptothricin) inactivated by plasmid pIE 636-encoded acetyltransferase: detection of N-acetyl-beta-lysine in the inactivated product].

    Science.gov (United States)

    Seltmann, G

    1985-12-01

    Nourseothricin (streptothricin) can be inactivated by an acetyl transferase synthesized by E. coli strains containing plasmid pIE 636. Nourseothricin inactivated in the presence of 14C-acetyl-coenzyme A was purified and submitted to partial acidic hydrolysis. By electrophoresis of the hydrolysate a 14C-containing substance moving only slowly towards the cathode could be isolated. This substance after complete hydrolysis yields only unlabelled beta-lysine.

  12. Studies on the biotin-binding site of avidin. Lysine residues involved in the active site.

    Science.gov (United States)

    Gitlin, G; Bayer, E A; Wilchek, M

    1987-01-01

    Egg-white avidin was treated with 1-fluoro-2,4-dinitrobenzene. Modification of an average of one lysine residue per avidin subunit caused the complete loss of biotin binding. Tryptic peptides obtained from the 2,4-dinitrophenylated avidin were fractionated by reversed-phase h.p.l.c. Three peptides contained the 2,4-dinitrophenyl group. Amino acid analysis revealed that lysine residues 45, 94 and 111 are modified and probably comprise part of the biotin-binding site. PMID:3109401

  13. Studies on the biotin-binding site of avidin. Lysine residues involved in the active site.

    OpenAIRE

    Gitlin, G; Bayer, E A; Wilchek, M

    1987-01-01

    Egg-white avidin was treated with 1-fluoro-2,4-dinitrobenzene. Modification of an average of one lysine residue per avidin subunit caused the complete loss of biotin binding. Tryptic peptides obtained from the 2,4-dinitrophenylated avidin were fractionated by reversed-phase h.p.l.c. Three peptides contained the 2,4-dinitrophenyl group. Amino acid analysis revealed that lysine residues 45, 94 and 111 are modified and probably comprise part of the biotin-binding site.

  14. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae.

    Science.gov (United States)

    Weinert, Brian T; Iesmantavicius, Vytautas; Moustafa, Tarek; Schölz, Christian; Wagner, Sebastian A; Magnes, Christoph; Zechner, Rudolf; Choudhary, Chunaram

    2014-01-01

    Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation accumulated in growth-arrested cells in a manner that depended on acetyl-CoA generation in distinct subcellular compartments. Mitochondrial acetylation levels correlated with acetyl-CoA concentration in vivo and acetyl-CoA acetylated lysine residues nonenzymatically in vitro. We developed a method to estimate acetylation stoichiometry and found that the vast majority of mitochondrial and cytoplasmic acetylation had a very low stoichiometry. However, mitochondrial acetylation occurred at a significantly higher basal level than cytoplasmic acetylation, consistent with the distinct acetylation dynamics and higher acetyl-CoA concentration in mitochondria. High stoichiometry acetylation occurred mostly on histones, proteins present in histone acetyltransferase and deacetylase complexes, and on transcription factors. These data show that a majority of acetylation occurs at very low levels in exponentially growing yeast and is uniformly affected by exposure to acetyl-CoA.

  15. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation

    DEFF Research Database (Denmark)

    Weinert, Brian T; Schölz, Christian; Wagner, Sebastian A;

    2013-01-01

    . cerevisiae), human (HeLa) cells, and mouse liver tissue, demonstrating widespread succinylation in diverse organisms. A majority of succinylation sites in bacteria, yeast, and mouse liver were acetylated at the same position. Quantitative analysis of succinylation in yeast showed that succinylation was globally...

  16. Role of lysine binding residues in the global folding of the lysC riboswitch.

    Science.gov (United States)

    Smith-Peter, Erich; Lamontagne, Anne-Marie; Lafontaine, Daniel A

    2015-01-01

    Riboswitches regulate gene expression by rearranging their structure upon metabolite binding. The lysine-sensing lysC riboswitch is a rare example of an RNA aptamer organized around a 5-way helical junction in which ligand binding is performed exclusively through nucleotides located at the junction core. We have probed whether the nucleotides involved in ligand binding play any role in the global folding of the riboswitch. As predicted, our findings indicate that ligand-binding residues are critical for the lysine-dependent gene regulation mechanism. We also find that these residues are not important for the establishment of key magnesium-dependent tertiary interactions, suggesting that folding and ligand recognition are uncoupled in this riboswitch for the formation of specific interactions. However, FRET assays show that lysine binding results in an additional conformational change, indicating that lysine binding may also participate in a specific folding transition. Thus, in contrast to helical junctions being primary determinants in ribozymes and rRNA folding, we speculate that the helical junction of the lysine-sensing lysC riboswitch is not employed as structural a scaffold to direct global folding, but rather has a different role in establishing RNA-ligand interactions required for riboswitch regulation. Our work suggests that helical junctions may adopt different functions such as the coordination of global architecture or the formation of specific ligand binding site.

  17. Regulation of translesion DNA synthesis: Posttranslational modification of lysine residues in key proteins.

    Science.gov (United States)

    McIntyre, Justyna; Woodgate, Roger

    2015-05-01

    Posttranslational modification of proteins often controls various aspects of their cellular function. Indeed, over the past decade or so, it has been discovered that posttranslational modification of lysine residues plays a major role in regulating translesion DNA synthesis (TLS) and perhaps the most appreciated lysine modification is that of ubiquitination. Much of the recent interest in ubiquitination stems from the fact that proliferating cell nuclear antigen (PCNA) was previously shown to be specifically ubiquitinated at K164 and that such ubiquitination plays a key role in regulating TLS. In addition, TLS polymerases themselves are now known to be ubiquitinated. In the case of human polymerase η, ubiquitination at four lysine residues in its C-terminus appears to regulate its ability to interact with PCNA and modulate TLS. Within the past few years, advances in global proteomic research have revealed that many proteins involved in TLS are, in fact, subject to a previously underappreciated number of lysine modifications. In this review, we will summarize the known lysine modifications of several key proteins involved in TLS; PCNA and Y-family polymerases η, ι, κ and Rev1 and we will discuss the potential regulatory effects of such modification in controlling TLS in vivo.

  18. Critical lysine residues of Klf4 required for protein stabilization and degradation

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Key-Hwan; Kim, So-Ra; Ramakrishna, Suresh; Baek, Kwang-Hyun, E-mail: baek@cha.ac.kr

    2014-01-24

    Highlights: • Klf4 undergoes the 26S proteasomal degradation by ubiquitination on its multiple lysine residues. • Essential Klf4 ubiquitination sites are accumulated between 190–263 amino acids. • A mutation of lysine at 232 on Klf4 elongates protein turnover. • Klf4 mutants dramatically suppress p53 expression both under normal and UV irradiated conditions. - Abstract: The transcription factor, Krüppel-like factor 4 (Klf4) plays a crucial role in generating induced pluripotent stem cells (iPSCs). As the ubiquitination and degradation of the Klf4 protein have been suggested to play an important role in its function, the identification of specific lysine sites that are responsible for protein degradation is of prime interest to improve protein stability and function. However, the molecular mechanism regulating proteasomal degradation of the Klf4 is poorly understood. In this study, both the analysis of Klf4 ubiquitination sites using several Klf4 deletion fragments and bioinformatics predictions showed that the lysine sites which are signaling for Klf4 protein degradation lie in its N-terminal domain (aa 1–296). The results also showed that Lys32, 52, 232, and 252 of Klf4 are responsible for the proteolysis of the Klf4 protein. These results suggest that Klf4 undergoes proteasomal degradation and that these lysine residues are critical for Klf4 ubiquitination.

  19. The Lysine Acetyltransferase GCN5 Is Required for iNKT Cell Development through EGR2 Acetylation

    Directory of Open Access Journals (Sweden)

    Yajun Wang

    2017-07-01

    Full Text Available The development of CD1d-restricted invariant natural killer T (iNKT cells, a population that is critical for both innate and adaptive immunity, is regulated by multiple transcription factors, but the molecular mechanisms underlying how the transcriptional activation of these factors are regulated during iNKT development remain largely unknown. We found that the histone acetyltransferase general control non-derepressible 5 (GCN5 is essential for iNKT cell development during the maturation stage. GCN5 deficiency blocked iNKT cell development in a cell-intrinsic manner. At the molecular level, GCN5 is a specific lysine acetyltransferase of early growth responsive gene 2 (EGR2, a transcription factor required for iNKT cell development. GCN5-mediated acetylation positively regulated EGR2 transcriptional activity, and both genetic and pharmacological GCN5 suppression specifically inhibited the transcription of EGR2 target genes in iNKT cells, including Runx1, promyelocytic leukemia zinc finger protein (PLZF, interleukin (IL-2Rb, and T-bet. Therefore, our study revealed GCN5-mediated EGR2 acetylation as a molecular mechanism that regulates iNKT development.

  20. Obesity increases histone H3 lysine 9 and 18 acetylation at Tnfa and Ccl2 genes in mouse liver.

    Science.gov (United States)

    Mikula, Michal; Majewska, Aneta; Ledwon, Joanna Karolina; Dzwonek, Artur; Ostrowski, Jerzy

    2014-12-01

    Obesity contributes to the development of non-alcoholic fatty liver disease (NAFLD), which is characterized by the upregulated expression of two key inflammatory mediators: tumor necrosis factor (Tnfa) and monocyte chemotactic protein 1 (Mcp1; also known as Ccl2). However, the chromatin make-up at these genes in the liver in obese individuals has not been explored. In this study, to identify obesity-mediated epigenetic changes at Tnfa and Ccl2, we used a murine model of obesity induced by a high-fat diet (HFD) and hyperphagic (ob/ob) mice. Chromatin immunoprecipitation (ChIP) assay was used to determine the abundance of permissive histone marks, namely histone H3 lysine 9 and 18 acetylation (H3K9/K18Ac), H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 36 trimethylation (H3K36me3), in conjunction with polymerase 2 RNA (Pol2) and nuclear factor (Nf)-κB recruitment in the liver. Additionally, to correlate the liver tissue-derived ChIP measurements with a robust in vitro transcriptional response at the Tnfa and Ccl2 genes, we used lipopolysaccharide (LPS) treatment to induce an inflammatory response in Hepa1-6 cells, a cell line derived from murine hepatocytes. ChIP revealed increased H3K9/K18Ac at Tnfa and Ccl2 in the obese mice, although the differences were only statistically significant for Tnfa (pgenes in the obese mice. By contrast, the acute treatment of Hepa1-6 cells with LPS significantly increased the H3K9/K18Ac marks, as well as Pol2 and Nf-κB recruitment at both genes, while the levels of H3K4me3 and H3K36me3 marks remained unaltered. These results demonstrate that increased Tnfa and Ccl2 expression in fatty liver at the chromatin level corresponds to changes in the level of histone H3 acetylation.

  1. Inactivation of eukaryotic initiation factor 5A (eIF5A) by specific acetylation of its hypusine residue by spermidine/spermine acetyltransferase 1 (SSAT1).

    Science.gov (United States)

    Lee, Seung Bum; Park, Jong Hwan; Folk, John E; Deck, Jason A; Pegg, Anthony E; Sokabe, Masaaki; Fraser, Christopher S; Park, Myung Hee

    2011-01-01

    eIF5A (eukaryotic translation initiation factor 5A) is the only cellular protein containing hypusine [Nϵ-(4-amino-2-hydroxybutyl)lysine]. eIF5A is activated by the post-translational synthesis of hypusine and the hypusine modification is essential for cell proliferation. In the present study, we report selective acetylation of the hypusine and/or deoxyhypusine residue of eIF5A by a key polyamine catabolic enzyme SSAT1 (spermidine/spermine-N1-acetyltransferase 1). This enzyme normally catalyses the N1-acetylation of spermine and spermidine to form acetyl-derivatives, which in turn are degraded to lower polyamines. Although SSAT1 has been reported to exert other effects in cells by its interaction with other cellular proteins, eIF5A is the first target protein specifically acetylated by SSAT1. Hypusine or deoxyhypusine, as the free amino acid, does not act as a substrate for SSAT1, suggesting a macromolecular interaction between eIF5A and SSAT1. Indeed, the binding of eIF5A and SSAT1 was confirmed by pull-down assays. The effect of the acetylation of hypusine on eIF5A activity was assessed by comparison of acetylated with non-acetylated bovine testis eIF5A in the methionyl-puromycin synthesis assay. The loss of eIF5A activity by this SSAT1-mediated acetylation confirms the strict structural requirement for the hypusine side chain and suggests a possible regulation of eIF5A by hypusine acetylation/deacetylation.

  2. Role of lysine and acidic amino acid residues on the insecticidal activity of Jackbean urease.

    Science.gov (United States)

    Real-Guerra, Rafael; Carlini, Célia Regina; Stanisçuaski, Fernanda

    2013-09-01

    Canavalia ensiformis has three isoforms of urease: Jackbean urease (JBU), Jackbean urease II and canatoxin. These isoforms present several biological activities, independent from the enzymatic property, such as entomotoxicity and antifungal properties. The entomotoxic activity is a property of the whole protein, as well as of a 10 kDa peptide released by insect digestive enzymes. Here we have used chemical modification to observe the influence of lysines and acidic residues on JBU enzymatic and insecticidal activities. Chemical modification of lysine residues was performed with dimethylamine-borane complex and formaldehyde, and acidic residues were modified by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and ethylenediamine. Derivatized ureases, called JBU-Lys (lysine-modified) and JBU-Ac (acidic residues-modified), were assayed for their biochemical and insecticidal properties. Neither modification altered significantly the kinetic parameters analyzed, indicating that no residue critical for the enzyme activity was affected and that the modifications did not incur in any significant structural alteration. On the other hand, both modifications reduced the toxic activity of the native protein fed to Dysdercus peruvianus. The changes observed in the entomotoxic property of the derivatized proteins reflect alterations in different steps of JBU's toxicity towards insects. JBU-Ac is not susceptible to hydrolysis by insect digestive enzymes, hence impairing the release of toxic peptide(s), while JBU-Lys is processed as the native protein. On the other hand, the antidiuretic effect of JBU on Rhodnius prolixus is altered in JBU-Lys, but not in JBU-Ac. Altogether, these data emphasize the role of lysine and acidic residues on the insecticidal properties of ureases.

  3. SucStruct: Prediction of succinylated lysine residues by using structural properties of amino acids.

    Science.gov (United States)

    López, Yosvany; Dehzangi, Abdollah; Lal, Sunil Pranit; Taherzadeh, Ghazaleh; Michaelson, Jacob; Sattar, Abdul; Tsunoda, Tatsuhiko; Sharma, Alok

    2017-03-28

    Post-Translational Modification (PTM) is a biological reaction which contributes to diversify the proteome. Despite many modifications with important roles in the cellular activity, lysine succinylation has recently emerged as an important PTM mark. It alters the chemical structure of lysines, leading to remarkable changes in the structure and function of proteins. Given the huge amount of proteins being sequenced in the post-genome era, the experimental detection of succinylated residues remains expensive, inefficient and time-consuming. Therefore, the development of computational tools for accurately predicting succinylated lysines is an urgent necessity. To date, several approaches have been proposed but their sensitivity has been reportedly poor. In this paper, we propose an approach that utilizes structural features of amino acids to improve lysine succinylation prediction. Succinylated and non-succinylated lysines were first retrieved from 670 proteins and characteristics such as accessible surface area, backbone torsion angles, and local structure conformations were incorporated. We used the k-nearest neighbors cleaning for dealing with class imbalance and designed a pruned decision tree for classification. Our predictor, referred as SucStruct (Succinylation using Structural features), proved to significantly improve performance when compared to previous predictors, with sensitivity, accuracy and Mathew's correlation coefficient equal to 0.7334-0.7946, 0.7444-0.7608 and 0.4884-0.5240, respectively.

  4. Bioconjugation of Oligodeoxynucleotides Carrying 1,4-Dicarbonyl Groups via Reductive Amination with Lysine Residues.

    Science.gov (United States)

    Yang, Bo; Jinnouchi, Akiko; Usui, Kazuteru; Katayama, Tsutomu; Fujii, Masayuki; Suemune, Hiroshi; Aso, Mariko

    2015-08-19

    We evaluated the efficacy of bioconjugation of oligodeoxynucleotides (ODNs) containing 1,4-dicarbonyl groups, a C4'-oxidized abasic site (OAS), and a newly designed 2'-methoxy analogue, via reductive amination with lysine residues. Dicarbonyls, aldehyde and ketone at C1- and C4-positions of deoxyribose in the ring-opened form of OAS allowed efficient reaction with amines. Kinetic studies indicated that reductive amination of OAS-containing ODNs with a proximal amine on the complementary strand proceeded 10 times faster than the corresponding reaction of an ODN containing an abasic site with C1-aldehyde. Efficient reductive amination between the DNA-binding domain of Escherichia coli DnaA protein and ODNs carrying OAS in the DnaA-binding sequence proceeded at the lysine residue in proximity to the phosphate group at the 5'-position of the OAS, in contrast to unsuccessful conjugation with abasic site ODNs, even though they have similar aldehydes. Theoretical calculation indicated that the C1-aldehyde of OAS was more accessible to the target lysine than that of the abasic site. These results demonstrate the potential utility of cross-linking strategies that use dicarbonyl-containing ODNs for the study of protein-nucleic acid interactions. Conjugation with a lysine-containing peptide that lacked specific affinity for ODN was also successful, further highlighting the advantages of 1,4-dicarbonyls.

  5. Computational prediction of methylation types of covalently modified lysine and arginine residues in proteins.

    Science.gov (United States)

    Deng, Wankun; Wang, Yongbo; Ma, Lili; Zhang, Ying; Ullah, Shahid; Xue, Yu

    2016-05-30

    Protein methylation is an essential posttranslational modification (PTM) mostly occurs at lysine and arginine residues, and regulates a variety of cellular processes. Owing to the rapid progresses in the large-scale identification of methylation sites, the available data set was dramatically expanded, and more attention has been paid on the identification of specific methylation types of modification residues. Here, we briefly summarized the current progresses in computational prediction of methylation sites, which provided an accurate, rapid and efficient approach in contrast with labor-intensive experiments. We collected 5421 methyllysines and methylarginines in 2592 proteins from the literature, and classified most of the sites into different types. Data analyses demonstrated that different types of methylated proteins were preferentially involved in different biological processes and pathways, whereas a unique sequence preference was observed for each type of methylation sites. Thus, we developed a predictor of GPS-MSP, which can predict mono-, di- and tri-methylation types for specific lysines, and mono-, symmetric di- and asymmetrical di-methylation types for specific arginines. We critically evaluated the performance of GPS-MSP, and compared it with other existing tools. The satisfying results exhibited that the classification of methylation sites into different types for training can considerably improve the prediction accuracy. Taken together, we anticipate that our study provides a new lead for future computational analysis of protein methylation, and the prediction of methylation types of covalently modified lysine and arginine residues can generate more useful information for further experimental manipulation.

  6. Nuclear Magnetic Resonance Observation of α-Synuclein Membrane Interaction by Monitoring the Acetylation Reactivity of Its Lysine Side Chains.

    Science.gov (United States)

    Lee, Jung Ho; Ying, Jinfa; Bax, Ad

    2016-09-06

    The interaction between α-synuclein (αS) protein and lipid membranes is key to its role in synaptic vesicle homeostasis and plays a role in initiating fibril formation, which is implicated in Parkinson's disease. The natural state of αS inside the cell is generally believed to be intrinsically disordered, but chemical cross-linking experiments provided evidence of a tetrameric arrangement, which was reported to be rich in α-helical secondary structure based on circular dichroism (CD). Cross-linking relies on chemical modification of the protein's Lys C(ε) amino groups, commonly by glutaraldehyde, or by disuccinimidyl glutarate (DSG), with the latter agent preferred for cellular assays. We used ultra-high-resolution homonuclear decoupled nuclear magnetic resonance experiments to probe the reactivity of the 15 αS Lys residues toward N-succinimidyl acetate, effectively half the DSG cross-linker, which results in acetylation of Lys. The intensities of both side chain and backbone amide signals of acetylated Lys residues provide direct information about the reactivity, showing a difference of a factor of 2.5 between the most reactive (K6) and the least reactive (K102) residue. The presence of phospholipid vesicles decreases reactivity of most Lys residues by up to an order of magnitude at high lipid:protein stoichiometries (500:1), but only weakly at low ratios. The decrease in Lys reactivity is found to be impacted by lipid composition, even for vesicles that yield similar αS CD signatures. Our data provide new insight into the αS-bilayer interaction, including the pivotal state in which the available lipid surface is limited. Protection of Lys C(ε) amino groups by αS-bilayer interaction will strongly impact quantitative interpretation of DSG cross-linking experiments.

  7. Inhibition of N-terminal lysines acetylation and transcription factor assembly by epirubicin induced deranged cell homeostasis.

    Directory of Open Access Journals (Sweden)

    Shahper N Khan

    Full Text Available Epirubicin (EPI, an anthracycline antitumour antibiotic, is a known intercalating and DNA damaging agent. Here, we study the molecular interaction of EPI with histones and other cellular targets. EPI binding with histone core protein was predicted with spectroscopic and computational techniques. The molecular distance r, between donor (histone H3 and acceptor (EPI was estimated using Förster's theory of non-radiation energy transfer and the detailed binding phenomenon is expounded. Interestingly, the concentration dependent reduction in the acetylated states of histone H3 K9/K14 was observed suggesting more repressed chromatin state on EPI treatment. Its binding site near N-terminal lysines is further characterized by thermodynamic determinants and molecular docking studies. Specific DNA binding and inhibition of transcription factor (Tf-DNA complex formation implicates EPI induced transcriptional inhibition. EPI also showed significant cell cycle arrest in drug treated cells. Chromatin fragmentation and loss of membrane integrity in EPI treated cells is suggestive of their commitment to cell death. This study provides an analysis of nucleosome dynamics during EPI treatment and provides a novel insight into its action.

  8. Histone H3 lysine 23 acetylation is associated with oncogene TRIM24 expression and a poor prognosis in breast cancer.

    Science.gov (United States)

    Ma, Li; Yuan, Lili; An, Jing; Barton, Michelle C; Zhang, Qingyuan; Liu, Zhaoliang

    2016-11-01

    Acetylated H3 lysine 23 (H3K23ac) is a specific histone post-translational modification recognized by oncoprotein TRIM24. However, it is not clear whether H3K23ac levels are correlated with TRIM24 expression and what role H3K23ac may have in cancer. In this study, we collected breast carcinoma samples from 121 patients and conducted immunohistochemistry to determine the levels of TRIM24 and H3K23ac in breast cancer. Our results demonstrated that TRIM24 expression is positively correlated with H3K23ac levels, and high levels of both TRIM24 and H3K23ac predict shorter overall survival of breast cancer patients. We also showed that both TRIM24 and H3K23ac are higher in HER2-positive patients, and their levels were positively correlated with HER2 levels in breast cancer. Moreover, TRIM24 expression is associated with estrogen receptor (ER) and progesterone receptor (PR) statuses in both our cohort and The Cancer Genome Atlas (TCGA) breast carcinoma. In summary, our results revealed an important role of TRIM24 and H3K23ac in breast cancer and provided further evidence that TRIM24 small-molecule inhibitors may benefit ER- and PR-negative or HER2-positive breast cancer patients.

  9. Lysine residues in N-terminal and C-terminal regions of human histone H2A are targets for biotinylation by biotinidase.

    Science.gov (United States)

    Chew, Yap Ching; Camporeale, Gabriela; Kothapalli, Nagarama; Sarath, Gautam; Zempleni, Janos

    2006-04-01

    In eukaryotic cell nuclei, DNA associates with the core histones H2A, H2B, H3 and H4 to form nucleosomal core particles. DNA binding to histones is regulated by posttranslational modifications of N-terminal tails (e.g., acetylation and methylation of histones). These modifications play important roles in the epigenetic control of chromatin structure. Recently, evidence that biotinidase and holocarboxylase synthetase (HCS) catalyze the covalent binding of biotin to histones has been provided. The primary aim of this study was to identify biotinylation sites in histone H2A and its variant H2AX. Secondary aims were to determine whether acetylation and methylation of histone H2A affect subsequent biotinylation and whether biotinidase and HCS localize to the nucleus in human cells. Biotinylation sites were identified using synthetic peptides as substrates for biotinidase. These studies provided evidence that K9 and K13 in the N-terminus of human histones H2A and H2AX are targets for biotinylation and that K125, K127 and K129 in the C-terminus of histone H2A are targets for biotinylation. Biotinylation of lysine residues was decreased by acetylation of adjacent lysines but was increased by dimethylation of adjacent arginines. The existence of biotinylated histone H2A in vivo was confirmed by using modification-specific antibodies. Antibodies to biotinidase and HCS localized primarily to the nuclear compartment, consistent with a role for these enzymes in regulating chromatin structure. Collectively, these studies have identified five novel biotinylation sites in human histones; histone H2A is unique among histones in that its biotinylation sites include amino acid residues from the C-terminus.

  10. Effects of lysine residues on structural characteristics and stability of tau proteins

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Myeongsang; Baek, Inchul; Choi, Hyunsung; Kim, Jae In; Na, Sungsoo, E-mail: nass@korea.ac.kr

    2015-10-23

    Pathological amyloid proteins have been implicated in neuro-degenerative diseases, specifically Alzheimer's, Parkinson's, Lewy-body diseases and prion related diseases. In prion related diseases, functional tau proteins can be transformed into pathological agents by environmental factors, including oxidative stress, inflammation, Aβ-mediated toxicity and covalent modification. These pathological agents are stable under physiological conditions and are not easily degraded. This un-degradable characteristic of tau proteins enables their utilization as functional materials to capturing the carbon dioxides. For the proper utilization of amyloid proteins as functional materials efficiently, a basic study regarding their structural characteristic is necessary. Here, we investigated the basic tau protein structure of wild-type (WT) and tau proteins with lysine residues mutation at glutamic residue (Q2K) on tau protein at atomistic scale. We also reported the size effect of both the WT and Q2K structures, which allowed us to identify the stability of those amyloid structures. - Highlights: • Lysine mutation effect alters the structure conformation and characteristic of tau. • Over the 15 layers both WT and Q2K models, both tau proteins undergo fractions. • Lysine mutation causes the increment of non-bonded energy and solvent accessible surface area. • Structural instability of Q2K model was proved by the number of hydrogen bonds analysis.

  11. Functional importance of motif I of pseudouridine synthases: mutagenesis of aligned lysine and proline residues.

    Science.gov (United States)

    Spedaliere, C J; Hamilton, C S; Mueller, E G

    2000-08-01

    On the basis of sequence alignments, the pseudouridine synthases were grouped into four families that share no statistically significant global sequence similarity, though some common sequence motifs were discovered [Koonin, E. V. (1996) Nucleic Acids. Res. 24, 2411-2415; Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762]. We have investigated the functional significance of these alignments by substituting the nearly invariant lysine and proline residues in Motif I of RluA and TruB, pseudouridine synthases belonging to different families. Contrary to our expectations, the altered enzymes display only very mild kinetic impairment. Substitution of the aligned lysine and proline residues does, however, reduce structural stability, consistent with a temperature sensitive phenotype that results from substitution of the cognate proline residue in Cbf5p, a yeast homologue of TruB [Zerbarjadian, Y., King, T., Fournier, M. J., Clarke, L., and Carbon, J. (1999) Mol. Cell. Biol. 19, 7461-7472]. Together, our data support a functional role for Motif I, as predicted by sequence alignments, though the effect of substituting the highly conserved residues was milder than we anticipated. By extrapolation, our findings also support the assignment of pseudouridine synthase function to certain physiologically important eukaryotic proteins that contain Motif I, including the human protein dyskerin, alteration of which leads to the disease dyskeratosis congenita.

  12. Structure-function relationships in scorpion neurotoxins. Identification of the supperreactive lysine residue in toxin I of Androctonus australis Hector.

    Science.gov (United States)

    Sampieri, F; Habersetzer-Rochat, C

    1978-07-21

    In a previous article (Habersetzer-Rochat, C. and Sampieri, R. (1976) Biochemistry 15, 2254--2261) it was demonstrated that the toxin I of the North African Scorpion Androctonus australis Hector was inactivated after reaction with iodoacetate; the toxicity loss in mice was correlated with the carboxymethylation of one superreactive residue. In the present work, alkylation of toxin I was performed with iodo[14C]-acetate. Hence, it was possible, after reduction, S-methylation and chymotryptic hydrolysis of this toxin, to isolate the peptide containing the labelled lysine residue. By automatic Edman degradation, this residue was identified as being the penultimate lysine at position 56 in the primary sequence. Comparison of three primary structures of scorpion neurotoxins and comparison in different kinds of activity seem to indicate that this lysine residue is mainly important for toxicity in mice.

  13. Superoxide reduction by a superoxide reductase lacking the highly conserved lysine residue.

    Science.gov (United States)

    Pinto, Ana F; Romão, Célia V; Pinto, Liliana C; Huber, Harald; Saraiva, Lígia M; Todorovic, Smilja; Cabelli, Diane; Teixeira, Miguel

    2015-01-01

    Superoxide reductases (SORs) are the most recently identified superoxide detoxification systems, being found in microorganisms from the three domains of life. These enzymes are characterized by a catalytic mononuclear iron site, with one cysteine and four histidine ligands of the ferrous active form. A lysine residue in the -EKHVP- motif, located close to the active site, has been considered to be essential for the enzyme function, by contributing to the positive surface patch that attracts the superoxide anion and by controlling the chemistry of the catalytic mechanism through a hydrogen bond network. However, we show here that this residue is substituted by non-equivalent amino acids in several putative SORs from Archaea and unicellular Eukarya. In this work, we focus on mechanistic and spectroscopic studies of one of these less common enzymes, the SOR from the hyperthermophilic Crenarchaeon Ignicoccus hospitalis. We employ pulse radiolysis fast kinetics and spectroscopic approaches to study the wild-type enzyme (-E23T24HVP-), and two mutants, T24K and E23A, the later mimicking enzymes lacking both the lysine and glutamate (a ferric ion ligand) of the motif. The efficiency of the wild-type protein and mutants in reducing superoxide is comparable to other SORs, revealing the robustness of these enzymes to single mutations.

  14. An MRM-based workflow for absolute quantitation of lysine-acetylated metabolic enzymes in mouse liver.

    Science.gov (United States)

    Xu, Leilei; Wang, Fang; Xu, Ying; Wang, Yi; Zhang, Cuiping; Qin, Xue; Yu, Hongxiu; Yang, Pengyuan

    2015-12-07

    As a key post-translational modification mechanism, protein acetylation plays critical roles in regulating and/or coordinating cell metabolism. Acetylation is a prevalent modification process in enzymes. Protein acetylation modification occurs in sub-stoichiometric amounts; therefore extracting biologically meaningful information from these acetylation sites requires an adaptable, sensitive, specific, and robust method for their quantification. In this work, we combine immunoassays and multiple reaction monitoring-mass spectrometry (MRM-MS) technology to develop an absolute quantification for acetylation modification. With this hybrid method, we quantified the acetylation level of metabolic enzymes, which could demonstrate the regulatory mechanisms of the studied enzymes. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of protein acetylation in physiology and pathophysiology.

  15. Progressive mitochondrial protein lysine acetylation and heart failure in a model of Friedreich’s ataxia cardiomyopathy

    Science.gov (United States)

    Stram, Amanda R.; Wagner, Gregory R.; Fogler, Brian D.; Pride, P. Melanie; Hirschey, Matthew D.; Payne, R. Mark

    2017-01-01

    Introduction The childhood heart disease of Friedreich’s Ataxia (FRDA) is characterized by hypertrophy and failure. It is caused by loss of frataxin (FXN), a mitochondrial protein involved in energy homeostasis. FRDA model hearts have increased mitochondrial protein acetylation and impaired sirtuin 3 (SIRT3) deacetylase activity. Protein acetylation is an important regulator of cardiac metabolism and loss of SIRT3 increases susceptibility of the heart to stress-induced cardiac hypertrophy and ischemic injury. The underlying pathophysiology of heart failure in FRDA is unclear. The purpose of this study was to examine in detail the physiologic and acetylation changes of the heart that occur over time in a model of FRDA heart failure. We predicted that increased mitochondrial protein acetylation would be associated with a decrease in heart function in a model of FRDA. Methods A conditional mouse model of FRDA cardiomyopathy with ablation of FXN (FXN KO) in the heart was compared to healthy controls at postnatal days 30, 45 and 65. We evaluated hearts using echocardiography, cardiac catheterization, histology, protein acetylation and expression. Results Acetylation was temporally progressive and paralleled evolution of heart failure in the FXN KO model. Increased acetylation preceded detectable abnormalities in cardiac function and progressed rapidly with age in the FXN KO mouse. Acetylation was also associated with cardiac fibrosis, mitochondrial damage, impaired fat metabolism, and diastolic and systolic dysfunction leading to heart failure. There was a strong inverse correlation between level of protein acetylation and heart function. Conclusion These results demonstrate a close relationship between mitochondrial protein acetylation, physiologic dysfunction and metabolic disruption in FRDA hypertrophic cardiomyopathy and suggest that abnormal acetylation contributes to the pathophysiology of heart disease in FRDA. Mitochondrial protein acetylation may represent a

  16. The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase

    Directory of Open Access Journals (Sweden)

    Rodrigues Valnês

    2009-01-01

    Full Text Available Abstract Background The shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis, the causative agent of tuberculosis, but absent in humans. M. tuberculosis aroE-encoded shikimate dehydrogenase catalyzes the forth reaction in the shikimate pathway. Structural and functional studies indicate that Lysine69 may be involved in catalysis and/or substrate binding in M. tuberculosis shikimate dehydrogenase. Investigation of the kinetic properties of mutant enzymes can bring important insights about the role of amino acid residues for M. tuberculosis shikimate dehydrogenase. Findings We have performed site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements and molecular modeling for both the wild-type M. tuberculosis shikimate dehydrogenase and the K69A mutant enzymes. The apparent steady-state kinetic parameters for the M. tuberculosis shikimate dehydrogenase were determined; the catalytic constant value for the wild-type enzyme (50 s-1 is 68-fold larger than that for the mutant K69A (0.73 s-1. There was a modest increase in the Michaelis-Menten constant for DHS (K69A = 76 μM; wild-type = 29 μM and NADPH (K69A = 30 μM; wild-type = 11 μM. The equilibrium dissociation constants for wild-type and K69A mutant enzymes are 32 (± 4 μM and 134 (± 21, respectively. Conclusion Our results show that the residue Lysine69 plays a catalytic role and is not involved in substrate binding for the M. tuberculosis shikimate dehydrogenase. These efforts on M. tuberculosis shikimate dehydrogenase catalytic mechanism determination should help the rational design of specific inhibitors, aiming at the development of antitubercular drugs.

  17. A conformational study of N -acetyl glucosamine derivatives utilizing residual dipolar couplings

    Science.gov (United States)

    Kramer, Markus; Kleinpeter, Erich

    2011-09-01

    The conformational analyses of six non-rigid N-acetyl glucosamine (NAG) derivatives employing residual dipolar couplings (RDCs) and NOEs together with molecular dynamics (MD) simulations are presented. Due to internal dynamics we had to consider different conformer ratios existing in solution. The good quality of the correlation between theoretically and experimentally obtained RDCs show the correctness of the calculated conformers even if the ratios derived from the MD simulations do not exactly meet the experimental data. If possible, the results were compared to former published data and commented.

  18. Lysine Acetylation Inhibits-Synuclein Fibrillation%乙酰化修饰抑制-synuclein的纤维化聚集

    Institute of Scientific and Technical Information of China (English)

    翟紫凝; 吴琼; 李从刚

    2016-01-01

    天然无结构蛋白a-synuclein(a-syn)的纤维化聚集是帕金森病的特征表现。静电相互作用已被证明会显著影响a-syn 的聚集。该文通过简单的赖氨酸乙酰化修饰改变蛋白的净电荷,研究静电效应对于a-syn 的构象和纤维化聚集的影响。核磁共振(NMR)实验结果表明乙酰化后的a-syn仍然是无序结构,而且展现出比野生型更加伸展的构象。由于N端和C端都高度带负电荷,结构打开会更加暴露NAC区域,静电排斥和疏水作用共同存在,但 ThT 荧光实验发现乙酰化修饰抑制了它的纤维化聚集,因此我们认为这里静电排斥占据主导作用。这种依赖电荷的作用机理会帮助我们更好地理解a-syn的纤维化聚集,而乙酰化修饰也提供了一种抑制聚集的新方法。%Fibrils of intrinsically disordered proteina-synuclein (a-syn) are hallmarks of Parkinson’s disease. Electrostatic interactions are known to contribute significantly ona-syn aggregation. Here we studied howa-syn conformation and fibrillation were affected by changing the net charge of the protein via acetylation of lysine side chains. NMR spectroscopy results showed that lysine-acetylateda-syn remained disordered, and showed a more extended conformation, relative to wild-type protein. Acetylation inhibiteda-syn fibrillation, revealed by thioflavin (ThT) fluorescence assay. The N- and C-terminals of the acetylated protein were highly negative charged, causing increased exposure of the non-amyloid-b component (NAC) region. It is proposed that, with the charge distribution in the acetylated protein, electrostatic repulsion, instead of hydrophobic effect, may contribute predominately to the aggregation. This charge-effect mechanism may constitute a new strategy to inhibita-syn fibrillation.

  19. Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions.

    Science.gov (United States)

    Weinert, Brian T; Moustafa, Tarek; Iesmantavicius, Vytautas; Zechner, Rudolf; Choudhary, Chunaram

    2015-11-03

    Acetylation is frequently detected on mitochondrial enzymes, and the sirtuin deacetylase SIRT3 is thought to regulate metabolism by deacetylating mitochondrial proteins. However, the stoichiometry of acetylation has not been studied and is important for understanding whether SIRT3 regulates or suppresses acetylation. Using quantitative mass spectrometry, we measured acetylation stoichiometry in mouse liver tissue and found that SIRT3 suppressed acetylation to a very low stoichiometry at its target sites. By examining acetylation changes in the liver, heart, brain, and brown adipose tissue of fasted mice, we found that SIRT3-targeted sites were mostly unaffected by fasting, a dietary manipulation that is thought to regulate metabolism through SIRT3-dependent deacetylation. Globally increased mitochondrial acetylation in fasted liver tissue, higher stoichiometry at mitochondrial acetylation sites, and greater sensitivity of SIRT3-targeted sites to chemical acetylation in vitro and fasting-induced acetylation in vivo, suggest a nonenzymatic mechanism of acetylation. Our data indicate that most mitochondrial acetylation occurs as a low-level nonenzymatic protein lesion and that SIRT3 functions as a protein repair factor that removes acetylation lesions from lysine residues.

  20. Differential Contributions of Ubiquitin-Modified APOBEC3G Lysine Residues to HIV-1 Vif-Induced Degradation.

    Science.gov (United States)

    Turner, Tiffany; Shao, Qiujia; Wang, Weiran; Wang, Yudi; Wang, Chenliang; Kinlock, Ballington; Liu, Bindong

    2016-08-28

    Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (A3G) is a host restriction factor that impedes HIV-1 replication. Viral integrity is salvaged by HIV-1 virion infectivity factor (Vif), which mediates A3G polyubiquitination and subsequent cellular depletion. Previous studies have implied that A3G polyubiquitination is essential for Vif-induced degradation. However, the contribution of polyubiquitination to the rate of A3G degradation remains unclear. Here, we show that A3G polyubiquitination is essential for degradation. Inhibition of ubiquitin-activating enzyme E1 by PYR-41 or blocking the formation of ubiquitin chains by over-expressing the lysine to arginine mutation of ubiquitin K48 (K48R) inhibited A3G degradation. Our A3G mutagenesis study showed that lysine residues 297, 301, 303, and 334 were not sufficient to render lysine-free A3G sensitive to Vif-mediated degradation. Our data also confirm that Vif could induce ubiquitin chain formation on lysine residues interspersed throughout A3G. Notably, A3G degradation relied on the lysine residues involved in polyubiquitination. Although A3G and the A3G C-terminal mutant interacted with Vif and were modified by ubiquitin chains, the latter remained more resistant to Vif-induced degradation. Furthermore, the A3G C-terminal mutant, but not the N-terminal mutant, maintained potent antiviral activity in the presence of Vif. Taken together, our results suggest that the location of A3G ubiquitin modification is a determinant for Vif-mediated degradation, implying that in addition to polyubiquitination, other factors may play a key role in the rate of A3G degradation.

  1. Lysine residues at the first and second KTKEGV repeats mediate α-Synuclein binding to membrane phospholipids.

    Science.gov (United States)

    Zarbiv, Yonaton; Simhi-Haham, Dganit; Israeli, Eitan; Elhadi, Suaad Abed; Grigoletto, Jessica; Sharon, Ronit

    2014-10-01

    While α-Synuclein (α-Syn) is mainly detected as a cytosolic protein, a portion of it is recovered bound to membranes. It is suggested that binding to membrane phospholipids controls α-Syn structure, physiology and pathogenesis. We aimed at investigating the role, of the positive charged lysine residues at the KTKEGV repeat motif, in mediating α-Syn associations with membrane phospholipids and in α-Syn oligomerization and aggregation. Specifically, two positive lysine (K) residues were replaced with two negative glutamic acid (E) residues at either the first or second KTKEGV repeat motifs. The effect of these mutations on membrane binding was determined by a quantitative phospholipid ELISA assay and compared to wild-type α-Syn and to the Parkinson's disease-causing mutations, A30P, E46K and A53T. We found that the K to E substitutions affected α-Syn binding to phospholipids. In addition, K to E substitutions resulted in a dramatically lower level of soluble α-Syn oligomers and larger intracellular inclusions. Together, our results suggest a critical role for lysine residues at the N-terminal repeat domain in the pathophysiology of α-Syn.

  2. Acetylation Reader Proteins: Linking Acetylation Signaling to Genome Maintenance and Cancer.

    Science.gov (United States)

    Gong, Fade; Chiu, Li-Ya; Miller, Kyle M

    2016-09-01

    Chromatin-based DNA damage response (DDR) pathways are fundamental for preventing genome and epigenome instability, which are prevalent in cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze the addition and removal of acetyl groups on lysine residues, a post-translational modification important for the DDR. Acetylation can alter chromatin structure as well as function by providing binding signals for reader proteins containing acetyl-lysine recognition domains, including the bromodomain (BRD). Acetylation dynamics occur upon DNA damage in part to regulate chromatin and BRD protein interactions that mediate key DDR activities. In cancer, DDR and acetylation pathways are often mutated or abnormally expressed. DNA damaging agents and drugs targeting epigenetic regulators, including HATs, HDACs, and BRD proteins, are used or are being developed to treat cancer. Here, we discuss how histone acetylation pathways, with a focus on acetylation reader proteins, promote genome stability and the DDR. We analyze how acetylation signaling impacts the DDR in the context of cancer and its treatments. Understanding the relationship between epigenetic regulators, the DDR, and chromatin is integral for obtaining a mechanistic understanding of genome and epigenome maintenance pathways, information that can be leveraged for targeting acetylation signaling, and/or the DDR to treat diseases, including cancer.

  3. Histone Acetylation in Fungal Pathogens of Plants

    Directory of Open Access Journals (Sweden)

    Junhyun Jeon

    2014-03-01

    Full Text Available Acetylation of histone lysine residues occurs in different organisms ranging from yeast to plants and mammals for the regulation of diverse cellular processes. With the identification of enzymes that create or reverse this modification, our understanding on histone acetylation has expanded at an amazing pace during the last two decades. In fungal pathogens of plants, however, the importance of such modification has only just begun to be appreciated in the recent years and there is a dearth of information on how histone acetylation is implicated in fungal pathogenesis. This review covers the current status of research related to histone acetylation in plant pathogenic fungi and considers relevant findings in the interaction between fungal pathogens and host plants. We first describe the families of histone acetyltransferases and deacetylases. Then we provide the cases where histone acetylation was investigated in the context of fungal pathogenesis. Finally, future directions and perspectives in epigenetics of fungal pathogenesis are discussed.

  4. A noncanonical function of sortase enables site-specific conjugation of small molecules to lysine residues in proteins.

    Science.gov (United States)

    Bellucci, Joseph J; Bhattacharyya, Jayanta; Chilkoti, Ashutosh

    2015-01-07

    We provide the first demonstration that isopeptide ligation, a noncanonical activity of the enzyme sortase A, can be used to modify recombinant proteins. This reaction was used in vitro to conjugate small molecules to a peptide, an engineered targeting protein, and a full-length monoclonal antibody with an exquisite level of control over the site of conjugation. Attachment to the protein substrate occurred exclusively through isopeptide bonds at a lysine ε-amino group within a specific amino acid sequence. This reaction allows more than one molecule to be site-specifically conjugated to a protein at internal sites, thereby overcoming significant limitations of the canonical native peptide ligation reaction catalyzed by sortase A. Our method provides a unique chemical ligation procedure that is orthogonal to existing methods, supplying a new method to site-specifically modify lysine residues that will be a valuable addition to the protein conjugation toolbox.

  5. Dynamic changes of histone H3 lysine 27 acetylation in pre-implantational pig embryos derived from somatic cell nuclear transfer.

    Science.gov (United States)

    Zhou, Naru; Cao, Zubing; Wu, Ronghua; Liu, Xing; Tao, Jia; Chen, Zhen; Song, Dandan; Han, Fei; Li, Yunsheng; Fang, Fugui; Zhang, Xiaorong; Zhang, Yunhai

    2014-08-01

    Histone H3 lysine 27 acetylation (H3K27ac) is an active epigenetic modification which has been revealed to be associated with active gene expression. It was hypothesized that H3K27ac might also participate in the porcine somatic reprogramming process during early development of SCNT-derived embryos. The spatial and temporal expression profiles of H3K27ac were investigated at different developmental stages in SCNT embryos compared with in vitro fertilization (IVF) and parthenogenetic activation (PA) counterparts. Specifically, results showed that amounts of H3K27ac gradually decreased from the earliest pronuclear stage to 8-cell stage, corresponding to the major embryonic genome activation (EGA), followed by re-acetylation of H3K27 from the morula stage onwards accompanying the first cell lineage specification in IVF embryos. Similar dynamic patterns of H3K27ac signal was observed at all developmental stages of porcine SCNT and PA embryos except for the hatched stage in which amounts of H3K27ac in SCNT and PA embryos was slightly less than that in IVF counterparts. Moreover, the gradual decrease of H3K27ac before EGA was demonstrated to be an active process independent of DNA replication, RNA and protein synthesis. The expression of HDAC1, HDAC2, MBD3 and CBP genes were well correlated with the dynamic changes of H3K27ac mark. Overall, these results indicate that H3K27ac is only defective in late SCNT blastocysts, and that the dynamic changes of this marker might also underlie the EGA and initial cell lineage specification during early embryo development.

  6. Elicitin-Induced Distal Systemic Resistance in Plants is Mediated Through the Protein-Protein Interactions Influenced by Selected Lysine Residues.

    Science.gov (United States)

    Uhlíková, Hana; Obořil, Michal; Klempová, Jitka; Šedo, Ondrej; Zdráhal, Zbyněk; Kašparovský, Tomáš; Skládal, Petr; Lochman, Jan

    2016-01-01

    Elicitins are a family of small proteins with sterol-binding activity that are secreted by Phytophthora and Pythium sp. classified as oomycete PAMPs. Although α- and β-elicitins bind with the same affinity to one high affinity binding site on the plasma membrane, β-elicitins (possessing 6-7 lysine residues) are generally 50- to 100-fold more active at inducing distal HR and systemic resistance than the α-isoforms (with only 1-3 lysine residues). To examine the role of lysine residues in elicitin biological activity, we employed site-directed mutagenesis to prepare a series of β-elicitin cryptogein variants with mutations on specific lysine residues. In contrast to direct infiltration of protein into leaves, application to the stem revealed a rough correlation between protein's charge and biological activity, resulting in protection against Phytophthora parasitica. A detailed analysis of proteins' movement in plants showed no substantial differences in distribution through phloem indicating differences in consequent apoplastic or symplastic transport. In this process, an important role of homodimer formation together with the ability to form a heterodimer with potential partner represented by endogenous plants LTPs is suggested. Our work demonstrates a key role of selected lysine residues in these interactions and stresses the importance of processes preceding elicitin recognition responsible for induction of distal systemic resistance.

  7. Elicitin-induced distal systemic resistance in plants is mediated through the protein-protein interactions influenced by selected lysine residues

    Directory of Open Access Journals (Sweden)

    Hana eUhlíková

    2016-02-01

    Full Text Available Elicitins are a family of small proteins with sterol-binding activity that are secreted by Phytophthora and Pythium spp. classified as oomycete PAMPs. Although alfa- and beta-elicitins bind with the same affinity to one high affinity binding site on the plasma membrane, beta-elicitins (possessing 6-7 lysine residues are generally 50- to 100-fold more active at inducing distal HR and systemic resistance than the alfa-isoforms (with only 1-3 lysine residues.To examine the role of lysine residues in elicitin biological activity, we employed site-directed mutagenesis to prepare a series of beta-elicitin cryptogein variants with mutations on specific lysine residues. In contrast to direct infiltration of protein into leaves, application to the stem revealed a rough correlation between protein’s charge and biological activity, resulting in protection against Phytophthora parasitica. A detailed analysis of proteins’ movement in plants showed no substantial differences in distribution through phloem indicating differences in consequent apoplastic or symplastic transport. In this process, an important role of homodimer formation together with the ability to form a heterodimer with potential partner represented by endogenous plants LTPs is suggested. Our work demonstrates a key role of selected lysine residues in these interactions and stresses the importance of processes preceding elicitin recognition responsible for induction of distal systemic resistance.

  8. Oxidative deamination of benzylamine and lysine residue in bovine serum albumin by green tea, black tea, and coffee.

    Science.gov (United States)

    Akagawa, Mitsugu; Shigemitsu, Tomoko; Suyama, Kyozo

    2005-10-01

    Oxidative deamination by various polyphenolic compounds is presumed to be due to the oxidative conversion of polyphenols to the corresponding quinones through autoxidation. Here we examined the oxidative deamination by the polyphenol-rich beverages green tea, black tea, and coffee at a physiological pH and temperature. Green tea, black tea, and coffee extracts oxidatively deaminated benzylamine and the lysine residues of bovine serum albumin to benzaldehyde and alpha-aminoadipic delta-semialdehyde residues, respectively, in sodium phosphate buffer (pH 7.4) at 37 degrees C in both the presence and absence of Cu2+, indicating the occurrence of an amine (lysyl) oxidase-like reaction. We also examined the effects of pH and metal ions on the reaction. The possible biological effects of drinking polyphenol-rich beverages on human are also discussed.

  9. Altered acetylation and succinylation profiles in Corynebacterium glutamicum in response to conditions inducing glutamate overproduction.

    Science.gov (United States)

    Mizuno, Yuta; Nagano-Shoji, Megumi; Kubo, Shosei; Kawamura, Yumi; Yoshida, Ayako; Kawasaki, Hisashi; Nishiyama, Makoto; Yoshida, Minoru; Kosono, Saori

    2016-02-01

    The bacterium Corynebacterium glutamicum is utilized during industrial fermentation to produce amino acids such as L-glutamate. During L-glutamate fermentation, C. glutamicum changes the flux of central carbon metabolism to favor L-glutamate production, but the molecular mechanisms that explain these flux changes remain largely unknown. Here, we found that the profiles of two major lysine acyl modifications were significantly altered upon glutamate overproduction in C. glutamicum; acetylation decreased, whereas succinylation increased. A label-free semi-quantitative proteomic analysis identified 604 acetylated proteins with 1328 unique acetylation sites and 288 succinylated proteins with 651 unique succinylation sites. Acetylation and succinylation targeted enzymes in central carbon metabolic pathways that are directly related to glutamate production, including the 2-oxoglutarate dehydrogenase complex (ODHC), a key enzyme regulating glutamate overproduction. Structural mapping revealed that several critical lysine residues in the ODHC components were susceptible to acetylation and succinylation. Furthermore, induction of glutamate production was associated with changes in the extent of acetylation and succinylation of lysine, suggesting that these modifications may affect the activity of enzymes involved in glutamate production. Deletion of phosphotransacetylase decreased the extent of protein acetylation in nonproducing condition, suggesting that acetyl phosphate-dependent acetylation is active in C. glutamicum. However, no effect was observed on the profiles of acetylation and succinylation in glutamate-producing condition upon disruption of acetyl phosphate metabolism or deacetylase homologs. It was considered likely that the reduced acetylation in glutamate-producing condition may reflect metabolic states where the flux through acid-producing pathways is very low, and substrates for acetylation do not accumulate in the cell. Succinylation would occur more

  10. Role of lysine and tryptophan residues in the biological activity of toxin VII (Ts gamma) from the scorpion Tityus serrulatus.

    Science.gov (United States)

    Hassani, O; Mansuelle, P; Cestèle, S; Bourdeaux, M; Rochat, H; Sampieri, F

    1999-02-01

    Toxin VII (TsVII), also known as Ts gamma, is the most potent neurotoxin in the venom of the Brazilian scorpion Tityus serrulatus. It has been purified to homogeneity using a new fast and efficient method. Chemical modification of TsVII with the tryptophan-specific reagent o-nitrophenylsulfenyl chloride yielded three modified derivatives (residues Trp39, Trp50 and Trp54). Acetylation of TsVII mostly generated the monoacetylated Lys12 derivative. No side reactions were detected, as indicated by endoproteinase Lys-C peptide mapping, Edman degradation and electrospray mass spectrometry. Circular dichroism and fluorimetric measurements showed that none of the chemical modifications altered the overall structure of the derivatives. The acetylation of Lys12 or the sulfenylation of Trp39 or Trp54 led to a loss of both toxicity in mice and apparent binding affinity for rat brain and cockroach synaptosomal preparations. Sulfenylation of Trp50, however, moderately affected the toxicity of TsVII in mice and had almost no effect on its binding properties. A 3-dimensional model of TsVII was constructed by homology modeling. It suggests that the most reactive residues (Lys12 and Trp39 and Trp54) are all important in the functional disruption of neuronal sodium channels by TsVII, and are close to each other in the hydrophobic conserved region.

  11. Cold-induced alteration in the global structure of the male sex chromosome of In(1)B$^{M2}$(reinverted) of Drosophila melanogaster is associated with increased acetylation of histone 4 at lysine 16

    Indian Academy of Sciences (India)

    S. Kulkarni-Shukla; A. P. Barge; R. S. Vartak; Anita Kar

    2008-12-01

    In Drosophila melanogaster, dosage compensation occurs through hypertranscription of sex-linked genes in males. The hypertranscription involves acetylation of histone 4 at lysine 16 (H4K16) on amale X-chromosome, brought about by a histone acetyltransferase encoded by the dosage compensation gene, males absent on the first (mof). We report a phenomenon in the strain In(1)B$^{M2}$(reinverted) of D. melanogaster where the global structure of the male X-chromosome can be altered at the third instar larval stage through a 4-h cold shock at 12±1°C. We show that the cold shock results in a transient hyperacetylation of H4K16 and an increased expression of MOF. Control proteins H4 acetylated at lysine 5, and the dosage compensation gene msl-2, do not show any change in expression after cold shock. Cytology of the male X-chromosome at different time points during cold shock and recovery, suggests that the hyperacetylation of H4 at lysine 16 causes the X-chromosome to corkscrew into itself, thereby achieving the cold-induced change in the higher order structure of the male polytene X-chromosome. Our studies suggest a role for H4K16 in maintaining the structure of the male X-chromosome in Drosophila.

  12. Phospho-N-Acetyl-Muramyl-Pentapeptide Translocase from Escherichia coli: Catalytic Role of Conserved Aspartic Acid Residues

    Science.gov (United States)

    Lloyd, Adrian J.; Brandish, Philip E.; Gilbey, Andrea M.; Bugg, Timothy D. H.

    2004-01-01

    Phospho-N-acetyl-muramyl-pentapeptide translocase (translocase 1) catalyzes the first of a sequence of lipid-linked steps that ultimately assemble the peptidoglycan layer of the bacterial cell wall. This essential enzyme is the target of several natural product antibiotics and has recently been the focus of antimicrobial drug discovery programs. The catalytic mechanism of translocase 1 is believed to proceed via a covalent intermediate formed between phospho-N-acetyl-muramyl-pentapeptide and a nucleophilic amino acid residue. Amino acid sequence alignments of the translocase 1 family and members of the related transmembrane phosphosugar transferase superfamily revealed only three conserved residues that possess nucleophilic side chains: the aspartic acid residues D115, D116, and D267. Here we report the expression and partial purification of Escherichia coli translocase 1 as a C-terminal hexahistidine (C-His6) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His6 fusions. Enzymatic analysis established that all three mutations eliminated translocase 1 activity, and this finding verified the essential role of these residues. By analogy with the structural environment of the double aspartate motif found in prenyl transferases, we propose a model whereby D115 and D116 chelate a magnesium ion that coordinates with the pyrophosphate bridge of the UDP-N-acetyl-muramyl-pentapeptide substrate and in which D267 therefore fulfills the role of the translocase 1 active-site nucleophile. PMID:14996806

  13. Lysine methylation: beyond histones

    Institute of Scientific and Technical Information of China (English)

    Xi Zhang; Hong Wen; Xiaobing Shi

    2012-01-01

    Posttranslational modifications (PTMs) of histone proteins,such as acetylation,methylation,phosphorylation,and ubiquitylation,play essential roles in regulating chromatin dynamics.Combinations of different modifications on the histone proteins,termed 'histone code' in many cases,extend the information potential of the genetic code by regulating DNA at the epigenetic level.Many PTMs occur on non-histone proteins as well as histones,regulating protein-protein interactions,stability,localization,and/or enzymatic activities of proteins involved in diverse cellular processes.Although protein phosphorylation,ubiquitylation,and acetylation have been extensively studied,only a few proteins other than histones have been reported that can be modified by lysine methylation.This review summarizes the current progress on lysine methylation of nonhistone proteins,and we propose that lysine methylation,like phosphorylation and acetylation,is a common PTM that regulates proteins in diverse cellular processes.

  14. Molecular Modeling of Differentially Phosphorylated Serine 10 and Acetylated lysine 9/14 of Histone H3 Regulates their Interactions with 14-3-3ζ, MSK1, and MKP1

    Science.gov (United States)

    Sharma, Ajit K.; Mansukh, Abhilasha; Varma, Ashok; Gadewal, Nikhil; Gupta, Sanjay

    2013-01-01

    Histone modifications occur in precise patterns, with several modifications known to affect the binding of proteins. These interactions affect the chromatin structure, gene regulation, and cell cycle events. The dual modifications on the H3 tail, serine10 phosphorylation, and lysine14 acetylation (H3Ser10PLys14Ac) are reported to be crucial for interaction with 14-3-3ζ. However, the mechanism by which H3Ser10P along with neighboring site-specific acetylation(s) is targeted by its regulatory proteins, including kinase and phosphatase, is not fully understood. We carried out molecular modeling studies to understand the interaction of 14-3-3ζ, and its regulatory proteins, mitogen-activated protein kinase phosphatase-1 (MKP1), and mitogen- and stress-activated protein kinase-1 (MSK1) with phosphorylated H3Ser10 alone or in combination with acetylated H3Lys9 and Lys14. In silico molecular association studies suggested that acetylated Lys14 and phosphorylated Ser10 of H3 shows the highest binding affinity towards 14-3-3ζ. In addition, acetylation of H3Lys9 along with Ser10PLys14Ac favors the interaction of the phosphatase, MKP1, for dephosphorylation of H3Ser10P. Further, MAP kinase, MSK1 phosphorylates the unmodified H3Ser10 containing N-terminal tail with maximum affinity compared to the N-terminal tail with H3Lys9AcLys14Ac. The data clearly suggest that opposing enzymatic activity of MSK1 and MKP1 corroborates with non-acetylated and acetylated, H3Lys9Lys14, respectively. Our in silico data highlights that site-specific phosphorylation (H3Ser10P) and acetylation (H3Lys9 and H3Lys14) of H3 are essential for the interaction with their regulatory proteins (MKP1, MSK1, and 14-3-3ζ) and plays a major role in the regulation of chromatin structure. PMID:24027420

  15. Maize root lectins mediate the interaction with Herbaspirillum seropedicae via N-acetyl glucosamine residues of lipopolysaccharides.

    Science.gov (United States)

    Balsanelli, Eduardo; Tuleski, Thalita Regina; de Baura, Valter Antonio; Yates, Marshall Geoffrey; Chubatsu, Leda Satie; Pedrosa, Fabio de Oliveira; de Souza, Emanuel Maltempi; Monteiro, Rose Adele

    2013-01-01

    Herbaspirillum seropedicae is a plant growth-promoting diazotrophic betaproteobacterium which associates with important crops, such as maize, wheat, rice and sugar-cane. We have previously reported that intact lipopolysaccharide (LPS) is required for H. seropedicae attachment and endophytic colonization of maize roots. In this study, we present evidence that the LPS biosynthesis gene waaL (codes for the O-antigen ligase) is induced during rhizosphere colonization by H. seropedicae. Furthermore a waaL mutant strain lacking the O-antigen portion of the LPS is severely impaired in colonization. Since N-acetyl glucosamine inhibits H. seropedicae attachment to maize roots, lectin-like proteins from maize roots (MRLs) were isolated and mass spectrometry (MS) analysis showed that MRL-1 and MRL-2 correspond to maize proteins with a jacalin-like lectin domain, while MRL-3 contains a B-chain lectin domain. These proteins showed agglutination activity against wild type H. seropedicae, but failed to agglutinate the waaL mutant strain. The agglutination reaction was severely diminished in the presence of N-acetyl glucosamine. Moreover addition of the MRL proteins as competitors in H. seropedicae attachment assays decreased 80-fold the adhesion of the wild type to maize roots. The results suggest that N-acetyl glucosamine residues of the LPS O-antigen bind to maize root lectins, an essential step for efficient bacterial attachment and colonization.

  16. Maize root lectins mediate the interaction with Herbaspirillum seropedicae via N-acetyl glucosamine residues of lipopolysaccharides.

    Directory of Open Access Journals (Sweden)

    Eduardo Balsanelli

    Full Text Available Herbaspirillum seropedicae is a plant growth-promoting diazotrophic betaproteobacterium which associates with important crops, such as maize, wheat, rice and sugar-cane. We have previously reported that intact lipopolysaccharide (LPS is required for H. seropedicae attachment and endophytic colonization of maize roots. In this study, we present evidence that the LPS biosynthesis gene waaL (codes for the O-antigen ligase is induced during rhizosphere colonization by H. seropedicae. Furthermore a waaL mutant strain lacking the O-antigen portion of the LPS is severely impaired in colonization. Since N-acetyl glucosamine inhibits H. seropedicae attachment to maize roots, lectin-like proteins from maize roots (MRLs were isolated and mass spectrometry (MS analysis showed that MRL-1 and MRL-2 correspond to maize proteins with a jacalin-like lectin domain, while MRL-3 contains a B-chain lectin domain. These proteins showed agglutination activity against wild type H. seropedicae, but failed to agglutinate the waaL mutant strain. The agglutination reaction was severely diminished in the presence of N-acetyl glucosamine. Moreover addition of the MRL proteins as competitors in H. seropedicae attachment assays decreased 80-fold the adhesion of the wild type to maize roots. The results suggest that N-acetyl glucosamine residues of the LPS O-antigen bind to maize root lectins, an essential step for efficient bacterial attachment and colonization.

  17. Acetylation modulates the STAT signaling code.

    Science.gov (United States)

    Wieczorek, Martin; Ginter, Torsten; Brand, Peter; Heinzel, Thorsten; Krämer, Oliver H

    2012-12-01

    A fascinating question of modern biology is how a limited number of signaling pathways generate biological diversity and crosstalk phenomena in vivo. Well-defined posttranslational modification patterns dictate the functions and interactions of proteins. The signal transducers and activators of transcription (STATs) are physiologically important cytokine-induced transcription factors. They are targeted by a multitude of posttranslational modifications that control and modulate signaling responses and gene expression. Beyond phosphorylation of serine and tyrosine residues, lysine acetylation has recently emerged as a critical modification regulating STAT functions. Interestingly, acetylation can determine STAT signaling codes by various molecular mechanisms, including the modulation of other posttranslational modifications. Here, we provide an overview on the acetylation of STATs and how this protein modification shapes cellular cytokine responses. We summarize recent advances in understanding the impact of STAT acetylation on cell growth, apoptosis, innate immunity, inflammation, and tumorigenesis. Furthermore, we discuss how STAT acetylation can be targeted by small molecules and we consider the possibility that additional molecules controlling STAT signaling are regulated by acetylation. Our review also summarizes evolutionary aspects and we show similarities between the acetylation-dependent control of STATs and other important molecules. We propose the concept that, similar to the 'histone code', distinct posttranslational modifications and their crosstalk orchestrate the functions and interactions of STAT proteins.

  18. Coactivator-dependent acetylation stabilizes members of the SREBP family of transcription factors.

    Science.gov (United States)

    Giandomenico, Valeria; Simonsson, Maria; Grönroos, Eva; Ericsson, Johan

    2003-04-01

    Members of the SREBP family of transcription factors control cholesterol and lipid homeostasis and play important roles during adipocyte differentiation. The transcriptional activity of SREBPs is dependent on the coactivators p300 and CBP. We now present evidence that SREBPs are acetylated by the intrinsic acetyltransferase activity of p300 and CBP. In SREBP1a, the acetylated lysine residue resides in the DNA-binding domain of the protein. Coexpression with p300 dramatically increases the expression of both SREBP1a and SREBP2, and this effect is dependent on the acetyltransferase activity of p300, indicating that acetylation of SREBPs regulates their stability. Indeed, acetylation or mutation of the acetylated lysine residue in SREBP1a stabilizes the protein. We demonstrate that the acetylated residue in SREBP1a is also targeted by ubiquitination and that acetylation inhibits this process. Thus, our studies define acetylation-dependent stabilization of transcription factors as a novel mechanism for coactivators to regulate gene expression.

  19. Modulation of Central Carbon Metabolism by Acetylation of Isocitrate Lyase in Mycobacterium tuberculosis

    Science.gov (United States)

    Bi, Jing; Wang, Yihong; Yu, Heguo; Qian, Xiaoyan; Wang, Honghai; Liu, Jun; Zhang, Xuelian

    2017-01-01

    Several enzymes involved in central carbon metabolism such as isocitrate lyase and phosphoenolpyruvate carboxykinase are key determinants of pathogenesis of Mycobacterium tuberculosis (M. tb). In this study, we found that lysine acetylation plays an important role in the modulation of central carbon metabolism in M. tb. Mutant of M. tb defective in sirtuin deacetylase exhibited improved growth in fatty acid-containing media. Global analysis of lysine acetylome of M. tb identified three acetylated lysine residues (K322, K331, and K392) of isocitrate lyase (ICL1). Using a genetically encoding system, we demonstrated that acetylation of K392 increased the enzyme activity of ICL1, whereas acetylation of K322 decreased its activity. Antibodies that specifically recognized acetyllysine at 392 and 322 of ICL1 were used to monitor the levels of ICL1 acetylation in M. tb cultures. The physiological significance of ICL1 acetylation was demonstrated by the observation that M. tb altered the levels of acetylated K392 in response to changes of carbon sources, and that acetylation of K392 affected the abundance of ICL1 protein. Our study has uncovered another regulatory mechanism of ICL1. PMID:28322251

  20. Acetylation at O-2 of arabinofuranose residues in feruloylated arabinoxylan from bamboo shoot cell-walls.

    Science.gov (United States)

    Ishii, T

    1991-01-01

    Hydrolysis of bamboo shoot cell-walls with Driselase (a fungal enzyme preparation) gave an arabinoxylan trisaccharide with ferulic and acetic acids as ester groups. The structure of this oligosaccharide was determined to be O-[2-O-acetyl-5-O-[E)-feruloyl)-alpha-L-arabinofuranosyl]-(1----3) -O-beta- D-xylopyranosyl-(1----4)-D-xylopyranose, on the basis of spectroscopy and methylation analysis.

  1. Global histone analysis by mass spectrometry reveals a high content of acetylated lysine residues in the malaria parasite Plasmodium falciparum

    DEFF Research Database (Denmark)

    Trelle, Morten Beck; Salcedo-Amaya, Adriana M; Cohen, Adrian

    2009-01-01

    Post-translational modifications (PTMs) of histone tails play a key role in epigenetic regulation of gene expression in a range of organisms from yeast to human, however, little is known about histone proteins from the parasite that causes malaria in humans, Plasmodium falciparum. We characterize...... comprehensive map of histone modifications in Plasmodium falciparum and highlight the utility of tandem MS for detailed analysis of peptides containing multiple PTMs....

  2. Histone H3 globular domain acetylation identifies a new class of enhancers.

    Science.gov (United States)

    Pradeepa, Madapura M; Grimes, Graeme R; Kumar, Yatendra; Olley, Gabrielle; Taylor, Gillian C A; Schneider, Robert; Bickmore, Wendy A

    2016-06-01

    Histone acetylation is generally associated with active chromatin, but most studies have focused on the acetylation of histone tails. Various histone H3 and H4 tail acetylations mark the promoters of active genes. These modifications include acetylation of histone H3 at lysine 27 (H3K27ac), which blocks Polycomb-mediated trimethylation of H3K27 (H3K27me3). H3K27ac is also widely used to identify active enhancers, and the assumption has been that profiling H3K27ac is a comprehensive way of cataloguing the set of active enhancers in mammalian cell types. Here we show that acetylation of lysine residues in the globular domain of histone H3 (lysine 64 (H3K64ac) and lysine 122 (H3K122ac)) marks active gene promoters and also a subset of active enhancers. Moreover, we find a new class of active functional enhancers that is marked by H3K122ac but lacks H3K27ac. This work suggests that, to identify enhancers, a more comprehensive analysis of histone acetylation is required than has previously been considered.

  3. Core promoter acetylation is not required for high transcription from the phosphoenolpyruvate carboxylase promoter in maize

    Directory of Open Access Journals (Sweden)

    Horst Ina

    2009-12-01

    Full Text Available Abstract Background Acetylation of promoter nucleosomes is tightly correlated and mechanistically linked to gene activity. However, transcription is not necessary for promoter acetylation. It seems, therefore, that external and endogenous stimuli control histone acetylation and by this contribute to gene regulation. Photosynthetic genes in plants are excellent models with which to study the connection between stimuli and chromatin modifications because these genes are strongly expressed and regulated by multiple stimuli that are easily manipulated. We have previously shown that acetylation of specific histone lysine residues on the photosynthetic phosphoenolpyruvate carboxylase (Pepc promoter in maize is controlled by light and is independent of other stimuli or gene activity. Acetylation of upstream promoter regions responds to a set of other stimuli which include the nutrient availability of the plant. Here, we have extended these studies by analysing histone acetylation during the diurnal and circadian rhythm of the plant. Results We show that histone acetylation of individual lysine residues is removed from the core promoter before the end of the illumination period which is an indication that light is not the only factor influencing core promoter acetylation. Deacetylation is accompanied by a decrease in gene activity. Pharmacological inhibition of histone deacetylation is not sufficient to prevent transcriptional repression, indicating that deacetylation is not controlling diurnal gene regulation. Variation of the Pepc promoter activity during the day is controlled by the circadian oscillator as it is maintained under constant illumination for at least 3 days. During this period, light-induced changes in histone acetylation are completely removed from the core promoter, although the light stimulus is continuously applied. However, acetylation of most sites on upstream promoter elements follows the circadian rhythm. Conclusion Our results

  4. Inhibition of Alkaline Phosphatase from Pearl Oyster Pinctada fucata by o-Phthalaldehyde: Involvement of Lysine and Histidine Residues at the Active Site

    Institute of Scientific and Technical Information of China (English)

    CHEN Hongtao; XIE Liping; YU Zhenyan; ZHANG Rongqing

    2005-01-01

    Alkaline phosphatase from Pinctada fucata was inactivated by o-phthalaldehyde (OPA). The inactivation followed pseudo first-order kinetics with a second rate constant of 0.167 (mmol/L)-1·min-1 at pH 7.5 and 25°C. A Tsou's plot analysis showed that inactivation occurred upon formation of one isoindole group. The OPA-modified enzyme lost the ability to bind with the specific affinity column and the presence of substrates or competitive inhibitors protected the enzyme from inactivation. The results revealed that the OPA-reaction site was at the enzyme substrate binding site. Prior modification of the enzyme by lysine or histidine specific reagent abolished formation of the isoindole derivatives, suggesting that lysine and histidine residues were involved in the OPA-induced inactivation. Taken together, OPA inactivated the alkaline phosphatase from Pinctada fucata by cross-linking lysine and histidine residues at the active site and formed an isoindole group at the substrate binding site of the enzyme.

  5. The effect of protein acetylation on the formation and processing of inclusion bodies and endogenous protein aggregates in Escherichia coli cells.

    Science.gov (United States)

    Kuczyńska-Wiśnik, Dorota; Moruno-Algara, María; Stojowska-Swędrzyńska, Karolina; Laskowska, Ewa

    2016-11-10

    Acetylation of lysine residues is a reversible post-translational modification conserved from bacteria to humans. Several recent studies have revealed hundreds of lysine-acetylated proteins in various bacteria; however, the physiological role of these modifications remains largely unknown. Since lysine acetylation changes the size and charge of proteins and thereby may affect their conformation, we assumed that lysine acetylation can stimulate aggregation of proteins, especially for overproduced recombinant proteins that form inclusion bodies. To verify this assumption, we used Escherichia coli strains that overproduce aggregation-prone VP1GFP protein. We found that in ΔackA-pta cells, which display diminished protein acetylation, inclusion bodies were formed with a delay and processed faster than in the wild-type cells. Moreover, in ΔackA-pta cells, inclusion bodies exhibited significantly increased specific GFP fluorescence. In CobB deacetylase-deficient cells, in which protein acetylation was enhanced, the formation of inclusion bodies was increased and their processing was significantly inhibited. Similar results were obtained with regard to endogenous protein aggregates formed during the late stationary phase in ΔackA-pta and ΔcobB cells. Our studies revealed that protein acetylation affected the aggregation of endogenous E. coli proteins and the yield, solubility, and biological activity of a model recombinant protein. In general, decreased lysine acetylation inhibited the formation of protein aggregates, whereas increased lysine acetylation stabilized protein aggregates. These findings should be considered during the designing of efficient strategies for the production of recombinant proteins in E. coli cells.

  6. Quantitating the specificity and selectivity of Gcn5-mediated acetylation of histone H3.

    Directory of Open Access Journals (Sweden)

    Yin-Ming Kuo

    Full Text Available Lysine acetyltransferases (KATs play a unique role in regulating gene transcription as well as maintaining the epigenetic state of the cell. KATs such as Gcn5 and p300/CBP can modify multiple residues on a single histone; however, order and specificity of acetylation can be altered by factors such as histone chaperones, subunit proteins or external stimulus. While the importance of acetylation is well documented, it has been difficult to quantitatively measure the specificity and selectivity of acetylation at different residues within a histone. In this paper, we demonstrate a label-free quantitative high throughput mass spectrometry-based assay capable of quantitatively monitoring all known acetylation sites of H3 simultaneously. Using this assay, we are able to analyze the steady-state enzyme kinetics of Gcn5, an evolutionarily conserved KAT. In doing so, we measured Gcn5-mediated acetylation at six residues (K14>K9 ≈ K23> K18> K27 ≈ K36 and the catalytic efficiency (k(cat/K(m for K9, K14, K18, and K23 as well as the nonenzymatic acetylation rate. We observed selectivity differences of up to -4 kcal/mol between K14 and K18, the highest and lowest measurable k(cat/K(m. These data provide a first look at quantitating the specificity and selectivity of multiple lysines on a single substrate (H3 by Gcn5.

  7. Piscidin-1-analogs with double L- and D-lysine residues exhibited different conformations in lipopolysaccharide but comparable anti-endotoxin activities

    Science.gov (United States)

    Kumar, Amit; Mahajan, Mukesh; Awasthi, Bhanupriya; Tandon, Anshika; Harioudh, Munesh Kumar; Shree, Sonal; Singh, Pratiksha; Shukla, Praveen Kumar; Ramachandran, Ravishankar; Mitra, Kalyan; Bhattacharjya, Surajit; Ghosh, Jimut Kanti

    2017-01-01

    To become clinically effective, antimicrobial peptides (AMPs) should be non-cytotoxic to host cells. Piscidins are a group of fish-derived AMPs with potent antimicrobial and antiendotoxin activities but limited by extreme cytotoxicity. We conjectured that introduction of cationic residue(s) at the interface of polar and non-polar faces of piscidins may control their insertion into hydrophobic mammalian cell membrane and thereby reducing cytotoxicity. We have designed several novel analogs of piscidin-1 by substituting threonine residue(s) with L and D-lysine residue(s). L/D-lysine-substituted analogs showed significantly reduced cytotoxicity but exhibited either higher or comparable antibacterial activity akin to piscidin-1. Piscidin-1-analogs demonstrated higher efficacy than piscidin-1 in inhibiting lipopolysaccharide (LPS)-induced pro-inflammatory responses in THP-1 cells. T15,21K-piscidin-1 (0.5 mg/Kg) and T15,21dK-piscidin-1 (1.0 mg/Kg) demonstrated 100% survival of LPS (12.0 mg/Kg)-administered mice. High resolution NMR studies revealed that both piscidin-1 and T15,21K-piscidin-1 adopted helical structures, with latter showing a shorter helix, higher amphipathicity and cationic residues placed at optimal distances to form ionic/hydrogen bond with lipid A of LPS. Remarkably, T15,21dK-piscidin-1 showed a helix-loop-helix structure in LPS and its interactions with LPS could be sustained by the distance of separation of side chains of R7 and D-Lys-15 which is close to the inter-phosphate distance of lipid A. PMID:28051162

  8. Autoacetylation of the MYST lysine acetyltransferase MOF protein.

    Science.gov (United States)

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

    2012-10-12

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

  9. Autoacetylation of the MYST Lysine Acetyltransferase MOF Protein*

    Science.gov (United States)

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

    2012-01-01

    The MYST family of histone acetyltransferases (HATs) plays critical roles in diverse cellular processes, such as the epigenetic regulation of gene expression. Lysine autoacetylation of the MYST HATs has recently received considerable attention. Nonetheless, the mechanism and function of the autoacetylation process are not well defined. To better understand the biochemical mechanism of MYST autoacetylation and the impact of autoacetylation on the cognate histone acetylation, we carried out detailed analyses of males-absent-on-the-first (MOF), a key member of the MYST family. A number of mutant MOF proteins were produced with point mutations at several key residues near the active site of the enzyme. Autoradiography and immunoblotting data showed that mutation of these residues affects the autoacetylation activity and HAT activity of MOF by various degrees demonstrating that MOF activity is highly sensitive to the chemical changes in those residues. We produced MOF protein in the deacetylated form by using a nonspecific lysine deacetylase. Interestingly, both the autoacetylation activity and the histone acetylation activity of the deacetylated MOF were found to be very close to that of wild-type MOF, suggesting that autoacetylation of MOF only marginally modulates the enzymatic activity. Also, we found that the autoacetylation rates of MOF and deacetylated MOF were much slower than the cognate substrate acetylation. Thus, autoacetylation does not seem to contribute to the intrinsic enzymatic activity in a significant manner. These data provide new insights into the mechanism and function of MYST HAT autoacetylation. PMID:22918831

  10. Accurate quantification of site-specific acetylation stoichiometry reveals the impact of sirtuin deacetylase CobB on the E. coli acetylome

    DEFF Research Database (Denmark)

    Weinert, Brian Tate; Satpathy, Shankha; Hansen, Bogi Karbech

    2017-01-01

    Lysine acetylation is a protein posttranslational modification (PTM) that occurs on thousands of lysine residues in diverse organisms from bacteria to humans. Accurate measurement of acetylation stoichiometry on a proteome-wide scale remains challenging. Most methods employ a comparison of chemic......Lysine acetylation is a protein posttranslational modification (PTM) that occurs on thousands of lysine residues in diverse organisms from bacteria to humans. Accurate measurement of acetylation stoichiometry on a proteome-wide scale remains challenging. Most methods employ a comparison...... used quantitative proteomic methods. Here we show that serial dilution of SILAC-labeled peptides (SD-SILAC) can be used to identify accurately quantified peptides and to estimate the quantification error rate. We applied SD-SILAC to determine absolute acetylation stoichiometry in exponentially......-growing and stationary-phase wild type and Sirtuin deacetylase CobB-deficient cells. To further analyze CobB-regulated sites under conditions of globally increased or decreased acetylation, we measured stoichiometry in phophotransacetylase (ptaΔ) and acetate kinase (ackAΔ) mutant strains in the presence and absence...

  11. Proteomic analysis of acetylation in thermophilic Geobacillus kaustophilus.

    Science.gov (United States)

    Lee, Dong-Woo; Kim, Dooil; Lee, Yong-Jik; Kim, Jung-Ae; Choi, Ji Young; Kang, Sunghyun; Pan, Jae-Gu

    2013-08-01

    Recent analysis of prokaryotic N(ε)-lysine-acetylated proteins highlights the posttranslational regulation of a broad spectrum of cellular proteins. However, the exact role of acetylation remains unclear due to a lack of acetylated proteome data in prokaryotes. Here, we present the N(ε)-lysine-acetylated proteome of gram-positive thermophilic Geobacillus kaustophilus. Affinity enrichment using acetyl-lysine-specific antibodies followed by LC-MS/MS analysis revealed 253 acetylated peptides representing 114 proteins. These acetylated proteins include not only common orthologs from mesophilic Bacillus counterparts, but also unique G. kaustophilus proteins, indicating that lysine acetylation is pronounced in thermophilic bacteria. These data complement current knowledge of the bacterial acetylproteome and provide an expanded platform for better understanding of the function of acetylation in cellular metabolism.

  12. Nano-electrospray tandem mass spectrometric analysis of the acetylation state of histones H3 and H4 in stationary phase in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Patterton Hugh G

    2011-07-01

    Full Text Available Abstract Background The involvement of histone acetylation in facilitating gene expression is well-established, particularly in the case of histones H3 and H4. It was previously shown in Saccharomyces cerevisiae that gene expression was significantly down-regulated and chromatin more condensed in stationary phase compared to exponential phase. We were therefore interested in establishing the acetylation state of histone H3 and H4 in stationary and in exponential phase, since the regulation of this modification could contribute to transcriptional shut-down and chromatin compaction during semi-quiescence. Results We made use of nano-spray tandem mass spectrometry to perform a precursor ion scan to detect an m/z 126 immonium ion, diagnostic of an Nε-acetylated lysine residue that allowed unambiguous identification of acetylated as opposed to tri-methylated lysine. The fragmentation spectra of peptides thus identified were searched with Mascot against the Swiss-Prot database, and the y-ion and b-ion fragmentation series subsequently analyzed for mass shifts compatible with acetylated lysine residues. We found that K9, K14 and K36 of histone H3 and K12 and K16 of histone H4 were acetylated in exponential phase (bulk histones, but could not detect these modifications in histones isolated from stationary phase cells at the sensitivity level of the mass spectrometer. The corresponding un-acetylated peptides were, however, observed. A significantly higher level of acetylation of these residues in exponential phase was confirmed by immuno-blotting. Conclusion H4K16 acetylation was previously shown to disrupt formation of condensed chromatin in vitro. We propose that de-acetylation of H4K16 allowed formation of condensed chromatin in stationary phase, and that acetylation of H3K9, H3K14, H3K36, and H4K12 reflected the active transcriptional state of the yeast genome in exponential phase.

  13. The Bacterial Two-Hybrid System Uncovers the Involvement of Acetylation in Regulating of Lrp Activity in Salmonella Typhimurium

    Science.gov (United States)

    Qin, Ran; Sang, Yu; Ren, Jie; Zhang, Qiufen; Li, Shuxian; Cui, Zhongli; Yao, Yu-Feng

    2016-01-01

    N𝜀-lysine acetylation is an abundant and important Post-translational modification in bacteria. We used the bacterial two-hybrid system to screen the genome library of the Salmonella Typhimurium to identify potential proteins involved in acetyltransferase Pat – or deacetylase CobB-mediated acetylation. Then, the in vitro (de)acetylation assays were used to validate the potential targets, such as STM14_1074, NrdF, RhaR. Lrp, a leucine-responsive regulatory protein and global regulator, was shown to interact with Pat. We further demonstrate that Lrp could be acetylated by Pat and deacetylated by NAD+-dependent CobB in vitro. Specifically, the conserved lysine residue 36 (K36) in helix-turn-helix (HTH) DNA-binding domain of Lrp was acetylated. Acetylation of K36 impaired the function of Lrp through altering the affinity with the target promoter. The mutation of K36 in chromosome mimicking acetylation enhanced the transcriptional level of itself and attenuated the mRNA levels of Lrp-regulated genes including fimA, which was confirmed by yeast agglutination assay. These findings demonstrate that the acetylation regulates the DNA-binding activity of Lrp, suggesting that acetylation modification of transcription factors is a conserved regulatory manner to modulate gene expression in bacteria and eukaryotes. PMID:27909434

  14. Binding Mode of Acetylated Histones to Bromodomains: Variations on a Common Motif.

    Science.gov (United States)

    Marchand, Jean-Rémy; Caflisch, Amedeo

    2015-08-01

    Bromodomains, epigenetic readers that recognize acetylated lysine residues in histone tails, are potential drug targets in cancer and inflammation. Herein we review the crystal structures of human bromodomains in complex with histone tails and analyze the main interaction motifs. The histone backbone is extended and occupies, in one of the two possible orientations, the bromodomain surface groove lined by the ZA and BC loops. The acetyl-lysine side chain is buried in the cavity between the four helices of the bromodomain, and its oxygen atom accepts hydrogen bonds from a structural water molecule and a conserved asparagine residue in the BC loop. In stark contrast to this common binding motif, a large variety of ancillary interactions emerge from our analysis. In 10 of 26 structures, a basic side chain (up to five residues up- or downstream in sequence with respect to the acetyl-lysine) interacts with the carbonyl groups of the C-terminal turn of helix αB. Furthermore, the complexes reveal many heterogeneous backbone hydrogen bonds (direct or water-bridged). These interactions contribute unselectively to the binding of acetylated histone tails to bromodomains, which provides further evidence that specific recognition is modulated by combinations of multiple histone modifications and multiple modules of the proteins involved in transcription. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. An eleven amino acid residue deletion expands the substrate specificity of acetyl xylan esterase II (AXE II) from Penicillium purpurogenum

    Science.gov (United States)

    Colombres, Marcela; Garate, José A.; Lagos, Carlos F.; Araya-Secchi, Raúl; Norambuena, Patricia; Quiroz, Soledad; Larrondo, Luis; Pérez-Acle, Tomas; Eyzaguirre, Jaime

    2008-01-01

    The soft-rot fungus Penicillium purpurogenum secretes to the culture medium a variety of enzymes related to xylan biodegradation, among them three acetyl xylan esterases (AXE I, II and III). AXE II has 207 amino acids; it belongs to family 5 of the carbohydrate esterases and its structure has been determined by X-ray crystallography at 0.9 Å resolution (PDB 1G66). The enzyme possesses the α/β hydrolase fold and the catalytic triad typical of serine esterases (Ser90, His187 and Asp175). AXE II can hydrolyze esters of a large variety of alcohols, but it is restricted to short chain fatty acids. An analysis of its three-dimensional structure shows that a loop that covers the active site may be responsible for this strict specificity. Cutinase, an enzyme that hydrolyzes esters of long chain fatty acids and shows a structure similar to AXE II, lacks this loop. In order to generate an AXE II with this broader specificity, the preparation of a mutant lacking residues involving this loop (Gly104 to Ala114) was proposed. A set of molecular simulation experiments based on a comparative model of the mutant enzyme predicted a stable structure. Using site-directed mutagenesis, the loop's residues have been eliminated from the AXE II cDNA. The mutant protein has been expressed in Aspergillus nidulans A722 and Pichia pastoris, and it is active towards a range of fatty acid esters of up to at least 14 carbons. The availability of an esterase with broader specificity may have biotechnological applications for the synthesis of sugar esters.

  16. Apolipoprotein A-I lysine modification: effects on helical content, lipid binding and cholesterol acceptor activity.

    Science.gov (United States)

    Brubaker, Gregory; Peng, Dao-Quan; Somerlot, Benjamin; Abdollahian, Davood J; Smith, Jonathan D

    2006-01-01

    We examined the role of the positively charged lysine residues in apoAI by chemical modification. Lysine modification by reductive methylation did not alter apoAI's net charge, secondary or tertiary structure as observed by circular dichroism and trytophan fluorescence, respectively, or have much impact on lipid binding or ABCA1-dependent cholesterol acceptor activity. Acetylation of lysine residues lowered the isoelectric point of apoAI, altered its secondary and tertiary structure, and led to a 40% decrease in cholesterol acceptor activity, while maintaining 93% of its lipid binding activity. Exhaustive lysine acetoacetylation lowered apoAI's isoelectric point, profoundly disrupted its secondary and tertiary structure, and led to 90% and 82% reductions in cholesterol acceptor and lipid binding activities, respectively. The dose-dependent acetoacetylation of an increasing proportion of apoAI lysine residues demonstrated that cholesterol acceptor activity was more sensitive to this modification than lipid binding activity, suggesting that apoAI lysine positive charges play an important role in ABCA1 mediated lipid efflux beyond the role needed to maintain alpha-helical content and lipid binding activity.

  17. The roles of selected arginine and lysine residues of TAFI (Pro-CPU) in its activation to TAFIa by the thrombin-thrombomodulin complex.

    Science.gov (United States)

    Wu, Chengliang; Kim, Paul Y; Manuel, Reg; Seto, Marian; Whitlow, Marc; Nagashima, Mariko; Morser, John; Gils, Ann; Declerck, Paul; Nesheim, Michael E

    2009-03-13

    Thrombomodulin (TM) increases the catalytic efficiency of thrombin (IIa)-mediated activation of thrombin-activable fibrinolysis inhibitor (TAFI) 1250-fold. Negatively charged residues of the C-loop of TM-EGF-like domain 3 are required for TAFI activation. Molecular models suggested several positively charged residues of TAFI with which the C-loop residues could interact. Seven TAFI mutants were constructed to determine if these residues are required for efficient TAFI activation. TAFI wild-type or mutants were activated in the presence or absence of TM and the kinetic parameters of TAFI activation were determined. When the three consecutive lysine residues in the activation peptide of TAFI were substituted with alanine (K42/43/44A), the catalytic efficiencies for TAFI activation with TM decreased 8-fold. When other positively charged surface residues of TAFI (Lys-133, Lys-211, Lys-212, Arg-220, Lys-240, or Arg-275) were mutated to alanine, the catalytic efficiencies for TAFI activation with TM decreased by 1.7-2.7-fold. All decreases were highly statistically significant. In the absence of TM, catalytic efficiencies ranged from 2.8-fold lower to 1.24-fold higher than wild-type. None of these, except the 2.8-fold lower value, was statistically significant. The average half-life of the TAFIa mutants was 8.1+/-0.6 min, and that of wild type was 8.4+/-0.3 min at 37 degrees C. Our data show that these residues are important in the activation of TAFI by IIa, especially in the presence of TM. Whether the mutated residues promote a TAFI-TM or TAFI-IIa interaction remains to be determined. In addition, these residues do not influence spontaneous inactivation of TAFIa.

  18. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Weinert, Brian Tate; Iesmantavicius, Vytautas; Moustafa, Tarek

    2014-01-01

    Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation...... acetylation stoichiometry and found that the vast majority of mitochondrial and cytoplasmic acetylation had a very low stoichiometry. However, mitochondrial acetylation occurred at a significantly higher basal level than cytoplasmic acetylation, consistent with the distinct acetylation dynamics and higher...... acetyl-CoA concentration in mitochondria. High stoichiometry acetylation occurred mostly on histones, proteins present in histone acetyltransferase and deacetylase complexes, and on transcription factors. These data show that a majority of acetylation occurs at very low levels in exponentially growing...

  19. In-vitro investigations of the speed of pyrrole formation of 2,5-hexanedione and 2,5-heptanedione with N{alpha}-acetyl-L-lysine as a precondition for a comparative assessment of the neurotoxic potentials of the two {gamma}-diketones; In-vitro-Untersuchungen zur Pyrrolbildungsgeschwindigkeit von 2,5-Hexandion und 2,5-Heptandion mit N{alpha}-Acetyl-L-lysin als Voraussetzung fuer eine vergleichende Abschaetzung der neurotoxischen Potentiale beider {gamma}-Diketone

    Energy Technology Data Exchange (ETDEWEB)

    Richter, M.F.

    1997-09-01

    N-hexane and n-heptane are important solvents. Chronic exposure to n-hexane causes polyneuropathies, which are attributed to the metabolite 2,5-hexanedione, a {gamma} diketone. As a basis for a comparative assessment of the neurotoxic potentials of 2,5-hexanedione and 2,5-heptanedione, an in-vitro test was developed and used to investigate the speed of pyrrole formation of the two {gamma} diketones in reacting with the {epsilon} amino group of N{alpha}-acetyl L-lysine. The speed of the formation of pyrrole was always directly proportional to the respective reactant concentration. It consequently is subject to a second-order kinetics. As a further result, the pyrrole formation speed of 2,5-heptanedione was found to be only half that of 2,5-hexanedione. The results lead to the conclusion that 2,5-heptanedione poses a smaller risk of developing peripheral neuropathy than 2,5-hexanedione. (orig./MG) [Deutsch] n-Hexan und n-Heptan sind wichtige Loesungsmittel. Chronische Exposition gegenueber n-Hexan ruft Polyneuropathien hervor, die auf den Metaboliten 2,5-Hexandion, ein {gamma}-Diketon, zurueckgefuehrt werden. Als Grundlage fuer eine vergleichende Abschaetzung der neurotoxischen Potentiale von 2,5-Hexandion und 2,5-Heptandion wurde in der vorliegenden Arbeit ein In-vitro-Test entwickelt, mit dem die Pyrrolbildungsgeschwindigkeiten der beiden {gamma}-Diketone mit der {epsilon}-Aminogruppe von N{alpha}-Acetyl-L-Iysin untersucht wurden. Die Pyrrolbildungsgeschwindigkeit war stets direkt proportional zur jeweiligen Reaktantenkonzentration. Somit unterliegt sie einer Kinetik 2. Ordnung. Weiterhin wurde gezeigt, dass die Pyrrolbildungsgeschwindigkeit fuer 2,5-Heptandion nur etwa halb so gross ist wie fuer 2,5-Hexandion. Aus den Ergebnissen wird gefolgert, dass das von 2,5-Heptandion ausgehende Risiko an peripheren Neuropathien zu erkranken geringer ist, als das von 2,5-Hexandion ausgehende. (orig./MG)

  20. The bacterial two-hybrid system uncovers the involvement of acetylation in regulating of Lrp activity in Salmonella Typhimurium

    Directory of Open Access Journals (Sweden)

    Ran Qin

    2016-11-01

    Full Text Available Nε-lysine acetylation is an abundant and important Post-translational modification in bacteria. We used the bacterial two-hybrid system to screen the genome library of the Salmonella Typhimurium to identify potential proteins involved in acetyltransferase Pat - or deacetylase CobB-mediated acetylation. Then, the in vitro (deacetylation assays were used to validate the potential targets, such as STM14_1074, NrdF, RhaR. Lrp, a leucine-responsive regulatory protein and global regulator, was shown to interact with Pat. We further demonstrate that Lrp could be acetylated by Pat and deacetylated by NAD+-dependent CobB in vitro. Specifically, the conserved lysine residue 36 (K36 in helix-turn-helix (HTH DNA-binding domain of Lrp was acetylated. Acetylation of K36 impaired the function of Lrp through altering the affinity with the target promoter. The mutation of K36 in chromosome mimicking acetylation enhanced the transcriptional level of itself and attenuated the mRNA levels of Lrp-regulated genes including fimA, which was confirmed by yeast agglutination assay. These findings demonstrate that the acetylation regulates the DNA-binding activity of Lrp, suggesting that acetylation modification of transcription factors is a conserved regulatory manner to modulate gene expression in bacteria and eukaryotes.

  1. 4-Acyl Pyrrole Derivatives Yield Novel Vectors for Designing Inhibitors of the Acetyl-Lysine Recognition Site of BRD4(1).

    Science.gov (United States)

    Hügle, Martin; Lucas, Xavier; Weitzel, Gerhard; Ostrovskyi, Dmytro; Breit, Bernhard; Gerhardt, Stefan; Einsle, Oliver; Günther, Stefan; Wohlwend, Daniel

    2016-02-25

    Several human diseases, including cancer, show altered signaling pathways resulting from changes in the activity levels of epigenetic modulators. In the past few years, small-molecule inhibitors against specific modulators, including the bromodomain and extra-terminal (BET) bromodomain family of acetylation readers, have shown early promise in the treatment of the genetically defined midline carcinoma and hematopoietic malignancies. We have recently developed a novel potent inhibitor of BET proteins, 1 (XD14[ Angew. Chem., Int. Ed. 2013, 52, 14055]), which exerts a strong inhibitory potential on the proliferation of specific leukemia cell lines. In the study presented here, we designed analogues of 1 to study the potential of substitutions on the 4-acyl pyrrole backbone to occupy additional sites within the substrate recognition site of BRD4(1). The compounds were profiled using ITC, DSF, and X-ray crystallography. We could introduce several substitutions that address previously untargeted areas of the substrate recognition site. This work may substantially contribute to the development of therapeutics with increased target specificity against BRD4-related malignancies.

  2. Acetylation of cyclin-dependent kinase 5 is mediated by GCN5

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Juhyung; Yun, Nuri; Kim, Chiho [Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749 (Korea, Republic of); Song, Min-Young; Park, Kang-Sik [Department of Physiology and Biomedical Science Institute, Kyung Hee University School of Medicine, Seoul 130-701 (Korea, Republic of); Oh, Young J., E-mail: yjoh@yonsei.ac.kr [Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749 (Korea, Republic of)

    2014-04-25

    Highlights: • Cyclin-dependent kinase 5 (CDK5) is present as an acetylated form. • CDK5 is acetylated by GCN5. • CDK5’s acetylation site is mapped at Lys33. • Its acetylation may affect CDK5’s kinase activity. - Abstract: Cyclin-dependent kinase 5 (CDK5), a member of atypical serine/threonine cyclin-dependent kinase family, plays a crucial role in pathophysiology of neurodegenerative disorders. Its kinase activity and substrate specificity are regulated by several independent pathways including binding with its activator, phosphorylation and S-nitrosylation. In the present study, we report that acetylation of CDK5 comprises an additional posttranslational modification within the cells. Among many candidates, we confirmed that its acetylation is enhanced by GCN5, a member of the GCN5-related N-acetyl-transferase family of histone acetyltransferase. Co-immunoprecipitation assay and fluorescent localization study indicated that GCN5 physically interacts with CDK5 and they are co-localized at the specific nuclear foci. Furthermore, liquid chromatography in conjunction with a mass spectrometry indicated that CDK5 is acetylated at Lys33 residue of ATP binding domain. Considering this lysine site is conserved among a wide range of species and other related cyclin-dependent kinases, therefore, we speculate that acetylation may alter the kinase activity of CDK5 via affecting efficacy of ATP coordination.

  3. Lysine221 is the general base residue of the isochorismate synthase from Pseudomonas aeruginosa (PchA) in a reaction that is diffusion limited.

    Science.gov (United States)

    Meneely, Kathleen M; Luo, Qianyi; Dhar, Prajnaparamita; Lamb, Audrey L

    2013-10-01

    The isochorismate synthase from Pseudomonas aeruginosa (PchA) catalyzes the conversion of chorismate to isochorismate, which is subsequently converted by a second enzyme (PchB) to salicylate for incorporation into the salicylate-capped siderophore pyochelin. PchA is a member of the MST family of enzymes, which includes the structurally homologous isochorismate synthases from Escherichia coli (EntC and MenF) and salicylate synthases from Yersinia enterocolitica (Irp9) and Mycobacterium tuberculosis (MbtI). The latter enzymes generate isochorismate as an intermediate before generating salicylate and pyruvate. General acid-general base catalysis has been proposed for isochorismate synthesis in all five enzymes, but the residues required for the isomerization are a matter of debate, with both lysine221 and glutamate313 proposed as the general base (PchA numbering). This work includes a classical characterization of PchA with steady state kinetic analysis, solvent kinetic isotope effect analysis and by measuring the effect of viscosogens on catalysis. The results suggest that isochorismate production from chorismate by the MST enzymes is the result of general acid-general base catalysis with a lysine as the base and a glutamic acid as the acid, in reverse protonation states. Chemistry is determined to not be rate limiting, favoring the hypothesis of a conformational or binding step as the slow step.

  4. Accurate Quantification of Site-specific Acetylation Stoichiometry Reveals the Impact of Sirtuin Deacetylase CobB on the E. coli Acetylome.

    Science.gov (United States)

    Weinert, Brian Tate; Satpathy, Shankha; Hansen, Bogi Karbech; Lyon, David; Jensen, Lars Juhl; Choudhary, Chunaram

    2017-05-01

    Lysine acetylation is a protein posttranslational modification (PTM) that occurs on thousands of lysine residues in diverse organisms from bacteria to humans. Accurate measurement of acetylation stoichiometry on a proteome-wide scale remains challenging. Most methods employ a comparison of chemically acetylated peptides to native acetylated peptides, however, the potentially large differences in abundance between these peptides presents a challenge for accurate quantification. Stable isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry (MS) is one of the most widely used quantitative proteomic methods. Here we show that serial dilution of SILAC-labeled peptides (SD-SILAC) can be used to identify accurately quantified peptides and to estimate the quantification error rate. We applied SD-SILAC to determine absolute acetylation stoichiometry in exponentially-growing and stationary-phase wild-type and Sirtuin deacetylase CobB-deficient cells. To further analyze CobB-regulated sites under conditions of globally increased or decreased acetylation, we measured stoichiometry in phophotransacetylase (ptaΔ) and acetate kinase (ackAΔ) mutant strains in the presence and absence of the Sirtuin inhibitor nicotinamide. We measured acetylation stoichiometry at 3,669 unique sites and found that the vast majority of acetylation occurred at a low stoichiometry. Manipulations that cause increased nonenzymatic acetylation by acetyl-phosphate (AcP), such as stationary-phase arrest and deletion of ackA, resulted in globally increased acetylation stoichiometry. Comparison to relative quantification under the same conditions validated our stoichiometry estimates at hundreds of sites, demonstrating the accuracy of our method. Similar to Sirtuin deacetylase 3 (SIRT3) in mitochondria, CobB suppressed acetylation to lower than median stoichiometry in WT, ptaΔ, and ackAΔ cells. Together, our results provide a detailed view of acetylation stoichiometry in E. coli

  5. Substitution of glutamate residue by lysine in the dimerization domain affects DNA binding ability of HapR by inducing structural deformity in the DNA binding domain.

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    Richa Singh

    Full Text Available HapR has been given the status of a high cell density master regulatory protein in Vibrio cholerae. Though many facts are known regarding its structural and functional aspects, much still can be learnt from natural variants of the wild type protein. This work aims at investigating the nature of functional inertness of a HapR natural variant harboring a substitution of a conserved glutamate residue at position 117 which participates in forming a salt bridge by lysine (HapRV2G-E(117K. Experimental evidence presented here reveals the inability of this variant to interact with various cognate promoters by in vitro gel shift assay. Furthermore, the elution profiles of HapRV2G-E(117K protein along with the wild type functional HapRV2G in size-exclusion chromatography as well as circular dichroism spectra did not reflect any significant differences in its structure, thereby indicating the intactness of dimer in the variant protein. To gain further insight into the global shape of the proteins, small angle X-ray scattering analysis (SAXS was performed. Intriguingly, increased radius of gyration of HapRV2G-E(117K of 27.5 Å in comparison to the wild type protein from SAXS data analyses implied a significant alteration in the global shape of the dimeric HapRV2G-E(117K protein. Structure reconstruction brought forth that the DNA binding domains were substantially "parted away" in this variant. Taken together, our data illustrates that substitution of the conserved glutamate residue by lysine in the dimerization domain induces separation of the two DNA binding domains from their native-like positioning without altering the dimeric status of HapR variant.

  6. Substitution of glutamate residue by lysine in the dimerization domain affects DNA binding ability of HapR by inducing structural deformity in the DNA binding domain.

    Science.gov (United States)

    Singh, Richa; Rathore, Yogendra Singh; Singh, Naorem Santa; Peddada, Nagesh; Ashish; Raychaudhuri, Saumya

    2013-01-01

    HapR has been given the status of a high cell density master regulatory protein in Vibrio cholerae. Though many facts are known regarding its structural and functional aspects, much still can be learnt from natural variants of the wild type protein. This work aims at investigating the nature of functional inertness of a HapR natural variant harboring a substitution of a conserved glutamate residue at position 117 which participates in forming a salt bridge by lysine (HapRV2G-E(117)K). Experimental evidence presented here reveals the inability of this variant to interact with various cognate promoters by in vitro gel shift assay. Furthermore, the elution profiles of HapRV2G-E(117)K protein along with the wild type functional HapRV2G in size-exclusion chromatography as well as circular dichroism spectra did not reflect any significant differences in its structure, thereby indicating the intactness of dimer in the variant protein. To gain further insight into the global shape of the proteins, small angle X-ray scattering analysis (SAXS) was performed. Intriguingly, increased radius of gyration of HapRV2G-E(117)K of 27.5 Å in comparison to the wild type protein from SAXS data analyses implied a significant alteration in the global shape of the dimeric HapRV2G-E(117)K protein. Structure reconstruction brought forth that the DNA binding domains were substantially "parted away" in this variant. Taken together, our data illustrates that substitution of the conserved glutamate residue by lysine in the dimerization domain induces separation of the two DNA binding domains from their native-like positioning without altering the dimeric status of HapR variant.

  7. Structural insights into acetylated-histone H4 recognition by the bromodomain-PHD finger module of human transcriptional coactivator CBP.

    Science.gov (United States)

    Plotnikov, Alexander N; Yang, Shuai; Zhou, Thomas Jiachi; Rusinova, Elena; Frasca, Antonio; Zhou, Ming-Ming

    2014-02-01

    Bromodomain functions as the acetyl-lysine binding domains to regulate gene transcription in chromatin. Bromodomains are rapidly emerging as new epigenetic drug targets for human diseases. However, owing to their transient nature and modest affinity, histone-binding selectivity of bromodomains has remained mostly elusive. Here, we report high-resolution crystal structures of the bromodomain-PHD tandem module of human transcriptional coactivator CBP bound to lysine-acetylated histone H4 peptides. The structures reveal that the PHD finger serves a structural role in the tandem module and that the bromodomain prefers lysine-acetylated motifs comprising a hydrophobic or aromatic residue at -2 and a lysine or arginine at -3 or -4 position from the acetylated lysine. Our study further provides structural insights into distinct modes of singly and diacetylated histone H4 recognition by the bromodomains of CBP and BRD4 that function differently as a transcriptional coactivator and chromatin organizer, respectively, explaining their distinct roles in control of gene expression in chromatin.

  8. In vitro phosphorylation and acetylation of the murine pocket protein Rb2/p130.

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    Muhammad Saeed

    Full Text Available The retinoblastoma protein (pRb and the related proteins Rb2/p130 and 107 represent the "pocket protein" family of cell cycle regulators. A key function of these proteins is the cell cycle dependent modulation of E2F-regulated genes. The biological activity of these proteins is controlled by acetylation and phosphorylation in a cell cycle dependent manner. In this study we attempted to investigate the interdependence of acetylation and phosphorylation of Rb2/p130 in vitro. After having identified the acetyltransferase p300 among several acetyltransferases to be associated with Rb2/p130 during S-phase in NIH3T3 cells in vivo, we used this enzyme and the CDK4 protein kinase for in vitro modification of a variety of full length Rb2/p130 and truncated versions with mutations in the acetylatable lysine residues 1079, 128 and 130. Mutation of these residues results in the complete loss of Rb2/p130 acetylation. Replacement of lysines by arginines strongly inhibits phosphorylation of Rb2/p130 by CDK4; the inhibitory effect of replacement by glutamines is less pronounced. Preacetylation of Rb2/p130 strongly enhances CDK4-catalyzed phosphorylation, whereas deacetylation completely abolishes in vitro phosphorylation. In contrast, phosphorylation completely inhibits acetylation of Rb2/p130 by p300. These results suggest a mutual interdependence of modifications in a way that acetylation primes Rb2/p130 for phosphorylation and only dephosphorylated Rb2/p130 can be subject to acetylation. Human papillomavirus 16-E7 protein, which increases acetylation of Rb2/p130 by p300 strongly reduces phosphorylation of this protein by CDK4. This suggests that the balance between phosphorylation and acetylation of Rb2/p130 is essential for its biological function in cell cycle control.

  9. p300-mediated acetylation of the Rothmund-Thomson-syndrome gene product RECQL4 regulates its subcellular localization

    DEFF Research Database (Denmark)

    Dietschy, Tobias; Shevelev, Igor; Pena Diaz, Javier

    2009-01-01

    RECQL4 belongs to the conserved RecQ family of DNA helicases, members of which play important roles in the maintenance of genome stability in all organisms that have been examined. Although genetic alterations in the RECQL4 gene are reported to be associated with three autosomal recessive disorders...... (Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes), the molecular role of RECQL4 still remains poorly understood. Here, we show that RECQL4 specifically interacts with the histone acetyltransferase p300 (also known as p300 HAT), both in vivo and in vitro, and that p300 acetylates one or more...... of the lysine residues at positions 376, 380, 382, 385 and 386 of RECQL4. Furthermore, we report that these five lysine residues lie within a short motif of 30 amino acids that is essential for the nuclear localization of RECQL4. Remarkably, the acetylation of RECQL4 by p300 in vivo leads to a significant shift...

  10. Contribution of a lysine residue in the first transmembrane segment to the selectivity filter region in the CFTR chloride channel.

    Science.gov (United States)

    Negoda, Alexander; El Hiani, Yassine; Cowley, Elizabeth A; Linsdell, Paul

    2017-02-21

    The anion selectivity and conductance of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are determined predominantly by interactions between permeant anions and the narrow region of the channel pore. This narrow region has therefore been described as functioning as the "selectivity filter" of the channel. Multiple pore-lining transmembrane segments (TMs) have previously been shown to contribute to the selectivity filter region. However, little is known about the three-dimensional organization of this region, or how multiple TMs combine to determine its functional properties. In the present study we have used patch clamp recording to identify changes in channel function associated with the formation of disulfide cross-links between cysteine residues introduced into different TMs within the selectivity filter. Cysteine introduced at position L102 in TM1 was able to form disulfide bonds with F337C and T338C in TM6, two positions that are known to play key roles in determining anion permeation properties. Consistent with this proximal arrangement of L102, F337 and T338, different mutations at L102 altered anion selectivity and conductance properties in a way that suggests that this residue plays an important role in determining selectivity filter function, albeit a much lesser role than that of F337. These results suggest an asymmetric three-dimensional arrangement of the key selectivity filter region of the pore, as well as having important implications regarding the molecular mechanism of anion permeation.

  11. SIRT1 Regulates Thyroid-Stimulating Hormone Release by Enhancing PIP5Kgamma Activity through Deacetylation of Specific Lysine Residues in Mammals.

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    Sayaka Akieda-Asai

    Full Text Available BACKGROUND: SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSH-containing granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5Kgamma was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268 in PIP5Kgamma and enhanced PIP5Kgamma enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kgamma knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kgamma, PI(4,5P(2, and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice. CONCLUSIONS/SIGNIFICANCE: Our findings indicated that the control of TSH release by the SIRT1-PIP5Kgamma pathway is important for regulating the metabolism of the whole body.

  12. ID4 regulates transcriptional activity of wild type and mutant p53 via K373 acetylation.

    Science.gov (United States)

    Morton, Derrick J; Patel, Divya; Joshi, Jugal; Hunt, Aisha; Knowell, Ashley E; Chaudhary, Jaideep

    2017-01-10

    Given that mutated p53 (50% of all human cancers) is over-expressed in many cancers, restoration of mutant p53 to its wild type biological function has been sought after as cancer therapy. The conformational flexibility has allowed to restore the normal biological function of mutant p53 by short peptides and small molecule compounds. Recently, studies have focused on physiological mechanisms such as acetylation of lysine residues to rescue the wild type activity of mutant p53. Using p53 null prostate cancer cell line we show that ID4 dependent acetylation promotes mutant p53 DNA-binding capabilities to its wild type consensus sequence, thus regulating p53-dependent target genes leading to subsequent cell cycle arrest and apoptosis. Specifically, by using wild type, mutant (P223L, V274F, R175H, R273H), acetylation mimics (K320Q and K373Q) and non-acetylation mimics (K320R and K373R) of p53, we identify that ID4 promotes acetylation of K373 and to a lesser extent K320, in turn restoring p53-dependent biological activities. Together, our data provides a molecular understanding of ID4 dependent acetylation that suggests a strategy of enhancing p53 acetylation at sites K373 and K320 that may serve as a viable mechanism of physiological restoration of mutant p53 to its wild type biological function.

  13. Conserved aspartate and lysine residues of RcsB are required for amylovoran biosynthesis, virulence, and DNA binding in Erwinia amylovora.

    Science.gov (United States)

    Ancona, Veronica; Chatnaparat, Tiyakhon; Zhao, Youfu

    2015-08-01

    In Erwinia amylovora, the Rcs phosphorelay system is essential for amylovoran production and virulence. To further understand the role of conserved aspartate residue (D56) in the phosphor receiver (PR) domain and lysine (K180) residue in the function domain of RcsB, amino acid substitutions of RcsB mutant alleles were generated by site-directed mutagenesis and complementation of various rcs mutants were performed. A D56E substitution of RcsB, which mimics the phosphorylation state of RcsB, complemented the rcsB mutant, resulting in increased amylovoran production and gene expression, reduced swarming motility, and restored pathogenicity. In contrast, D56N and K180A or K180Q substitutions of RcsB did not complement the rcsB mutant. Electrophoresis mobility shift assays showed that D56E, but not D56N, K180Q and K180A substitutions of RcsB bound to promoters of amsG and flhD, indicating that both D56 and K180 are required for DNA binding. Interestingly, the RcsBD56E allele could also complement rcsAB, rcsBC and rcsABCD mutants with restored virulence and increased amylovoran production, indicating that RcsB phosphorylation is essential for virulence of E. amylovora. In addition, mutations of T904 and A905, but not phosphorylation mimic mutation of D876 in the PR domain of RcsC, constitutively activate the Rcs system, suggesting that phosphor transfer is required for activating the Rcs system and indicating both A905 and T904 are required for the phosphatase activity of RcsC. Our results demonstrated that RcsB phosphorylation and dephosphorylation, phosphor transfer from RcsC are essential for the function of the Rcs system, and also suggested that constitutive activation of the Rcs system could reduce the fitness of E. amylovora.

  14. Aspirin inhibits glucose‑6‑phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites.

    Science.gov (United States)

    Ai, Guoqiang; Dachineni, Rakesh; Kumar, D Ramesh; Alfonso, Lloyd F; Marimuthu, Srinivasan; Bhat, G Jayarama

    2016-08-01

    Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first reaction in the pentose phosphate pathway, and generates ribose sugars, which are required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosphate (NADPH), which is important for neutralization of oxidative stress. The expression of G6PD is elevated in several types of tumor, including colon, breast and lung cancer, and has been implicated in cancer cell growth. Our previous study demonstrated that exposure of HCT 116 human colorectal cancer cells to aspirin caused acetylation of G6PD, and this was associated with a decrease in its enzyme activity. In the present study, this observation was expanded to HT‑29 colorectal cancer cells, in order to compare aspirin‑mediated acetylation of G6PD and its activity between HCT 116 and HT‑29 cells. In addition, the present study aimed to determine the acetylation targets of aspirin on recombinant G6PD to provide an insight into the mechanisms of inhibition. The results demonstrated that the extent of G6PD acetylation was significantly higher in HCT 116 cells compared with in HT‑29 cells; accordingly, a greater reduction in G6PD enzyme activity was observed in the HCT 116 cells. Mass spectrometry analysis of aspirin‑acetylated G6PD (isoform a) revealed that aspirin acetylated a total of 14 lysine residues, which were dispersed throughout the length of the G6PD protein. One of the important amino acid targets of aspirin included lysine 235 (K235, in isoform a) and this corresponds to K205 in isoform b, which has previously been identified as being important for catalysis. Acetylation of G6PD at several sites, including K235 (K205 in isoform b), may mediate inhibition of G6PD activity, which may contribute to the ability of aspirin to exert anticancer effects through decreased synthesis of ribose sugars and NADPH.

  15. Histone H3 lysine 14 (H3K14) acetylation facilitates DNA repair in a positioned nucleosome by stabilizing the binding of the chromatin Remodeler RSC (Remodels Structure of Chromatin).

    Science.gov (United States)

    Duan, Ming-Rui; Smerdon, Michael J

    2014-03-21

    Histone H3 acetylation is induced by UV damage in yeast and may play an important role in regulating the repair of UV photolesions in nucleosome-loaded genomic loci. However, it remains elusive how H3 acetylation facilitates repair. We generated a strongly positioned nucleosome containing homogeneously acetylated H3 at Lys-14 (H3K14ac) and investigated possible mechanisms by which H3K14 acetylation modulates repair. We show that H3K14ac does not alter nucleosome unfolding dynamics or enhance the repair of UV-induced cyclobutane pyrimidine dimers by UV photolyase. Importantly, however, nucleosomes with H3K14ac have a higher affinity for purified chromatin remodeling complex RSC (Remodels the Structure of Chromatin) and show greater cyclobutane pyrimidine dimer repair compared with unacetylated nucleosomes. Our study indicates that, by anchoring RSC, H3K14 acetylation plays an important role in the unfolding of strongly positioned nucleosomes during repair of UV damage.

  16. PLMD: An updated data resource of protein lysine modifications.

    Science.gov (United States)

    Xu, Haodong; Zhou, Jiaqi; Lin, Shaofeng; Deng, Wankun; Zhang, Ying; Xue, Yu

    2017-05-20

    Post-translational modifications (PTMs) occurring at protein lysine residues, or protein lysine modifications (PLMs), play critical roles in regulating biological processes. Due to the explosive expansion of the amount of PLM substrates and the discovery of novel PLM types, here we greatly updated our previous studies, and presented a much more integrative resource of protein lysine modification database (PLMD). In PLMD, we totally collected and integrated 284,780 modification events in 53,501 proteins across 176 eukaryotes and prokaryotes for up to 20 types of PLMs, including ubiquitination, acetylation, sumoylation, methylation, succinylation, malonylation, glutarylation, glycation, formylation, hydroxylation, butyrylation, propionylation, crotonylation, pupylation, neddylation, 2-hydroxyisobutyrylation, phosphoglycerylation, carboxylation, lipoylation and biotinylation. Using the data set, a motif-based analysis was performed for each PLM type, and the results demonstrated that different PLM types preferentially recognize distinct sequence motifs for the modifications. Moreover, various PLMs synergistically orchestrate specific cellular biological processes by mutual crosstalks with each other, and we totally found 65,297 PLM events involved in 90 types of PLM co-occurrences on the same lysine residues. Finally, various options were provided for accessing the data, while original references and other annotations were also present for each PLM substrate. Taken together, we anticipated the PLMD database can serve as a useful resource for further researches of PLMs. PLMD 3.0 was implemented in PHP + MySQL and freely available at http://plmd.biocuckoo.org. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  17. The valine and lysine residues in the conserved FxVTxK motif are important for the function of phylogenetically distant plant cellulose synthases.

    Science.gov (United States)

    Slabaugh, Erin; Scavuzzo-Duggan, Tess; Chaves, Arielle; Wilson, Liza; Wilson, Carmen; Davis, Jonathan K; Cosgrove, Daniel J; Anderson, Charles T; Roberts, Alison W; Haigler, Candace H

    2016-05-01

    Cellulose synthases (CESAs) synthesize the β-1,4-glucan chains that coalesce to form cellulose microfibrils in plant cell walls. In addition to a large cytosolic (catalytic) domain, CESAs have eight predicted transmembrane helices (TMHs). However, analogous to the structure of BcsA, a bacterial CESA, predicted TMH5 in CESA may instead be an interfacial helix. This would place the conserved FxVTxK motif in the plant cell cytosol where it could function as a substrate-gating loop as occurs in BcsA. To define the functional importance of the CESA region containing FxVTxK, we tested five parallel mutations in Arabidopsis thaliana CESA1 and Physcomitrella patens CESA5 in complementation assays of the relevant cesa mutants. In both organisms, the substitution of the valine or lysine residues in FxVTxK severely affected CESA function. In Arabidopsis roots, both changes were correlated with lower cellulose anisotropy, as revealed by Pontamine Fast Scarlet. Analysis of hypocotyl inner cell wall layers by atomic force microscopy showed that two altered versions of Atcesa1 could rescue cell wall phenotypes observed in the mutant background line. Overall, the data show that the FxVTxK motif is functionally important in two phylogenetically distant plant CESAs. The results show that Physcomitrella provides an efficient model for assessing the effects of engineered CESA mutations affecting primary cell wall synthesis and that diverse testing systems can lead to nuanced insights into CESA structure-function relationships. Although CESA membrane topology needs to be experimentally determined, the results support the possibility that the FxVTxK region functions similarly in CESA and BcsA.

  18. Structural insights into cellulolytic and chitinolytic enzymes revealing crucial residues of insect β-N-acetyl-D-hexosaminidase.

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    Tian Liu

    Full Text Available The chemical similarity of cellulose and chitin supports the idea that their corresponding hydrolytic enzymes would bind β-1,4-linked glucose residues in a similar manner. A structural and mutational analysis was performed for the plant cellulolytic enzyme BGlu1 from Oryza sativa and the insect chitinolytic enzyme OfHex1 from Ostrinia furnacalis. Although BGlu1 shows little amino-acid sequence or topological similarity with OfHex1, three residues (Trp(490, Glu(328, Val(327 in OfHex1, and Trp(358, Tyr(131 and Ile(179 in BGlu1 were identified as being conserved in the +1 sugar binding site. OfHex1 Glu(328 together with Trp(490 was confirmed to be necessary for substrate binding. The mutant E328A exhibited a 8-fold increment in K(m for (GlcNAc(2 and a 42-fold increment in K(i for TMG-chitotriomycin. A crystal structure of E328A in complex with TMG-chitotriomycin was resolved at 2.5 Å, revealing the obvious conformational changes of the catalytic residues (Glu(368 and Asp(367 and the absence of the hydrogen bond between E328A and the C3-OH of the +1 sugar. V327G exhibited the same activity as the wild-type, but acquired the ability to efficiently hydrolyse β-1,2-linked GlcNAc in contrast to the wild-type. Thus, Glu(328 and Val(327 were identified as important for substrate-binding and as glycosidic-bond determinants. A structure-based sequence alignment confirmed the spatial conservation of these three residues in most plant cellulolytic, insect and bacterial chitinolytic enzymes.

  19. Interaction of L-lysine and soluble elastin with the semicarbazide-sensitive amine oxidase in the context of its vascular-adhesion and tissue maturation functions.

    LENUS (Irish Health Repository)

    Olivieri, Aldo

    2010-04-01

    The copper-containing quinoenzyme semicarbazide-sensitive amine oxidase (EC 1.4.3.21; SSAO) is a multifunctional protein. In some tissues, such as the endothelium, it also acts as vascular-adhesion protein 1 (VAP-1), which is involved in inflammatory responses and in the chemotaxis of leukocytes. Earlier work had suggested that lysine might function as a recognition molecule for SSAO\\/VAP-1. The present work reports the kinetics of the interaction of L-lysine and some of its derivatives with SSAO. Binding was shown to be saturable, time-dependent but reversible and to cause uncompetitive inhibition with respect to the amine substrate. It was also specific, since D-lysine, L-lysine ethyl ester and epsilon-acetyl-L-lysine, for example, did not bind to the enzyme. The lysine-rich protein soluble elastin bound to the enzyme relatively tightly, which may have relevance to the reported roles of SSAO in maintaining the extracellular matrix (ECM) and in the maturation of elastin. Our data show that lysyl residues are not oxidized by SSAO, but they bind tightly to the enzyme in the presence of hydrogen peroxide. This suggests that binding in vivo of SSAO to lysyl residues in physiological targets might be regulated in the presence of H(2)O(2), formed during the oxidation of a physiological SSAO substrate, yet to be identified.

  20. Luminal localization of α-tubulin K40 acetylation by cryo-EM analysis of fab-labeled microtubules.

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    Virupakshi Soppina

    Full Text Available The αβ-tubulin subunits of microtubules can undergo a variety of evolutionarily-conserved post-translational modifications (PTMs that provide functional specialization to subsets of cellular microtubules. Acetylation of α-tubulin residue Lysine-40 (K40 has been correlated with increased microtubule stability, intracellular transport, and ciliary assembly, yet a mechanistic understanding of how acetylation influences these events is lacking. Using the anti-acetylated tubulin antibody 6-11B-1 and electron cryo-microscopy, we demonstrate that the K40 acetylation site is located inside the microtubule lumen and thus cannot directly influence events on the microtubule surface, including kinesin-1 binding. Surprisingly, the monoclonal 6-11B-1 antibody recognizes both acetylated and deacetylated microtubules. These results suggest that acetylation induces structural changes in the K40-containing loop that could have important functional consequences on microtubule stability, bending, and subunit interactions. This work has important implications for acetylation and deacetylation reaction mechanisms as well as for interpreting experiments based on 6-11B-1 labeling.

  1. Host determinant residue lysine 627 lies on the surface of a discrete, folded domain of influenza virus polymerase PB2 subunit.

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    Franck Tarendeau

    Full Text Available Understanding how avian influenza viruses adapt to human hosts is critical for the monitoring and prevention of future pandemics. Host specificity is determined by multiple sites in different viral proteins, and mutation of only a limited number of these sites can lead to inter-species transmission. Several of these sites have been identified in the viral polymerase, the best characterised being position 627 in the PB2 subunit. Efficient viral replication at the relatively low temperature of the human respiratory tract requires lysine 627 rather than the glutamic acid variant found systematically in avian viruses. However, the molecular mechanism by which any of these host specific sites determine host range are unknown, although adaptation to host factors is frequently evoked. We used ESPRIT, a library screening method, to identify a new PB2 domain that contains a high density of putative host specific sites, including residue 627. The X-ray structure of this domain (denoted the 627-domain exhibits a novel fold with the side-chain of Lys627 solvent exposed. The structure of the K627E mutated domain shows no structural differences but the charge reversal disrupts a striking basic patch on the domain surface. Five other recently proposed host determining sites of PB2 are also located on the 627-domain surface. The structure of the complete C-terminal region of PB2 comprising the 627-domain and the previously identified NLS-domain, which binds the host nuclear import factor importin alpha, was also determined. The two domains are found to pack together with a largely hydrophilic interface. These data enable a three-dimensional mapping of approximately half of PB2 sites implicated in cross-species transfer onto a single structural unit. Their surface location is consistent with roles in interactions with other viral proteins or host factors. The identification and structural characterization of these well-defined PB2 domains will help design

  2. Investigation of the acetylation mechanism by GCN5 histone acetyltransferase.

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    Junfeng Jiang

    Full Text Available The histone acetylation of post-translational modification can be highly dynamic and play a crucial role in regulating cellular proliferation, survival, differentiation and motility. Of the enzymes that mediate post-translation modifications, the GCN5 of the histone acetyltransferase (HAT proteins family that add acetyl groups to target lysine residues within histones, has been most extensively studied. According to the mechanism studies of GCN5 related proteins, two key processes, deprotonation and acetylation, must be involved. However, as a fundamental issue, the structure of hGCN5/AcCoA/pH3 remains elusive. Although biological experiments have proved that GCN5 mediates the acetylation process through the sequential mechanism pathway, a dynamic view of the catalytic process and the molecular basis for hGCN5/AcCoA/pH3 are still not available and none of theoretical studies has been reported to other related enzymes in HAT family. To explore the molecular basis for the catalytic mechanism, computational approaches including molecular modeling, molecular dynamic (MD simulation and quantum mechanics/molecular mechanics (QM/MM simulation were carried out. The initial hGCN5/AcCoA/pH3 complex structure was modeled and a reasonable snapshot was extracted from the trajectory of a 20 ns MD simulation, with considering post-MD analysis and reported experimental results. Those residues playing crucial roles in binding affinity and acetylation reaction were comprehensively investigated. It demonstrated Glu80 acted as the general base for deprotonation of Lys171 from H3. Furthermore, the two-dimensional QM/MM potential energy surface was employed to study the sequential pathway acetylation mechanism. Energy barriers of addition-elimination reaction in acetylation obtained from QM/MM calculation indicated the point of the intermediate ternary complex. Our study may provide insights into the detailed mechanism for acetylation reaction of GCN5, and has

  3. Effects of temperature on the elimination of benzocaine and acetylated benzocaine residues from the edible fillet of rainbow trout (Oncorhynchus mykiss)

    Science.gov (United States)

    Stehly, G.R.; Meinertz, J.R.; Gingerich, W.H.

    2000-01-01

    The effect of temperature (7 degrees C and 16 degrees C) on the extent of accumulation and the elimination of benzocaine (BNZ) and its metabolite, acetylated benzocaine (AcBNZ), in the fillet tissue of rainbow trout was investigated Residues were measured after bath exposure to an anesthetizing concentration of benzocaine (30 mg/l for 5 min) followed by a maintenance concentration (15 mg/l for 30 min). Immediately after exposure, the BNZ concentration in fillet tissue was approximately 27 mu g/g at both temperatures; AcBNZ was 0.3 mu g/g at 7 degrees C and 0.6 mu g/g at 16 degrees C. The rates for elimination (alpha and beta) of BNZ and AcBNZ were not significantly different between the two temperatures. Terminal half-lives of elimination for BNZ were 1.62 h at 7 degrees C and 1.63 h at 16 degrees C; half-lives for AcBNZ were 2.36 h at 7 degrees C and 2.77 h at 16 degrees C.

  4. N-alpha-terminal acetylation of histone H4 regulates arginine methylation and ribosomal DNA silencing.

    Directory of Open Access Journals (Sweden)

    Vassia Schiza

    Full Text Available Post-translational modifications of histones play a key role in DNA-based processes, like transcription, by modulating chromatin structure. N-terminal acetylation is unique among the numerous histone modifications because it is deposited on the N-alpha amino group of the first residue instead of the side-chain of amino acids. The function of this modification and its interplay with other internal histone marks has not been previously addressed. Here, we identified N-terminal acetylation of H4 (N-acH4 as a novel regulator of arginine methylation and chromatin silencing in Saccharomyces cerevisiae. Lack of the H4 N-alpha acetyltransferase (Nat4 activity results specifically in increased deposition of asymmetric dimethylation of histone H4 arginine 3 (H4R3me2a and in enhanced ribosomal-DNA silencing. Consistent with this, H4 N-terminal acetylation impairs the activity of the Hmt1 methyltransferase towards H4R3 in vitro. Furthermore, combinatorial loss of N-acH4 with internal histone acetylation at lysines 5, 8 and 12 has a synergistic induction of H4R3me2a deposition and rDNA silencing that leads to a severe growth defect. This defect is completely rescued by mutating arginine 3 to lysine (H4R3K, suggesting that abnormal deposition of a single histone modification, H4R3me2a, can impact on cell growth. Notably, the cross-talk between N-acH4 and H4R3me2a, which regulates rDNA silencing, is induced under calorie restriction conditions. Collectively, these findings unveil a molecular and biological function for H4 N-terminal acetylation, identify its interplay with internal histone modifications, and provide general mechanistic implications for N-alpha-terminal acetylation, one of the most common protein modifications in eukaryotes.

  5. Intra- and inter-nucleosomal interactions of the histone H4 tail revealed with a human nucleosome core particle with genetically-incorporated H4 tetra-acetylation.

    Science.gov (United States)

    Wakamori, Masatoshi; Fujii, Yoshifumi; Suka, Noriyuki; Shirouzu, Mikako; Sakamoto, Kensaku; Umehara, Takashi; Yokoyama, Shigeyuki

    2015-11-26

    Post-translational modifications (PTMs) of histones, such as lysine acetylation of the N-terminal tails, play crucial roles in controlling gene expression. Due to the difficulty in reconstituting site-specifically acetylated nucleosomes with crystallization quality, structural analyses of histone acetylation are currently performed using synthesized tail peptides. Through engineering of the genetic code, translation termination, and cell-free protein synthesis, we reconstituted human H4-mono- to tetra-acetylated nucleosome core particles (NCPs), and solved the crystal structures of the H4-K5/K8/K12/K16-tetra-acetylated NCP and unmodified NCP at 2.4 Å and 2.2 Å resolutions, respectively. The structure of the H4-tetra-acetylated NCP resembled that of the unmodified NCP, and the DNA wrapped the histone octamer as precisely as in the unmodified NCP. However, the B-factors were significantly increased for the peripheral DNAs near the N-terminal tail of the intra- or inter-nucleosomal H4. In contrast, the B-factors were negligibly affected by the H4 tetra-acetylation in histone core residues, including those composing the acidic patch, and at H4-R23, which interacts with the acidic patch of the neighboring NCP. The present study revealed that the H4 tetra-acetylation impairs NCP self-association by changing the interactions of the H4 tail with DNA, and is the first demonstration of crystallization quality NCPs reconstituted with genuine PTMs.

  6. Molecular and structural insight into lysine selection on substrate and ubiquitin lysine 48 by the ubiquitin-conjugating enzyme Cdc34

    DEFF Research Database (Denmark)

    Suryadinata, Randy; Holien, Jessica K; Yang, George

    2013-01-01

    The attachment of ubiquitin (Ub) to lysines on substrates or itself by ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes results in protein ubiquitination. Lysine selection is important for generating diverse substrate-Ub structures and targeting proteins to different fates; however......, the mechanisms of lysine selection are not clearly understood. The positioning of lysine(s) toward the E2/E3 active site and residues proximal to lysines are critical in their selection. We investigated determinants of lysine specificity of the ubiquitin-conjugating enzyme Cdc34, toward substrate and Ub lysines....... Evaluation of the relative importance of different residues positioned -2, -1, +1 and +2 toward ubiquitination of its substrate, Sic1, on lysine 50 showed that charged residues in the -1 and -2 positions negatively impact on ubiquitination. Modeling suggests that charged residues at these positions alter...

  7. Spatial Memory Consolidation is Associated with Induction of Several Lysine-Acetyltransferase (Histone Acetyltransferase) Expression Levels and H2B/H4 Acetylation-Dependent Transcriptional Events in the Rat Hippocampus

    Science.gov (United States)

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

    2010-01-01

    Numerous genetic studies have shown that the CREB-binding protein (CBP) is an essential component of long-term memory formation, through its histone acetyltransferase (HAT) function. E1A-binding protein p300 and p300/CBP-associated factor (PCAF) have also recently been involved in memory formation. By contrast, only a few studies have reported on acetylation modifications during memory formation, and it remains unclear as to how the system is regulated during this dynamic phase. We investigated acetylation-dependent events and the expression profiles of these HATs during a hippocampus-dependent task taxing spatial reference memory in the Morris water maze. We found a specific increase in H2B and H4 acetylation in the rat dorsal hippocampus, while spatial memory was being consolidated. This increase correlated with the degree of specific acetylated histones enrichment on some memory/plasticity-related gene promoters. Overall, a global increase in HAT activity was measured during this memory consolidation phase, together with a global increase of CBP, p300, and PCAF expression. Interestingly, these regulations were altered in a model of hippocampal denervation disrupting spatial memory consolidation, making it impossible for the hippocampus to recruit the CBP pathway (CBP regulation and acetylated-H2B-dependent transcription). CBP has long been thought to be present in limited concentrations in the cells. These results show, for the first time, that CBP, p300, and PCAF are dynamically modulated during the establishment of a spatial memory and are likely to contribute to the induction of a specific epigenetic tagging of the genome for hippocampus-dependent (spatial) memory consolidation. These findings suggest the use of HAT-activating molecules in new therapeutic strategies of pathological aging, Alzheimer's disease, and other neurodegenerative disorders. PMID:20811339

  8. CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis.

    Science.gov (United States)

    Cazzalini, Ornella; Sommatis, Sabrina; Tillhon, Micol; Dutto, Ilaria; Bachi, Angela; Rapp, Alexander; Nardo, Tiziana; Scovassi, A Ivana; Necchi, Daniela; Cardoso, M Cristina; Stivala, Lucia A; Prosperi, Ennio

    2014-07-01

    The proliferating cell nuclear antigen (PCNA) protein serves as a molecular platform recruiting and coordinating the activity of factors involved in multiple deoxyribonucleic acid (DNA) transactions. To avoid dangerous genome instability, it is necessary to prevent excessive retention of PCNA on chromatin. Although PCNA functions during DNA replication appear to be regulated by different post-translational modifications, the mechanism regulating PCNA removal and degradation after nucleotide excision repair (NER) is unknown. Here we report that CREB-binding protein (CBP), and less efficiently p300, acetylated PCNA at lysine (Lys) residues Lys13,14,77 and 80, to promote removal of chromatin-bound PCNA and its degradation during NER. Mutation of these residues resulted in impaired DNA replication and repair, enhanced the sensitivity to ultraviolet radiation, and prevented proteolytic degradation of PCNA after DNA damage. Depletion of both CBP and p300, or failure to load PCNA on DNA in NER deficient cells, prevented PCNA acetylation and degradation, while proteasome inhibition resulted in accumulation of acetylated PCNA. These results define a CBP and p300-dependent mechanism for PCNA acetylation after DNA damage, linking DNA repair synthesis with removal of chromatin-bound PCNA and its degradation, to ensure genome stability.

  9. Obesity, cancer, and acetyl-CoA metabolism

    OpenAIRE

    Lee, Joyce V.; Shah, Supriya A.; Wellen, Kathryn E.

    2013-01-01

    As rates of obesity soar in the Unites States and around the world, cancer attributed to obesity has emerged as major threat to public health. The link between obesity and cancer can be attributed in part to the state of chronic inflammation that develops in obesity. Acetyl-CoA production and protein acetylation patterns are highly sensitive to metabolic state and are significantly altered in obesity. In this article, we explore the potential role of nutrient-sensitive lysine acetylation in r...

  10. E2F family members are differentially regulated by reversible acetylation

    DEFF Research Database (Denmark)

    Marzio, G; Wagener, C; Gutierrez, M I;

    2000-01-01

    The six members of the E2F family of transcription factors play a key role in the control of cell cycle progression by regulating the expression of genes involved in DNA replication and cell proliferation. E2F-1, -2, and -3 belong to a structural and functional subfamily distinct from those...... of the other E2F family members. Here we report that E2F-1, -2, and -3, but not E2F-4, -5, and -6, associate with and are acetylated by p300 and cAMP-response element-binding protein acetyltransferases. Acetylation occurs at three conserved lysine residues located at the N-terminal boundary of their DNA...

  11. Oligo-lysine Induced Formation of Silica Particles in Neutral Silicate Solution

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Oligo-(lysine)n (n = 1-4) containing different numbers of lysine residues was used to induce the condensation of silicic acid to form silica particles in neutral silicate solution. It was found that the condensation rate and the formation of silica particles are dependent on the number of lysine residues in an oligo-lysine. Oligo-lysine with more lysine residues can link more silicic acid together to form a matrix that promotes the effective aggregation of the condensed silica pieces to form large silica particles.

  12. Histone Acetylation near the Nucleosome Dyad Axis Enhances Nucleosome Disassembly by RSC and SWI/SNF.

    Science.gov (United States)

    Chatterjee, Nilanjana; North, Justin A; Dechassa, Mekonnen Lemma; Manohar, Mridula; Prasad, Rashmi; Luger, Karolin; Ottesen, Jennifer J; Poirier, Michael G; Bartholomew, Blaine

    2015-12-01

    Signaling associated with transcription activation occurs through posttranslational modification of histones and is best exemplified by lysine acetylation. Lysines are acetylated in histone tails and the core domain/lateral surface of histone octamers. While acetylated lysines in histone tails are frequently recognized by other factors referred to as "readers," which promote transcription, the mechanistic role of the modifications in the lateral surface of the histone octamer remains unclear. By using X-ray crystallography, we found that acetylated lysines 115 and 122 in histone H3 are solvent accessible, but in biochemical assays they appear not to interact with the bromodomains of SWI/SNF and RSC to enhance recruitment or nucleosome mobilization, as previously shown for acetylated lysines in H3 histone tails. Instead, we found that acetylation of lysines 115 and 122 increases the predisposition of nucleosomes for disassembly by SWI/SNF and RSC up to 7-fold, independent of bromodomains, and only in conjunction with contiguous nucleosomes. Thus, in combination with SWI/SNF and RSC, acetylation of lateral surface lysines in the histone octamer serves as a crucial regulator of nucleosomal dynamics distinct from the histone code readers and writers.

  13. Hypochlorite-induced damage to proteins: formation of nitrogen-centred radicals from lysine residues and their role in protein fragmentation.

    Science.gov (United States)

    Hawkins, C L; Davies, M J

    1998-01-01

    Stimulated monocytes and neutrophils generate hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl damages proteins by reaction with amino acid side-chains or backbone cleavage. Little information is available about the mechanisms and intermediates involved in these reactions. EPR spin trapping has been employed to identify radicals on proteins, peptides and amino acids after treatment with HOCl. Reaction with HOCl gives both high- and low-molecular-mass nitrogen-centred, protein-derived radicals; the yield of the latter increases with both higher HOCl:protein ratios and enzymic digestion. These radicals, which arise from lysine side-chain amino groups, react with ascorbate, glutathione and Trolox. Reaction of HOCl-treated proteins with excess methionine eliminates radical formation, which is consistent with lysine-derived chloramines (via homolysis of N-Cl bonds) being the radical source. Incubation of HOCl-treated proteins, after removal of excess oxidant, gives rise to both nitrogen-centred radicals, over a period of hours, and time-dependent fragmentation of the protein. Treatment with excess methionine or antioxidants (Trolox, ascorbate, glutathione) protects against fragmentation; urate and bilirubin do not. Chloramine formation and nitrogen-centred radicals are therefore key species in HOCl-induced protein fragmentation. PMID:9620862

  14. Detection of histone acetylation levels in the dorsal hippocampus reveals early tagging on specific residues of H2B and H4 histones in response to learning.

    Science.gov (United States)

    Bousiges, Olivier; Neidl, Romain; Majchrzak, Monique; Muller, Marc-Antoine; Barbelivien, Alexandra; Pereira de Vasconcelos, Anne; Schneider, Anne; Loeffler, Jean-Philippe; Cassel, Jean-Christophe; Boutillier, Anne-Laurence

    2013-01-01

    The recent literature provides evidence that epigenetic mechanisms such as DNA methylation and histone modification are crucial to gene transcription linked to synaptic plasticity in the mammalian brain--notably in the hippocampus--and memory formation. We measured global histone acetylation levels in the rat hippocampus at an early stage of spatial or fear memory formation. We found that H3, H4 and H2B underwent differential acetylation at specific sites depending on whether rats had been exposed to the context of a task without having to learn or had to learn about a place or fear therein: H3K9K14 acetylation was mostly responsive to any experimental conditions compared to naive animals, whereas H2B N-terminus and H4K12 acetylations were mostly associated with memory for either spatial or fear learning. Altogether, these data suggest that behavior/experience-dependent changes differently regulate specific acetylation modifications of histones in the hippocampus, depending on whether a memory trace is established or not: tagging of H3K9K14 could be associated with perception/processing of testing-related manipulations and context, thereby enhancing chromatin accessibility, while tagging of H2B N-terminus tail and H4K12 could be more closely associated with the formation of memories requiring an engagement of the hippocampus.

  15. Detection of histone acetylation levels in the dorsal hippocampus reveals early tagging on specific residues of H2B and H4 histones in response to learning.

    Directory of Open Access Journals (Sweden)

    Olivier Bousiges

    Full Text Available The recent literature provides evidence that epigenetic mechanisms such as DNA methylation and histone modification are crucial to gene transcription linked to synaptic plasticity in the mammalian brain--notably in the hippocampus--and memory formation. We measured global histone acetylation levels in the rat hippocampus at an early stage of spatial or fear memory formation. We found that H3, H4 and H2B underwent differential acetylation at specific sites depending on whether rats had been exposed to the context of a task without having to learn or had to learn about a place or fear therein: H3K9K14 acetylation was mostly responsive to any experimental conditions compared to naive animals, whereas H2B N-terminus and H4K12 acetylations were mostly associated with memory for either spatial or fear learning. Altogether, these data suggest that behavior/experience-dependent changes differently regulate specific acetylation modifications of histones in the hippocampus, depending on whether a memory trace is established or not: tagging of H3K9K14 could be associated with perception/processing of testing-related manipulations and context, thereby enhancing chromatin accessibility, while tagging of H2B N-terminus tail and H4K12 could be more closely associated with the formation of memories requiring an engagement of the hippocampus.

  16. A Quantitative Study on the in-vitro and in-vivo Acetylation of High Mobility Group A1 Proteins

    OpenAIRE

    Zhang, Qingchun; Zhang, Kangling; Zou, Yan; Perna, Avi; Wang, Yinsheng

    2007-01-01

    High mobility group (HMG) A1 proteins are subject to a number of post-translational modifications, which may regulate their function in gene transcription and other cellular processes. We examined, by using mass spectrometry, the acetylation of HMGA1a and HMGA1b proteins induced by histone acetyltransferases p300 and PCAF in vitro and in PC-3 human prostate cancer cells in vivo. It turned out that five lysine residues in HMGA1a, i.e., Lys-14, Lys-64, Lys-66, Lys-70, and Lys-73, could be acety...

  17. Tetranectin-binding site on plasminogen kringle 4 involves the lysine-binding pocket and at least one additional amino acid residue

    DEFF Research Database (Denmark)

    Graversen, Jonas Heilskov; Sigurskjold, B W; Thøgersen, H C

    2000-01-01

    that all amino acid residues of plasminogen kringle 4 found to be involved in t-AMCHA binding are also involved in binding tetranectin. Notably, one amino acid residue of plasminogen kringle 4, Arg 32, not involved in binding t-AMCHA, is critical for binding tetranectin. We also find that Asp 57 and Asp 55......, we analyze the interaction of wild-type and six single-residue mutants of recombinant plasminogen kringle 4 expressed in Escherichia coli with the recombinant C-type lectin domain of tetranectin and trans-aminomethyl-cyclohexanoic acid (t-AMCHA) using isothermal titration calorimetry. We find...

  18. The impact of fixatives on the binding of lectins to N-acetyl-glucosamine residues of human syncytiotrophoblast: a quantitative histochemical study

    DEFF Research Database (Denmark)

    Høyer, P E; Kirkeby, S

    1996-01-01

    binding to N-acetyl-galactosamine, mannose, galactose, and fucose was also significantly higher in sections from tissues fixed in an acid fixative compared with a neutral buffered fixative. Unfixed cryosections revealed a considerably lower degree of specific lectin binding compared with sections from...

  19. Probing China's Lysine Market

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ The lysine sector in China developed further in 2006. Both the capacity and the output hit new highs and China had a major impact on the global lysine market. The import amount of lysine satisfied only a very small portion of the domestic market's demand.

  20. A novel MeCP2 acetylation site regulates interaction with ATRX and HDAC1.

    Science.gov (United States)

    Pandey, Somnath; Simmons, Glenn E; Malyarchuk, Svitlana; Calhoun, Tara N; Pruitt, Kevin

    2015-09-01

    Methyl-CpG-binding protein-2 (MeCP2) regulates gene expression by recruiting SWI/SNF DNA helicase/ATPase (ATRX) and Histone Deacetylase-1 (HDAC1) to methylated gene regions and modulates heterochromatin association by interacting with Heterochromatin protein-1. As MeCP2 contributes to tumor suppressor gene silencing and its mutation causes Rett Syndrome, we investigated how novel post-translational-modification contributes to its function. Herein we report that upon pharmacological inhibition of SIRT1 in RKO colon and MCF-7 breast cancer cells, endogenous MeCP2 is acetylated at sites critical for binding to DNA and transcriptional regulators. We created an acetylation mimetic mutation in MeCP2 and found it to possess decreased binding to ATRX and HDAC1. Conditions inducing MeCP2 acetylation do not alter its promoter occupancy at a subset of target genes analyzed, but do cause decreased binding to ATRX and HDAC1. We also report here that a specific inhibitor of SIRT1, IV, can be used to selectively decrease H3K27me3 repressive marks on a subset of repressed target gene promoters analyzed. Lastly, we show that RKO cells over-expressing MeCP2 mutant show reduced proliferation compared to those over-expressing MeCP2-wildtype. Our study demonstrates the importance of acetylated lysine residues and suggests their key role in regulating MeCP2 function and its ability to bind transcriptional regulators.

  1. Structural basis for the site-specific incorporation of lysine derivatives into proteins.

    Directory of Open Access Journals (Sweden)

    Veronika Flügel

    Full Text Available Posttranslational modifications (PTMs of proteins determine their structure-function relationships, interaction partners, as well as their fate in the cell and are crucial for many cellular key processes. For instance chromatin structure and hence gene expression is epigenetically regulated by acetylation or methylation of lysine residues in histones, a phenomenon known as the 'histone code'. Recently it was shown that these lysine residues can furthermore be malonylated, succinylated, butyrylated, propionylated and crotonylated, resulting in significant alteration of gene expression patterns. However the functional implications of these PTMs, which only differ marginally in their chemical structure, is not yet understood. Therefore generation of proteins containing these modified amino acids site specifically is an important tool. In the last decade methods for the translational incorporation of non-natural amino acids using orthogonal aminoacyl-tRNA synthetase (aaRS:tRNAaaCUA pairs were developed. A number of studies show that aaRS can be evolved to use non-natural amino acids and expand the genetic code. Nevertheless the wild type pyrrolysyl-tRNA synthetase (PylRS from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of non-natural amino acids. Here we present structural data on the wild type enzyme in complex with adenylated ε-N-alkynyl-, ε-N-butyryl-, ε-N-crotonyl- and ε-N-propionyl-lysine providing insights into the plasticity of the PylRS active site. This shows that given certain key features in the non-natural amino acid to be incorporated, directed evolution of this enzyme is not necessary for substrate tolerance.

  2. Changes to biological activity following acetylation of dendrotoxin I from Dendroaspis polylepis (black mamba).

    Science.gov (United States)

    Harvey, A L; Rowan, E G; Vatanpour, H; Engström, A; Westerlund, B; Karlsson, E

    1997-08-01

    The potassium channel blocker dendrotoxin I was acetylated with acetic anhydride. Mono-acetyl derivatives of all seven lysine residues (N-terminus blocked) and a di-derivative were isolated by chromatography on the cation-exchanger Bio-Rex 70 and reversed-phase high-performance liquid chromatography. The derivative acetyl-Lys 29 and the di-derivative of Tyr 24 and Lys 28 had more than 1000 times lower affinity than the native toxin as determined by inhibition of the 125I-dendrotoxin binding to synaptosomal membranes from rat brain. Lys 29 is part of the triplet Lys-Lys-Lys (28-30) which also occurs in the homologous alpha-dendrotoxin where the triplet is not in the functional site, as shown by site-directed mutagenesis. Acetylation of Lys 29 may have produced large structural perturbations that inactivated the toxin. Acetylation of Lys 28 alone had little effect, but the toxin became almost inactive when both Lys 28 and Tyr 24 were modified. Ten experiments were conducted under similar conditions, but a derivative of Tyr 24 was obtained only three times. In these cases the toxin apparently had a different structure, with Tyr 24 accessible to the reagent. This may depend on freeze-drying, which can alter the structure of proteins. The third derivative with low activity was acetyl-Lys 5, with affinity decreased 20-fold. Lys 5 has a protruding side-chain that does not interact with any other group in the toxin molecule. Therefore, Lys 5 is probably part of the functional site for dendrotoxin's binding to the voltage-dependent K+ channels.

  3. An update on histone lysine methylation in plants

    Institute of Scientific and Technical Information of China (English)

    Yu Yu; Zhongyuan Bu; Wen-Hui Shen; Aiwu Dong

    2009-01-01

    Histone methylation plays crucial roles in epigenetic regulation.The SET domain proteins are now recognized as generally having methyltransferase activity targeted to specific lysine residues of histones.The enzymes and their specific histone lysine methylation have enormous impacts on the regulation of chromatin structure and function.In this review,we discuss recent advances made on histone lysine methylations and their diverse functions in plant growth and development.

  4. Helix stability in succinylated and acetylated ovalbumins: effect of high pH, urea and guanidine hydrochloride.

    Science.gov (United States)

    Batra, P P; Uetrecht, D

    1990-08-01

    Previous studies (Batra, P.P., Roebuck, M.A. and Uetrecht, D. (1990) J. Protein Chem. 9, 37-44) showed that succinylation or acetylation of 75% of the lysine residues has little effect on the secondary structure of ovalbumin. The acylation of the remaining 25% lysine residues, which apparently are partially buried, results in a substantial loss of the helical structure. These conformational changes may be due not only to electrostatic repulsions introduced by succinylation or acetylation of the positively charged epsilon-amino groups but also to steric hindrance, since an increase in the ionic strength failed to reverse the loss of the helical structure. An increase in pH to 12.2 results in a complete helix-to-coil transition in the maximally succinylated ovalbumin (but not in the partially succinylated or in any of the acetylated ovalbumins including the maximally acetylated derivative), perhaps because it is most expanded and its molecular interior most accessible to solvent as succinylation replaces +1 charge of epsilon-amino group with a -1 charge so that a net of -2 charge per succinyl group is placed on the protein molecule. This helix-to-coil transition in the maximally succinylated ovalbumin induced by high pH is fully reversed by increasing the ionic strength, indicating that only electrostatic effects are responsible for this disruption. Studies have also shown that although there is no loss of the helical structure until after the 75% surface lysine residues have been acylated, the helical structure does become progressively destabilized with increasing degree of modification, a conclusion drawn from urea unfolding curves. This destabilization of the helical structure is due primarily to electrostatic effects, as an increase in the ionic strength led to an increase in the urea transition mid-point. Unlike urea, the guanidine hydrochloride unfolding curves indicate that the transition mid-point for the native protein, as well as for the maximally

  5. Myofibril growth during cardiac hypertrophy is regulated through dual phosphorylation and acetylation of the actin capping protein CapZ.

    Science.gov (United States)

    Lin, Ying-Hsi; Warren, Chad M; Li, Jieli; McKinsey, Timothy A; Russell, Brenda

    2016-08-01

    The mechanotransduction signaling pathways initiated in heart muscle by increased mechanical loading are known to lead to long-term transcriptional changes and hypertrophy, but the rapid events for adaptation at the sarcomeric level are not fully understood. The goal of this study was to test the hypothesis that actin filament assembly during cardiomyocyte growth is regulated by post-translational modifications (PTMs) of CapZβ1. In rapidly hypertrophying neonatal rat ventricular myocytes (NRVMs) stimulated by phenylephrine (PE), two-dimensional gel electrophoresis (2DGE) of CapZβ1 revealed a shift toward more negative charge. Consistent with this, mass spectrometry identified CapZβ1 phosphorylation on serine-204 and acetylation on lysine-199, two residues which are near the actin binding surface of CapZβ1. Ectopic expression of dominant negative PKCɛ (dnPKCɛ) in NRVMs blunted the PE-induced increase in CapZ dynamics, as evidenced by the kinetic constant (Kfrap) of fluorescence recovery after photobleaching (FRAP), and concomitantly reduced phosphorylation and acetylation of CapZβ1. Furthermore, inhibition of class I histone deacetylases (HDACs) increased lysine-199 acetylation on CapZβ1, which increased Kfrap of CapZ and stimulated actin dynamics. Finally, we show that PE treatment of NRVMs results in decreased binding of HDAC3 to myofibrils, suggesting a signal-dependent mechanism for the regulation of sarcomere-associated CapZβ1 acetylation. Taken together, this dual regulation through phosphorylation and acetylation of CapZβ1 provides a novel model for the regulation of myofibril growth during cardiac hypertrophy. Copyright © 2016. Published by Elsevier Inc.

  6. Installation of site-specific methylation into histones using methyl lysine analogs.

    Science.gov (United States)

    Simon, Matthew D

    2010-04-01

    Chromatin structure is influenced by post-translational modifications on histones, the principal basic protein component of chromatin. In order to study one of these modifications, lysine methylation, in the context of reconstituted chromatin, this unit describes the installation of analogs of methyl lysine residues into recombinant histones. The modification site is specified by mutating the lysine of interest to cysteine. The mutant histones are expressed and purified, and the cysteine residue alkylated to produce N-methyl aminoethylcysteine, an isosteric analog of methyl lysine. Using different alkylating reagents, it is possible to install analogs of mono-, di-, or trimethyl lysine. While these analogs are not identical to methyl lysine residues, they show similar biochemical properties to their natural counterparts. The ease of synthesis of methyl lysine analog (MLA) histones, especially on a large scale, makes them particularly useful reagents for studying the effects of histone lysine methylation on chromatin structure, biophysics and biochemistry. (c) 2010 by John Wiley & Sons, Inc.

  7. Acetylation of malate dehydrogenase 1 promotes adipogenic differentiation via activating its enzymatic activity.

    Science.gov (United States)

    Kim, Eun Young; Kim, Won Kon; Kang, Hyo Jin; Kim, Jeong-Hoon; Chung, Sang J; Seo, Yeon Soo; Park, Sung Goo; Lee, Sang Chul; Bae, Kwang-Hee

    2012-09-01

    Acetylation is one of the most crucial post-translational modifications that affect protein function. Protein lysine acetylation is catalyzed by acetyltransferases, and acetyl-CoA functions as the source of the acetyl group. Additionally, acetyl-CoA plays critical roles in maintaining the balance between carbohydrate metabolism and fatty acid synthesis. Here, we sought to determine whether lysine acetylation is an important process for adipocyte differentiation. Based on an analysis of the acetylome during adipogenesis, various proteins displaying significant quantitative changes were identified by LC-MS/MS. Of these identified proteins, we focused on malate dehydrogenase 1 (MDH1). The acetylation level of MDH1 was increased up to 6-fold at the late stage of adipogenesis. Moreover, overexpression of MDH1 in 3T3-L1 preadipocytes induced a significant increase in the number of cells undergoing adipogenesis. The introduction of mutations to putative lysine acetylation sites showed a significant loss of the ability of cells to undergo adipogenic differentiation. Furthermore, the acetylation of MDH1 dramatically enhanced its enzymatic activity and subsequently increased the intracellular levels of NADPH. These results clearly suggest that adipogenic differentiation may be regulated by the acetylation of MDH1 and that the acetylation of MDH1 is one of the cross-talk mechanisms between adipogenesis and the intracellular energy level.

  8. Mechanistic insights into the regulation of metabolic enzymes by acetylation

    Science.gov (United States)

    2012-01-01

    The activity of metabolic enzymes is controlled by three principle levels: the amount of enzyme, the catalytic activity, and the accessibility of substrates. Reversible lysine acetylation is emerging as a major regulatory mechanism in metabolism that is involved in all three levels of controlling metabolic enzymes and is altered frequently in human diseases. Acetylation rivals other common posttranslational modifications in cell regulation not only in the number of substrates it modifies, but also the variety of regulatory mechanisms it facilitates. PMID:22826120

  9. Mutational, kinetic, and NMR studies of the roles of conserved glutamate residues and of lysine-39 in the mechanism of the MutT pyrophosphohydrolase.

    Science.gov (United States)

    Harris, T K; Wu, G; Massiah, M A; Mildvan, A S

    2000-02-22

    The MutT enzyme catalyzes the hydrolysis of nucleoside triphosphates (NTP) to NMP and PP(i) by nucleophilic substitution at the rarely attacked beta-phosphorus. The solution structure of the quaternary E-M(2+)-AMPCPP-M(2+) complex indicated that conserved residues Glu-53, -56, -57, and -98 are at the active site near the bound divalent cation possibly serving as metal ligands, Lys-39 is positioned to promote departure of the NMP leaving group, and Glu-44 precedes helix I (residues 47-59) possibly stabilizing this helix which contributes four catalytic residues to the active site [Lin, J. , Abeygunawardana, C., Frick, D. N., Bessman, M. J., and Mildvan, A. S. (1997) Biochemistry 36, 1199-1211]. To test these proposed roles, the effects of mutations of each of these residues on the kinetic parameters and on the Mn(2+), Mg(2+), and substrate binding properties were examined. The largest decreases in k(cat) for the Mg(2+)-activated enzyme of 10(4.7)- and 10(2.6)-fold were observed for the E53Q and E53D mutants, respectively, while 97-, 48-, 25-, and 14-fold decreases were observed for the E44D, E56D, E56Q, and E44Q mutations, respectively. Smaller effects on k(cat) were observed for mutations of Glu-98 and Lys-39. For wild type MutT and its E53D and E44D mutants, plots of log(k(cat)) versus pH exhibited a limiting slope of 1 on the ascending limb and then a hump, i.e., a sharply defined maximum near pH 8 followed by a plateau, yielding apparent pK(a) values of 7.6 +/- 0.3 and 8.4 +/- 0.4 for an essential base and a nonessential acid catalyst, respectively, in the active quaternary MutT-Mg(2+)-dGTP-Mg(2+) complex. The pK(a) of 7.6 is assigned to Glu-53, functioning as a base catalyst in the active quaternary complex, on the basis of the disappearance of the ascending limb of the pH-rate profile of the E53Q mutant, and its restoration in the E53D mutant with a 10(1.9)-fold increase in (k(cat))(max). The pK(a) of 8.4 is assigned to Lys-39 on the basis of the disappearance

  10. Histone H4 acetylation required for chromatin decompaction during DNA replication.

    Science.gov (United States)

    Ruan, Kun; Yamamoto, Takaharu G; Asakawa, Haruhiko; Chikashige, Yuji; Kimura, Hiroshi; Masukata, Hisao; Haraguchi, Tokuko; Hiraoka, Yasushi

    2015-07-30

    Faithful DNA replication is a prerequisite for cell proliferation. Several cytological studies have shown that chromosome structures alter in the S-phase of the cell cycle. However, the molecular mechanisms behind the alteration of chromosome structures associated with DNA replication have not been elucidated. Here, we investigated chromatin structures and acetylation of specific histone residues during DNA replication using the meiotic nucleus of the fission yeast Schizosaccharomyces pombe. The S. pombe meiotic nucleus provides a unique opportunity for measuring the levels of compaction of chromatin along the chromosome in a defined orientation. By direct measurement of chromatin compaction in living cells, we demonstrated that decompaction of chromatin occurs during meiotic DNA replication. This chromatin decompaction was suppressed by depletion of histone acetyltransferase Mst1 or by arginine substitution of specific lysine residues (K8 and K12) of histone H4. These results suggest that acetylation of histone H4 residues K8 and K12 plays a critical role in loosening chromatin structures during DNA replication.

  11. Structure and Histone Binding Properties of the Vps75-Rtt109 Chaperone-Lysine Acetyltransferase Complex

    Energy Technology Data Exchange (ETDEWEB)

    Su, Dan; Hu, Qi; Zhou, Hui; Thompson, James R.; Xu, Rui-Ming; Zhang, Zhiguo; Mer, Georges (Mayo); (Chinese Aca. Sci.)

    2011-11-02

    The histone chaperone Vps75 presents the remarkable property of stimulating the Rtt109-dependent acetylation of several histone H3 lysine residues within (H3-H4){sub 2} tetramers. To investigate this activation mechanism, we determined x-ray structures of full-length Vps75 in complex with full-length Rtt109 in two crystal forms. Both structures show similar asymmetric assemblies of a Vps75 dimer bound to an Rtt109 monomer. In the Vps75-Rtt109 complexes, the catalytic site of Rtt109 is confined to an enclosed space that can accommodate the N-terminal tail of histone H3 in (H3-H4){sub 2}. Investigation of Vps75-Rtt109-(H3-H4)2 and Vps75-(H3-H4)2 complexes by NMR spectroscopy-probed hydrogen/deuterium exchange suggests that Vps75 guides histone H3 in the catalytic enclosure. These findings clarify the basis for the enhanced acetylation of histone H3 tail residues by Vps75-Rtt109.

  12. Aspirin acetylates wild type and mutant p53 in colon cancer cells: identification of aspirin acetylated sites on recombinant p53.

    Science.gov (United States)

    Ai, Guoqiang; Dachineni, Rakesh; Kumar, D Ramesh; Marimuthu, Srinivasan; Alfonso, Lloyd F; Bhat, G Jayarama

    2016-05-01

    Aspirin's ability to inhibit cell proliferation and induce apoptosis in cancer cell lines is considered to be an important mechanism for its anti-cancer effects. We previously demonstrated that aspirin acetylated the tumor suppressor protein p53 at lysine 382 in MDA-MB-231 human breast cancer cells. Here, we extended these observations to human colon cancer cells, HCT 116 harboring wild type p53, and HT-29 containing mutant p53. We demonstrate that aspirin induced acetylation of p53 in both cell lines in a concentration-dependent manner. Aspirin-acetylated p53 was localized to the nucleus. In both cell lines, aspirin induced p21(CIP1). Aspirin also acetylated recombinant p53 (rp53) in vitro suggesting that it occurs through a non-enzymatic chemical reaction. Mass spectrometry analysis and immunoblotting identified 10 acetylated lysines on rp53, and molecular modeling showed that all lysines targeted by aspirin are surface exposed. Five of these lysines are localized to the DNA-binding domain, four to the nuclear localization signal domain, and one to the C-terminal regulatory domain. Our results suggest that aspirin's anti-cancer effect may involve acetylation and activation of wild type and mutant p53 and induction of target gene expression. This is the first report attempting to characterize p53 acetylation sites targeted by aspirin.

  13. Elucidating the effects of arginine and lysine on a monoclonal antibody C-terminal lysine variation in CHO cell cultures.

    Science.gov (United States)

    Zhang, Xintao; Tang, Hongping; Sun, Ya-Ting; Liu, Xuping; Tan, Wen-Song; Fan, Li

    2015-08-01

    C-terminal lysine variants are commonly observed in monoclonal antibodies (mAbs) and found sensitive to process conditions, especially specific components in culture medium. The potential roles of media arginine (Arg) and lysine (Lys) in mAb heavy chain C-terminal lysine processing were investigated by monitoring the lysine variant levels under various Arg and Lys concentrations. Both Arg and Lys were found to significantly affect lysine variant level. Specifically, lysine variant level increased from 18.7 to 31.8 % when Arg and Lys concentrations were increased from 2 to 10 mM. Since heterogeneity of C-terminal lysine residues is due to the varying degree of proteolysis by basic carboxypeptidases (Cps), enzyme (basic Cps) level, pH conditions, and product (Arg and Lys) inhibition, which potentially affect the enzymatic reaction, were investigated under various Arg and Lys conditions. Enzyme level and pH conditions were found not to account for the different lysine variant levels, which was evident from the minimal variation in transcription level and intracellular pH. On the other hand, product inhibition effect of Arg and Lys on basic Cps was evident from the notable intracellular and extracellular Arg and Lys concentrations comparable with Ki values (inhibition constant) of basic Cps and further confirmed by cell-free assays. Additionally, a kinetic study of lysine variant level during the cell culture process enabled further characterization of the C-terminal lysine processing.

  14. Acetylation of pea isolate in a torus microreactor.

    Science.gov (United States)

    Legrand, J; Guéguen, J; Berot, S; Popineau, Y; Nouri, L

    1997-02-20

    Acetylation, which acts on the amino groups of proteins, allows to increase the solubility and the emulsifying properties of pea isolate. Acetylation by acetic anhydride was carried out in a torus microreactor in semibatch and continuous conditions. The mixing characteristics, obtained by a residence time distribution (RTD) method, are the same in batch and continuous processes. The maximum acetylation degree reached by the torus reactor is higher than with the stirred reactor. Torus reactors are more efficient than stirred ones as shown by a conversion efficiency, defined by the quantity of modified lysine groups by consumed acetic anhydride. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 409-414, 1997.

  15. Phosphorylation and Acetylation of Acyl-CoA Synthetase- I

    DEFF Research Database (Denmark)

    Frahm, Jennifer L; Li, Lei O; Grevengoed, Trisha J

    2011-01-01

    Long chain acyl-CoA synthetase 1 (ACSL1) contributes 50 to 90% of total ACSL activity in liver, adipose tissue, and heart and appears to direct the use of long chain fatty acids for energy. Although the functional importance of ACSL1 is becoming clear, little is understood about its post...... and acetylated amino acids by mass spectrometry. We then compared these results to the post-translational modifications observed in vivo in liver and brown adipose tissue after mice were fasted or exposed to a cold environment. We identified universal N-terminal acetylation, 15 acetylated lysines, and 25...

  16. Impact of a High-fat Diet on Tissue Acyl-CoA and Histone Acetylation Levels.

    Science.gov (United States)

    Carrer, Alessandro; Parris, Joshua L D; Trefely, Sophie; Henry, Ryan A; Montgomery, David C; Torres, AnnMarie; Viola, John M; Kuo, Yin-Ming; Blair, Ian A; Meier, Jordan L; Andrews, Andrew J; Snyder, Nathaniel W; Wellen, Kathryn E

    2017-02-24

    Cellular metabolism dynamically regulates the epigenome via availability of the metabolite substrates of chromatin-modifying enzymes. The impact of diet on the metabolism-epigenome axis is poorly understood but could alter gene expression and influence metabolic health. ATP citrate-lyase produces acetyl-CoA in the nucleus and cytosol and regulates histone acetylation levels in many cell types. Consumption of a high-fat diet (HFD) results in suppression of ATP citrate-lyase levels in tissues such as adipose and liver, but the impact of diet on acetyl-CoA and histone acetylation in these tissues remains unknown. Here we examined the effects of HFD on levels of acyl-CoAs and histone acetylation in mouse white adipose tissue (WAT), liver, and pancreas. We report that mice consuming a HFD have reduced levels of acetyl-CoA and/or acetyl-CoA:CoA ratio in these tissues. In WAT and the pancreas, HFD also impacted the levels of histone acetylation; in particular, histone H3 lysine 23 acetylation was lower in HFD-fed mice. Genetic deletion of Acly in cultured adipocytes also suppressed acetyl-CoA and histone acetylation levels. In the liver, no significant effects on histone acetylation were observed with a HFD despite lower acetyl-CoA levels. Intriguingly, acetylation of several histone lysines correlated with the acetyl-CoA: (iso)butyryl-CoA ratio in liver. Butyryl-CoA and isobutyryl-CoA interacted with the acetyltransferase P300/CBP-associated factor (PCAF) in liver lysates and inhibited its activity in vitro This study thus provides evidence that diet can impact tissue acyl-CoA and histone acetylation levels and that acetyl-CoA abundance correlates with acetylation of specific histone lysines in WAT but not in the liver.

  17. Effect of dietary lysine on hepatic lysine catabolism in broilers

    Science.gov (United States)

    Lysine is frequently a first- or second-limiting amino acid in poultry diets. Improving the efficiency of lysine use for protein synthesis would effectively lower the lysine requirement and decrease feed costs. Understanding how lysine is degraded and how the degradation is regulated would identif...

  18. Systemic lupus erythematosus patients contain significantly less igm against mono-methylated lysine than healthy subjects.

    Directory of Open Access Journals (Sweden)

    Sha Guo

    Full Text Available Post-translational modifications on proteins are important in biological processes but may create neo-epitopes that induce autoimmune responses. In this study, we measured the serum IgG and IgM response to a set of non-modified or acetyl- and methyl-modified peptides corresponding to residues 1-19 of the histone 3 N-terminal tail in systemic lupus erythematosus (SLE patients and healthy subjects. Our results indicated that the SLE patients and healthy subjects produced antibodies (Abs to the peptides, but the two groups had different Ab isotype and epitope preferences. Abs to the non-modified form, H31-19, were of the IgG isotype and produced by SLE patients. They could not recognize the scrambled H31-19, which contained the same amino acid composition but a different sequence as H31-19. In comparison, healthy subjects in general did not produce IgG against H31-19. However, about 70% of the healthy subjects produced IgM Abs against mono-methylated K9 of H31-19 (H31-19K9me. Our further studies revealed that ε-amine mono-methylated lysine could completely inhibit the IgM binding to H31-19K9me, but lysine had no inhibitory effect. In addition, the IgM Abs could bind peptides containing a mono-methylated lysine residue but with totally different sequences. Thus, mono-methylated lysine was the sole epitope for the IgM. Interestingly, SLE patients had much lower levels of this type of IgM. There was no obvious correlation between the IgM levels and disease activity and the decreased IgM was unlikely caused by medical treatments.We also found that the IgM Abs were not polyreactive to dsDNA, ssDNA, lipopolysaccharide (LPS or insulin and they did not exist in umbilical cord serum, implying that they were not natural Abs. The IgM Abs against mono-methylated lysine are present in healthy subjects but are significantly lower in SLE patients, suggesting a distinct origin of production and special physiological functions.

  19. HDAC inhibitors induce global changes in histone lysine and arginine methylation and alter expression of lysine demethylases.

    Science.gov (United States)

    Lillico, Ryan; Sobral, Marina Gomez; Stesco, Nicholas; Lakowski, Ted M

    2016-02-01

    Histone deacetylase (HDAC) inhibitors are cancer treatments that inhibit the removal of the epigenetic modification acetyllysine on histones, resulting in altered gene expression. Such changes in expression may influence other histone epigenetic modifications. We describe a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify lysine acetylation and methylation and arginine methylation on histones extracted from cultured cells treated with HDAC inhibitors. The HDAC inhibitors vorinostat, mocetinostat and entinostat induced 400-600% hyperacetylation in HEK 293 and K562 cells. All HDAC inhibitors decreased histone methylarginines in HEK 293 cells but entinostat produced dose dependent reductions in asymmetric dimethylarginine, not observed in K562 cells. Vorinostat produced increases in histone lysine methylation and decreased expression of some lysine demethylases (KDM), measured by quantitative PCR. Entinostat had variable effects on lysine methylation and decreased expression of some KDM while increasing expression of others. Mocetinostat produced dose dependent increases in histone lysine methylation by LC-MS/MS. This was corroborated with a multiplex colorimetric assay showing increases in histone H3 lysine 4, 9, 27, 36 and 79 methylation. Increases in lysine methylation were correlated with dose dependent decreases in the expression of seven KDM. Mocetinostat functions as an HDAC inhibitor and a de facto KDM inhibitor.

  20. Global Lysine Acetylome Analysis of Desiccated Somatic Embryos of Picea asperata

    Science.gov (United States)

    Xia, Yan; Jing, Danlong; Kong, Lisheng; Zhang, Jianwei; OuYang, Fangqun; Zhang, Hanguo; Wang, Junhui; Zhang, Shougong

    2016-01-01

    Partial desiccation treatment (PDT) promotes the germination capacity of conifer somatic embryos. Lysine acetylation (LysAc) is a dynamic and reversible post-translational modification that plays a key role in many biological processes including metabolic pathways and stress response. To investigate the functional impact of LysAc in the response of Picea asperata somatic embryos to PDT, we performed a global lysine acetylome analysis. Here, combining antibody-based affinity enrichment and high-resolution mass spectrometry, we identified and validated 1079 acetylation sites in 556 acetylated proteins from P. asperata somatic embryos during PDT. These data represent a novel large-scale dataset of lysine-acetylated proteins from the conifer family. Intensive bioinformatics analysis of the Gene Ontology of molecular functions demonstrated that lysine-acetylated proteins were mainly associated with binding, catalytic activities, and structural molecular activities. Functional characterization of the acetylated proteins revealed that in the desiccated somatic embryos, LysAc is mainly involved in the response to stress and central metabolism. Accordingly, the majority of these interacting proteins were also highly enriched in ribosome, proteasome, spliceosome, and carbon metabolism clusters. This work provides the most comprehensive profile of LysAc for a coniferous species obtained to date and facilitates the systematic study of the physiological role of LysAc in desiccated somatic embryos of P. asperata. PMID:28066480

  1. Ubiquitination of Notch1 is regulated by MAML1-mediated p300 acetylation of Notch1

    Energy Technology Data Exchange (ETDEWEB)

    Popko-Scibor, Anita E.; Lindberg, Mikael J.; Hansson, Magnus L.; Holmlund, Teresa [Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, 171 77 Stockholm (Sweden); Wallberg, Annika E., E-mail: Annika.Wallberg@ki.se [Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, 171 77 Stockholm (Sweden)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer p300 acetylates conserved lysines within Notch1 C-terminal nuclear localization signal. Black-Right-Pointing-Pointer MAML1 and CSL, components of Notch transcription complex, increase Notch acetylation. Black-Right-Pointing-Pointer MAML1-dependent acetylation of Notch1 by p300 decreases the ubiquitination of Notch1. Black-Right-Pointing-Pointer CDK8 inhibits Notch acetylation and Notch transcription enhanced by p300. -- Abstract: Earlier studies demonstrated the involvement of the p300 histone acetyltransferase in Notch signaling but the precise mechanisms by which p300 might modulate Notch function remains to be investigated. In this study, we show that p300 acetylates Notch1 ICD in cell culture assay and in vitro, and conserved lysines located within the Notch C-terminal nuclear localization signal are essential for Notch acetylation. MAML1 and CSL, which are components of the Notch transcription complex, enhance Notch acetylation and we suggest that MAML1 increases Notch acetylation by potentiating p300 autoacetylation. Furthermore, MAML1-dependent acetylation of Notch1 ICD by p300 decreases the ubiquitination of Notch1 ICD in cellular assays. CDK8 has been shown to target Notch1 for ubiquitination and proteosomal degradation. We show that CDK8 inhibits Notch acetylation and Notch transcription enhanced by p300. Therefore, we speculate that acetylation of Notch1 might be a mechanism to regulate Notch activity by interfering with ubiquitin-dependent pathways.

  2. Generation of acetyllysine antibodies and affinity enrichment of acetylated peptides.

    Science.gov (United States)

    Guan, Kun-Liang; Yu, Wei; Lin, Yan; Xiong, Yue; Zhao, Shimin

    2010-09-01

    Lysine acetylation has emerged as one of the major post-translational modifications, as indicated by its roles in chromatin remodeling, activation of transcription factors and, most recently, regulation of metabolic enzymes. Identification of acetylation sites in a protein is the first essential step for functional characterization of acetylation in physiological regulation. However, the study of the acetylome is hindered by the lack of suitable physical and biochemical properties of the acetyl group and existence of high-abundance acetylated histones in the cell, and needs a robust method to overcome these problems. Here we present protocols for (i) using chemically acetylated ovalbumin and synthetic acetylated peptide to generate a pan-acetyllysine antibody and a site-specific antibody to Lys288-acetylated argininosuccinate lyase, respectively; (ii) using subcellular fractionation to reduce highly abundant acetylated histones; and (iii) using acetyllysine antibody affinity purification and mass spectrometry to characterize acetylome of human liver tissue. The entire characterization procedure takes ∼2-3 d to complete.

  3. Acetylation site specificities of lysine deacetylase inhibitors in human cells

    DEFF Research Database (Denmark)

    Schölz, Christian; Weinert, Brian Tate; Wagner, Sebastian A;

    2015-01-01

    that the effects of the pan-sirtuin inhibitor nicotinamide are primarily mediated by SIRT1 inhibition. Furthermore, we confirmed that the effects of tubacin and bufexamac on cytoplasmic proteins result from inhibition of HDAC6. Bufexamac also triggered an HDAC6-independent, hypoxia-like response by stabilizing HIF...

  4. [The effect of the raw protein supply on the lysine requirements of young pigs of 12-40 kg. 1. Report. Feeding studies with wheat-peanut extraction residue rations].

    Science.gov (United States)

    Schüler, D; Bodenstein, K H; Hennig, A

    1976-09-01

    10 feedings trials were carried out with a total of more than 500 pigs weighing 12 to 40 kgs. To investigate the lysine needs of growing pigs, the animals were fed rations of wheat + extracted ground nut meal. Different food mixtures were tested containing 5 levels of crude protein (19%, 17%, 15%, 13% and 11% of the dry feed. Within each crude protein level 6 graded lysine supplements were added to the ration. The trial showed that the lysine requirements of the weaned pigs were in a decisive measure determined by the percentage proportion of crude protein contained in the ration. The crude protein portion may be calculated (for the liveweight range tested) by using the following regression equation: y=0.28+0.075x (y=lysine requirements expressed as % of the air-dried ration; x=percentage proportion of crude protein in the ration). Rations containing only protein sources of vegetable origin, with a minimum protein content of 15%, produced the same daily weight gains (520 g) as a standard diet, if the lysine demands were met through the supplementation of synthetic lysine.

  5. The fasted/fed mouse metabolic acetylome: N6-acetylation differences suggest acetylation coordinates organ-specific fuel switching.

    Science.gov (United States)

    Yang, Li; Vaitheesvaran, Bhavapriya; Hartil, Kirsten; Robinson, Alan J; Hoopmann, Michael R; Eng, Jimmy K; Kurland, Irwin J; Bruce, James E

    2011-09-02

    The elucidation of extra-nuclear lysine acetylation has been of growing interest, as the cosubstrate for acetylation, acetyl CoA, is at a key metabolic intersection. Our hypothesis was that mitochondrial and cytoplasmic protein acetylation may be part of a fasted/re-fed feedback control system for the regulation of the metabolic network in fuel switching, where acetyl CoA would be provided by fatty acid oxidation, or glycolysis, respectively. To test this, we characterized the mitochondrial and cytoplasmic acetylome in various organs that have a high metabolic rate relative to their mass, and/or switch fuels, under fasted and re-fed conditions (brain, kidney, liver, skeletal muscle, heart muscle, white and brown adipose tissues). Using immunoprecipitation, coupled with LC-MS/MS label free quantification, we show there is a dramatic variation in global quantitative profiles of acetylated proteins from different organs. In total, 733 acetylated peptides from 337 proteins were identified and quantified, out of which 31 acetylated peptides from the metabolic proteins that may play organ-specific roles were analyzed in detail. Results suggest that fasted/re-fed acetylation changes coordinated by organ-specific (de)acetylases in insulin-sensitive versus -insensitive organs may underlie fuel use and switching. Characterization of the tissue-specific acetylome should increase understanding of metabolic conditions wherein normal fuel switching is disrupted, such as in Type II diabetes.

  6. Site-specific acetylation of ISWI by GCN5

    Directory of Open Access Journals (Sweden)

    Chioda Mariacristina

    2007-08-01

    Full Text Available Abstract Background The tight organisation of eukaryotic genomes as chromatin hinders the interaction of many DNA-binding regulators. The local accessibility of DNA is regulated by many chromatin modifying enzymes, among them the nucleosome remodelling factors. These enzymes couple the hydrolysis of ATP to disruption of histone-DNA interactions, which may lead to partial or complete disassembly of nucleosomes or their sliding on DNA. The diversity of nucleosome remodelling factors is reflected by a multitude of ATPase complexes with distinct subunit composition. Results We found further diversification of remodelling factors by posttranslational modification. The histone acetyltransferase GCN5 can acetylate the Drosophila remodelling ATPase ISWI at a single, conserved lysine, K753, in vivo and in vitro. The target sequence is strikingly similar to the N-terminus of histone H3, where the corresponding lysine, H3K14, can also be acetylated by GCN5. The acetylated form of ISWI represents a minor species presumably associated with the nucleosome remodelling factor NURF. Conclusion Acetylation of histone H3 and ISWI by GCN5 is explained by the sequence similarity between the histone and ISWI around the acetylation site. The common motif RKT/SxGx(KacxPR/K differs from the previously suggested GCN5/PCAF recognition motif GKxxP. This raises the possibility of co-regulation of a nucleosome remodelling factor and its nucleosome substrate through acetylation of related epitopes and suggests a direct crosstalk between two distinct nucleosome modification principles.

  7. Histone deacetylase 1 reduces NO production in endothelial cells via lysine deacetylation of NO synthase 3

    Science.gov (United States)

    Hyndman, Kelly A.; Ho, Dao H.; Sega, Martiana F.

    2014-01-01

    The lysine acetylation state of nonhistone proteins may be regulated through histone deacetylases (HDACs). Evidence suggests that nitric oxide (NO) synthase 3 (NOS3; endothelial NOS) is posttranslationally lysine acetylated, leading to increased NO production in the endothelium. We tested the hypothesis that NOS3 is lysine acetylated and that upregulated HDAC1-mediated deacetylation leads to reduced NO production in endothelial cells. We determined that NOS3 is basally lysine acetylated in cultured bovine aortic endothelial cells (BAECs). In BAECs, HDAC1 is expressed in the nucleus and cytosol and forms a novel protein-protein interaction with NOS3. Overexpression of HDAC1 in BAECs resulted in a significant reduction in NOS3 lysine acetylation (control = 1.0 ± 0.1 and HDAC1 = 0.59 ± 0.08 arbitrary units, P NOS3 acetylation level, yet increased basal nitrite production (730.6 ± 99.1 pmol·mg−1·h−1) and further exaggerated increases in endothelin-1 stimulated nitrite production (1276.9 ± 288.2 pmol·mg−1·h−1) was observed. Moreover, overexpression or knockdown of HDAC1 resulted in no significant effect on NOS3 protein expression or NOS3 phosphorylation sites T497, S635, or S1179. Thus these data indicate that upregulated HDAC1 decreases NOS3 activity, most likely through direct lysine deacetylation of NOS3. We propose that HDAC1-mediated deacetylation of NOS3 may represent a novel target for endothelial dysfunction. PMID:25015965

  8. ß-Lysine discrimination by lysyl-tRNA synthetase

    DEFF Research Database (Denmark)

    Gilreath, Marla S; Roy, Hervé; Bullwinkle, Tammy J

    2011-01-01

    guided by the PoxA structure. A233S LysRS behaved as wild type with a-lysine, while the G469A and A233S/G469A variants decreased stable a-lysyl-adenylate formation. A233S LysRS recognized ß-lysine better than wildtype, suggesting a role for this residue in discriminating a- and ß-amino acids. Both...

  9. Involvement of histone acetylation in the regulation of choline acetyltransferase gene in NG108-15 neuronal cells.

    Science.gov (United States)

    Aizawa, Shu; Yamamuro, Yutaka

    2010-03-01

    Post-translational modification of histone such as acetylation of N-terminal of lysine residues influences gene expression by modulating the accessibility of specific transcription factors to the promoter region, and is essential for a wide variety of cellular processes in the development of individual tissues, including the brain. However, few details concerning the acquisition of specific neurotransmitter phenotype have been obtained. In the present study, we investigated the possible involvement of histone acetylation in the gene expression of choline acetyltransferase (ChAT), a specific marker for cholinergic neuron and its function, in NG108-15 neuronal cells as an in vitro model of cholinergic neuron. Treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA), which induces global histone hyper-acetylation of the cells, resulted in marked increase in the expression of ChAT gene in proliferating NG108-15 cells. Furthermore, RT-PCR analysis using primer pairs for individual variants of ChAT mRNA (R1-4, N1, and M type) revealed that M type, not R1-4 and N1 type, ChAT mRNA were mainly transcribed, and chromatin immunoprecipitation assay indicated that the promoter region of M type ChAT gene was highly acetylated, in the dibutyryl cyclic AMP-induced neuronal differentiation of NG108-15 cells. The present findings demonstrate that the acquisition of neurotransmitter phenotype is epigenetically, at least the hyper-acetylation on the core promoter region of ChAT gene, regulated in NG108-15 neuronal cells.

  10. Acetylation of Cavin-1 Promotes Lipolysis in White Adipose Tissue.

    Science.gov (United States)

    Zhou, Shui-Rong; Guo, Liang; Wang, Xu; Liu, Yang; Peng, Wan-Qiu; Liu, Yuan; Wei, Xiang-Bo; Dou, Xin; Ding, Meng; Lei, Qun-Ying; Qian, Shu-Wen; Li, Xi; Tang, Qi-Qun

    2017-08-15

    White adipose tissue (WAT) serves as a reversible energy storage depot in the form of lipids in response to nutritional status. Cavin-1, an essential component in the biogenesis of caveolae, is a positive regulator of lipolysis in adipocytes. However, molecular mechanisms of cavin-1 in the modulation of lipolysis remain poorly understood. Here, we showed that cavin-1 was acetylated at lysines 291, 293, and 298 (3K), which were under nutritional regulation in WAT. We further identified GCN5 as the acetyltransferase and Sirt1 as the deacetylase of cavin-1. Acetylation-mimetic 3Q mutants of cavin-1 augmented fat mobilization in 3T3-L1 adipocytes and zebrafish. Mechanistically, acetylated cavin-1 preferentially interacted with hormone-sensitive lipase and recruited it to the caveolae, thereby promoting lipolysis. Our findings shed light on the essential role of cavin-1 in regulating lipolysis in an acetylation-dependent manner in WAT. Copyright © 2017 American Society for Microbiology.

  11. Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level

    DEFF Research Database (Denmark)

    Danielsen, Jannie M R; Sylvestersen, Kathrine B; Bekker-Jensen, Simon;

    2011-01-01

    The covalent attachment of ubiquitin to proteins regulates numerous processes in eukaryotic cells. Here we report the identification of 753 unique lysine ubiquitylation sites on 471 proteins using higher-energy collisional dissociation on the LTQ Orbitrap Velos. In total 5756 putative ubiquitin...... substrates were identified. Lysine residues targeted by the ubiquitin-ligase system show no unique sequence feature. Surface accessible lysine residues located in ordered secondary regions, surrounded by smaller and positively charged amino acids are preferred sites of ubiquitylation. Lysine ubiquitylation...

  12. Biosynthesis and turnover of O-acetyl and N-acetyl groups in the gangliosides of human melanoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Manzi, A.E.; Sjoberg, E.R.; Diaz, S.; Varki, A.

    1990-08-05

    We and others previously described the melanoma-associated oncofetal glycosphingolipid antigen 9-O-acetyl-GD3, a disialoganglioside O-acetylated at the 9-position of the outer sialic acid residue. We have now developed methods to examine the biosynthesis and turnover of disialogangliosides in cultured melanoma cells and in Golgi-enriched vesicles from these cells. O-Acetylation was selectively expressed on di- and trisialogangliosides, but not on monosialogangliosides, nor on glycoprotein-bound sialic acids. Double-labeling of cells with (3H)acetate and (14C)glucosamine introduced easily detectable labels into each of the components of the ganglioside molecules. Pulse-chase studies of such doubly labeled molecules indicated that the O-acetyl groups turn over faster than the parent molecule. When Golgi-enriched vesicles from these cells were incubated with (acetyl-3H)acetyl-coenzyme A, the major labeled products were disialogangliosides. (Acetyl-3H)O-acetyl groups were found at both the 7- and the 9-positions, indicating that both 7-O-acetyl GD3 and 9-O-acetyl GD3 were synthesized by the action of O-acetyltransferase(s) on endogenous GD3. Analysis of the metabolically labeled molecules confirmed the existence of both 7- and 9-O-acetylated GD3 in the intact cells. Surprisingly, the major 3H-labeled product of the in vitro labeling reaction was not O-acetyl-GD3, but GD3, with the label exclusively in the sialic acid residues. Fragmentation of the labeled sialic acids by enzymatic and chemical methods showed that the 3H-label was exclusively in (3H)N-acetyl groups. Analyses of the double-labeled sialic acids from intact cells also showed that the 3H-label from (3H)acetate was exclusively in the form of (3H)N-acetyl groups, whereas the 14C-label was at the 4-position.

  13. O-acetylated oligosaccharides from pectins of potato tuber cell walls.

    Science.gov (United States)

    Ishii, T

    1997-04-01

    Acetylated trigalacturonides and rhamnogalacturonan I (RG-I)-derived oligosaccharides were isolated from a Driselase digest of potato tuber cell walls by ion-exchange and size-exclusion chromatography. The oligosaccharides were structurally characterized by fast atom bombardment-mass spectroscopy, nuclear magnetic resonance spectroscopy, and glycosyl-linkage composition analysis. One trigalacturonide contained a single acetyl group at O-3 of the reducing galacturonic acid residue. A second trigalacturonide contained two acetyl substituents, which were located on O-3 or O-4 of the nonreducing galacturonic acid residue and O-3 of the reducing galacturonic acid residue. RG-I backbone-derived oligomers had acetyl groups at O-2 of the galacturonic acid residues. Some of these galacturonic acid residues were O-acetylated at both O-2 and O-3 positions. Rhamnosyl residues of RG-I oligomers were not acetylated.

  14. Sumoylation of poly(ADP-ribose) polymerase 1 inhibits its acetylation and restrains transcriptional coactivator function.

    Science.gov (United States)

    Messner, Simon; Schuermann, David; Altmeyer, Matthias; Kassner, Ingrid; Schmidt, Darja; Schär, Primo; Müller, Stefan; Hottiger, Michael O

    2009-11-01

    Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated nuclear protein and functions as a molecular stress sensor. At the cellular level, PARP1 has been implicated in a wide range of processes, such as maintenance of genome stability, cell death, and transcription. PARP1 functions as a transcriptional coactivator of nuclear factor kappaB (NF-kappaB) and hypoxia inducible factor 1 (HIF1). In proteomic studies, PARP1 was found to be modified by small ubiquitin-like modifiers (SUMOs). Here, we characterize PARP1 as a substrate for modification by SUMO1 and SUMO3, both in vitro and in vivo. PARP1 is sumoylated at the single lysine residue K486 within its automodification domain. Interestingly, modification of PARP1 with SUMO does not affect its ADP-ribosylation activity but completely abrogates p300-mediated acetylation of PARP1, revealing an intriguing crosstalk of sumoylation and acetylation on PARP1. Genetic complementation of PARP1-depleted cells with wild-type and sumoylation-deficient PARP1 revealed that SUMO modification of PARP1 restrains its transcriptional coactivator function and subsequently reduces gene expression of distinct PARP1-regulated target genes.

  15. Effects of lysine-induced acute renal failure in dogs.

    Science.gov (United States)

    Asanuma, Kentaro; Adachi, Kenji; Sugimoto, Tetsuro; Chiba, Shuichi

    2006-05-01

    This study investigates the effects of lysine-induced acute renal failure. Female dogs received a lysine hydrochloride (lysine) of 4500 mg/kg/day (3.75 ml/kg/hr) for 3 consecutive days. The dogs were observed for clinical signs. Body weights were recorded, food consumption and water consumption calculated, and urinalysis and blood biochemistry were performed daily. Plasma samples for amino acid determinations were obtained from all dogs, which were necropsied on Day 3. Histopathological examinations were done on all test animals. Compound-related findings include the following. Blood biochemistry results showed increases in ammonia, blood urea nitrogen, blood urea nitrogen/creatinine ratio, and creatinine. Urinary changes consisted of increases in urine volume, total protein, albumin, gamma-glutamyl transpeptidase, and N-acetyl-beta-D-glucosaminidase. In addition, macroscopic findings consisted of pale, congested capsule; microscopic findings consisted of hypertrophy of proximal convoluted tubule (mainly S1 segment), and degeneration/desquamation of urinary tubule (mainly S3 segment with hyaline casts) in the kidney. From these findings, it can be concluded that lysine is nephrotoxic in dogs. Nephrotoxicity of lysine may relate to direct tubular toxicity and to tubular obstruction.

  16. Specificity of the chromodomain Y chromosome family of chromodomains for lysine-methylated ARK(S/T) motifs.

    Science.gov (United States)

    Fischle, Wolfgang; Franz, Henriette; Jacobs, Steven A; Allis, C David; Khorasanizadeh, Sepideh

    2008-07-11

    Previous studies have shown two homologous chromodomain modules in the HP1 and Polycomb proteins exhibit discriminatory binding to related methyllysine residues (embedded in ARKS motifs) of the histone H3 tail. Methylated ARK(S/T) motifs have recently been identified in other chromatin factors (e.g. linker histone H1.4 and lysine methyltransferase G9a). These are thought to function as peripheral docking sites for the HP1 chromodomain. In vertebrates, HP1-like chromodomains are also present in the chromodomain Y chromosome (CDY) family of proteins adjacent to a putative catalytic motif. The human genome encodes three CDY family proteins, CDY, CDYL, and CDYL2. These have putative functions ranging from establishment of histone H4 acetylation during spermiogenesis to regulation of transcription co-repressor complexes. To delineate the biochemical functions of the CDY family chromodomains, we analyzed their specificity of methyllysine recognition. We detected substantial differences among these factors. The CDY chromodomain exhibits discriminatory binding to lysine-methylated ARK(S/T) motifs, whereas the CDYL2 chromodomain binds with comparable strength to multiple ARK(S/T) motifs. Interestingly, subtle amino acid changes in the CDYL chromodomain prohibit such binding interactions in vitro and in vivo. However, point mutations can rescue binding. In support of the in vitro binding properties of the chromodomains, the full-length CDY family proteins exhibit substantial variability in chromatin localization. Our studies underscore the significance of subtle sequence differences in a conserved signaling module for diverse epigenetic regulatory pathways.

  17. Autoimmune regulator is acetylated by transcription coactivator CBP/p300

    Energy Technology Data Exchange (ETDEWEB)

    Saare, Mario, E-mail: mario.saare@ut.ee [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia); Rebane, Ana [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia); SIAF, Swiss Institute of Allergy and Asthma Research, University of Zuerich, Davos (Switzerland); Rajashekar, Balaji; Vilo, Jaak [BIIT, Bioinformatics, Algorithmics and Data Mining group, Institute of Computer Science, University of Tartu, Tartu (Estonia); Peterson, Paert [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia)

    2012-08-15

    The Autoimmune Regulator (AIRE) is a regulator of transcription in the thymic medulla, where it controls the expression of a large set of peripheral-tissue specific genes. AIRE interacts with the transcriptional coactivator and acetyltransferase CBP and synergistically cooperates with it in transcriptional activation. Here, we aimed to study a possible role of AIRE acetylation in the modulation of its activity. We found that AIRE is acetylated in tissue culture cells and this acetylation is enhanced by overexpression of CBP and the CBP paralog p300. The acetylated lysines were located within nuclear localization signal and SAND domain. AIRE with mutations that mimicked acetylated K243 and K253 in the SAND domain had reduced transactivation activity and accumulated into fewer and larger nuclear bodies, whereas mutations that mimicked the unacetylated lysines were functionally similar to wild-type AIRE. Analogously to CBP, p300 localized to AIRE-containing nuclear bodies, however, the overexpression of p300 did not enhance the transcriptional activation of AIRE-regulated genes. Further studies showed that overexpression of p300 stabilized the AIRE protein. Interestingly, gene expression profiling revealed that AIRE, with mutations mimicking K243/K253 acetylation in SAND, was able to activate gene expression, although the affected genes were different and the activation level was lower from those regulated by wild-type AIRE. Our results suggest that the AIRE acetylation can influence the selection of AIRE activated genes. -- Highlights: Black-Right-Pointing-Pointer AIRE is acetylated by the acetyltransferases p300 and CBP. Black-Right-Pointing-Pointer Acetylation occurs between CARD and SAND domains and within the SAND domain. Black-Right-Pointing-Pointer Acetylation increases the size of AIRE nuclear dots. Black-Right-Pointing-Pointer Acetylation increases AIRE protein stability. Black-Right-Pointing-Pointer AIRE acetylation mimic regulates a different set of AIRE

  18. Chemical tools for unraveling the substrate specificity of the lysine deacylase enzymes

    DEFF Research Database (Denmark)

    Madsen, Andreas Stahl; Olsen, Christian Adam

    The lysine deacylase (KDAC) enzymes catalyze hydrolytic removal of acyl functionalities from theε-amino group of lysine residues ina variety of proteins including histones, and KDAC-mediated deacetylation of proteins has been established as a key epigeneticandmetabolic regulator. Recent studies h......-dependent HDACs 1–11 as well as NAD + -dependent sirtuins (SIRT1–7) will be discussed....

  19. Intrinsic Tau Acetylation Is Coupled to Auto-Proteolytic Tau Fragmentation.

    Directory of Open Access Journals (Sweden)

    Todd J Cohen

    Full Text Available Tau proteins are abnormally aggregated in a range of neurodegenerative tauopathies including Alzheimer's disease (AD. Recently, tau has emerged as an extensively post-translationally modified protein, among which lysine acetylation is critical for normal tau function and its pathological aggregation. Here, we demonstrate that tau isoforms have different propensities to undergo lysine acetylation, with auto-acetylation occurring more prominently within the lysine-rich microtubule-binding repeats. Unexpectedly, we identified a unique intrinsic property of tau in which auto-acetylation induces proteolytic tau cleavage, thereby generating distinct N- and C-terminal tau fragments. Supporting a catalytic reaction-based mechanism, mapping and mutagenesis studies showed that tau cysteines, which are required for acetyl group transfer, are also essential for auto-proteolytic tau processing. Further mass spectrometry analysis identified the C-terminal 2nd and 4th microtubule binding repeats as potential sites of auto-cleavage. The identification of acetylation-mediated auto-proteolysis provides a new biochemical mechanism for tau self-regulation and warrants further investigation into whether auto-catalytic functions of tau are implicated in AD and other tauopathies.

  20. Novel mechanisms for superoxide-scavenging activity of human manganese superoxide dismutase determined by the K68 key acetylation site.

    Science.gov (United States)

    Lu, Jiaqi; Cheng, Kuoyuan; Zhang, Bo; Xu, Huan; Cao, Yuanzhao; Guo, Fei; Feng, Xudong; Xia, Qing

    2015-08-01

    Superoxide is the primary reactive oxygen species generated in the mitochondria. Manganese superoxide dismutase (SOD2) is the major enzymatic superoxide scavenger present in the mitochondrial matrix and one of the most crucial reactive oxygen species-scavenging enzymes in the cell. SOD2 is activated by sirtuin 3 (SIRT3) through NAD(+)-dependent deacetylation. However, the exact acetylation sites of SOD2 are ambiguous and the mechanisms underlying the deacetylation-mediated SOD2 activation largely remain unknown. We are the first to characterize SOD2 mutants of the acetylation sites by investigating the relative enzymatic activity, structures, and electrostatic potential of SOD2 in this study. These SOD2 mutations affected the superoxide-scavenging activity in vitro and in HEK293T cells. The lysine 68 (K68) site is the most important acetylation site contributing to SOD2 activation and plays a role in cell survival after paraquat treatment. The molecular basis underlying the regulation of SOD2 activity by K68 was investigated in detail. Molecular dynamics simulations revealed that K68 mutations induced a conformational shift of residues located in the active center of SOD2 and altered the charge distribution on the SOD2 surface. Thus, the entry of the superoxide anion into the coordinated core of SOD2 was inhibited. Our results provide a novel mechanistic insight, whereby SOD2 acetylation affects the structure and charge distribution of SOD2, its tetramerization, and p53-SOD2 interactions of SOD2 in the mitochondria, which may play a role in nuclear-mitochondrial communication during aging.

  1. An acetylation switch controls TDP-43 function and aggregation propensity

    Science.gov (United States)

    Cohen, Todd J.; Hwang, Andrew W.; Restrepo, Clark R.; Yuan, Chao-Xing; Trojanowski, John Q.; Lee, Virginia M.Y.

    2015-01-01

    TDP-43 pathology is a disease hallmark that characterizes amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). Although a critical role for TDP-43 as an RNA-binding protein has emerged, the regulation of TDP-43 function is poorly understood. Here we identify lysine acetylation as a novel post-translational modification controlling TDP-43 function and aggregation. We provide evidence that TDP-43 acetylation impairs RNA-binding and promotes accumulation of insoluble, hyper-phosphorylated TDP-43 species that largely resemble pathological inclusions in ALS and FTLD-TDP. Moreover, biochemical and cell-based assays identify oxidative stress as a signaling cue that promotes acetylated TDP-43 aggregates that are readily engaged by the cellular defense machinery. Importantly, acetylated TDP-43 lesions are found in ALS patient spinal cord, indicating that aberrant TDP-43 acetylation and loss of RNA binding are linked to TDP-43 proteinopathy. Thus, modulating TDP-43 acetylation represents a plausible strategy to fine-tune TDP-43 activity, which could provide new therapeutic avenues for TDP-43 proteinopathies. PMID:25556531

  2. Accurate in silico identification of species-specific acetylation sites by integrating protein sequence-derived and functional features

    Science.gov (United States)

    Li, Yuan; Wang, Mingjun; Wang, Huilin; Tan, Hao; Zhang, Ziding; Webb, Geoffrey I.; Song, Jiangning

    2014-07-01

    Lysine acetylation is a reversible post-translational modification, playing an important role in cytokine signaling, transcriptional regulation, and apoptosis. To fully understand acetylation mechanisms, identification of substrates and specific acetylation sites is crucial. Experimental identification is often time-consuming and expensive. Alternative bioinformatics methods are cost-effective and can be used in a high-throughput manner to generate relatively precise predictions. Here we develop a method termed as SSPKA for species-specific lysine acetylation prediction, using random forest classifiers that combine sequence-derived and functional features with two-step feature selection. Feature importance analysis indicates functional features, applied for lysine acetylation site prediction for the first time, significantly improve the predictive performance. We apply the SSPKA model to screen the entire human proteome and identify many high-confidence putative substrates that are not previously identified. The results along with the implemented Java tool, serve as useful resources to elucidate the mechanism of lysine acetylation and facilitate hypothesis-driven experimental design and validation.

  3. Characterization and crystal structure of lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase (cDHDPS) protein.

    Science.gov (United States)

    Rice, Elena A; Bannon, Gary A; Glenn, Kevin C; Jeong, Soon Seog; Sturman, Eric J; Rydel, Timothy J

    2008-12-15

    The lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase enzyme (cDHDPS) was recently successfully introduced into maize plants to enhance the level of lysine in the grain. To better understand lysine insensitivity of the cDHDPS, we expressed, purified, kinetically characterized the protein, and solved its X-ray crystal structure. The cDHDPS enzyme has a fold and overall structure that is highly similar to other DHDPS proteins. A noteworthy feature of the active site is the evidence that the catalytic lysine residue forms a Schiff base adduct with pyruvate. Analyses of the cDHDPS structure in the vicinity of the putative binding site for S-lysine revealed that the allosteric binding site in the Escherichia coli DHDPS protein does not exist in cDHDPS due to three non-conservative amino acids substitutions, and this is likely why cDHDPS is not feedback inhibited by lysine.

  4. Acetylation of N-terminus and two internal amino acids is dispensable for degradation of a protein that aberrantly engages the endoplasmic reticulum translocon

    Directory of Open Access Journals (Sweden)

    Sarah M. Engle

    2017-08-01

    Full Text Available Conserved homologues of the Hrd1 ubiquitin ligase target for degradation proteins that persistently or aberrantly engage the endoplasmic reticulum translocon, including mammalian apolipoprotein B (apoB; the major protein component of low-density lipoproteins and the artificial yeast protein Deg1-Sec62. A complete understanding of the molecular mechanism by which translocon-associated proteins are recognized and degraded may inform the development of therapeutic strategies for cholesterol-related pathologies. Both apoB and Deg1-Sec62 are extensively post-translationally modified. Mass spectrometry of a variant of Deg1-Sec62 revealed that the protein is acetylated at the N-terminal methionine and two internal lysine residues. N-terminal and internal acetylation regulates the degradation of a variety of unstable proteins. However, preventing N-terminal and internal acetylation had no detectable consequence for Hrd1-mediated proteolysis of Deg1-Sec62. Our data highlight the importance of empirically validating the role of post-translational modifications and sequence motifs on protein degradation, even when such elements have previously been demonstrated sufficient to destine other proteins for destruction.

  5. Genome-wide integration on transcription factors, histone acetylation and gene expression reveals genes co-regulated by histone modification patterns.

    Directory of Open Access Journals (Sweden)

    Yayoi Natsume-Kitatani

    Full Text Available N-terminal tails of H2A, H2B, H3 and H4 histone families are subjected to posttranslational modifications that take part in transcriptional regulation mechanisms, such as transcription factor binding and gene expression. Regulation mechanisms under control of histone modification are important but remain largely unclear, despite of emerging datasets for comprehensive analysis of histone modification. In this paper, we focus on what we call genetic harmonious units (GHUs, which are co-occurring patterns among transcription factor binding, gene expression and histone modification. We present the first genome-wide approach that captures GHUs by combining ChIP-chip with microarray datasets from Saccharomyces cerevisiae. Our approach employs noise-robust soft clustering to select patterns which share the same preferences in transcription factor-binding, histone modification and gene expression, which are all currently implied to be closely correlated. The detected patterns are a well-studied acetylation of lysine 16 of H4 in glucose depletion as well as co-acetylation of five lysine residues of H3 with H4 Lys12 and H2A Lys7 responsible for ribosome biogenesis. Furthermore, our method further suggested the recognition of acetylated H4 Lys16 being crucial to histone acetyltransferase ESA1, whose essential role is still under controversy, from a microarray dataset on ESA1 and its bypass suppressor mutants. These results demonstrate that our approach allows us to provide clearer principles behind gene regulation mechanisms under histone modifications and detect GHUs further by applying to other microarray and ChIP-chip datasets. The source code of our method, which was implemented in MATLAB (http://www.mathworks.com/, is available from the supporting page for this paper: http://www.bic.kyoto-u.ac.jp/pathway/natsume/hm_detector.htm.

  6. (R)-β-lysine-modified elongation factor P functions in translation elongation

    DEFF Research Database (Denmark)

    Bullwinkle, Tammy J; Zou, S Betty; Rajkovic, Andrei

    2013-01-01

    Post-translational modification of bacterial elongation factor P (EF-P) with (R)-β-lysine at a conserved lysine residue activates the protein in vivo and increases puromycin reactivity of the ribosome in vitro. The additional hydroxylation of EF-P at the same lysine residue by the YfcM protein has......-(β)-lysyl-EF-P showed 30% increased puromycin reactivity but no differences in dipeptide synthesis rates when compared with the β-lysylated form. Unlike disruption of the other genes required for EF-P modification, deletion of yfcM had no phenotypic consequences in Salmonella. Taken together, our findings indicate...

  7. High glucose-induced oxidative stress represses sirtuin deacetylase expression and increases histone acetylation leading to neural tube defects.

    Science.gov (United States)

    Yu, Jingwen; Wu, Yanqing; Yang, Peixin

    2016-05-01

    Aberrant epigenetic modifications are implicated in maternal diabetes-induced neural tube defects (NTDs). Because cellular stress plays a causal role in diabetic embryopathy, we investigated the possible role of the stress-resistant sirtuin (SIRT) family histone deacetylases. Among the seven sirtuins (SIRT1-7), pre-gestational maternal diabetes in vivo or high glucose in vitro significantly reduced the expression of SIRT 2 and SIRT6 in the embryo or neural stem cells, respectively. The down-regulation of SIRT2 and SIRT6 was reversed by superoxide dismutase 1 (SOD1) over-expression in the in vivo mouse model of diabetic embryopathy and the SOD mimetic, tempol and cell permeable SOD, PEGSOD in neural stem cell cultures. 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a superoxide generating agent, mimicked high glucose-suppressed SIRT2 and SIRT6 expression. The acetylation of histone 3 at lysine residues 56 (H3K56), H3K14, H3K9, and H3K27, putative substrates of SIRT2 and SIRT6, was increased by maternal diabetes in vivo or high glucose in vitro, and these increases were blocked by SOD1 over-expression or tempol treatment. SIRT2 or SIRT6 over-expression abrogated high glucose-suppressed SIRT2 or SIRT6 expression, and prevented the increase in acetylation of their histone substrates. The potent sirtuin activator (SRT1720) blocked high glucose-increased histone acetylation and NTD formation, whereas the combination of a pharmacological SIRT2 inhibitor and a pan SIRT inhibitor mimicked the effect of high glucose on increased histone acetylation and NTD induction. Thus, diabetes in vivo or high glucose in vitro suppresses SIRT2 and SIRT6 expression through oxidative stress, and sirtuin down-regulation-induced histone acetylation may be involved in diabetes-induced NTDs. The mechanism underlying pre-gestational diabetes-induced neural tube defects (NTDs) is still elusive. Our study unravels a new epigenetic mechanism in which maternal diabetes-induced oxidative stress represses

  8. Label-free quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways.

    Science.gov (United States)

    Rardin, Matthew J; Newman, John C; Held, Jason M; Cusack, Michael P; Sorensen, Dylan J; Li, Biao; Schilling, Birgit; Mooney, Sean D; Kahn, C Ronald; Verdin, Eric; Gibson, Bradford W

    2013-04-16

    Large-scale proteomic approaches have identified numerous mitochondrial acetylated proteins; however in most cases, their regulation by acetyltransferases and deacetylases remains unclear. Sirtuin 3 (SIRT3) is an NAD(+)-dependent mitochondrial protein deacetylase that has been shown to regulate a limited number of enzymes in key metabolic pathways. Here, we use a rigorous label-free quantitative MS approach (called MS1 Filtering) to analyze changes in lysine acetylation from mouse liver mitochondria in the absence of SIRT3. Among 483 proteins, a total of 2,187 unique sites of lysine acetylation were identified after affinity enrichment. MS1 Filtering revealed that lysine acetylation of 283 sites in 136 proteins was significantly increased in the absence of SIRT3 (at least twofold). A subset of these sites was independently validated using selected reaction monitoring MS. These data show that SIRT3 regulates acetylation on multiple proteins, often at multiple sites, across several metabolic pathways including fatty acid oxidation, ketogenesis, amino acid catabolism, and the urea and tricarboxylic acid cycles, as well as mitochondrial regulatory proteins. The widespread modification of key metabolic pathways greatly expands the number of known substrates and sites that are targeted by SIRT3 and establishes SIRT3 as a global regulator of mitochondrial protein acetylation with the capability of coordinating cellular responses to nutrient status and energy homeostasis.

  9. GmPHD5 acts as an important regulator for crosstalk between histone H3K4 di-methylation and H3K14 acetylation in response to salinity stress in soybean

    Directory of Open Access Journals (Sweden)

    Wu Tao

    2011-12-01

    Full Text Available Abstract Background Accumulated evidence suggest that specific patterns of histone posttranslational modifications (PTMs and their crosstalks may determine transcriptional outcomes. However, the regulatory mechanisms of these "histone codes" in plants remain largely unknown. Results In this study, we demonstrate for the first time that a salinity stress inducible PHD (plant homeodomain finger domain containing protein GmPHD5 can read the "histone code" underlying the methylated H3K4. GmPHD5 interacts with other DNA binding proteins, including GmGNAT1 (an acetyl transferase, GmElongin A (a transcription elongation factor and GmISWI (a chromatin remodeling protein. Our results suggest that GmPHD5 can recognize specific histone methylated H3K4, with preference to di-methylated H3K4. Here, we illustrate that the interaction between GmPHD5 and GmGNAT1 is regulated by the self-acetylation of GmGNAT1, which can also acetylate histone H3. GmGNAT1 exhibits a preference toward acetylated histone H3K14. These results suggest a histone crosstalk between methylated H3K4 and acetylated H3K14. Consistent to its putative roles in gene regulation under salinity stress, we showed that GmPHD5 can bind to the promoters of some confirmed salinity inducible genes in soybean. Conclusion Here, we propose a model suggesting that the nuclear protein GmPHD5 is capable of regulating the crosstalk between histone methylation and histone acetylation of different lysine residues. Nevertheless, GmPHD5 could also recruit chromatin remodeling factors and transcription factors of salt stress inducible genes to regulate their expression in response to salinity stress.

  10. Acetylation dynamics of human nuclear proteins during the ionizing radiation-induced DNA damage response

    DEFF Research Database (Denmark)

    Bennetzen, Martin; Andersen, J.S.; Lasen, D.H.

    2013-01-01

    -dependent posttranslational modifications (PT Ms). To complement our previous analysis of IR-induced temporal dynamics of nuclear phosphoproteome, we now identify a range of human nuclear proteins that are dynamically regulated by acetylation, and predominantly deacetylation, during IR-induced DDR by using mass spectrometry......-based proteomic approaches. Apart from cataloging acetylation sites through SILAC proteomic analyses before IR and at 5 and 60 min after IR exposure of U2OS cells, we report that: (1) key components of the transcriptional machinery, such as EP 300 and CREBBP, are dynamically acetylated; (2) that nuclear...... to assess lysine acetylation status and thereby validate the mass spectrometry data. We thus present evidence that nuclear proteins, including those known to regulate cellular functions via epigenetic modifications of histones, are regulated by (de)acetylation in a timely manner upon cell's exposure...

  11. Histone acetylation, acetyltransferases, and ataxia--alteration of histone acetylation and chromatin dynamics is implicated in the pathogenesis of polyglutamine-expansion disorders.

    Science.gov (United States)

    McCullough, Shaun D; Grant, Patrick A

    2010-01-01

    Eukaryotic chromosomal DNA is packaged into nucleosomes to form a dynamic structure known as chromatin. The compaction of DNA within chromatin poses a unique hindrance with regards to the accessibility of the DNA to enzymes involved in replication, transcriptional regulation, and repair. The physical structure and physiological activity of chromatin are regulated through a diverse set of posttranslational modifications, histone exchange, and structural remodeling. Of the covalent chromatin modifications, the acetylation of lysine residues within histone proteins by acetyltransferase enzymes, such as GCN5, is one of the most prevalent and important steps in the regulation of chromatin function. Alteration of histone acetyltransferase activity can easily result in the dysregulation of gene transcription and ultimately the onset of a disease state. Many transcription factors contain polyglutamine regions within their primary sequence. Mutations resulting in the elongation of these polyglutamine tracts are associated with a disease family known as the polyglutamine expansion disorders. Spinocerebellar ataxia type 7 (SCA7) is one of the nine diseases that are grouped in this family and is caused by polyglutamine expansion of the ataxin-7 protein, which is a component of the GCN5-containing human SAGA histone acetyltransferase complex. Mutation of ataxin-7 in this manner has been shown to disrupt the structural integrity of the SAGA complex and result in aberrant chromatin acetylation patterns at the promoters of genes involved in the normal function of tissues that are affected by the disease. The specific aspects of molecular pathology are not currently understood; however, studies carried out in laboratory systems ranging from the budding yeast Saccharomyces cerevisiae to transgenic mouse models and cultured human cells are poised to allow for the elucidation of disease mechanisms and subsequent therapeutic approaches.

  12. Enhanced Amelioration of High-Fat Diet-Induced Fatty Liver by Docosahexaenoic Acid and Lysine Supplementations

    Directory of Open Access Journals (Sweden)

    Hsin-Yu Lin

    2014-01-01

    Full Text Available Fatty liver disease is the most common pathological condition in the liver. Here, we generated high-fat diet-(HFD- induced nonalcoholic fatty liver disease (NAFLD in mice and tested the effects of docosahexaenoic acid (DHA and lysine during a four-week regular chow (RCfeeding. Our results showed that 1% lysine and the combination of 1% lysine + 1% DHA reduced body weight. Moreover, serum triglyceride levels were reduced by 1% DHA and 1% lysine, whereas serum alanine transaminase activity was reduced by 1% DHA and 1% DHA + 0.5% lysine. Switching to RC reduced hepatic lipid droplet accumulation, which was further reduced by the addition of DHA or lysine. Furthermore, the mRNA expressions of hepatic proinflammatory cytokines were suppressed by DHA and combinations of DHA + lysine, whereas the mRNA for the lipogenic gene, acetyl-CoA carboxylase 1 (ACC1, was suppressed by DHA. In the gonadal adipose tissues, combinations of DHA and lysine inhibited mRNA expression of lipid metabolism-associated genes, including ACC1, fatty acid synthase, lipoprotein lipase, and perilipin. In conclusion, the present study demonstrated that, in conjunction with RC-induced benefits, supplementation with DHA or lysine further ameliorated the high-fat diet-induced NAFLD and provided an alternative strategy to treat, and potentially prevent, NAFLD.

  13. Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon.

    Science.gov (United States)

    Nguyen, T Van; Lee, J Eugene; Sweredoski, Michael J; Yang, Seung-Joo; Jeon, Seung-Je; Harrison, Joseph S; Yim, Jung-Hyuk; Lee, Sang Ghil; Handa, Hiroshi; Kuhlman, Brian; Jeong, Ji-Seon; Reitsma, Justin M; Park, Chul-Seung; Hess, Sonja; Deshaies, Raymond J

    2016-03-17

    Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.

  14. Acetylation control of cardiac fatty acid β-oxidation and energy metabolism in obesity, diabetes, and heart failure.

    Science.gov (United States)

    Fukushima, Arata; Lopaschuk, Gary D

    2016-12-01

    Alterations in cardiac energy metabolism are an important contributor to the cardiac pathology associated with obesity, diabetes, and heart failure. High rates of fatty acid β-oxidation with cardiac insulin resistance represent a cardiac metabolic hallmark of diabetes and obesity, while a marginal decrease in fatty acid oxidation and a prominent decrease in insulin-stimulated glucose oxidation are commonly seen in the early stages of heart failure. Alterations in post-translational control of energy metabolic processes have recently been identified as an important contributor to these metabolic changes. In particular, lysine acetylation of non-histone proteins, which controls a diverse family of mitochondrial metabolic pathways, contributes to the cardiac energy derangements seen in obesity, diabetes, and heart failure. Lysine acetylation is controlled both via acetyltransferases and deacetylases (sirtuins), as well as by non-enzymatic lysine acetylation due to increased acetyl CoA pool size or dysregulated nicotinamide adenine dinucleotide (NAD(+)) metabolism (which stimulates sirtuin activity). One of the important mitochondrial acetylation targets are the fatty acid β-oxidation enzymes, which contributes to alterations in cardiac substrate preference during the course of obesity, diabetes, and heart failure, and can ultimately lead to cardiac dysfunction in these disease states. This review will summarize the role of lysine acetylation and its regulatory control in the context of mitochondrial fatty acid β-oxidation. The functional contribution of cardiac protein lysine acetylation to the shift in cardiac energy substrate preference that occurs in obesity, diabetes, and especially in the early stages of heart failure will also be reviewed. This article is part of a Special Issue entitled: The role of post-translational protein modifications on heart and vascular metabolism edited by Jason R.B. Dyck & Jan F.C. Glatz.

  15. Histone Acylation beyond Acetylation: Terra Incognita in Chromatin Biology

    Directory of Open Access Journals (Sweden)

    Sophie Rousseaux

    2015-04-01

    Full Text Available Histone acetylation, one of the first and best studied histone post-translational modifications (PTMs, as well as the factors involved in its deposition (writers, binding (readers and removal (erasers, have been shown to act at the heart of regulatory circuits controlling essential cellular functions. The identification of a variety of competing histone lysine-modifying acyl groups including propionyl, butyryl, 2-hydroxyisobutyryl, crotonyl, malonyl, succinyl and glutaryl, raises numerous questions on their functional significance, the molecular systems that manage their establishment, removal and interplay with the well-known acetylation-based mechanisms. Detailed and large-scale investigations of two of these new histone PTMs, crotonylation and 2-hydroxyisobutyrylation, along with histone acetylation, in the context of male genome programming, where stage-specific gene expression programs are switched on and off in turn, have shed light on their functional contribution to the epigenome for the first time. These initial investigations fired many additional questions, which remain to be explored. This review surveys the major results taken from these two new histone acylations and discusses the new biology that is emerging based on the diversity of histone lysine acylations.

  16. Effects of fortified lysine on the amino acid profile and sensory qualities of deep-fried and dried noodles.

    Science.gov (United States)

    Polpuech, C; Chavasit, V; Srichakwal, P; Paniangvait, P

    2011-08-01

    Lysine fortification of wheat flour has been used toward reducing protein energy malnutrition in developing countries. The feasibility of fortifying instant noodles with lysine was evaluated based on sensory qualities and the residual lysine content. Fifty grams of deep-fried and dried instant noodles were fortified with 0.23 and 0.21 g lysine, respectively. The production temperatures used for deep-frying were 165-175 degrees C and for drying, 80-105 degrees C; these are the temperatures used in the industrial production of both kinds of noodles. Lysine fortification was then performed at the local factories using the commercial production lines and packaging for both types of instant noodles. Both fortified and unfortified deep-fried and dried instant noodles were stored at 50 degrees C under fluorescent light for 2 and 4 months, respectively. The fortified products were tested for residual lysine content and sensory qualities as compared with unfortified noodles. The results show fortified products from the tested processing temperatures were all accepted. After storage, significant losses of lysine were not found in both types of noodles analysed. The lysine-fortified noodles had amino acid scores of 102% and 122%, respectively. After 2 months, the sensory quality of fortified deep-fried noodles was still acceptable; however, the dried noodles turned to an unacceptable dark colour. This study shows that it is feasible to fortify deep-fried instant noodles with lysine, though lysine fortification exhibited an undesirable colour in the dried instant noodles after storage.

  17. Effect of heat damage in an autoclave on the reactive lysine contents of soy products and corn distillers dried grains with solubles. Use of the results to check on lysine damage in common qualities of these ingredients.

    Science.gov (United States)

    Fontaine, Johannes; Zimmer, Ulrike; Moughan, Paul J; Rutherfurd, Shane M

    2007-12-26

    The suitability of the homoarginine reaction for determining the reactive lysine in soy products and corn distillers dried grain with solubles (DDGS) was tested. For this purpose, some batches were subjected to deliberate heat damage for up to 30 min in an autoclave with 135 degrees C hot steam, and the samples were analyzed for total lysine and reactive lysine. In addition, 84 samples of common soy and 80 samples of corn DDGS were tested for their content of total and reactive lysine, and the contents were compared with those of the autoclave tests. For soy products conclusive results were obtained. In the case of heat treatment, both total lysine and reactive lysine decrease, but the latter is clearly a more sensitive indicator of lysine damage. Most normal products are quite similar, with toasting-induced damage to reactive lysine of ca. 15% compared to untoasted beans. The cause of the constantly occurring residual lysine after guanidination and the poorer reaction balance in the case of damage were explained. For common DDGS samples, however, less favorable results were obtained. Reactive and total lysine decreased almost in parallel due to heat damage, showing a great gap between them. Results showed indeed that variation of total and reactive lysine in DDGS is high, proving that its production conditions are not yet optimal for a feed ingredient.

  18. Final report on the safety assessment of acetyl triethyl citrate, acetyl tributyl citrate, acetyl trihexyl citrate, and acetyl trioctyl citrate.

    Science.gov (United States)

    Johnson, Wilbur

    2002-01-01

    Acetyl Triethyl Citrate, Acetyl Tributyl Citrate, Acetyl Trihexyl Citrate, and Acetyl Trioctyl Citrate all function as plasticizers in cosmetics. Additionally, the Trihexyl and Trioctyl forms are described as skin-conditioning agents-emollients, although there are currently no reported uses of Acetyl Trihexyl Citrate or Acetyl Trioctyl Citrate. Acetyl Triethyl Citrate and Acetyl Tributyl Citrate are used in nail products at concentrations up to 7%. Recognizing that there are no reported uses of Acetyl Trihexyl or Trioctyl Citrate, if they were to be used in the future, their concentration of use is expected to be no higher than that reported for Acetyl Triethyl and Tributyl Citrate. These ingredients were sufficiently similar in structure that safety test data on one were considered applicable to all. Approximately 99% of orally administered Acetyl Tributyl Citrate is excreted-intermediate metabolites include acetyl citrate, monobutyl citrate, acetyl monobutyl citrate, dibutyl citrate, and acetyl dibutyl citrate. In acute, short-term, subchronic, and chronic feeding studies, these ingredients were relatively nontoxic. Differences from controls were either not statistically significant or not related to any organ toxicity. Ocular exposures produced moderate reactions that cleared by 48 hours after instillation. Dermal application was not toxic in rabbits. In a guinea pig maximization test, Acetyl Triethyl Citrate was a sensitizer whereas Acetyl Tributyl Citrate was not. Limited clinical testing of Acetyl Triethyl Citrate and Acetyl Tributyl Citrate was negative for both skin irritation and sensitization. These clinical data were considered more relevant than the guinea pig maximization data, suggesting to the Cosmetic Ingredient Review Expert Panel that none of these ingredients would be a sensitizer. Physiologic effects noted with intravenous delivery of Acetyl Triethyl Citrate or Acetyl Tributyl Citrate include dose-related decreases in blood pressure and

  19. Acetylation of mitochondrial proteins by GCN5L1 promotes enhanced fatty acid oxidation in the heart.

    Science.gov (United States)

    Thapa, Dharendra; Zhang, Manling; Manning, Janet R; Guimarães, Danielle A; Stoner, Michael W; O'Doherty, Robert M; Shiva, Sruti; Scott, Iain

    2017-08-01

    Lysine acetylation is a reversible posttranslational modification and is particularly important in the regulation of mitochondrial metabolic enzymes. Acetylation uses acetyl-CoA derived from fuel metabolism as a cofactor, thereby linking nutrition to metabolic activity. In the present study, we investigated how mitochondrial acetylation status in the heart is controlled by food intake and how these changes affect mitochondrial metabolism. We found that there was a significant increase in cardiac mitochondrial protein acetylation in mice fed a long-term high-fat diet and that this change correlated with an increase in the abundance of the mitochondrial acetyltransferase-related protein GCN5L1. We showed that the acetylation status of several mitochondrial fatty acid oxidation enzymes (long-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, and hydroxyacyl-CoA dehydrogenase) and a pyruvate oxidation enzyme (pyruvate dehydrogenase) was significantly upregulated in high-fat diet-fed mice and that the increase in long-chain and short-chain acyl-CoA dehydrogenase acetylation correlated with increased enzymatic activity. Finally, we demonstrated that the acetylation of mitochondrial fatty acid oxidation proteins was decreased after GCN5L1 knockdown and that the reduced acetylation led to diminished fatty acid oxidation in cultured H9C2 cells. These data indicate that lysine acetylation promotes fatty acid oxidation in the heart and that this modification is regulated in part by the activity of GCN5L1.NEW & NOTEWORTHY Recent research has shown that acetylation of mitochondrial fatty acid oxidation enzymes has greatly contrasting effects on their activity in different tissues. Here, we provide new evidence that acetylation of cardiac mitochondrial fatty acid oxidation enzymes by GCN5L1 significantly upregulates their activity in diet-induced obese mice. Copyright © 2017 the American Physiological Society.

  20. Multiple roles for acetylation in the interaction of p300 HAT with ATF-2.

    Science.gov (United States)

    Karanam, Balasubramanyam; Wang, Ling; Wang, Dongxia; Liu, Xin; Marmorstein, Ronen; Cotter, Robert; Cole, Philip A

    2007-07-17

    The transcriptional coactivator paralogues p300 and CBP contain acetyltransferase domains (HAT) and catalyze the lysine acetylation of histones and other proteins as an important aspect of their functions. Prior studies revealed that the basic leucine zipper domain (b-ZIP) of transcription factor ATF-2 (also called CRE-BP1) can interact with the CBP HAT domain. In this study, we have examined the ATF-2 b-ZIP interaction with the p300 HAT domain and shown that p300 HAT autoacetylation can enhance the binding affinity. Pull-down assays revealed that hyperacetylated p300 HAT is more efficiently retained by immobilized ATF-2 b-ZIP than hypoacetylated p300 HAT. Loop deleted p300 HAT lacking autoacetylation was retained about as well as hyperacetylated p300 HAT, suggesting that the loop and ATF-2 compete for p300 HAT binding. While ATF-2 b-ZIP is a weak inhibitor of hypoacetylated p300 HAT acetylation of a histone H4 peptide, hyperacetylated p300 HAT is much more potently inhibited by ATF-2 b-ZIP. Moreover, we showed that ATF-2 b-ZIP could serve as an acetyltransferase substrate for p300 HAT. Using mass spectrometry, two p300 HAT lysine acetylation sites were mapped in ATF-2 b-ZIP. Immunoprecipitation-Western blot analysis with anti-acetyl-lysine antibody revealed that ATF-2 can undergo reversible acetylation in vivo. Mutational analysis of the two ATF-2 b-ZIP acetylation sites revealed their potential contributions to ATF-2-mediated transcriptional activation. Taken together, these studies suggest multiple roles for protein acetylation in the regulation of transcription by p300/CBP and ATF-2.

  1. Reactivity of consecutive basic amino acid residues in peptides.

    Science.gov (United States)

    Bayle-Lacoste, M; De Tinguy-Moreaud, E; Geoffre, S; Neuzil, E

    1987-03-01

    Different tetrapeptides of general formula L-Ala-X-X-Gly, possessing a basic doublet in the second and third position (X = Arg or Lys), have been synthesized as free or N-acetylated molecules. The chemical reactivity of the arginine guanidino group and of the lysine epsilon-amino group were studied using respectively the Sakaguchi and the ortho-diacetylbenzene reactions, in the tetrapeptides as well as in related molecules. In both cases, the colour yield is markedly influenced by the length of the polypeptide chain and by the relative positions of the arginine and lysine residues, suggesting the occurrence of intramolecular bonds within the tetrapeptide molecule. Tryptic hydrolysis of the tetrapeptides was followed by evaluating the amino acids or peptides which appear to be specific for the different possible cleavages at the arginyl or at the lysyl bonds. The susceptibility to trypsin of the carboxylic group of the second basic amino acid decreases progressively in the order Lys-Arg greater than Arg-Arg much greater than Lys-Lys greater than Arg-Lys, which shows a fair correlation with the intra-cellular cleavage of the bonds observed during the processing of preproteins of of the precursors of several physiologically active peptides.

  2. Roles for H2A.Z and its acetylation in GAL1 transcription and gene induction, but not GAL1-transcriptional memory.

    Directory of Open Access Journals (Sweden)

    Jeffrey E Halley

    Full Text Available H2A.Z is a histone H2A variant conserved from yeast to humans, and is found at 63% of promoters in Saccharomyces cerevisiae. This pattern of localization suggests that H2A.Z is somehow important for gene expression or regulation. H2A.Z can be acetylated at up to four lysine residues on its amino-terminal tail, and acetylated-H2A.Z is enriched in chromatin containing promoters of active genes. We investigated whether H2A.Z's role in GAL1 gene regulation and gene expression depends on H2A.Z acetylation. Our findings suggested that H2A.Z functioned both in gene regulation and in gene expression and that only its role in gene regulation depended upon its acetylation. Our findings provided an alternate explanation for results that were previously interpreted as evidence that H2A.Z plays a role in GAL1 transcriptional memory. Additionally, our findings provided new insights into the phenotypes of htz1Delta mutants: in the absence of H2A.Z, the SWR1 complex, which deposits H2A.Z into chromatin, was deleterious to the cell, and many of the phenotypes of cells lacking H2A.Z were due to the SWR1 complex's activity rather than to the absence of H2A.Z per se. These results highlight the need to reevaluate all studies on the phenotypes of cells lacking H2A.Z.

  3. Roles for H2A.Z and Its Acetylation in GAL1 Transcription and Gene Induction, but Not GAL1-Transcriptional Memory

    Science.gov (United States)

    Halley, Jeffrey E.; Kaplan, Tommy; Wang, Alice Y.; Kobor, Michael S.; Rine, Jasper

    2010-01-01

    H2A.Z is a histone H2A variant conserved from yeast to humans, and is found at 63% of promoters in Saccharomyces cerevisiae. This pattern of localization suggests that H2A.Z is somehow important for gene expression or regulation. H2A.Z can be acetylated at up to four lysine residues on its amino-terminal tail, and acetylated-H2A.Z is enriched in chromatin containing promoters of active genes. We investigated whether H2A.Z's role in GAL1 gene regulation and gene expression depends on H2A.Z acetylation. Our findings suggested that H2A.Z functioned both in gene regulation and in gene expression and that only its role in gene regulation depended upon its acetylation. Our findings provided an alternate explanation for results that were previously interpreted as evidence that H2A.Z plays a role in GAL1 transcriptional memory. Additionally, our findings provided new insights into the phenotypes of htz1Δ mutants: in the absence of H2A.Z, the SWR1 complex, which deposits H2A.Z into chromatin, was deleterious to the cell, and many of the phenotypes of cells lacking H2A.Z were due to the SWR1 complex's activity rather than to the absence of H2A.Z per se. These results highlight the need to reevaluate all studies on the phenotypes of cells lacking H2A.Z. PMID:20582323

  4. Auto-acetylation on K289 is not essential for HopZ1a-mediated plant defense suppression

    Directory of Open Access Journals (Sweden)

    Jose Sebastian Rufian

    2015-07-01

    Full Text Available The Pseudomonas syringae type III-secreted effector HopZ1a is a member of the HopZ / YopJ superfamily of effectors that triggers immunity in Arabidopsis. We have previously shown that HopZ1a suppresses both local (effector-triggered immunity, ETI and systemic immunity (systemic acquired resistance, SAR triggered by the heterologous effector AvrRpt2. HopZ1a has been shown to possess acetyltransferase activity, and this activity is essential to trigger immunity in Arabidopsis. HopZ1a acetyltransferase activity has been reported to require the auto-acetylation of the effector on a specific lysine (K289 residue. In this paper we analyze the relevance of autoacetylation of lysine residue 289 in HopZ1a ability to suppress plant defenses, and on the light of the results obtained, we also revise its relevance for HopZ1a avirulence activity. Our results indicate that, while the HopZ1aK289R mutant is impaired to some degree in its virulence and avirulence activities, is by no means phenotypically equivalent to the catalytically inactive HopZ1aC216A, since it is still able to trigger a defense response that induces detectable macroscopic HR and effectively protects Arabidopsis from infection, reducing growth of P. syringae within the plant. We also present evidence that the HopZ1aK289R mutant still displays virulence activities, partially suppressing both ETI and SAR.

  5. AIRE acetylation and deacetylation: effect on protein stability and transactivation activity.

    Science.gov (United States)

    Incani, Federica; Serra, Maria; Meloni, Alessandra; Cossu, Carla; Saba, Luisella; Cabras, Tiziana; Messana, Irene; Rosatelli, Maria C

    2014-08-27

    The AIRE protein plays a remarkable role as a regulator of central tolerance by controlling the promiscuous expression of tissue-specific antigens in thymic medullary epithelial cells. Defects in AIRE gene cause the autoimmune polyendocrinopathy- candidiasis-ectodermal dystrophy, a rare disease frequent in Iranian Jews, Finns, and Sardinian population. In this study, we have precisely mapped, by mass spectrometry experiments, the sites of protein acetylation and, by mutagenesis assays, we have described a set of acetylated lysines as being crucial in influencing the subcellular localization of AIRE. Furthermore, we have also determined that the de-acetyltransferase enzymes HDAC1-2 are involved in the lysine de-acetylation of AIRE. On the basis of our results and those reported in literature, we propose a model in which lysines acetylation increases the stability of AIRE in the nucleus. In addition, we observed that the interaction of AIRE with deacetylases complexes inhibits its transcriptional activity and is probably responsible for the instability of AIRE, which becomes more susceptible to degradation in the proteasome.

  6. Solution structure of the second bromodomain of Brd2 and its specific interaction with acetylated histone tails

    Directory of Open Access Journals (Sweden)

    Wu Jihui

    2007-09-01

    Full Text Available Abstract Background Brd2 is a transcriptional regulator and belongs to BET family, a less characterized novel class of bromodomain-containing proteins. Brd2 contains two tandem bromodomains (BD1 and BD2, 46% sequence identity in the N-terminus and a conserved motif named ET (extra C-terminal domain at the C-terminus that is also present in some other bromodomain proteins. The two bromodomains have been shown to bind the acetylated histone H4 and to be responsible for mitotic retention on chromosomes, which is probably a distinctive feature of BET family proteins. Although the crystal structure of Brd2 BD1 is reported, no structure features have been characterized for Brd2 BD2 and its interaction with acetylated histones. Results Here we report the solution structure of human Brd2 BD2 determined by NMR. Although the overall fold resembles the bromodomains from other proteins, significant differences can be found in loop regions, especially in the ZA loop in which a two amino acids insertion is involved in an uncommon π-helix, termed πD. The helix πD forms a portion of the acetyl-lysine binding site, which could be a structural characteristic of Brd2 BD2 and other BET bromodomains. Unlike Brd2 BD1, BD2 is monomeric in solution. With NMR perturbation studies, we have mapped the H4-AcK12 peptide binding interface on Brd2 BD2 and shown that the binding was with low affinity (2.9 mM and in fast exchange. Using NMR and mutational analysis, we identified several residues important for the Brd2 BD2-H4-AcK12 peptide interaction and probed the potential mechanism for the specific recognition of acetylated histone codes by Brd2 BD2. Conclusion Brd2 BD2 is monomeric in solution and dynamically interacts with H4-AcK12. The additional secondary elements in the long ZA loop may be a common characteristic of BET bromodomains. Surrounding the ligand-binding cavity, five aspartate residues form a negatively charged collar that serves as a secondary binding site

  7. Dysregulation of AKT Pathway by SMYD2-Mediated Lysine Methylation on PTEN

    OpenAIRE

    Makoto Nakakido; Zhenzhong Deng; Takehiro Suzuki; Naoshi Dohmae; Yusuke Nakamura; Ryuji Hamamoto

    2015-01-01

    Phosphatase and tensin homologue (PTEN), one of the well-characterized tumor suppressor proteins, counteracts the phosphatidylinositol 3-kinase-AKT pathway through its unique lipid phosphatase activity. The functions of PTEN are regulated by a variety of posttranslational modifications such as acetylation, oxidation, ubiquitylation, phosphorylation, and SUMOylation. However, methylation of PTEN has not been reported so far. In this study, we demonstrated that the oncogenic protein lysine meth...

  8. Digestible lysine requirements of broilers

    Directory of Open Access Journals (Sweden)

    LEP Bernal

    2014-03-01

    Full Text Available Modern broilers have been submitted to continuous genetic improvement, and therefore, their nutritional requirements must be constantly updated to ensure their performance. Two experiments were carried out to evaluate different digestible lysine levels for starter (1021 days and grower (22-35 days phases. The experiments were carried out with male and female Cobb 500 broilers, distributed according to a randomized block experimental design in a 5x2 factorial arrangement (5 increasing digestible lysine levels x 2 sexes, totaling 10 treatments, with 8 replicates of 22 and 20 birds during the starter and grower phase, respectively. Digestible lysine levels of 1.06, 1.12, 1.18, 1.24, and 1.30 were used in the starter diets (10-21 days and 0.9, 0.98, 1.04, 1.10, and 1.16% in the grower diets (22-35 days. Based on the statistical analyses of the evaluated performance parameters, digestible lysine requirements for maximum performance were determined as 1.22% for males and 1.24% for females in the starter phase, and 1.16% for both sexes in the grower phase. Carcass and performance results indicate that digestible lysine requirements vary with sex and evaluated production parameter. Considering the most relevant broiler production parameters, in 22- to 35-d-old males, digestible lysine requirement for breast meat yield (1.16% was higher than those for feed conversion ratio (1.07% and weight gain (1.05%.

  9. Acetylation of the pro-apoptotic factor, p53 in the hippocampus following cerebral ischemia and modulation by estrogen.

    Directory of Open Access Journals (Sweden)

    Limor Raz

    Full Text Available BACKGROUND: Recent studies demonstrate that acetylation of the transcription factor, p53 on lysine(373 leads to its enhanced stabilization/activity and increased susceptibility of cells to stress. However, it is not known whether acetylation of p53 is altered in the hippocampus following global cerebral ischemia (GCI or is regulated by the hormone, 17β-estradiol (17β-E(2, and thus, this study examined these issues. METHODOLOGY/PRINCIPAL FINDINGS: The study revealed that Acetyl p53-Lysine(373 levels were markedly increased in the hippocampal CA1 region after GCI at 3 h, 6 h and 24 h after reperfusion, an effect strongly attenuated by 17β-E(2. 17β-E(2 also enhanced interaction of p53 with the ubiquitin ligase, Mdm2, increased ubiquitination of p53, and induced its down-regulation, as well as attenuated elevation of the p53 transcriptional target, Puma. We also observed enhanced acetylation of p53 at a different lysine (Lys(382 at 3 h after reperfusion, and 17β-E(2 also markedly attenuated this effect. Furthermore, administration of an inhibitor of CBP/p300 acetyltransferase, which acetylates p53, was strongly neuroprotective of the CA1 region following GCI. In long-term estrogen deprived (LTED animals, the ability of 17β-E(2 to attenuate p53 acetylation was lost, and intriguingly, Acetyl p53-Lysine(373 levels were markedly elevated in sham (non-ischemic LTED animals. Finally, intracerebroventricular injections of Gp91ds-Tat, a specific NADPH oxidase (NOX2 inhibitor, but not the scrambled tat peptide control (Sc-Tat, attenuated acetylation of p53 and reduced levels of Puma following GCI. CONCLUSIONS/SIGNIFICANCE: The studies demonstrate that p53 undergoes enhanced acetylation in the hippocampal CA1 region following global cerebral ischemia, and that the neuroprotective agent, 17β-E(2, markedly attenuates the ischemia-induced p53 acetylation. Furthermore, following LTED, the suppressive effect of 17β-E(2 on p53 acetylation is lost, and p53

  10. Location of the O-acetyl substituents on a nonasaccharide repeating unit of sycamore extracellular xyloglucan.

    Science.gov (United States)

    York, W S; Oates, J E; van Halbeek, H; Darvill, A G; Albersheim, P; Tiller, P R; Dell, A

    1988-02-15

    The locations of the O-acetyl substituents on the major nonasaccharide repeating unit of the xyloglucan isolated from sycamore extracellular polysaccharides were determined by a combination of analytical methods, including f.a.b.-m.s. and 1H-n.m.r. spectroscopy. The O-2-linked-beta-D-galactosyl residue of the nonasaccharide was found to be the dominant site of O-acetyl substitution. Both mono-O-acetylated and di-O-acetylated beta-D-galactosyl residues were detected. The degree of O-acetylation of the beta-D-galactosyl residue, was estimated by 1H-n.m.r. spectroscopy to be 55-60% at O-6, 15-20% at O-4, and 20-25% at O-3. 1H-n.m.r. spectroscopy also indicated that approximately 50% of the beta-D-galactosyl residues are mono-O-acetylated, 25-30% are di-O-acetylated, and 20% are not acetylated.

  11. Dyslipidemic Diet-Induced Monocyte “Priming” and Dysfunction in Non-Human Primates Is Triggered by Elevated Plasma Cholesterol and Accompanied by Altered Histone Acetylation

    Directory of Open Access Journals (Sweden)

    John D. Short

    2017-08-01

    Full Text Available Monocytes and the recruitment of monocyte-derived macrophages into sites of inflammation play a key role in atherogenesis and other chronic inflammatory diseases linked to cardiometabolic syndrome and obesity. Previous studies from our group have shown that metabolic stress promotes monocyte priming, i.e., enhanced adhesion and accelerated chemotaxis of monocytes in response to chemokines, both in vitro and in dyslipidemic LDLR−/− mice. We also showed that metabolic stress-induced monocyte dysfunction is, at least to a large extent caused by the S-glutathionylation, inactivation, and subsequent degradation of mitogen-activated protein kinase phosphatase 1. Here, we analyzed the effects of a Western-style, dyslipidemic diet (DD, which was composed of high levels of saturated fat, cholesterol, and simple sugars, on monocyte (dysfunction in non-human primates (NHPs. We found that similar to mice, a DD enhances monocyte chemotaxis in NHP within 4 weeks, occurring concordantly with the onset of hypercholesterolemia but prior to changes in triglycerides, blood glucose, monocytosis, or changes in monocyte subset composition. In addition, we identified transitory decreases in the acetylation of histone H3 at the lysine residues 18 and 23 in metabolically primed monocytes, and we found that monocyte priming was correlated with the acetylation of histone H3 at lysine 27 after an 8-week DD regimen. Our data show that metabolic stress promotes monocyte priming and hyper-chemotactic responses in NHP. The histone modifications accompanying monocyte priming in primates suggest a reprogramming of the epigenetic landscape, which may lead to dysregulated responses and functionalities in macrophages derived from primed monocytes that are recruited to sites of inflammation.

  12. Available lysine in canned fish

    OpenAIRE

    Rao, D. Ramananda; Gadre, Ujjwala V.

    1984-01-01

    Otolithus argenteus was canned in brine by heat processing at two different steam pressures either at 0.70 kg/cm super(2) or 1.05 kg/cm super(2) for 25 minutes. The nutritive value of canned fish as evaluated by the total nitrogen and available lysine did not alter much either during heat processing or during storage over a period of nine months at 28 degree plus or minus 5 degree C.

  13. BIOLOGICAL ADHESIVES. Adaptive synergy between catechol and lysine promotes wet adhesion by surface salt displacement.

    Science.gov (United States)

    Maier, Greg P; Rapp, Michael V; Waite, J Herbert; Israelachvili, Jacob N; Butler, Alison

    2015-08-01

    In physiological fluids and seawater, adhesion of synthetic polymers to solid surfaces is severely limited by high salt, pH, and hydration, yet these conditions have not deterred the evolution of effective adhesion by mussels. Mussel foot proteins provide insights about adhesive adaptations: Notably, the abundance and proximity of catecholic Dopa (3,4-dihydroxyphenylalanine) and lysine residues hint at a synergistic interplay in adhesion. Certain siderophores—bacterial iron chelators—consist of paired catechol and lysine functionalities, thereby providing a convenient experimental platform to explore molecular synergies in bioadhesion. These siderophores and synthetic analogs exhibit robust adhesion energies (E(ad) ≥-15 millijoules per square meter) to mica in saline pH 3.5 to 7.5 and resist oxidation. The adjacent catechol-lysine placement provides a "one-two punch," whereby lysine evicts hydrated cations from the mineral surface, allowing catechol binding to underlying oxides.

  14. Identification and characterization of lysine-methylated sites on histones and non-histone proteins.

    Science.gov (United States)

    Lee, Tzong-Yi; Chang, Cheng-Wei; Lu, Cheng-Tzung; Cheng, Tzu-Hsiu; Chang, Tzu-Hao

    2014-06-01

    Protein methylation is a kind of post-translational modification (PTM), and typically takes place on lysine and arginine amino acid residues. Protein methylation is involved in many important biological processes, and most recent studies focused on lysine methylation of histones due to its critical roles in regulating transcriptional repression and activation. Histones possess highly conserved sequences and are homologous in most species. However, there is much less sequence conservation among non-histone proteins. Therefore, mechanisms for identifying lysine-methylated sites may greatly differ between histones and non-histone proteins. Nevertheless, this point of view was not considered in previous studies. Here we constructed two support vector machine (SVM) models by using lysine-methylated data from histones and non-histone proteins for predictions of lysine-methylated sites. Numerous features, such as the amino acid composition (AAC) and accessible surface area (ASA), were used in the SVM models, and the predictive performance was evaluated using five-fold cross-validations. For histones, the predictive sensitivity was 85.62% and specificity was 80.32%. For non-histone proteins, the predictive sensitivity was 69.1% and specificity was 88.72%. Results showed that our model significantly improved the predictive accuracy of histones compared to previous approaches. In addition, features of the flanking region of lysine-methylated sites on histones and non-histone proteins were also characterized and are discussed. A gene ontology functional analysis of lysine-methylated proteins and correlations of lysine-methylated sites with other PTMs in histones were also analyzed in detail. Finally, a web server, MethyK, was constructed to identify lysine-methylated sites. MethK now is available at http://csb.cse.yzu.edu.tw/MethK/.

  15. Effect of bacteriophage lysin on lysogens

    Institute of Scientific and Technical Information of China (English)

    Balaji Subramanyam; Vanaja Kumar

    2011-01-01

    Objective: To study the effect of phage lysin on the growth of lysogens. Methods: Sputum specimens processed by modified Petroff's method were respectively treated with phagebiotics in combination with lysin and lysin alone. The specimens were incubated at 37℃ for 4 days. At the end of day 1, 2, 3 and day 4, the specimens were streaked on blood agar plates and incubated at 37℃ for 18-24 hours. The growth of normal flora observed after day 1 was considered as lysogens.Results:When specimens treated with lysin alone, lysogen formation was avoided and normal flora was controlled. Conclusions: Lysin may have no effect on the growth of lysogens. Sputum specimens treated with phagebiotics-lysin showed the growth of lysogens.

  16. Mass Spectrometry Analysis of Lysine Posttranslational Modifications of Tau Protein from Alzheimer's Disease Brain.

    Science.gov (United States)

    Thomas, Stefani N; Yang, Austin J

    2017-01-01

    Recent advances in mass spectrometry (MS)-based proteomics have greatly facilitated the robust identification and quantification of posttranslational modifications (PTMs), including those that are present at substoichiometric site occupancies. The abnormal posttranslational modification and accumulation of the microtubule-associated protein tau has been implicated in the pathogenesis of Alzheimer's disease (AD), and it is thought that the primary mode of regulation of tau occurs through PTMs. Several studies have been published regarding tau phosphorylation; however, other tau PTMs such as ubiquitylation, acetylation, methylation, oxidation, sumoylation, nitration, and glycosylation have not been analyzed as extensively. The comprehensive detection and delineation of these PTMs is critical for drug target discovery and validation. Lysine-directed PTMs including ubiquitylation, acetylation, and methylation play key regulatory roles with respect to the rates of tau turnover and aggregation. MS-based analytical approaches have been used to gain insight into the tau lysine-directed PTM signature that is most closely associated with neurofibrillary lesion formation. This chapter provides details pertaining to the liquid chromatography tandem mass spectrometry (LC-MS/MS)-based analysis of the lysine-directed posttranslational modification of tau.

  17. PCAF-primed EZH2 acetylation regulates its stability and promotes lung adenocarcinoma progression

    Institute of Scientific and Technical Information of China (English)

    Wan Junhu; Chin Y Eugene; Zhang Hongquan; Zhan Jun; Li Shuai; Ma Ji; Xu Weizhi; Liu Chang; Xue Xiaowei; Xie Yuping; Fang Weigang

    2015-01-01

    Enhancer of zeste homolog 2 ( EZH2 ) is a key epigenetic regulator that catalyzes the trimethyla-tion of H3K27 and is modulated by post-translational modifications (PTMs). However, the precise regulation of EZH2 PTMs remains elusive. We, herein, report that EZH2 is acetylated by acetyltransferase P300/CBP-associat-ed factor (PCAF) and is deacetylated by deacetylase SIRT1. We identified that PCAF interacts with and acetylates EZH2 mainly at lysine 348 (K348). Mechanistically, K348 acetylation decreases EZH2 phosphorylation at T345 and T487 and increases EZH2 stability without disrupting the formation of polycomb repressive complex 2 ( PRC2 ) . Functionally, EZH2 K348 acetylation enhances its capacity in suppression of the target genes and promotes lung cancer cell migration and invasion. Further, elevated EZH2 K348 acetylation in lung adenocarcinoma patients pre-dicts a poor prognosis. Our findings define a new mechanism underlying EZH2 modulation by linking EZH2 acety-lation to its phosphorylation that stabilizes EZH2 and promotes lung adenocarcinoma progression.

  18. Biological function and regulation of histone and non-histone lysine methylation in response to DNA damage

    Institute of Scientific and Technical Information of China (English)

    Yongcan Chen; Wei-Guo Zhu

    2016-01-01

    DNA damage response (DDR) signaling network is initiated to protect cells from various exogenous and endogenous damage resources.Timely and accurate regulation of DDR proteins is required for distinct DNA damage repair pathways.Post-translational modifications of histone and non-histone proteins play a vital role in the DDR factor foci formation and signaling pathway.Phosphorylation,ubiquitylation,SUMOylation,neddylation,poly(ADP-ribosyl)ation,acetylation,and methylation are all involved in the spatial-temporal regulation of DDR,among which phosphorylation and ubiquitylation are well studied.Studies in the past decade also revealed extensive roles of lysine methylation in response to DNA damage.Lysine methylation is finely regulated by plenty of lysine methyltransferases,lysine demethylases,and can be recognized by proteins with chromodomain,plant homeodomain,Tudor domain,malignant brain tumor domain,or prolinetryptophan-tryptophan-proline domain.In this review,we outline the dynamics and regulation of histone lysine methylation at canonical (H3K4,H3K9,H3K27,H3K36,H3K79,and H4K20) and non-canonical sites after DNA damage,and discuss their context-specific functions in DDR protein recruitment or extraction,chromatin environment establishment,and transcriptional regulation.We also present the emerging advances of lysine methylation in non-histone proteins during DDR.

  19. SHORT COMMUNICATION ACETYLATION AND OXYGENATION ...

    African Journals Online (AJOL)

    a

    mild conditions and in processes that are environmentally benign. ... All the products were characterized by a comparison of their spectral and .... In conclusion, an eco-friendly, clean, and cheap heterogeneous catalytic acetylation and.

  20. Hemoglobin Labeled by Radioactive Lysine

    Science.gov (United States)

    Bale, W. F.; Yuile, C. L.; DeLaVergne, L.; Miller, L. L.; Whipple, G. H.

    1949-12-08

    This paper reports on the utilization of tagged epsilon carbon of DL-lysine by a dog both anemic and hypoproteinemic due to repeated bleeding plus a diet low in protein. The experiment extended over period of 234 days, a time sufficient to indicate an erythrocyte life span of at least 115 days based upon the rate of replacement of labeled red cell proteins. The proteins of broken down red cells seem not to be used with any great preference for the synthesis of new hemoglobin.

  1. Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemicellulose

    DEFF Research Database (Denmark)

    Biely, Peter; Cziszarava, Maria; Agger, Jane W.

    2014-01-01

    prominent target is the 3-O-acetyl group at the non-reducing terminal Xylp residues of linear neutral xylooligosaccharides or on aldouronic acids carrying MeGlcA at the non-reducing terminus. Deacetylation of the non-reducing end sugar may involve migration of acetyl groups to position 4, which also serves...... as substrate of the TrCE16 esterase. Conclusion Concerted action of CtGH10 xylanase, an AcXE and TrCE16 AcE resulted in close to complete deacetylation of neutral xylooligosaccharides, whereas substitution with MeGlcA prevents removal of acetyl groups from only a small fraction of the aldouronic acids...

  2. Experiment list: SRX186658 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available e. It remains unknown whether acetylation has can have different consequences depending on the specific lysi...ns unknown whether acetylation has can have different consequences depending on the specific lysine residue

  3. Experiment list: SRX186748 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available It remains unknown whether acetylation can have different consequences depending on the specific lysine resi...n whether acetylation can have different consequences depending on the specific lysine residue targeted. In

  4. Small molecule inhibitors of histone deacetylases and acetyltransferases as potential therapeutics in oncology

    NARCIS (Netherlands)

    van den Bosch, Teatske; Leus, Niek; Timmerman, Tirza; Dekker, Frans

    2016-01-01

    Uncontrolled cell proliferation and resistance to apoptosis in cancer are, among others, regulated by post-translational modifications of histone proteins. The most investigated type of histone modification is lysine acetylation. Histone acetyltransferases (HATs), acetylate histone lysine residues,

  5. Antioxidant activity of carbocysteine lysine salt monohydrate.

    Science.gov (United States)

    Pinamonti, S; Venturoli, L; Leis, M; Chicca, M; Barbieri, A; Sostero, S; Ravenna, F; Daffonchio, L; Novellini, R; Ciaccia, A

    2001-09-01

    Reactive oxygen radicals are involved in many respiratory diseases, including chronic obstructive pulmonary disease (COPD). Carbocysteine lysine salt monohydrate (CLS) is a mucoactive drug effective in the treatment of bronchopulmonary diseases characterized by mucus alterations, including COPD. In the present study, the antioxidant activity of CLS was studied in vitro in three different oxygen radical producing systems, i.e. bronchoalveolar lavages (BAL) from patients affected by COPD, ultrasound treated human serum and cultured human lung endothelial cells challenged with elastase. BAL, exposed or not to different concentrations of CLS (1.5-30 mM), was assayed for free radical content by fluorometric analysis of DNA unwinding (FADU) or by cytochrome c reduction kinetics. Human serum was treated with ultrasound in the presence or absence of CLS (1.5, 2.5 mM) or N-acetyl cysteine (NAC; 4, 5 mM) and assayed for free radical content by FADU. Human endothelial cells cultured in vitro from pulmonary artery were incubated with elastase (0.3 IU/mL), in the presence or absence of glutathione (GSH; 0.65 mM) or CLS (0.16 mM). The supernatant was tested for cytochrome c reduction kinetics whereas cell homogenates were assessed for xanthine oxidase (XO) content by SDS-PAGE. Results showed that CLS is more effective as an in vitro scavenger in comparison to GSH and NAC. CLS reduced the damage of DNA from healthy donors exposed to COPD-BAL and was able to quench clastogenic activity induced in human serum by exposure to ultrasound at concentrations as low as 2.5 mM. NAC protect DNA from radical damage, starting from 5 mM. In human lung endothelial cells cultured in presence of elastase, CLS (0.16 mM) decreased xanthine oxidase activity. These results suggest that CLS could act by interfering with the conversion of xanthine dehydrogenase into superoxide-producing xanthine oxidase. The antioxidant activity of CLS could contribute to its therapeutic activity by reducing radical

  6. PENILAIAN PENGARUH PENAMBAHAN LYSINE PADA NASI

    Directory of Open Access Journals (Sweden)

    Ignatius Tarwotjo

    2012-11-01

    Full Text Available Pengaruh penambahan lysine pada mutu protein nasi dilakukan pada tikus putih dengan mengukur Protein Efficiency Ratio. Nasi dan Nasi dengan sayur beserta laukpauk, seperti dikonsumsi oleh kebanyakan keluarga di Indonesia, yang berasnya lebih dulu ditambahi butiran premix berisi lysine, thiamine dan riboflavin ternaya menghasilkan Protein Efficiency Ratio lebih tinggi dari pada yang tidak ditambahi.

  7. Relationship between lunasin's sequence and its inhibitory activity of histones H3 and H4 acetylation.

    Science.gov (United States)

    Hernández-Ledesma, Blanca; Hsieh, Chia-Chien; de Lumen, Ben O

    2011-07-01

    Dysfunction of histone acetyltransferases (HATs) or histone deacetylases (HDACs) involved in histones acetylation has been associated with cancer. Inhibitors of these enzymes are becoming potential targets for new therapies. This study reports by Western-Blot analysis, that peptide lunasin is mainly an in vitro inhibitor of histone H4 acetylation by P300/cAMP-response element-binding protein (CBP)-associated factor (PCAF), with IC₅₀ values dependent on the lysine position sensitive to be acetylated (0.83 μM (H4-Lys 8), 1.27 μM (H4-Lys 12) and 0.40 μM (H4-Lys 5, 8, 12, 16)). Lunasin is also capable of inhibiting H3 acetylation (IC₅₀ of 5.91 μM (H3-Lys 9) and 7.81 μM (H3-Lys 9, 14)). Studies on structure-activity relationship establish that lunasin's sequence are essential for inhibiting H4 acetylation whereas poly-D sequence is the main active sequence responsible for H3 acetylation inhibition. Lunasin also inhibits H3 and H4 acetylation and cell proliferation (IC₅₀ of 181 μM) in breast cancer MDA-MB-231 cells. Moreover, this peptide decreases expression of cyclins and cyclin dependent kinases-4 and -6, implicated in cell cycle pathways. Results from this study demonstrates lunasin's role as modulator of histone acetylation and protein expression that might contribute on its chemopreventive properties against breast cancer. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Engineering a Lysine-ON Riboswitch for Metabolic Control of Lysine Production in Corynebacterium glutamicum.

    Science.gov (United States)

    Zhou, Li-Bang; Zeng, An-Ping

    2015-12-18

    Riboswitches are natural RNA elements that regulate gene expression by binding a ligand. Here, we demonstrate the possibility of altering a natural lysine-OFF riboswitch from Eschericia coli (ECRS) to a synthetic lysine-ON riboswitch and using it for metabolic control. To this end, a lysine-ON riboswitch library was constructed using tetA-based dual genetic selection. After screening the library, the functionality of the selected lysine-ON riboswitches was examined using a report gene, lacZ. Selected lysine-ON riboswitches were introduced into the lysE gene (encoding a lysine transport protein) of Corynebacterium glutamicum and used to achieve dynamic control of lysine transport in a recombinant lysine-producing strain, C. glutamicum LPECRS, which bears a deregulated aspartokinase and a lysine-OFF riboswitch for dynamic control of the enzyme citrate synthase. Batch fermentation results of the strains showed that the C. glutamicum LPECRS strain with an additional lysine-ON riboswitch for the control of lysE achieved a 21% increase in the yield of lysine compared to that of the C. glutamicum LPECRS strain and even a 89% increase in yield compared to that of the strain with deregulated aspartokinase. This work provides a useful approach to generate lysine-ON riboswitches for C. glutamicum metabolic engineering and demonstrates for the first time a synergetic effect of lysine-ON and -OFF riboswitches for improving lysine production in this industrially important microorganism. The approach can be used to dynamically control other genes and can be applied to other microorganisms.

  9. Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media.

    Science.gov (United States)

    Christensen, David G; Orr, James S; Rao, Christopher V; Wolfe, Alan J

    2017-03-15

    Complex media are routinely used to cultivate diverse bacteria. However, this complexity can obscure the factors that govern cell growth. While studying protein acetylation in buffered tryptone broth supplemented with glucose (TB7-glucose), we observed that Escherichia coli did not fully consume glucose prior to stationary phase. However, when we supplemented this medium with magnesium, the glucose was completely consumed during exponential growth, with concomitant increases in cell number and biomass but reduced cell size. Similar results were observed with other sugars and other peptide-based media, including lysogeny broth. Magnesium also limited cell growth for Vibrio fischeri and Bacillus subtilis in TB7-glucose. Finally, magnesium supplementation reduced protein acetylation. Based on these results, we conclude that growth in peptide-based media is magnesium limited. We further conclude that magnesium supplementation can be used to tune protein acetylation without genetic manipulation. These results have the potential to reduce potentially deleterious acetylated isoforms of recombinant proteins without negatively affecting cell growth.IMPORTANCE Bacteria are often grown in complex media. These media are thought to provide the nutrients necessary to grow bacteria to high cell densities. In this work, we found that peptide-based media containing a sugar are magnesium limited for bacterial growth. In particular, magnesium supplementation is necessary for the bacteria to use the sugar for cell growth. Interestingly, in the absence of magnesium supplementation, the bacteria still consume the sugar. However, rather than use it for cell growth, the bacteria instead use the sugar to acetylate lysines on proteins. As lysine acetylation may alter the activity of proteins, this work demonstrates how lysine acetylation can be tuned through magnesium supplementation. These findings may be useful for recombinant protein production, when acetylated isoforms are to be avoided

  10. Site-Specific Conjugation to Native and Engineered Lysines in Human Immunoglobulins by Microbial Transglutaminase.

    Science.gov (United States)

    Spidel, Jared L; Vaessen, Benjamin; Albone, Earl F; Cheng, Xin; Verdi, Arielle; Kline, J Bradford

    2017-09-20

    The use of microbial transglutaminase (MTG) to produce site-specific antibody-drug conjugates (ADCs) has thus far focused on the transamidation of engineered acyl donor glutamine residues in an antibody based on the hypothesis that the lower specificity of MTG for acyl acceptor lysines may result in the transamidation of multiple native lysine residues, thereby yielding heterogeneous products. We investigated the utilization of native IgG lysines as acyl acceptor sites for glutamine-based acyl donor substrates. Of the approximately 80 lysines in multiple recombinant IgG monoclonal antibodies (mAbs), none were transamidated. Because recombinant mAbs lack the C-terminal Lys447 due to cleavage by carboxypeptidase B in the production cell host, we explored whether blocking the cleavage of Lys447 by the addition of a C-terminal amino acid could result in transamidation of Lys447 by a variety of acyl donor substrates. MTG efficiently transamidated Lys447 in the presence of any nonacidic, nonproline amino acid residue at position 448. Lysine scanning mutagenesis throughout the antibody further revealed several transamidation sites in both the heavy- and light-chain constant regions. Additionally, scanning mutagenesis of the hinge region in a Fab' fragment revealed sites of transamidation that were not reactive in the context of the full-length mAb. Here, we demonstrate the utility of single lysine substitutions and the C-terminal Lys447 for engineering efficient acyl acceptor sites suitable for site-specific conjugation to a range of glutamine-based acyl donor substrates.

  11. Patterns of methyl and O-acetyl esterification in spinach pectins: new complexity.

    Science.gov (United States)

    Perrone, Paola; Hewage, Chandralal M; Thomson, Adam R; Bailey, Kevin; Sadler, Ian H; Fry, Stephen C

    2002-05-01

    Driselase-digestion of cell walls from suspension-cultures of spinach (Spinacia oleracea L.), followed by anion-exchange chromatography, gel-permeation chromatography, preparative paper chromatography and preparative paper electrophoresis, yielded ten uronic acid-containing products in addition to free galacturonic acid (GalA). These included 4-O-methylglucuronic acid, alpha-L-rhamnopyranosyl-(1-->4)-D-glucuronic acid and several oligosaccharides containing GalA residues. The structures were unambiguously determined by a combination of 1- and 2-dimensional NMR spectroscopic techniques. Five of the six homogalacturonan-derived oligosaccharides purified contained 3-O-acetyl-GalA residues; however, methyl-esterified GalA residues occurred adjacent to both 2-O-acetyl-GalA and 3-O-acetyl-GalA residues. An acetylated, rhamnogalacturonan-I-derived oligosaccharide that was purified also contained 3-O-acetyl-GalA residues. Taken together with published data, our findings indicate considerable diversity in the patterns of pectin esterification. The implications for the action of pectin esterases are discussed.

  12. MEC-17 deficiency leads to reduced α-tubulin acetylation and impaired migration of cortical neurons.

    Science.gov (United States)

    Li, Lei; Wei, Dan; Wang, Qiong; Pan, Jing; Liu, Rong; Zhang, Xu; Bao, Lan

    2012-09-12

    Neuronal migration is a fundamental process during the development of the cerebral cortex and is regulated by cytoskeletal components. Microtubule dynamics can be modulated by posttranslational modifications to tubulin subunits. Acetylation of α-tubulin at lysine 40 is important in regulating microtubule properties, and this process is controlled by acetyltransferase and deacetylase. MEC-17 is a newly discovered α-tubulin acetyltransferase that has been found to play a major role in the acetylation of α-tubulin in different species in vivo. However, the physiological function of MEC-17 during neural development is largely unknown. Here, we report that MEC-17 is critical for the migration of cortical neurons in the rat. MEC-17 was strongly expressed in the cerebral cortex during development. MEC-17 deficiency caused migratory defects in the cortical projection neurons and interneurons, and perturbed the transition of projection neurons from the multipolar stage to the unipolar/bipolar stage in the intermediate zone of the cortex. Furthermore, knockdown of α-tubulin deacetylase HDAC6 or overexpression of tubulin(K40Q) to mimic acetylated α-tubulin could reduce the migratory and morphological defects caused by MEC-17 deficiency in cortical projection neurons. Thus, MEC-17, which regulates the acetylation of α-tubulin, appears to control the migration and morphological transition of cortical neurons. This finding reveals the importance of MEC-17 and α-tubulin acetylation in cortical development.

  13. The Effect of Various Zinc Binding Groups on Inhibition of Histone Deacetylases 1–11

    DEFF Research Database (Denmark)

    Madsen, Andreas Stahl; Kristensen, Helle M. E.; Lanz, Gyrithe;

    2014-01-01

    Histone deacetylases (HDACs) have the ability to cleave the acetyl groups of ε‐N‐acetylated lysine residues in a variety of proteins. Given that human cells contain thousands of different acetylated lysine residues, HDACS may regulate a wide variety of processes including some implicated in condi......Histone deacetylases (HDACs) have the ability to cleave the acetyl groups of ε‐N‐acetylated lysine residues in a variety of proteins. Given that human cells contain thousands of different acetylated lysine residues, HDACS may regulate a wide variety of processes including some implicated...

  14. Global proteomic analysis of protein acetylation affecting metabolic regulation in Daphnia pulex.

    Science.gov (United States)

    Kwon, Oh Kwang; Sim, Juhee; Kim, Sun Ju; Oh, Hye Ryeung; Nam, Doo Hyun; Lee, Sangkyu

    2016-02-01

    Daphnia (Daphnia pulex) is a small planktonic crustacean and a key constituent of aquatic ecosystems. It is generally used as a model organism to study environmental toxic problems. In the past decade, genomic and proteomic datasets of Daphnia have been developed. The proteomic dataset allows for the investigation of toxicological effects in the context of "Daphnia proteomics," resulting in greater insights for toxicological research. To exploit Daphnia for ecotoxicological research, information on the post-translational modification (PTM) of proteins is necessary, as this is a critical regulator of biological processes. Acetylation of lysine (Kac) is a reversible and highly regulated PTM that is associated with diverse biological functions. However, a comprehensive description of Kac in Daphnia is not yet available. To understand the cellular distribution of lysine acetylation in Daphnia, we identified 98 acetylation sites in 65 proteins by immunoprecipitation using an anti-acetyllysine antibody and a liquid chromatography system supported by mass spectroscopy. We identified 28 acetylated sites related to metabolic proteins and six acetylated enzymes associated with the TCA cycle in Daphnia. From GO and KEGG enrichment analyses, we showed that Kac in D. pulex is highly enriched in proteins associated with metabolic processes. Our data provide the first global analysis of Kac in D. pulex and is an important resource for the functional analysis of Kac in this organism. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  15. Post-translational modification by acetylation regulates the mitochondrial carnitine/acylcarnitine transport protein.

    Science.gov (United States)

    Giangregorio, Nicola; Tonazzi, Annamaria; Console, Lara; Indiveri, Cesare

    2017-02-01

    The carnitine/acylcarnitine transporter (CACT; SLC25A20) mediates an antiport reaction allowing entry of acyl moieties in the form of acylcarnitines into the mitochondrial matrix and exit of free carnitine. The transport function of CACT is crucial for the β-oxidation pathway. In this work, it has been found that CACT is partially acetylated in rat liver mitochondria as demonstrated by anti-acetyl-lys antibody immunostaining. Acetylation was reversed by the deacetylase Sirtuin 3 in the presence of NAD(+). After treatment of the mitochondrial extract with the deacetylase, the CACT activity, assayed in proteoliposomes, increased. The half-saturation constant of the CACT was not influenced, while the V max was increased by deacetylation. Sirtuin 3 was not able to deacetylate the CACT when incubation was performed in intact mitoplasts, indicating that the acetylation sites are located in the mitochondrial matrix. Prediction on the localization of acetylated residues by bioinformatics correlates well with the experimental data. Recombinant CACT treated with acetyl-CoA was partially acetylated by non-enzymatic mechanism with a corresponding decrease of transport activity. The experimental data indicate that acetylation of CACT inhibits its transport activity, and thus may contribute to the regulation of the mitochondrial β-oxidation pathway.

  16. Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation

    DEFF Research Database (Denmark)

    Lombard, David B; Alt, Frederick W; Cheng, Hwei-Ling;

    2007-01-01

    Homologs of the Saccharomyces cerevisiae Sir2 protein, sirtuins, promote longevity in many organisms. Studies of the sirtuin SIRT3 have so far been limited to cell culture systems. Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble...... mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial...... deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis...

  17. The growing landscape of lysine acetylation links metabolism and cell signalling

    DEFF Research Database (Denmark)

    Choudhary, Chuna Ram; Weinert, Brian Tate; Nishida, Yuya

    2014-01-01

    , implicating it in many biological processes through the regulation of protein interactions, activity and localization. In addition, proteins are frequently modified by other types of acylations, such as formylation, butyrylation, propionylation, succinylation, malonylation, myristoylation, glutarylation...

  18. Reactive lysine content in commercially available pet foods

    NARCIS (Netherlands)

    Rooijen, van C.; Bosch, G.; Poel, van der A.F.B.; Wierenga, P.A.; Alexander, L.; Hendriks, W.H.

    2014-01-01

    The Maillard reaction can occur during processing of pet foods. During this reaction, the e-amino group of lysine reacts with reducing sugars to become unavailable for metabolism. The aim of the present study was to determine the reactive lysine (RL; the remaining available lysine) to total lysine (

  19. Oligo(L-lysine)-induced titanium dioxide: Effects of consecutive lysine on precipitation

    Science.gov (United States)

    Ahn, Sungjun; Park, Sangwoo; Lee, Sang-Yup

    2011-11-01

    Biomineralization of metal oxide utilizes biomolecular substances, such as peptides and proteins, to induce mineralization of metal precursors in a mild aqueous solution. In this study, we investigated biomineralization of an abiological substance, titanium dioxide (TiO 2), by oligo(L-lysine). Specifically, we systemically studied the influence of the number of consecutive lysine on TiO 2 precipitation. Oligo(L-lysine) was chosen as a homopeptide lysine source whose lysine quantity was adjusted. When oligo(L-lysine) contains more than three consecutive lysine, it induces notably fast precipitation, while single and dilysine do not readily form TiO 2 precipitates. Precipitation of TiO 2 was promoted with the length of oligo(L-lysine). The oligo(L-lysine) was associated with TiO 2 precipitate, which was confirmed by spectroscopic and thermogravitational analyses. The outcomes of this research provide a plausible rationale for explaining precipitation of the Ti precursor that is highly dependent on peptide sequences.

  20. A Novel Staphylococcus Podophage Encodes a Unique Lysin with Unusual Modular Design

    Science.gov (United States)

    Cater, Katie; Dandu, Vidya Sree; Bari, S. M. Nayeemul; Lackey, Kim; Everett, Gabriel F. K.

    2017-01-01

    ABSTRACT Drug-resistant staphylococci, particularly Staphylococcus aureus and Staphylococcus epidermidis, are leading causes of hospital-acquired infections. Bacteriophages and their peptidoglycan hydrolytic enzymes (lysins) are currently being explored as alternatives to conventional antibiotics; however, only a limited diversity of staphylococcal phages and their lysins has yet been characterized. Here, we describe a novel staphylococcal phage and its lysins. Bacteriophage Andhra is the first reported S. epidermidis phage belonging to the family Podoviridae. Andhra possesses an 18,546-nucleotide genome with 20 open reading frames. BLASTp searches revealed that gene product 10 (gp10) and gp14 harbor putative catalytic domains with predicted peptidase and amidase activities, characteristic functions of phage lysins. We purified these proteins and show that both Andhra_gp10 and Andhra_gp14 inhibit growth and degrade cell walls of diverse staphylococci, with Andhra_gp10 exhibiting more robust activity against the panel of cell wall substrates tested. Site-directed mutagenesis of its predicted catalytic residues abrogated the activity of Andhra_gp10, consistent with the presence of a catalytic CHAP domain on its C terminus. The active site location combined with the absence of an SH3b cell wall binding domain distinguishes Andhra_gp10 from the majority of staphylococcal lysins characterized to date. Importantly, close homologs of Andhra_gp10 are present in related staphylococcal podophages, and we propose that these constitute a new class of phage-encoded lysins. Altogether, our results reveal insights into the biology of a rare family of staphylococcal phages while adding to the arsenal of antimicrobials with potential for therapeutic use. IMPORTANCE The spread of antibiotic resistance among bacterial pathogens is inciting a global public health crisis. Drug-resistant Staphylococcus species, especially S. aureus and S. epidermidis, have emerged in both hospital

  1. Crystal structures of lysine-preferred racemases, the non-antibiotic selectable markers for transgenic plants.

    Directory of Open Access Journals (Sweden)

    Hsin-Mao Wu

    Full Text Available Lysine racemase, a pyridoxal 5'-phosphate (PLP-dependent amino acid racemase that catalyzes the interconversion of lysine enantiomers, is valuable to serve as a novel non-antibiotic selectable marker in the generation of transgenic plants. Here, we have determined the first crystal structure of a lysine racemase (Lyr from Proteus mirabilis BCRC10725, which shows the highest activity toward lysine and weaker activity towards arginine. In addition, we establish the first broad-specificity amino acid racemase (Bar structure from Pseudomonas putida DSM84, which presents not only the highest activity toward lysine but also remarkably broad substrate specificity. A complex structure of Bar-lysine is also established here. These structures demonstrate the similar fold of alanine racemase, which is a head-to-tail homodimer with each protomer containing an N-terminal (α/β(8 barrel and a C-terminal β-stranded domain. The active-site residues are located at the protomer interface that is a funnel-like cavity with two catalytic bases, one from each protomer, and the PLP binding site is at the bottom of this cavity. Structural comparisons, site-directed mutagenesis, kinetic, and modeling studies identify a conserved arginine and an adjacent conserved asparagine that fix the orientation of the PLP O3 atom in both structures and assist in the enzyme activity. Furthermore, side chains of two residues in α-helix 10 have been discovered to point toward the cavity and define the substrate specificity. Our results provide a structural foundation for the design of racemases with pre-determined substrate specificity and for the development of the non-antibiotic selection system in transgenic plants.

  2. Gamma scintigraphy of {sup 111}In-labelled branched chain polypeptides (BCP) with a poly(L-lysine) backbone in mice with mammary carcinoma. Effect of charge on biodistribution and tumour imaging potential

    Energy Technology Data Exchange (ETDEWEB)

    Pimm, M.V.; Gribben, S.J. [University of Nottingham (United Kingdom); Perkins, A.C.; Mezo, G.; Gaal, D.; Hudecz, F.

    1995-11-01

    Radiolabelled synthetic branched chain polypeptides (BCP) represent a new and novel range of materials with potential as radiopharmaceuticals. Preliminary imaging studies have been undertaken with {sup 111}In-labelled BCP in mice with subcutaneously transplanted mammary carcinoma. Four polypeptides each with a poly(L-lysine) backbone and side chains of DL-alanine residues were studied. These were AK, which is polycationic, EAK which is amphoteric, having additional glutamic acid residues at the end of the side chains, and AcEAK (anionic) and SucEAK (highly polyanionic) where the terminal glutamic acid amino groups were acetylated or succinylated respectively. Radiolabelling was achieved by previous conjugation with DTPA. Serial images up to 48 hours showed marked retention of {sup 111}In-labelled polycationic AK and polyanionic SucEAK in the liver and spleen, with renal uptake also being visible in the case of AK. {sup 111}In-labelled EAK and AcEAK showed longer blood survival with some liver uptake, but tumour uptake was also visualized by 24 hours with both of these polypeptides. These studies demonstrate the feasibility of using {sup 111}In-labelled synthetic branched chain polypeptides as radiopharmaceuticals for gamma scintigraphy and the visualization of tumours by modification of the side chain structure. These materials warrant further study. (author).

  3. Lysine requirement of growing male Pekin ducks.

    Science.gov (United States)

    Bons, A; Timmler, R; Jeroch, H

    2002-12-01

    1. One growth experiment and one balance test were conducted to study the response to increasing levels of dietary lysine supplementation in male Pekin ducks with special reference to the growth periods from 1 to 3 weeks and 4 to 7 weeks of age. 2. Two different low-lysine diets were used as basal diets in both periods. The basal lysine levels were 7.6 g/kg (d 1 to 21) and 6.2 g/kg (d 22 to 49) and the ranges in lysine concentration were 7.6 to 12.6 g/kg (d 1 to 21) and 6.2 to 11.2 g/kg (d 22 to 49). 3. Growth performance, feed conversion efficiency and meat yield increased (P < 0.05) with increasing lysine concentration (requirement defined as 95% of the asymptote). 4. It is concluded that the dietary lysine concentration should be 0.93 g/MJ nitrogen corrected apparent metabolisable energy (AMEN) (11.7 g/kg) for the starter period (until d 21) and 0.75 g/MJ AMEN (10.0 g/kg) for the grower period (from d 22 onwards).

  4. Antibacterial activity of a newly developed peptide-modified lysin against Acinetobacter baumannii and Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Hang eYang

    2015-12-01

    Full Text Available The global emergence of multidrug-resistant (MDR bacteria is a growing threat to public health worldwide. Natural bacteriophage lysins are promising alternatives in the treatment of infections caused by Gram-positive pathogens, but not Gram-negative ones, like Acinetobacter baumannii and Pseudomonas aeruginosa, due to the barriers posed by their outer membranes. Recently, modifying a natural lysin with an antimicrobial peptide was found able to break the barriers, and to kill Gram-negative pathogens. Herein, a new peptide-modified lysin (PlyA was constructed by fusing the cecropin A peptide residues 1–8 (KWKLFKKI with the OBPgp279 lysin and its antibacterial activity was studied. PlyA showed good and broad antibacterial activities against logarithmic phase A. baumannii and P. aeruginosa, but much reduced activities against the cells in stationary phase. Addition of outer membrane permeabilizers (EDTA and citric acid could enhance the antibacterial activity of PlyA against stationary phase cells. Finally, no antibacterial activity of PlyA could be observed in some bio-matrices, such as culture media, milk, and sera. In conclusion, we reported here a novel peptide-modified lysin with significant antibacterial activity against both logarithmic (without OMPs and stationary phase (with OMPs A. baumannii and P. aeruginosa cells in buffer, but further optimization is needed to achieve broad activity in diverse bio-matrices.

  5. Structure of lysine adducts with 16 alpha-hydroxyestrone and cortisol.

    Science.gov (United States)

    Bucala, R; Ulrich, P C; Chait, B T; Bencsath, F A; Cerami, A

    1986-07-01

    Recent studies indicate that steroids containing a vicinal hydroxyketone moiety can react with proteins both in vitro and in vivo to form covalent addition products. This reaction is non-enzymatic and occurs via the Heyns rearrangement of an initial Schiff base adduct between the steroid carbonyl and the epsilon-amino group of lysine residues. The present study describes the synthesis, isolation, and structural analysis of model adducts prepared by the incubation of 16 alpha-hydroxyesterone or cortisol with NaCNBH3 and lysine derivatives blocked in the N alpha-position. The product formed from the reaction of 16 alpha-hydroxyesterone and lysine was found to have the structure predicted for a reduced Schiff base between these molecules. A stable, cortisol-lysine adduct was similarly synthesized and isolated. This conjugate was found not to be the expected reduced Schiff base but rather a C-20 cyano amine. This compound most likely was formed by the nucleophilic addition of cyanide during the course of the incubation. The observation that the cortisol-lysine Schiff base is not reducible with NaCNBH3 accounts for the observation that the incorporation rate of glucocorticoids into proteins is not increased by the presence of NaCNBH3.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Linya You

    2015-03-01

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

  9. Differences in lysine pKa values may be used to improve NMR signal dispersion in reductively methylated proteins

    Energy Technology Data Exchange (ETDEWEB)

    Abraham, Sherwin J. [University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics (United States); Kobayashi, Tomoyoshi; John Solaro, R. [University of Illinois at Chicago, Department of Physiology and Biophysics, Center for Cardiovascular Research (United States); Gaponenko, Vadim [University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics (United States)], E-mail: vadimg@uic.edu

    2009-04-15

    Reductive methylation of lysine residues in proteins offers a way to introduce {sup 13}C methyl groups into otherwise unlabeled molecules. The {sup 13}C methyl groups on lysines possess favorable relaxation properties that allow highly sensitive NMR signal detection. One of the major limitations in the use of reductive methylation in NMR is the signal overlap of {sup 13}C methyl groups in NMR spectra. Here we show that the uniform influence of the solvent on chemical shifts of exposed lysine methyl groups could be overcome by adjusting the pH of the buffering solution closer to the pKa of lysine side chains. Under these conditions, due to variable pKa values of individual lysine side chains in the protein of interest different levels of lysine protonation are observed. These differences are reflected in the chemical shift differences of methyl groups in reductively methylated lysines. We show that this approach is successful in four different proteins including Ca{sup 2+}-bound Calmodulin, Lysozyme, Ca{sup 2+}-bound Troponin C, and Glutathione S-Transferase. In all cases significant improvement in NMR spectral resolution of methyl signals in reductively methylated proteins was obtained. The increased spectral resolution helps with more precise characterization of protein structural rearrangements caused by ligand binding as shown by studying binding of Calmodulin antagonist trifluoperazine to Calmodulin. Thus, this approach may be used to increase resolution in NMR spectra of {sup 13}C methyl groups on lysine residues in reductively methylated proteins that enhances the accuracy of protein structural assessment.

  10. Identification and functional characterization of N-terminally acetylated proteins in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Sandra Goetze

    2009-11-01

    Full Text Available Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (XPX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (XPX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species.

  11. Bioavailability of free lysine and protein-bound lysine from casein and fishmeal in juvenile turbot (Psetta maxima).

    Science.gov (United States)

    Kroeckel, Saskia; Dietz, Carsten; Schulz, Carsten; Susenbeth, Andreas

    2015-03-14

    In the present study, a linear regression analysis between lysine intake and lysine retention was conducted to investigate the efficiency of lysine utilisation (k(Lys)) at marginal lysine intake of either protein-bound or free lysine sources in juvenile turbot (Psetta maxima). For this purpose, nine isonitrogenous and isoenergetic diets were formulated to contain 2·25-4·12 g lysine/100 g crude protein (CP) to ensure that lysine was the first-limiting amino acid in all diets. The basal diet contained 2·25 g lysine/100 g CP. Graded levels of casein (Cas), fishmeal (FM) and L-lysine HCl (Lys) were added to the experimental diets to achieve stepwise lysine increments. A total of 240 fish (initial weight 50·1 g) were hand-fed all the experimental diets once daily until apparent satiation over a period of 56 d. Feed intake was significantly affected by dietary lysine concentration rather than by dietary lysine source. Specific growth rate increased significantly at higher lysine concentrations (PCas, Lys or FM were 0·833, 0·857 and 0·684, respectively. The bioavailability of lysine from the respective lysine sources was determined by a slope-ratio approach. The bioavailability of lysine (relative to the reference lysine source Cas) from FM and Lys was 82·1 and 103 %, respectively. Nutrient requirement for maintenance was in the range of 16·7-23·4 mg/kg(0·8) per d, and did not differ between the treatments. There were no significant differences in lysine utilisation efficiency or bioavailability of protein-bound or crystalline lysine from the respective sources observed when lysine was confirmed to be the first-limiting nutrient.

  12. Acetate supplementation modulates brain histone acetylation and decreases interleukin-1β expression in a rat model of neuroinflammation

    Directory of Open Access Journals (Sweden)

    Soliman Mahmoud L

    2012-03-01

    Full Text Available Abstract Background Long-term acetate supplementation reduces neuroglial activation and cholinergic cell loss in a rat model of lipopolysaccharide-induced neuroinflammation. Additionally, a single dose of glyceryl triacetate, used to induce acetate supplementation, increases histone H3 and H4 acetylation and inhibits histone deacetylase activity and histone deacetylase-2 expression in normal rat brain. Here, we propose that the therapeutic effect of acetate in reducing neuroglial activation is due to a reversal of lipopolysaccharide-induced changes in histone acetylation and pro-inflammatory cytokine expression. Methods In this study, we examined the effect of a 28-day-dosing regimen of glyceryl triacetate, to induce acetate supplementation, on brain histone acetylation and interleukin-1β expression in a rat model of lipopolysaccharide-induced neuroinflammation. The effect was analyzed using Western blot analysis, quantitative real-time polymerase chain reaction and enzymic histone deacetylase and histone acetyltransferase assays. Statistical analysis was performed using one-way analysis of variance, parametric or nonparametric when appropriate, followed by Tukey's or Dunn's post-hoc test, respectively. Results We found that long-term acetate supplementation increased the proportion of brain histone H3 acetylated at lysine 9 (H3K9, histone H4 acetylated at lysine 8 and histone H4 acetylated at lysine 16. However, unlike a single dose of glyceryl triacetate, long-term treatment increased histone acetyltransferase activity and had no effect on histone deacetylase activity, with variable effects on brain histone deacetylase class I and II expression. In agreement with this hypothesis, neuroinflammation reduced the proportion of brain H3K9 acetylation by 50%, which was effectively reversed with acetate supplementation. Further, in rats subjected to lipopolysaccharide-induced neuroinflammation, the pro-inflammatory cytokine interleukin-1β protein

  13. Swelling of acetylated wood in organic liquids

    CERN Document Server

    Obataya, E; Obataya, Eiichi; Gril, Joseph

    2005-01-01

    To investigate the affinity of acetylated wood for organic liquids, Yezo spruce wood specimens were acetylated with acetic anhydride, and their swelling in various liquids were compared to those of untreated specimens. The acetylated wood was rapidly and remarkably swollen in aprotic organic liquids such as benzene and toluene in which the untreated wood was swollen only slightly and/or very slowly. On the other hand, the swelling of wood in water, ethylene glycol and alcohols remained unchanged or decreased by the acetylation. Consequently the maximum volume of wood swollen in organic liquids was always larger than that in water. The effect of acetylation on the maximum swollen volume of wood was greater in liquids having smaller solubility parameters. The easier penetration of aprotic organic liquids into the acetylated wood was considered to be due to the scission of hydrogen bonds among the amorphous wood constituents by the substitution of hydroxyl groups with hydrophobic acetyl groups.

  14. Flow properties of acetylated chickpea protein dispersions.

    Science.gov (United States)

    Liu, Li H; Hung, Tran V

    2010-06-01

    Chickpea protein concentrate was acetylated with acetic anhydride at 5 levels. Acetylated chickpea protein (ACP) dispersions at 3 levels (6%, 45%, and 49%) were chosen for this flow property study. Effects of protein concentration, temperature, concentrations of salt addition and particularly, degree of acetylation on these properties were examined. Compared with native chickpea proteins, the ACP dispersions exhibited a strong shear thinning behavior. Within measured temperature range (15 to 55 degrees C), the apparent viscosities of native chickpea protein dispersions were temperature independent; those of ACP dispersions were thermally affected. The flow index (n), consistency coefficient (m), apparent yield stress, and apparent viscosities of ACP dispersions increased progressively up to 45% acetylation but decreased at 49% acetylation level. Conformational studies by gel filtration suggested that chickpea proteins were associated or polymerized at up to 45% acetylation but the associated subunits gradually dissociated to smaller units at higher levels (49%) of acetylation.

  15. Rewiring AMPK and mitochondrial retrograde signaling for metabolic control of aging and histone acetylation in respiratory-defective cells.

    Science.gov (United States)

    Friis, R Magnus N; Glaves, John Paul; Huan, Tao; Li, Liang; Sykes, Brian D; Schultz, Michael C

    2014-04-24

    Abnormal respiratory metabolism plays a role in numerous human disorders. We find that regulation of overall histone acetylation is perturbed in respiratory-incompetent (ρ(0)) yeast. Because histone acetylation is highly sensitive to acetyl-coenzyme A (acetyl-CoA) availability, we sought interventions that suppress this ρ(0) phenotype through reprogramming metabolism. Nutritional intervention studies led to the discovery that genetic coactivation of the mitochondrion-to-nucleus retrograde (RTG) response and the AMPK (Snf1) pathway prevents abnormal histone deacetylation in ρ(0) cells. Metabolic profiling of signaling mutants uncovered links between chromatin-dependent phenotypes of ρ(0) cells and metabolism of ATP, acetyl-CoA, glutathione, branched-chain amino acids, and the storage carbohydrate trehalose. Importantly, RTG/AMPK activation reprograms energy metabolism to increase the supply of acetyl-CoA to lysine acetyltransferases and extend the chronological lifespan of ρ(0) cells. Our results strengthen the framework for rational design of nutrient supplementation schemes and drug-discovery initiatives aimed at mimicking the therapeutic benefits of dietary interventions.

  16. Rewiring AMPK and Mitochondrial Retrograde Signaling for Metabolic Control of Aging and Histone Acetylation in Respiratory-Defective Cells

    Directory of Open Access Journals (Sweden)

    R. Magnus N. Friis

    2014-04-01

    Full Text Available Abnormal respiratory metabolism plays a role in numerous human disorders. We find that regulation of overall histone acetylation is perturbed in respiratory-incompetent (ρ0 yeast. Because histone acetylation is highly sensitive to acetyl-coenzyme A (acetyl-CoA availability, we sought interventions that suppress this ρ0 phenotype through reprogramming metabolism. Nutritional intervention studies led to the discovery that genetic coactivation of the mitochondrion-to-nucleus retrograde (RTG response and the AMPK (Snf1 pathway prevents abnormal histone deacetylation in ρ0 cells. Metabolic profiling of signaling mutants uncovered links between chromatin-dependent phenotypes of ρ0 cells and metabolism of ATP, acetyl-CoA, glutathione, branched-chain amino acids, and the storage carbohydrate trehalose. Importantly, RTG/AMPK activation reprograms energy metabolism to increase the supply of acetyl-CoA to lysine acetyltransferases and extend the chronological lifespan of ρ0 cells. Our results strengthen the framework for rational design of nutrient supplementation schemes and drug-discovery initiatives aimed at mimicking the therapeutic benefits of dietary interventions.

  17. Long-term exposure to a ‘safe’ dose of bisphenol A reduced protein acetylation in adult rat testes

    Science.gov (United States)

    Chen, Zhuo; Zuo, Xuezhi; He, Dongliang; Ding, Shibin; Xu, Fangyi; Yang, Huiqin; Jin, Xin; Fan, Ying; Ying, Li; Tian, Chong; Ying, Chenjiang

    2017-01-01

    Bisphenol A (BPA), a typical environmental endocrine-disrupting chemical, induces epigenetic inheritance. Whether histone acetylation plays a role in these effects of BPA is largely unknown. Here, we investigated histone acetylation in male rats after long-term exposure to a ‘safe’ dose of BPA. Twenty adult male rats received either BPA (50 μg/kg·bw/day) or a vehicle diet for 35 weeks. Decreased protein lysine-acetylation levels at approximately ~17 kDa and ~25 kDa, as well as decreased histone acetylation of H3K9, H3K27 and H4K12, were detected by Western blot analysis of testes from the treated rats compared with controls. Additionally, increased protein expression of deacetylase Sirt1 and reduced binding of Sirt1, together with increased binding of estrogen receptor β (ERβ) to caveolin-1 (Cav-1), a structural protein component of caveolar membranes, were detected in treated rats compared with controls. Moreover, decreased acetylation of Cav-1 was observed in the treated rats for the first time. Our study showed that long-term exposure to a ‘safe’ dose of BPA reduces histone acetylation in the male reproductive system, which may be related to the phenotypic paternal-to-offspring transmission observed in our previous study. The evidence also suggested that these epigenetic effects may be meditated by Sirt1 via competition with ERβ for binding to Cav-1.

  18. Long-term exposure to a ‘safe’ dose of bisphenol A reduced protein acetylation in adult rat testes

    Science.gov (United States)

    Chen, Zhuo; Zuo, Xuezhi; He, Dongliang; Ding, Shibin; Xu, Fangyi; Yang, Huiqin; Jin, Xin; Fan, Ying; Ying, Li; Tian, Chong; Ying, Chenjiang

    2017-01-01

    Bisphenol A (BPA), a typical environmental endocrine-disrupting chemical, induces epigenetic inheritance. Whether histone acetylation plays a role in these effects of BPA is largely unknown. Here, we investigated histone acetylation in male rats after long-term exposure to a ‘safe’ dose of BPA. Twenty adult male rats received either BPA (50 μg/kg·bw/day) or a vehicle diet for 35 weeks. Decreased protein lysine-acetylation levels at approximately ~17 kDa and ~25 kDa, as well as decreased histone acetylation of H3K9, H3K27 and H4K12, were detected by Western blot analysis of testes from the treated rats compared with controls. Additionally, increased protein expression of deacetylase Sirt1 and reduced binding of Sirt1, together with increased binding of estrogen receptor β (ERβ) to caveolin-1 (Cav-1), a structural protein component of caveolar membranes, were detected in treated rats compared with controls. Moreover, decreased acetylation of Cav-1 was observed in the treated rats for the first time. Our study showed that long-term exposure to a ‘safe’ dose of BPA reduces histone acetylation in the male reproductive system, which may be related to the phenotypic paternal-to-offspring transmission observed in our previous study. The evidence also suggested that these epigenetic effects may be meditated by Sirt1 via competition with ERβ for binding to Cav-1. PMID:28067316

  19. Lysine-Rich Proteins in High-Lysine Hordeum Vulgare Grain

    DEFF Research Database (Denmark)

    Ingversen, J.; Køie, B.

    1973-01-01

    The salt-soluble proteins in barley grain selected for high-lysine content (Hiproly, CI 7115 and the mutants 29 and 86) and of a control (Carlsberg II) with normal lysine content, contain identical major proteins as determined by MW and electrophoretic mobility. The concentration of a protein group...

  20. Histone lysine methylation: critical regulator of memory and behavior.

    Science.gov (United States)

    Jarome, Timothy J; Lubin, Farah D

    2013-01-01

    Histone lysine methylation is a well-established transcriptional mechanism for the regulation of gene expression changes in eukaryotic cells and is now believed to function in neurons of the central nervous system to mediate the process of memory formation and behavior. In mature neurons, methylation of histone proteins can serve to both activate and repress gene transcription. This is in stark contrast to other epigenetic modifications, including histone acetylation and DNA methylation, which have largely been associated with one transcriptional state in the brain. In this review, we discuss the evidence for histone methylation mechanisms in the coordination of complex cognitive processes such as long-term memory formation and storage. In addition, we address the current literature highlighting the role of histone methylation in intellectual disability, addiction, schizophrenia, autism, depression, and neurodegeneration. Further, we discuss histone methylation within the context of other epigenetic modifications and the potential advantages of exploring this newly identified mechanism of cognition, emphasizing the possibility that this molecular process may provide an alternative locus for intervention in long-term psychopathologies that cannot be clearly linked to genes or environment alone.

  1. Distribution of the O-acetyl groups and β-galactofuranose units in galactoxylomannans of the opportunistic fungus Cryptococcus neoformans.

    Science.gov (United States)

    Previato, Jose O; Vinogradov, Evgeny; Maes, Emmanuel; Fonseca, Leonardo M; Guerardel, Yann; Oliveira, Priscila A V; Mendonça-Previato, Lucia

    2016-12-16

    Galactoxylomannans (GalXMs) are a mixture of neutral and acidic capsular polysaccharides produced by the opportunistic fungus Cryptococcus neoformans that exhibit potent suppressive effects on the host immune system. Previous studies describing the chemical structure of C. neoformans GalXMs have reported species without O-acetyl substituents. Herein we describe that C. neoformans grown in capsule-inducing medium produces highly O-acetylated GalXMs. The location of the O-acetyl groups was determined by nuclear magnetic resonance (NMR) spectroscopy. In the neutral GalXM (NGalXM), 80% of 3-linked mannose (α-Manp) residues present in side chains are acetylated at the O-2 position. In the acidic GalXM also termed glucuronoxylomannogalactan (GXMGal), 85% of the 3-linked α-Manp residues are acetylated either in the O-2 (75%) or in the O-6 (25%) position, but O-acetyl groups are not present at both positions simultaneously. In addition, NMR spectroscopy and methylation analysis showed that β-galactofuranose (β-Galf) units are linked to O-2 and O-3 positions of nonbranched α-galactopyranose (α-Galp) units present in the GalXMs backbone chain. These findings highlight new structural features of C. neoformans GalXMs. Among these features, the high degree of O-acetylation is of particular interest, since O-acetyl group-containing polysaccharides are known to possess a range of immunobiological activities.

  2. Structural characterization of the acetylated heteroxylan from the natural hybrid Paulownia elongata/Paulownia fortunei.

    Science.gov (United States)

    Gonçalves, Virgínia M F; Evtuguin, Dmitry V; Domingues, M Rosário M

    2008-02-04

    The heteroxylan from the hybrid Paulownia elongata/Paulownia fortunei is an O-acetyl-(4-O-methylglucurono)xylan with an acetylation degree (DS) of 0.59 and a molecular weight (M(w)) of 29 kDa. The heteroxylan backbone is composed by (1-->4)-linked beta-d-xylopyranosyl units (Xylp) partially ramified with terminal (1-->2)-linked 4-O-methyl-alpha-D-glucuronosyl (MeGlcpA) and a small proportion of alpha-D-glucuronosyl (GlcpA) residues in a molar ratio of Xylp:(MeGlcpA+GlcpA) of 20:1. Roughly half of the beta-D-xylopyranosyl units in the backbone are acetylated: 3-O-acetylated (22 mol %), 2-O-acetylated (23 mol %) or 2,3-di-O-acetylated (7 mol %). ESI-MS and MALDI-MS studies of partially hydrolyzed heteroxylan revealed a random distribution of O-Ac and MeGlcpA within the backbone. However, the frequency of substitution with O-Ac along the backbone is not uniform and the molecular regions that did not contain MeGlcpA substituents possessed an acetylation degree significantly lower than the average DS of the xylan.

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  4. Acetylation Is Crucial for p53-Mediated Ferroptosis and Tumor Suppression

    Directory of Open Access Journals (Sweden)

    Shang-Jui Wang

    2016-10-01

    Full Text Available Although previous studies indicate that loss of p53-mediated cell cycle arrest, apoptosis, and senescence does not completely abrogate its tumor suppression function, it is unclear how the remaining activities of p53 are regulated. Here, we have identified an acetylation site at lysine K98 in mouse p53 (or K101 for human p53. Whereas the loss of K98 acetylation (p53K98R alone has very modest effects on p53-mediated transactivation, simultaneous mutations at all four acetylation sites (p534KR: K98R+ 3KR[K117R+K161R+K162R] completely abolish its ability to regulate metabolic targets, such as TIGAR and SLC7A11. Notably, in contrast to p533KR, p534KR is severely defective in suppressing tumor growth in mouse xenograft models. Moreover, p534KR is still capable of inducing the p53-Mdm2 feedback loop, but p53-dependent ferroptotic responses are markedly abrogated. Together, these data indicate the critical role of p53 acetylation in ferroptotic responses and its remaining tumor suppression activity.

  5. Acetylation of VGLL4 Regulates Hippo-YAP Signaling and Postnatal Cardiac Growth.

    Science.gov (United States)

    Lin, Zhiqiang; Guo, Haidong; Cao, Yuan; Zohrabian, Sylvia; Zhou, Pingzhu; Ma, Qing; VanDusen, Nathan; Guo, Yuxuan; Zhang, Jin; Stevens, Sean M; Liang, Feng; Quan, Qimin; van Gorp, Pim R; Li, Amy; Dos Remedios, Cristobal; He, Aibin; Bezzerides, Vassilios J; Pu, William T

    2016-11-21

    Binding of the transcriptional co-activator YAP with the transcription factor TEAD stimulates growth of the heart and other organs. YAP overexpression potently stimulates fetal cardiomyocyte (CM) proliferation, but YAP's mitogenic potency declines postnatally. While investigating factors that limit YAP's postnatal mitogenic activity, we found that the CM-enriched TEAD1 binding protein VGLL4 inhibits CM proliferation by inhibiting TEAD1-YAP interaction and by targeting TEAD1 for degradation. Importantly, VGLL4 acetylation at lysine 225 negatively regulated its binding to TEAD1. This developmentally regulated acetylation event critically governs postnatal heart growth, since overexpression of an acetylation-refractory VGLL4 mutant enhanced TEAD1 degradation, limited neonatal CM proliferation, and caused CM necrosis. Our study defines an acetylation-mediated, VGLL4-dependent switch that regulates TEAD stability and YAP-TEAD activity. These insights may improve targeted modulation of TEAD-YAP activity in applications from cardiac regeneration to cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Snorkeling of lysine side chains in transmembrane helices: how easy can it get?

    Science.gov (United States)

    Strandberg, Erik; Killian, J Antoinette

    2003-06-05

    Transmembrane segments of proteins are often flanked by lysine residues. The side chains of these residues may snorkel, i.e. they may bury themselves with their aliphatic part in the hydrophobic region of the lipid bilayer, while positioning the charged amino group in the more polar interface. Here we estimate the free energy cost of snorkeling from thermodynamical calculations based on studies with synthetic transmembrane peptides [Strandberg et al. (2002) Biochemistry 41, 7190-7198]. The value is estimated to be between 0.07 and 0.7 kcal mol(-1) for a lysine side chain. This very low value indicates that snorkeling may be a common process, which should be taken into consideration both in experimental and in theoretical studies on protein-lipid interactions.

  7. NMR determination of lysine pKa values in the Pol lambda lyase domain: mechanistic implications.

    Science.gov (United States)

    Gao, Guanghua; DeRose, Eugene F; Kirby, Thomas W; London, Robert E

    2006-02-14

    The base excision repair (BER) process requires removal of an abasic deoxyribose-5-phosphate group, a catalytic activity that has been demonstrated for the N-terminal 8 kDa domain of DNA polymerase beta (Pol beta), and for the homologous domain of DNA polymerase lambda (Pol lambda). Previous studies have demonstrated that this activity results from formation of a Schiff base adduct of the abasic deoxyribose C-1' with a lysine residue (K312 in the case of Pol lambda), followed by a beta-elimination reaction. To better understand the underlying chemistry, we have determined pKa values for the lysine residues in the Pol lambda lyase domain labeled with [epsilon-13C]lysine. At neutral pH, the H(epsilon) protons on 3 of the 10 lysine residues in this domain, K287, K291, and K312, exhibit chemical shift inequivalence that results from immobilization of the lysyl side chains. For K287 and K291, this results from the K287-E261 and K291-E298 salt bridge interactions, while for K312, immobilization apparently results from steric and hydrogen-bonding interactions that constrain the position of the lysine side chain. The pKa value of K312 is depressed to 9.58, a value indicating that at physiological pH K312 will exist predominantly in the protonated form. Titration of the domain with hairpin DNA containing a 5'-tetrahydrofuran terminus to model the abasic site produced shifts of the labeled lysine resonances that were in fast exchange but appeared to be complete at a stoichiometry of approximately 1:1.3, consistent with a dissociation constant of approximately 1 microM. The epsilon-proton shifts of K273 were the most sensitive to the addition of the DNA, apparently due to changes in the relative orientation between K273 and W274 in the DNA complex. The average pKa values increased by 0.55, consistent with the formation of some DNA-lysine salt bridges and with the general pH increase expected to result from a reduction in the net positive charge of the complex. A general

  8. Lysine methylation regulates the pRb tumour suppressor protein.

    Science.gov (United States)

    Munro, S; Khaire, N; Inche, A; Carr, S; La Thangue, N B

    2010-04-22

    The pRb tumour suppressor protein has a central role in coordinating early cell cycle progression. An important level of control imposed on pRb occurs through post-translational modification, for example, phosphorylation. We describe here a new level of regulation on pRb, mediated through the targeted methylation of lysine residues, by the methyltransferase Set7/9. Set7/9 methylates the C-terminal region of pRb, both in vitro and in cells, and methylated pRb interacts with heterochromatin protein HP1. pRb methylation is required for pRb-dependent cell cycle arrest and transcriptional repression, as well as pRb-dependent differentiation. Our results indicate that methylation can influence the properties of pRb, and raise the interesting possibility that methylation modulates pRb tumour suppressor activity.

  9. A mechanism-based potent sirtuin inhibitor containing Nε-thiocarbamoyl-lysine (TuAcK)

    OpenAIRE

    2011-01-01

    In the current study, we have identified Nε-thiocarbamoyl-lysine (TuAcK) as a general sirtuin inhibitory warhead which was shown to be able to confer potent sirtuin inhibition. This inhibition was also shown to be mechanism-based in that the TuAck residue was able to be processed by a sirtuin enzyme with the formation of a stalled S-alkylamidate intermediate.

  10. Occurrence of naturally acetylated lignin units.

    Science.gov (United States)

    Del Río, José C; Marques, Gisela; Rencoret, Jorge; Martínez, Angel T; Gutiérrez, Ana

    2007-07-11

    This work examines the occurrence of native acetylated lignin in a large set of vascular plants, including both angiosperms and gymnosperms, by a modification of the so-called Derivatization Followed by Reductive Cleavage (DFRC) method. Acetylated lignin units were found in the milled wood lignins of all angiosperms selected for this study, including mono- and eudicotyledons, but were absent in the gymnosperms analyzed. In some plants (e.g., abaca, sisal, kenaf, or hornbeam), lignin acetylation occurred at a very high extent, exceeding 45% of the uncondensed (alkyl-aryl ether linked) syringyl lignin units. Acetylation was observed exclusively at the gamma-carbon of the lignin side chain and predominantly on syringyl units, although a predominance of acetylated guaiacyl over syringyl units was observed in some plants. In all cases, acetylation appears to occur at the monomer stage, and sinapyl and coniferyl acetates seem to behave as real lignin monomers participating in lignification.

  11. SET7/9 Catalytic Mutants Reveal the Role of Active Site Water Molecules in Lysine Multiple Methylation

    Energy Technology Data Exchange (ETDEWEB)

    Del Rizzo, Paul A.; Couture, Jean-François; Dirk, Lynnette M.A.; Strunk, Bethany S.; Roiko, Marijo S.; Brunzelle, Joseph S.; Houtz, Robert L.; Trievel, Raymond C. (Michigan); (NWU); (Kentucky)

    2010-11-15

    SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine {epsilon}-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di- and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine {epsilon}-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated {epsilon}-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes.

  12. Deregulation of histone lysine methyltransferases contributes to oncogenic transformation of human bronchoepithelial cells

    Directory of Open Access Journals (Sweden)

    Yoda Satoshi

    2008-11-01

    Full Text Available Abstract Background Alterations in the processing of the genetic information in carcinogenesis result from stable genetic mutations or epigenetic modifications. It is becoming clear that nucleosomal histones are central to proper gene expression and that aberrant DNA methylation of genes and histone methylation plays important roles in tumor progression. To date, several histone lysine methyltransferases (HKMTs have been identified and histone lysine methylation is now considered to be a critical regulator of transcription. However, still relatively little is known about the role of HKMTs in tumorigenesis. Results We observed differential HKMT expression in a lung cancer model in which normal human bronchial epithelial (NHBE cells expressing telomerase, SV40 large T antigen, and Ras were immortal, formed colonies in soft agar, and expressed specific HKMTs for H3 lysine 9 and 27 residues but not for H3 lysine 4 residue. Modifications in the H3 tails affect the binding of proteins to the histone tails and regulate protein function and the position of lysine methylation marks a gene to be either activated or repressed. In the present study, suppression by siRNA of HKMTs (EZH2, G9A, SETDB1 and SUV39H1 that are over-expressed in immortalized and transformed cells lead to reduced cell proliferation and much less anchorage-independent colony growth. We also found that the suppression of H3-K9, G9A and SUV39H1 induced apoptosis and the suppression of H3-K27, EZH2 caused G1 arrest. Conclusion Our results indicate the potential of these HKMTs in addition to the other targets for epigenetics such as DNMTs and HDACs to be interesting therapeutic targets.

  13. Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions

    DEFF Research Database (Denmark)

    Weinert, Brian T; Moustafa, Tarek; Iesmantavicius, Vytautas

    2015-01-01

    Acetylation is frequently detected on mitochondrial enzymes, and the sirtuin deacetylase SIRT3 is thought to regulate metabolism by deacetylating mitochondrial proteins. However, the stoichiometry of acetylation has not been studied and is important for understanding whether SIRT3 regulates...... or suppresses acetylation. Using quantitative mass spectrometry, we measured acetylation stoichiometry in mouse liver tissue and found that SIRT3 suppressed acetylation to a very low stoichiometry at its target sites. By examining acetylation changes in the liver, heart, brain, and brown adipose tissue...... of fasted mice, we found that SIRT3-targeted sites were mostly unaffected by fasting, a dietary manipulation that is thought to regulate metabolism through SIRT3-dependent deacetylation. Globally increased mitochondrial acetylation in fasted liver tissue, higher stoichiometry at mitochondrial acetylation...

  14. Acetylation of woody lignocellulose: significance and regulation

    Directory of Open Access Journals (Sweden)

    Prashant Mohan-Anupama Pawar

    2013-05-01

    Full Text Available Non-cellulosic cell wall polysaccharides constitute approximately one quarter of usable biomass for human exploitation. In contrast to cellulose, these components are usually substituted by O-acetyl groups, which affect their properties and interactions with other polymers, thus affecting their solubility and extractability. However, details of these interactions are still largely obscure. Moreover, polysaccharide hydrolysis to constituent monosaccharides, is hampered by the presence of O-acetyl groups, necessitating either enzymatic (esterase or chemical de-acetylation, increasing the costs and chemical consumption. Reduction of polysaccharide acetyl content in planta is a way to modify lignocellulose towards improved saccharification. In this review we: 1 summarize literature on lignocellulose acetylation in different tree species, 2 present data and current hypotheses concerning the role of O-acetylation in determining woody lignocellulose properties, 3 describe plant proteins involved in lignocellulose O-acetylation, 4 give examples of microbial enzymes capable to de-acetylate lignocellulose, and 5 discuss prospects for exploiting these enzymes in planta to modify xylan acetylation.

  15. Acetylation of Chinese bamboo flour and thermoplasticity

    Institute of Scientific and Technical Information of China (English)

    LI Xue-fang; CHEN Qin-hui; LIN Jin-huo; ZHUO Dong-xian; WU Xiu-ling

    2008-01-01

    Chinese bamboo flour was chemically modified by acetylation with acetic anhydride by using trichloroacetic acid as an activation agent and the optimized condition for acetylation of bamboo flour was determined as the trichloroacetic acid amount 6.0 g per 1.5-g bamboo flour, ultrasosonication duration 40 min and the reaction time 1 h at 65℃. The composition, microstructure and thermal behavior of acetylated bamboo flour were preliminarily characterized by FT-IR, DSC and SEM etc. The acetylated bamboo flour can be molded into sheets at 130℃ and 10 MPa, indicating the modified bamboo flour possesses thermalplastic performance.

  16. Acetylation regulates Jun protein turnover in Drosophila.

    Science.gov (United States)

    Zhang, Daoyong; Suganuma, Tamaki; Workman, Jerry L

    2013-11-01

    C-Jun is a major transcription factor belonging to the activating protein 1 (AP-1) family. Phosphorylation has been shown to be critical for c-Jun activation and stability. Here, we report that Jra, the Drosophila Jun protein, is acetylated in vivo. We demonstrate that the acetylation of Jra leads to its rapid degradation in response to osmotic stress. Intriguingly, we also found that Jra phosphorylation antagonized its acetylation, indicating the opposite roles of acetylation and phosphorylation in Jra degradation process under osmotic stress. Our results provide new insights into how c-Jun proteins are precisely regulated by the interplay of different posttranslational modifications.

  17. A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein.

    Directory of Open Access Journals (Sweden)

    Sriram Sokalingam

    Full Text Available Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering.

  18. Efficient Production of Enantiopure d-Lysine from l-Lysine by a Two-Enzyme Cascade System

    Directory of Open Access Journals (Sweden)

    Xin Wang

    2016-10-01

    Full Text Available The microbial production of d-lysine has been of great interest as a medicinal raw material. Here, a two-step process for d-lysine production from l-lysine by the successive microbial racemization and asymmetric degradation with lysine racemase and decarboxylase was developed. The whole-cell activities of engineered Escherichia coli expressing racemases from the strains Proteus mirabilis (LYR and Lactobacillus paracasei (AAR were first investigated comparatively. When the strain BL21-LYR with higher racemization activity was employed, l-lysine was rapidly racemized to give dl-lysine, and the d-lysine yield was approximately 48% after 0.5 h. Next, l-lysine was selectively catabolized to generate cadaverine by lysine decarboxylase. The comparative analysis of the decarboxylation activities of resting whole cells, permeabilized cells, and crude enzyme revealed that the crude enzyme was the best biocatalyst for enantiopure d-lysine production. The reaction temperature, pH, metal ion additive, and pyridoxal 5′-phosphate content of this two-step production process were subsequently optimized. Under optimal conditions, 750.7 mmol/L d-lysine was finally obtained from 1710 mmol/L l-lysine after 1 h of racemization reaction and 0.5 h of decarboxylation reaction. d-lysine yield could reach 48.8% with enantiomeric excess (ee ≥ 99%.

  19. Specific synthesis of neurostatin and gangliosides O-acetylated in the outer sialic acids using a sialate transferase.

    Directory of Open Access Journals (Sweden)

    Lorenzo Romero-Ramírez

    Full Text Available Gangliosides are sialic acid containing glycosphingolipids, commonly found on the outer leaflet of the plasma membrane. O-acetylation of sialic acid hydroxyl groups is one of the most common modifications in gangliosides. Studies on the biological activity of O-acetylated gangliosides have been limited by their scarcity in nature. This comparatively small change in ganglioside structure causes major changes in their physiological properties. When the ganglioside GD1b was O-acetylated in the outer sialic acid, it became the potent inhibitor of astroblast and astrocytoma proliferation called Neurostatin. Although various chemical and enzymatic methods to O-acetylate commercial gangliosides have been described, O-acetylation was nonspecific and produced many side-products that reduced the yield. An enzyme with O-acetyltransferase activity (SOAT has been previously cloned from the bacteria Campylobacter jejuni. This enzyme catalyzed the acetylation of oligosaccharide-bound sialic acid, with high specificity for terminal alpha-2,8-linked residues. Using this enzyme and commercial gangliosides as starting material, we have specifically O-acetylated the gangliosides' outer sialic acids, to produce the corresponding gangliosides specifically O-acetylated in the sialic acid bound in alpha-2,3 and alpha-2,8 residues. We demonstrate here that O-acetylation occurred specifically in the C-9 position of the sialic acid. In summary, we present a new method of specific O-acetylation of ganglioside sialic acids that permits the large scale preparation of these modified glycosphingolipids, facilitating both, the study of their mechanism of antitumoral action and their use as therapeutic drugs for treating glioblastoma multiform (GBM patients.

  20. Histone H4 N-terminal acetylation in Kasumi-1 cells treated with depsipeptide determined by acetic acid-urea polyacrylamide gel electrophoresis, amino acid coded mass tagging, and mass spectrometry.

    Science.gov (United States)

    Zhang, Liwen; Su, Xiaodan; Liu, Shujun; Knapp, Amy R; Parthun, Mark R; Marcucci, Guido; Freitas, Michael A

    2007-01-01

    Disrupted patterns of acetylation and deacetylation of core histones play an important role in silencing transcription of hematopoietic important genes in acute myeloid leukemia (AML). A thorough investigation of these mechanisms and the response to pharmacologic modifiers will provide a better understanding of the role of histone acetylation in leukemogenesis. We describe here an analytical approach that combines acid urea polyacrylamide gel electrophoresis (AU-PAGE), amino acid coded mass tagging (AACM), and mass spectrometry (MS) for the investigation of histone acetylation patterns. The combined approach was used to follow the dynamics of H4 acetylation in Kasumi-1 cells harboring the fusion gene AML1/ETO shown to aberrantly recruit histone deacetylases (HDACs). The histones in Kasumi-1 cells were labeled by growing the cells in media in which lysine was replaced with stable isotope-labeled lysine (Lys-D4). Labeled and unlabeled cells were treated with depsipeptide and analyzed at different time points (0, 4, 8, 12, 24, and 48 h). The cells were mixed, the histone was extracted, and acetylated H4 isoforms were separated using AU-PAGE before in-gel trypsin digestion. The digests were analyzed by MALDI-TOF MS. Peptides were identified by mass and isotope pattern. LC-MS/MS of Arg-C digests were also performed to verify the acetylation pattern for H4. The major pattern of acetylation was determined as follows: initial acetylation at K16, followed by acetylation at K12, and finally acetylation of either K8 and/or K5.

  1. Histone H4 N-Terminal Acetylation in Kasumi-1 Cells Treated with Depsipeptide Determined by Acetic Acid–Urea olyacrylamide Gel Electrophoresis, Amino Acid Coded Mass Tagging, and Mass Spectrometry

    Science.gov (United States)

    Zhang, Liwen; Su, Xiaodan; Liu, Shujun; Knapp, Amy R.; Parthun, Mark R.; Marcucci, Guido; Freitas, Michael A.

    2008-01-01

    Disrupted patterns of acetylation and deacetylation of core histones play an important role in silencing transcription of hematopoietic important genes in acute myeloid leukemia (AML). A thorough investigation of these mechanisms and the response to pharmacologic modifiers will provide a better understanding of the role of histone acetylation in leukemogenesis. We describe here an analytical approach that combines acid urea polyacrylamide gel electrophoresis (AU-PAGE), amino acid coded mass tagging (AACM), and mass spectrometry (MS) for the investigation of histone acetylation patterns. The combined approach was used to follow the dynamics of H4 acetylation in Kasumi-1 cells harboring the fusion gene AML1/ETO shown to aberrantly recruit histone deacetylases (HDACs). The histones in Kasumi-1 cells were labeled by growing the cells in media in which lysine was replaced with stable isotope-labeled lysine (Lys-D4). Labeled and unlabeled cells were treated with depsipeptide and analyzed at different time points (0, 4, 8, 12, 24, and 48 h). The cells were mixed, the histone was extracted, and acetylated H4 isoforms were separated using AU-PAGE before in-gel trypsin digestion. The digests were analyzed by MALDI-TOF MS. Peptides were identified by mass and isotope pattern. LC–MS/MS of Arg-C digests were also performed to verify the acetylation pattern for H4. The major pattern of acetylation was determined as follows: initial acetylation at K16, followed by acetylation at K12, and finally acetylation of either K8 and/or K5. PMID:17203951

  2. Radioactive Lysine in Protein Metabolism Studies

    Science.gov (United States)

    Miller, L. L.; Bale, W. F.; Yuile, C. L.; Masters, R. E.; Tishkoff, G. H.; Whipple,, G. H.

    1950-01-09

    Studies of incorporation of DL-lysine in various body proteins of the dog; the time course of labeled blood proteins; and apparent rate of disappearance of labeled plasma proteins for comparison of behavior of the plasma albumin and globulin fractions; shows more rapid turn over of globulin fraction.

  3. Lysine and arginine requirements of Salminus brasiliensis

    Directory of Open Access Journals (Sweden)

    Jony Koji Dairiki

    2013-08-01

    Full Text Available The objective of this work was to determine the dietary lysine (DL and dietary arginine (DA requirements of dourado (Salminus brasiliensis, through dose-response trials using the amino acid profiles of whole carcasses as a reference. Two experiments were carried out in a completely randomized design (n=4. In the first experiment, groups of 12 feed-conditioned dourado juveniles (11.4±0.2 g were stocked in 60 L cages placed in 300 L plastic indoor tanks in a closed circulation system. Fish were fed for 60 days on diets containing 1.0, 1.5, 2.0, 2.5, 3.0, or 3.5 % dietary lysine. In the second experiment, dourado juveniles (27.0±0.8 g were fed for 60 days on semipurified diets containing arginine at 1.0, 1.5, 2.0, 2.5 or 3.0%, in similar conditions to those of the first experiment. Optimal DL requirements, as determined by broken-line analysis method for final weight, weight gain and specific growth rate, were 2.15% DL or 5% lysine in dietary protein, and 1.48% DA or 3.43% arginine in dietary protein. The best feed conversion ratio is attained with 2.5% DL or 5.8% lysine in dietary protein and 1.4% DA or 3.25% arginine in dietary protein.

  4. Lysine kinetics in preterm infants: the importance of enteral feeding

    NARCIS (Netherlands)

    S.R.D. van der Schoor (Sophie); P.J. Reeds; F. Stellaard; J.L.D. Wattimena (Josias); P.J.J. Sauer (Pieter); H.A. Büller (Hans); J.B. van Goudoever (Hans)

    2004-01-01

    textabstractINTRODUCTION: Lysine is the first limiting essential amino acid in the diet of newborns. First pass metabolism by the intestine of dietary lysine has a direct effect on systemic availability. We investigated whether first pass lysine metabolism in the intestine is high

  5. Lysine kinetics in preterm infants : the importance of enteral feeding

    NARCIS (Netherlands)

    van der Schoor, SRD; Reeds, PJ; Stellaard, F; Wattimena, JDL; Sauer, PJJ; Buller, HA; van Goudoever, JB

    2004-01-01

    Introduction: Lysine is the first limiting essential amino acid in the diet of newborns. First pass metabolism by the intestine of dietary lysine has a direct effect on systemic availability. We investigated whether first pass lysine metabolism in the intestine is high in preterm infants, particular

  6. Inhibition of SIRT1 Catalytic Activity Increases p53 Acetylation but Does Not Alter Cell Survival following DNA Damage

    Science.gov (United States)

    Solomon, Jonathan M.; Pasupuleti, Rao; Xu, Lei; McDonagh, Thomas; Curtis, Rory; DiStefano, Peter S.; Huber, L. Julie

    2006-01-01

    Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. In this report, we used EX-527, a novel, potent, and specific small-molecule inhibitor of SIRT1 catalytic activity to examine the role of SIRT1 in p53 acetylation and cell survival after DNA damage. Treatment with EX-527 dramatically increased acetylation at lysine 382 of p53 after different types of DNA damage in primary human mammary epithelial cells and several cell lines. Significantly, inhibition of SIRT1 catalytic activity by EX-527 had no effect on cell growth, viability, or p53-controlled gene expression in cells treated with etoposide. Acetyl-p53 was also increased by the histone deacetylase (HDAC) class I/II inhibitor trichostatin A (TSA). EX-527 and TSA acted synergistically to increase acetyl-p53 levels, confirming that p53 acetylation is regulated by both SIRT1 and HDACs. While TSA alone reduced cell survival after DNA damage, the combination of EX-527 and TSA had no further effect on cell viability and growth. These results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells. PMID:16354677

  7. Investigation of acetyl migrations in furanosides

    Directory of Open Access Journals (Sweden)

    Migaud ME

    2006-07-01

    Full Text Available Abstract Standard reaction conditions for the desilylation of acetylated furanoside (riboside, arabinoside and xyloside derivatives facilitate acyl migration. Conditions which favour intramolecular and intermolecular mechanisms have been identified with intermolecular transesterifications taking place under mild basic conditions when intramolecular orthoester formations are disfavoured. In acetyl ribosides, acyl migration could be prevented when desilylation was catalysed by cerium ammonium nitrate.

  8. Analysis of acetylated wood by electron microscopy

    NARCIS (Netherlands)

    Sander, C.; Beckers, E.P.J.; Militz, H.; Veenendaal, van W.

    2003-01-01

    The properties of acetylated solid wood were investigated earlier, in particular the anti-shrink efficiency and the resistance against decay. This study focuses on the possible changes and damage to the wood structure due to an acetylation process leading to weight per cent gains of up to 20%. Elect

  9. The Acetylation of Starch by Reactive Extrusion

    NARCIS (Netherlands)

    Graaf, Robbert A. de; Broekroelofs, Annet; Janssen, Léon P.B.M.

    1998-01-01

    Potato starch has been acetylated in a counter rotating twin screw extruder using vinylacetate and sodium hydroxide. The desired starch acetylation reaction is accompanied by an undesired parallel base catalysed hydrolysis reaction of vinylacetate and a consecutive hydrolysis reaction of the acetyla

  10. Purification and characterization of the acetyl-CoA synthetase from Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    Ru Li; Jing Gu; Peng Chen; Zhiping Zhang; Jiaoyu Deng; XianEn Zhang

    2011-01-01

    Acetyl-CoA (AcCoA) synthetase (Acs) catalyzes the conversion of acetate into AcCoA,which is involved in many catabolic and anabolic pathways.Although this enzyme has been studied for many years in many organisms,the properties of Mycobacterium tuberculosis Acs and the regulation of its activity remain unknown.Here,the putative acs gene of M.tuberculosis H37Rv (Mt-Acs) was expressed as a fusion protein with 6×His-tag on the C-terminus in Escherichia coli.The recombinant Mt-Acs protein was successfully purified and then its enzymatic characteristics were analyzed.The optimal pH and temperature,and the kinetic parameters of Mt-Acs were determined.To investigate whether Mt-Acs is regulated by lysine acetylation as reported for Salmonella enterica Acs,its mutant K617R was also generated.Determination of the enzymatic activity suggests that Lys-617 is critical for its function.We further demonstrated that Mt-Acs underwent auto-acetylation with acetate but not with AcCoA as the acetyl donor,which resulted in the decrease of its activity.CoA,the substrate for AcCoA formation,inhibited the auto-acetylation.Furthermore,the silent information regulator (Sir2) of M.tuberculosis (Mt-Sir2) could catalyze Mt-Acs deacetylation,which resulted in activation of Acs.These results may provide more insights into the physiological roles of Mt-Acs in M.tuberculosis central metabolism.

  11. Impact of N-terminal acetylation of α-synuclein on its random coil and lipid binding properties.

    Science.gov (United States)

    Maltsev, Alexander S; Ying, Jinfa; Bax, Ad

    2012-06-26

    N-Terminal acetylation of α-synuclein (aS), a protein implicated in the etiology of Parkinson's disease, is common in mammals. The impact of this modification on the protein's structure and dynamics in free solution and on its membrane binding properties has been evaluated by high-resolution nuclear magnetic resonance and circular dichroism (CD) spectroscopy. While no tetrameric form of acetylated aS could be isolated, N-terminal acetylation resulted in chemical shift perturbations of the first 12 residues of the protein that progressively decreased with the distance from the N-terminus. The directions of the chemical shift changes and small changes in backbone (3)J(HH) couplings are consistent with an increase in the α-helicity of the first six residues of aS, although a high degree of dynamic conformational disorder remains and the helical structure is sampled <20% of the time. Chemical shift and (3)J(HH) data for the intact protein are virtually indistinguishable from those recorded for the corresponding N-terminally acetylated and nonacetylated 15-residue synthetic peptides. An increase in α-helicity at the N-terminus of aS is supported by CD data on the acetylated peptide and by weak medium-range nuclear Overhauser effect contacts indicative of α-helical character. The remainder of the protein has chemical shift values that are very close to random coil values and indistinguishable between the two forms of the protein. No significant differences in the fibrillation kinetics were observed between acetylated and nonacetylated aS. However, the lipid binding properties of aS are strongly impacted by acetylation and exhibit distinct behavior for the first 12 residues, indicative of an initiation role for the N-terminal residues in an "initiation-elongation" process of binding to the membrane.

  12. Insights into the epigenetic mechanisms involving histone lysine methylation and demethylation in ischemia induced damage and repair has therapeutic implication.

    Science.gov (United States)

    Chakravarty, Sumana; Jhelum, Priya; Bhat, Unis Ahmad; Rajan, Wenson D; Maitra, Swati; Pathak, Salil S; Patel, Anant B; Kumar, Arvind

    2017-01-01

    Cerebral ischemic stroke is one of the leading causes of death and disability worldwide. Therapeutic interventions to minimize ischemia-induced neural damage are limited due to poor understanding of molecular mechanisms mediating complex pathophysiology in stroke. Recently, epigenetic mechanisms mostly histone lysine (K) acetylation and deacetylation have been implicated in ischemic brain damage and have expanded the dimensions of potential therapeutic intervention to the systemic/local administration of histone deacetylase inhibitors. However, the role of other epigenetic mechanisms such as histone lysine methylation and demethylation in stroke-induced damage and subsequent recovery process is elusive. Here, we established an Internal Carotid Artery Occlusion (ICAO) model in CD1 mouse that resulted in mild to moderate level of ischemic damage to the striatum, as suggested by magnetic resonance imaging (MRI), TUNEL and histopathological staining along with an evaluation of neurological deficit score (NDS), grip strength and rotarod performance. The molecular investigations show dysregulation of a number of histone lysine methylases (KMTs) and few of histone lysine demethylases (KDMs) post-ICAO with significant global attenuation in the transcriptionally repressive epigenetic mark H3K9me2 in the striatum. Administration of Dimethyloxalylglycine (DMOG), an inhibitor of KDM4 or JMJD2 class of histone lysine demethylases, significantly ameliorated stroke-induced NDS by restoring perturbed H3K9me2 levels in the ischemia-affected striatum. Overall, these results highlight the novel role of epigenetic regulatory mechanisms controlling the epigenetic mark H3K9me2 in mediating the stroke-induced striatal damage and subsequent repair following mild to moderate cerebral ischemia.

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

    National Research Council Canada - National Science Library

    Wapenaar, Hannah; Dekker, Frank J

    2016-01-01

    Histone acetyltransferases (HATs) are epigenetic enzymes that install acetyl groups onto lysine residues of cellular proteins such as histones, transcription factors, nuclear receptors, and enzymes...

  14. Lysine conservation and context in TGFbeta and Wnt signaling suggest new targets and general themes for posttranslational modification.

    Science.gov (United States)

    Konikoff, Charlotte E; Wisotzkey, Robert G; Newfeld, Stuart J

    2008-10-01

    TGFbeta and Wnt pathways play important roles in the development of animals from sponges to humans. In both pathways posttranslational modification as a means of regulating their function, such as lysine modification by ubiquitination and sumoylation, has been observed. However, a gap exists between the immunological observation of posttranslational modification and the identification of the target lysine. To fill this gap, we conducted a phylogenetic analysis of lysine conservation and context in TGFbeta and Wnt pathway receptors and signal transducers and suggest numerous high-probability candidates for posttranslational modification. Further comparison of results from both pathways suggests two general features for biochemical regulation of intercellular signaling: receptors are less frequent targets for modification than signal transduction agonists, and a lysine adjacent to an upstream hydrophobic residue may be a preferred context for modification. Overall the results suggest numerous applications for an evolutionary approach to the biochemical regulation of developmental pathways, including (1) streamlining of the identification of the target lysine, (2) determination of when members of a multigene family acquire distinct activities, (3) application to any conserved protein family, and (4) application to any modification of a specific amino acid.

  15. Differences in lysine adduction by acrolein and methyl vinyl ketone: implications for cytotoxicity in cultured hepatocytes.

    Science.gov (United States)

    Kaminskas, Lisa M; Pyke, Simon M; Burcham, Philip C

    2005-11-01

    Acrolein is a highly toxic environmental pollutant that readily alkylates the epsilon-amino group of lysine residues in proteins. In model systems, such chemistry involves sequential addition of two acrolein molecules to a given nitrogen, forming bis-Michael-adducted species that undergo aldol condensation and dehydration to form Nepsilon-(3-formyl-3,4-dehydropiperidino)lysine. Whether this ability to form cyclic adducts participates in the toxicity of acrolein is unknown. To address this issue, we compared the chemistry of protein adduction by acrolein to that of its close structural analogue methyl vinyl ketone, expecting that the alpha-methyl group would hinder the intramolecular cyclization of any bis-adducted species formed by methyl vinyl ketone. Both acrolein and methyl vinyl ketone displayed comparable protein carbonylating activity during in vitro studies with the model protein bovine serum albumin, confirming the alpha,beta,-unsaturated bond of both compounds is an efficient Michael acceptor for protein nucleophiles. However, differences in adduction chemistry became apparent during the use of electrospray ionization-MS to monitor reaction products in a lysine-containing peptide after modification by each compound. For example, although a Schiff base adduct was detected following reaction of the peptide with acrolein, an analogous species was not formed by methyl vinyl ketone. Furthermore, while ions corresponding to mono- and bis-Michael adducts were detected at the N-terminus and lysine residues following peptide modification by both carbonyls, only acrolein modification generated ions attributable to cyclic adducts. Despite these differences in adduction chemistry, in mouse hepatocytes, the two compounds exhibited very comparable abilities to induce rapid, concentration-dependent cell death as well as protein carbonylation. These findings suggest that the acute toxicity of short-chain alpha,beta-unsaturated carbonyl compounds involves their ability to

  16. Inhibition of lysine-specific demethylase 1 by polyamine analogues results in reexpression of aberrantly silenced genes.

    Science.gov (United States)

    Huang, Yi; Greene, Eriko; Murray Stewart, Tracy; Goodwin, Andrew C; Baylin, Stephen B; Woster, Patrick M; Casero, Robert A

    2007-05-08

    Epigenetic chromatin modification is a major regulator of eukaryotic gene expression, and aberrant epigenetic silencing of gene expression contributes to tumorigenesis. Histone modifications include acetylation, phosphorylation, and methylation, resulting in a combination of histone marks known collectively as the histone code. The chromatin marks at a given promoter determine, in part, whether specific promoters are in an open/active conformation or closed/repressed conformation. Dimethyl-lysine 4 histone H3 (H3K4me2) is a transcription-activating chromatin mark at gene promoters, and demethylation of this mark by the lysine-specific demethylase 1 (LSD1), a homologue of polyamine oxidases, may broadly repress gene expression. We now report that novel biguanide and bisguanidine polyamine analogues are potent inhibitors of LSD1. These analogues inhibit LSD1 in human colon carcinoma cells and affect a reexpression of multiple, aberrantly silenced genes important in the development of colon cancer, including members of the secreted frizzle-related proteins (SFRPs) and the GATA family of transcription factors. Furthermore, we demonstrate by chromatin immunoprecipitation analysis that the reexpression is concurrent with increased H3K4me2 and acetyl-H3K9 marks, decreased H3K9me1 and H3K9me2 repressive marks. We thus define important new agents for reversing aberrant repression of gene transcription.

  17. Catalytic roles of lysines (K9, K27, K31) in the N-terminal domain in human adenylate kinase by random site-directed mutagenesis.

    Science.gov (United States)

    Ayabe, T; Park, S K; Takenaka, H; Sumida, M; Uesugi, S; Takenaka, O; Hamada, M

    1996-11-01

    To elucidate lysine residues in the N-terminal domain of human cytosolic adenylate kinase (hAK1, EC 2.7.4.3), random site-directed mutagenesis of K9, K27, and K31 residues was performed, and six mutants were analyzed by steady-state kinetics. K9 residue may play an important role in catalysis by interacting with AMP2-. K27 and K31 residues appear to play a functional role in catalysis by interacting with MgATP2-. In human AK, the epsilon-amino group in the side chain of these lysine residues would be essential for phosphoryl transfer between MgATP2- and AMP2- during transition state.

  18. Efficient acetylation of primary amines and amino acids in environmentally benign brine solution using acetyl chloride

    Indian Academy of Sciences (India)

    Kaushik Basu; Suchandra Chakraborty; Achintya Kumar Sarkar; Chandan Saha

    2013-05-01

    Acetyl chloride is one of the most commonly available and cheap acylating agent but its high reactivity and concomitant instability in water precludes its use to carry out acetylation in aqueous medium. The present methodology illustrates the efficient acetylation of primary amines and amino acids in brine solution by means of acetyl chloride under weakly basic condition in the presence of sodium acetate and/or triethyl amine followed by trituration with aqueous saturated bicarbonate solution. This effort represents the first efficient use of this most reactive but cheap acetylating agent to acetylate amines in excellent yields in aqueous medium. This is a potentially useful green chemical transformation where reaction takes place in environment-friendly brine solution leading to easy work-up and isolation of the amide derivatives. Mechanistic rationale of this methodology is also important.

  19. Lysine fortification: past, present, and future.

    Science.gov (United States)

    Pellett, Peter L; Ghosh, Shibani

    2004-06-01

    Fortification with lysine to improve the protein value of human diets that are heavily based on cereals has received support from the results of these recent studies [1,2]. Support also comes from examination of average food and nutrient availability data derived from food balance sheets. Whereas nutritional status is influenced by the nutrient content of foods consumed in relation to need, the requirements for protein and amino acids are influenced by many additional factors [10, 12, 14, 28, 29]. These include age, sex, body size, physical activity, growth, pregnancy and lactation, infection, and the efficiency of nutrient utilization. Even if the immune response was influenced by the added lysine, adequate water and basic sanitation would remain essential. Acute and chronic undernutrition and most micronutrient deficiencies primarily affect poor and deprived people who do not have access to food of adequate nutritional value, live in unsanitary environments without access to clean water and basic services, and lack access to appropriate education and information [30]. A further variable is the possible interaction between protein and food energy availability [31]. This could affect the protein value of diets when food energy is limiting to a significant degree. Thus, the additional effects of food energy deficiency on protein utilization could well be superimposed on the very poorest. The improvement of dietary diversity must be the long-term aim, with dietary fortification considered only a short-term solution. The former should take place as wealth improves and the gaps between rich and poor diminish. Although such changes are taking place, they are highly uneven. Over the last several decades, increases have occurred in the availability of food energy, total protein, and animal protein for both developed and developing countries. However, for the very poorest developing countries over the same period, changes have been almost nonexistent, and the values for

  20. Charge neutralization of the central lysine cluster in prion protein (PrP) promotes PrP(Sc)-like folding of recombinant PrP amyloids.

    Science.gov (United States)

    Groveman, Bradley R; Kraus, Allison; Raymond, Lynne D; Dolan, Michael A; Anson, Kelsie J; Dorward, David W; Caughey, Byron

    2015-01-09

    The structure of the infectious form of prion protein, PrP(Sc), remains unclear. Most pure recombinant prion protein (PrP) amyloids generated in vitro are not infectious and lack the extent of the protease-resistant core and solvent exclusion of infectious PrP(Sc), especially within residues ∼90-160. Polyanionic cofactors can enhance infectivity and PrP(Sc)-like characteristics of such fibrils, but the mechanism of this enhancement is unknown. In considering structural models of PrP(Sc) multimers, we identified an obstacle to tight packing that might be overcome with polyanionic cofactors, namely, electrostatic repulsion between four closely spaced cationic lysines within a central lysine cluster of residues 101-110. For example, in our parallel in-register intermolecular β-sheet model of PrP(Sc), not only would these lysines be clustered within the 101-110 region of the primary sequence, but they would have intermolecular spacings of only ∼4.8 Å between stacked β-strands. We have now performed molecular dynamics simulations predicting that neutralization of the charges on these lysine residues would allow more stable parallel in-register packing in this region. We also show empirically that substitution of these clustered lysine residues with alanines or asparagines results in recombinant PrP amyloid fibrils with extended proteinase-K resistant β-sheet cores and infrared spectra that are more reminiscent of bona fide PrP(Sc). These findings indicate that charge neutralization at the central lysine cluster is critical for the folding and tight packing of N-proximal residues within PrP amyloid fibrils. This charge neutralization may be a key aspect of the mechanism by which anionic cofactors promote PrP(Sc) formation.

  1. Exploring lysine riboswitch for metabolic flux control and improvement of L-lysine synthesis in Corynebacterium glutamicum.

    Science.gov (United States)

    Zhou, Li-Bang; Zeng, An-Ping

    2015-06-19

    Riboswitch, a regulatory part of an mRNA molecule that can specifically bind a metabolite and regulate gene expression, is attractive for engineering biological systems, especially for the control of metabolic fluxes in industrial microorganisms. Here, we demonstrate the use of lysine riboswitch and intracellular l-lysine as a signal to control the competing but essential metabolic by-pathways of lysine biosynthesis. To this end, we first examined the natural lysine riboswitches of Eschericia coli (ECRS) and Bacillus subtilis (BSRS) to control the expression of citrate synthase (gltA) and thus the metabolic flux in the tricarboxylic acid (TCA) cycle in E. coli. ECRS and BSRS were then successfully used to control the gltA gene and TCA cycle activity in a lysine producing strain Corynebacterium glutamicum LP917, respectively. Compared with the strain LP917, the growth of both lysine riboswitch-gltA mutants was slower, suggesting a reduced TCA cycle activity. The lysine production was 63% higher in the mutant ECRS-gltA and 38% higher in the mutant BSRS-gltA, indicating a higher metabolic flux into the lysine synthesis pathway. This is the first report on using an amino acid riboswitch for improvement of lysine biosynthesis. The lysine riboswitches can be easily adapted to dynamically control other essential but competing metabolic pathways or even be engineered as an "on-switch" to enhance the metabolic fluxes of desired metabolic pathways.

  2. Intracellular Acetyl Unit Transport in Fungal Carbon Metabolism

    NARCIS (Netherlands)

    Strijbis, K.; Distel, B.

    2010-01-01

    Acetyl coenzyme A (acetyl-CoA) is a central metabolite in carbon and energy metabolism. Because of its amphiphilic nature and bulkiness, acetyl-CoA cannot readily traverse biological membranes. In fungi, two systems for acetyl unit transport have been identified: a shuttle dependent on the carrier c

  3. Antimicrobial activity of chicken NK-lysin against Eimeria sporozoites.

    Science.gov (United States)

    Hong, Yeong H; Lillehoj, Hyun S; Siragusa, Gregory R; Bannerman, Douglas D; Lillehoj, Erik P

    2008-06-01

    NK-lysin is an antimicrobial and antitumor polypeptide that is considered to play an important role in innate immunity. Chicken NK-lysin is a member of the saposin-like protein family and exhibits potent antitumor cell activity. To evaluate the antimicrobial properties of chicken NK-lysin, we examined its ability to reduce the viability of various bacterial strains and two species of Eimeria parasites. Culture supernatants from COS7 cells transfected with a chicken NK-lysin cDNA and His-tagged purified NK-lysin from the transfected cells both showed high cytotoxic activity against Eimeria acervulina and Eimeria maxima sporozoites. In contrast, no bactericidal activity was observed. Further studies using synthetic peptides derived from NK-lysin may be useful for pharmaceutical and agricultural uses in the food animal industry.

  4. p53 Acetylation: Regulation and Consequences

    Directory of Open Access Journals (Sweden)

    Sara M. Reed

    2014-12-01

    Full Text Available Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer.

  5. p53 Acetylation: Regulation and Consequences

    Energy Technology Data Exchange (ETDEWEB)

    Reed, Sara M. [Department of Pharmacology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Medical Scientist Training Program, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Quelle, Dawn E., E-mail: dawn-quelle@uiowa.edu [Department of Pharmacology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Medical Scientist Training Program, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Department of Pathology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States)

    2014-12-23

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites) as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis) to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer.

  6. Biological activity of acetylated phenolic compounds.

    Science.gov (United States)

    Fragopoulou, Elizabeth; Nomikos, Tzortzis; Karantonis, Haralabos C; Apostolakis, Constantinos; Pliakis, Emmanuel; Samiotaki, Martina; Panayotou, George; Antonopoulou, Smaragdi

    2007-01-10

    In recent years an effort has been made to isolate and identify biologically active compounds that are included in the Mediterranean diet. The existence of naturally occurring acetylated phenolics, as well as studies with synthetic ones, provide evidence that acetyl groups could be correlated with their biological activity. Platelet activating factor (PAF) is implicated in atherosclerosis, whereas its inhibitors seem to play a protective role against cardiovascular disease. The aim of this study was to examine the biological activity of resveratrol and tyrosol and their acetylated derivatives as inhibitors of PAF-induced washed rabbit platelet aggregation. Acetylation of resveratrol and tyrosol was performed, and separation was achieved by HPLC. Acetylated derivatives were identified by negative mass spectrometry. The data showed that tyrosol and its monoacetylated derivatives act as PAF inhibitors, whereas diacetylated derivatives induce platelet aggregation. Resveratrol and its mono- and triacetylated derivatives exert similar inhibitory activity, whereas the diacetylated ones are more potent inhibitors. In conclusion, acetylated phenolics exert the same or even higher antithrombotic activity compared to the biological activity of the initial one.

  7. Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106

    Energy Technology Data Exchange (ETDEWEB)

    Su, Dan; Hu, Qi; Li, Qing; Thompson, James R; Cui, Gaofeng; Fazly, Ahmed; Davies, Brian A; Botuyan, Maria Victoria; Zhang, Zhiguo; Mer, Georges [Mayo

    2013-04-08

    Dynamic variations in the structure of chromatin influence virtually all DNA-related processes in eukaryotes and are controlled in part by post-translational modifications of histones. One such modification, the acetylation of lysine 56 (H3K56ac) in the amino-terminal α-helix (αN) of histone H3, has been implicated in the regulation of nucleosome assembly during DNA replication and repair, and nucleosome disassembly during gene transcription. In Saccharomyces cerevisiae, the histone chaperone Rtt106 contributes to the deposition of newly synthesized H3K56ac-carrying H3-H4 complex on replicating DNA, but it is unclear how Rtt106 binds H3-H4 and specifically recognizes H3K56ac as there is no apparent acetylated lysine reader domain in Rtt106. Here, we show that two domains of Rtt106 are involved in a combinatorial recognition of H3-H4. An N-terminal domain homodimerizes and interacts with H3-H4 independently of acetylation while a double pleckstrin-homology (PH) domain binds the K56-containing region of H3. Affinity is markedly enhanced upon acetylation of K56, an effect that is probably due to increased conformational entropy of the αN helix of H3. Our data support a mode of interaction where the N-terminal homodimeric domain of Rtt106 intercalates between the two H3-H4 components of the (H3-H4)2 tetramer while two double PH domains in the Rtt106 dimer interact with each of the two H3K56ac sites in (H3-H4)2. We show that the Rtt106-(H3-H4)2 interaction is important for gene silencing and the DNA damage response.

  8. Structural Basis for p53 Lys120-Acetylation-Dependent DNA-Binding Mode.

    Science.gov (United States)

    Vainer, Radion; Cohen, Sarit; Shahar, Anat; Zarivach, Raz; Arbely, Eyal

    2016-07-31

    Normal cellular homeostasis depends on tight regulation of gene expression, which requires the modulation of transcription factors' DNA-binding specificity. That said, the mechanisms that allow transcription factors to distinguish between closely related response elements following different cellular signals are not fully understood. In the tumor suppressor protein p53, acetylation of loop L1 residue Lys120 within the DNA-binding domain has been shown to promote the transcription of proapoptotic genes such as bax. Here, we report the crystal structures of Lys120-acetylated p53 DNA-binding domain in complex with a consensus response element and with the natural BAX response element. Our structural analyses reveal that Lys120 acetylation expands the conformational space of loop L1 in the DNA-bound state. Loop L1 flexibility is known to increase p53's DNA-binding specificity, and Lys120-acetylation-dependent conformational changes in loop L1 enable the formation of sequence-dependent DNA-binding modes for p53. Furthermore, binding to the natural BAX response element is accompanied by global conformational changes, deformation of the DNA helical structure, and formation of an asymmetric tetrameric complex. Based on these findings, we suggest a model for p53's Lys120 acetylation-dependent DNA-binding mode. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Pasting properties and (chemical) fine structure of acetylated yellow pea starch is affected by acetylation reagent type and granule size

    NARCIS (Netherlands)

    Huang, J.; Schols, H.A.; Jin, Z.; Sulmann, E.; Voragen, A.G.J.

    2007-01-01

    Yellow pea starch was fractionated into small and large size granule fractions and then modified with acetic anhydride and vinyl acetate (acetylation after sieving) or first acetylated in the same way and then fractionated into small and large size fractions (acetylation before sieving). Acetylation

  10. Acetylation of TUG protein promotes the accumulation of GLUT4 glucose transporters in an insulin-responsive intracellular compartment.

    Science.gov (United States)

    Belman, Jonathan P; Bian, Rachel R; Habtemichael, Estifanos N; Li, Don T; Jurczak, Michael J; Alcázar-Román, Abel; McNally, Leah J; Shulman, Gerald I; Bogan, Jonathan S

    2015-02-13

    Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD(+)-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake.

  11. Histone H4 Lysine 20 methylation

    DEFF Research Database (Denmark)

    Jørgensen, Stine; Schotta, Gunnar; Sørensen, Claus Storgaard

    2013-01-01

    of histones have emerged as key regulators of genomic integrity. Intense research during the past few years has revealed histone H4 lysine 20 methylation (H4K20me) as critically important for the biological processes that ensure genome integrity, such as DNA damage repair, DNA replication and chromatin...... instability, demonstrating the important functions of H4K20 methylation in genome maintenance. In this review, we explain molecular mechanisms underlying these defects and discuss novel ideas for furthering our understanding of genome maintenance in higher eukaryotes....

  12. Optimization of lysine metabolism in Corynebacterium glutamicum

    DEFF Research Database (Denmark)

    Rytter, Jakob Vang

    the project intends to eliminate. PGI catalyzes the conversion of alpha-D-glucose-6-phosphate to fructose-6-phosphate just downstream of the branch in the glycolysis, but it also catalyzes the reverse reaction. It is unknown whether up- or down-regulation of the pgi is required to increase the flux through......, and increased NADPH availability is therefore a potential way to enhance lysine production. The generation of NADPH is mainly located in the pentose phosphate pathway (PPP). Using the genome scale model the phosphoglucoisomerase enzyme (PGI) has been identified as a possible bottleneck in the metabolism, which...

  13. MRG15 activates the cdc2 promoter via histone acetylation in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Pena, AndreAna N., E-mail: andreana.pena@gmail.com [Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, TX (United States); Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX (United States); Tominaga, Kaoru; Pereira-Smith, Olivia M. [Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, TX (United States); Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX (United States)

    2011-07-01

    Chromatin remodeling is required for transcriptional activation and repression. MRG15 (MORF4L1), a chromatin modulator, is a highly conserved protein and is present in complexes containing histone acetyltransferases (HATs) as well as histone deacetylases (HDACs). Loss of expression of MRG15 in mice and Drosophila results in embryonic lethality and fibroblast and neural stem/progenitor cells cultured from Mrg15 null mouse embryos exhibit marked proliferative defects when compared with wild type cells. To determine the role of MRG15 in cell cycle progression we performed chromatin immunoprecipitation with an antibody to MRG15 on normal human fibroblasts as they entered the cell cycle from a quiescent state, and analyzed various cell cycle gene promoters. The results demonstrated a 3-fold increase in MRG15 occupancy at the cdc2 promoter during S phase of the cell cycle and a concomitant increase in acetylated histone H4. H4 lysine 12 was acetylated at 24 h post-serum stimulation while there was no change in acetylation of lysine 16. HDAC1 and 2 were decreased at this promoter during cell cycle progression. Over-expression of MRG15 in HeLa cells activated a cdc2 promoter-reporter construct in a dose-dependent manner, whereas knockdown of MRG15 resulted in decreased promoter activity. In order to implicate HAT activity, we treated cells with the HAT inhibitor anacardic acid and determined that HAT inhibition results in loss of expression of cdc2 mRNA. Further, chromatin immunoprecipitation with Tip60 localizes the protein to the same 110 bp stretch of the cdc2 promoter pulled down by MRG15. Additionally, we determined that cotransfection of MRG15 with the known associated HAT Tip60 had a cooperative effect in activating the cdc2 promoter. These results suggest that MRG15 is acting in a HAT complex involving Tip60 to modify chromatin via acetylation of histone H4 at the cdc2 promoter to activate transcription.

  14. HDAC6 deacetylates p53 at lysines 381/382 and differentially coordinates p53-induced apoptosis.

    Science.gov (United States)

    Ryu, Hyun-Wook; Shin, Dong-Hee; Lee, Dong Hoon; Choi, Junjeong; Han, Gyoonhee; Lee, Kang Young; Kwon, So Hee

    2017-04-10

    HDAC6-selective inhibitors represent promising new cancer therapeutic agents, but their precise mechanisms of action are not well understood. In particular, p53's role in HDAC6 inhibitor-induced effects has not been fully elucidated. In this study, we show that an HDAC6-selective inhibitor, A452, increased wild-type p53 levels by destabilizing MDM2, but decreased mutant p53 by inducing MDM2 and inhibiting Hsp90-mutant p53 complex formation. Interestingly, HDAC6 levels inversely correlated with p53 acetylation at lysines 381/382 associated with p53 functional activation. A452 blocked HDAC6 nuclear localization, resulting in increased levels of acetylated p53 at Lys381/382. HDAC6 bound to the C-terminal region of p53 via its deacetylase domain. A452 disrupted the HDAC6-Hsp90 chaperone machinery via Hsp90 acetylation and degradation. Furthermore, it chemosensitized cancer cells to the Hsp90 inhibitor 17-AAG. Overall, silencing of HDAC6 showed similar effects. These findings suggest that the anticancer action of HDAC6 inhibitors requires p53 and Hsp90 and targeting of HDAC6 may represent a new therapeutic strategy for cancers regardless of p53's mutation status.

  15. Triple therapy with pyridoxine, arginine supplementation and dietary lysine restriction in pyridoxine-dependent epilepsy: Neurodevelopmental outcome.

    Science.gov (United States)

    Coughlin, Curtis R; van Karnebeek, Clara D M; Al-Hertani, Walla; Shuen, Andrew Y; Jaggumantri, Sravan; Jack, Rhona M; Gaughan, Sommer; Burns, Casey; Mirsky, David M; Gallagher, Renata C; Van Hove, Johan L K

    2015-01-01

    Pyridoxine-dependent epilepsy (PDE) is an epileptic encephalopathy characterized by response to pharmacologic doses of pyridoxine. PDE is caused by deficiency of α-aminoadipic semialdehyde dehydrogenase resulting in impaired lysine degradation and subsequent accumulation of α-aminoadipic semialdehyde. Despite adequate seizure control with pyridoxine monotherapy, 75% of individuals with PDE have significant developmental delay and intellectual disability. We describe a new combined therapeutic approach to reduce putative toxic metabolites from impaired lysine metabolism. This approach utilizes pyridoxine, a lysine-restricted diet to limit the substrate that leads to neurotoxic metabolite accumulation and L-arginine to compete for brain lysine influx and liver mitochondrial import. We report the developmental and biochemical outcome of six subjects who were treated with this triple therapy. Triple therapy reduced CSF, plasma, and urine biomarkers associated with neurotoxicity in PDE. The addition of arginine supplementation to children already treated with dietary lysine restriction and pyridoxine further reduced toxic metabolites, and in some subjects appeared to improve neurodevelopmental outcome. Dietary lysine restriction was associated with improved seizure control in one subject, and the addition of arginine supplementation increased the objective motor outcome scale in two twin siblings, illustrating the contribution of each component of this treatment combination. Optimal results were noted in the individual treated with triple therapy early in the course of the disease. Residual disease symptoms could be related to early injury suggested by initial MR imaging prior to initiation of treatment or from severe epilepsy prior to diagnosis. This observational study reports the use of triple therapy, which combines three effective components in this rare condition, and suggests that early diagnosis and treatment with this new triple therapy may ameliorate the

  16. File list: Oth.Unc.50.Crotonyl_lysine.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.50.Crotonyl_lysine.AllCell mm9 TFs and others Crotonyl lysine Unclassified ...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Unc.50.Crotonyl_lysine.AllCell.bed ...

  17. File list: Oth.Pan.05.Crotonyl_lysine.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Pan.05.Crotonyl_lysine.AllCell mm9 TFs and others Crotonyl lysine Pancreas http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Pan.05.Crotonyl_lysine.AllCell.bed ...

  18. File list: Oth.Plc.20.Crotonyl_lysine.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Plc.20.Crotonyl_lysine.AllCell mm9 TFs and others Crotonyl lysine Placenta http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Plc.20.Crotonyl_lysine.AllCell.bed ...

  19. File list: Oth.Unc.10.Crotonyl_lysine.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.10.Crotonyl_lysine.AllCell mm9 TFs and others Crotonyl lysine Unclassified ...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Unc.10.Crotonyl_lysine.AllCell.bed ...

  20. File list: Oth.Unc.20.Crotonyl_lysine.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.20.Crotonyl_lysine.AllCell mm9 TFs and others Crotonyl lysine Unclassified ...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Unc.20.Crotonyl_lysine.AllCell.bed ...

  1. File list: Oth.Pan.50.Crotonyl_lysine.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Pan.50.Crotonyl_lysine.AllCell mm9 TFs and others Crotonyl lysine Pancreas http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Pan.50.Crotonyl_lysine.AllCell.bed ...

  2. File list: Oth.Plc.50.Crotonyl_lysine.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Plc.50.Crotonyl_lysine.AllCell mm9 TFs and others Crotonyl lysine Placenta http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Plc.50.Crotonyl_lysine.AllCell.bed ...

  3. File list: Oth.Prs.10.Crotonyl_lysine.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Prs.10.Crotonyl_lysine.AllCell mm9 TFs and others Crotonyl lysine Prostate http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Prs.10.Crotonyl_lysine.AllCell.bed ...

  4. File list: Oth.Prs.05.Crotonyl_lysine.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Prs.05.Crotonyl_lysine.AllCell mm9 TFs and others Crotonyl lysine Prostate http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Prs.05.Crotonyl_lysine.AllCell.bed ...

  5. File list: Oth.Prs.20.Crotonyl_lysine.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Prs.20.Crotonyl_lysine.AllCell mm9 TFs and others Crotonyl lysine Prostate http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Prs.20.Crotonyl_lysine.AllCell.bed ...

  6. File list: Oth.Plc.05.Crotonyl_lysine.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Plc.05.Crotonyl_lysine.AllCell mm9 TFs and others Crotonyl lysine Placenta http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Plc.05.Crotonyl_lysine.AllCell.bed ...

  7. File list: Oth.Pan.10.Crotonyl_lysine.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Pan.10.Crotonyl_lysine.AllCell mm9 TFs and others Crotonyl lysine Pancreas http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Pan.10.Crotonyl_lysine.AllCell.bed ...

  8. File list: Oth.Plc.10.Crotonyl_lysine.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Plc.10.Crotonyl_lysine.AllCell mm9 TFs and others Crotonyl lysine Placenta http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Plc.10.Crotonyl_lysine.AllCell.bed ...

  9. File list: Oth.Prs.50.Crotonyl_lysine.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Prs.50.Crotonyl_lysine.AllCell mm9 TFs and others Crotonyl lysine Prostate http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Prs.50.Crotonyl_lysine.AllCell.bed ...

  10. File list: Oth.Unc.05.Crotonyl_lysine.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.05.Crotonyl_lysine.AllCell mm9 TFs and others Crotonyl lysine Unclassified ...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Unc.05.Crotonyl_lysine.AllCell.bed ...

  11. Experiment list: SRX067403 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available general, though, there appears to be high redundancy. Histone acetylation is notable for susceptibility to s...n whether acetylation can have different consequences depending on the specific lysine residue targeted. In

  12. Experiment list: SRX185813 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available er acetylation has can have different consequences depending on the specific lysine residue targeted. In general, though, there appea...rs to be high redundancy. Histone acetylation is notable

  13. Quantification of Nε-(2-Furoylmethyl)-L-lysine (furosine), Nε-(Carboxymethyl)-L-lysine (CML), Nε-(Carboxyethyl)-L-lysine (CEL) and total lysine through stable isotope dilution assay and tandem mass spectrometry

    NARCIS (Netherlands)

    Troise, A.D.; Fiore, A.; Wiltafsky, M.; Fogliano, V.

    2015-01-01

    The control of Maillard reaction (MR) is a key point to ensure processed foods quality. Due to the presence of a primary amino group on its side chain, lysine is particularly prone to chemical modifications with the formation of Amadori products (AP), Nε-(Carboxymethyl)-L-lysine (CML),

  14. STUDY OF LYSINE AND ALANINE DELIVERANCE THROUGH POLYPYRROLE MEMBRANE

    Directory of Open Access Journals (Sweden)

    Adhitasari Suratman

    2010-06-01

    Full Text Available Electropolymerization processes of pyrrole and the usage of polypyrrole membrane as lysine and alanine deliverance have been studied by cyclic voltammetry technique. Polypyrrole membrane was prepared by electropolymerization processes of pyrrole in water based solvent containing sodium perchlorate as supporting electrolyte. Electropolymerization processes were carried out within potential range of 0-1100 mV vs Ag/AgCl reference electrode and at the scanning rate of 100 mV/s. In this study, lysine and alanine have been used as molecules which could easily be loaded on and released from polypyrrole membrane. The presence of lysine or alanine during electropolymerization process reduced the rate of electropolymerization of polypyrrole. In lysine or alanine transfer processes into polypyrrole membrane, the interaction between polypyrrole and lysine or alanine showed by the curve of E½ oxidation in respect of - log C. It proved that the E½ oxidation shifted to more positive potential showed by the increasing of concentration of lysine or alanine. Beside that, voltammetric responses of lysine and alanine transfered into polypyrrole membrane were found to be Nernstian. The results indicated that polypyrrole could be used as a sensor of lysine and alanine.   Keywords: Electropolymerization, polypyrrole membrane, voltammetry technique

  15. Digestible lysine levels in diets supplemented with ractopamine

    Directory of Open Access Journals (Sweden)

    Evelar de Oliveira Souza

    2011-10-01

    Full Text Available In order evaluate digestible lysine levels in diets supplemented with 20 ppm of ractopamine on the performance and carcass traits, 64 barrows with high genetic potential at finishing phase were allotted in a completely randomized block design with four digestible lysine levels (0.80, 0.90, 1.00, and 1.10%, eight replicates and two pigs per experimental unit. Initial body weight and pigs' kinship were used as criteria in the blocks formation. Diets were mainly composed of corn and soybean meal supplemented with minerals, vitamins and amino acids to meet pigs' nutritional requirements at the finishing phase, except for digestible lysine. No effect of digestible lysine levels was observed in animal performance. The digestible lysine intake increased linearly by increasing the levels of digestible lysine in the diets. Carcass traits were not influenced by the dietary levels of digestible lysine. The level of 0.80% of digestible lysine in diets supplemented with 20 ppm ractopamine meets the nutritional requirements of castrated male pigs during the finishing phase.

  16. Bioavailability of lysine in heat-treated foods and feedstuffs

    NARCIS (Netherlands)

    McArtney Rutherfurd, S.

    2010-01-01

    During the processing of foodstuffs, lysine can react with other compounds present to form nutritionally unavailable derivatives, the most common example of which are Maillard products. Maillard products can cause serious problems when determining the available lysine content of processed foods or f

  17. Bioavailability of lysine in heat-treated foods and feedstuffs

    NARCIS (Netherlands)

    McArtney Rutherfurd, S.

    2010-01-01

    During the processing of foodstuffs, lysine can react with other compounds present to form nutritionally unavailable derivatives, the most common example of which are Maillard products. Maillard products can cause serious problems when determining the available lysine content of processed foods or

  18. Creative lysins: Listeria and the engineering of antimicrobial enzymes.

    Science.gov (United States)

    Van Tassell, Maxwell L; Angela Daum, M; Kim, Jun-Seob; Miller, Michael J

    2016-02-01

    Cell wall lytic enzymes have been of increasing interest as antimicrobials for targeting Gram-positive spoilage and pathogenic bacteria, largely due to the development of strains resistant to antibiotics and bacteriophage therapy. Such lysins show considerable promise against Listeria monocytogenes, a primary concern in food-processing environments, but there is room for improvement via protein engineering. Advances in antilisterial applications could benefit from recent developments in lysin biotechnology that have largely targeted other organisms. Herein we present various considerations for the future development of lysins, including environmental factors, cell physiology concerns, and dynamics of protein architecture. Our goal is to review key developments in lysin biotechnology to provide a contextual framework for the current models of lysin-cell interactions and highlight key considerations for the characterization and design of novel lytic enzymes.

  19. Probing the binding of 8-Acetyl-7-hydroxycoumarin to human serum albumin by spectroscopic methods and molecular modeling

    Science.gov (United States)

    Li, Daojin; Ji, Baoming; Sun, Hairui

    2009-07-01

    Interaction of 8-Acetyl-7-hydroxycoumarin with human serum albumin (HSA) at pH 7.40 has been investigated at 291, 301 and 310 K, respectively, employing the steady fluorescence, circular dichroism (CD) and molecular modeling methods. The quenching mechanism and binding constants were determined by the fluorescence quenching experiments. Thermodynamic data showed that 8-Acetyl-7-hydroxycoumarin was included in the hydrophobic cavity of HSA via hydrophobic interactions. The result of CD indicated that the binding of 8-Acetyl-7-hydroxycoumarin to HSA causes a slight conformational change of the protein. Furthermore, upon binding with HSA, the fluorescence spectra of the 8-Acetyl-7-hydroxycoumarin exhibits appreciable hypsochromic shift associated with an enhancement in the fluorescence intensity. The binding constant ( K) and the standard free energy change (Δ G0) have been also calculated according to the fluorescence data of the ligand, which is in good agreement with the values determined by fluorescence quenching data of HSA. Computational mapping of the possible binding sites of 8-Acetyl-7-hydroxycoumarin revealed that the molecule was bound in the large hydrophobic cavity of subdomain IIA mainly by the hydrophobic interaction and also by the hydrogen bonding interactions between 8-Acetyl-7-hydroxycoumarin and the residues His 242, Arg 222, and Arg 218.

  20. Residuation theory

    CERN Document Server

    Blyth, T S; Sneddon, I N; Stark, M

    1972-01-01

    Residuation Theory aims to contribute to literature in the field of ordered algebraic structures, especially on the subject of residual mappings. The book is divided into three chapters. Chapter 1 focuses on ordered sets; directed sets; semilattices; lattices; and complete lattices. Chapter 2 tackles Baer rings; Baer semigroups; Foulis semigroups; residual mappings; the notion of involution; and Boolean algebras. Chapter 3 covers residuated groupoids and semigroups; group homomorphic and isotone homomorphic Boolean images of ordered semigroups; Dubreil-Jacotin and Brouwer semigroups; and loli

  1. Coarse-grained simulations of hemolytic peptide δ-lysin interacting with a POPC bilayer.

    Science.gov (United States)

    King, Mariah J; Bennett, Ashley L; Almeida, Paulo F; Lee, Hee-Seung

    2016-12-01

    δ-lysin, secreted by a Gram-positive bacterium Staphylococcus aureus, is a 26-residue membrane active peptide that shares many common features with antimicrobial peptides (AMPs). However, it possesses a few unique features that differentiate itself from typical AMPs. In particular, δ-lysin has zero net charge, even though it has many charged residues, and it preferentially lyses eukaryotic cells over bacterial cells. Here, we present the results of coarse-grained molecular dynamics simulations of δ-lysin interacting with a zwitterionic membrane over a wide range of peptide concentrations. When the peptides concentration is low, spontaneous dimerization of peptides is observed on the membrane surface, but deep insertion of peptides or pore formation was not observed. However, the calculated free energy of peptide insertion suggests that a small fraction of peptides is likely to be present inside the membrane at the peptide concentrations typically seen in dye efflux experiments. When the simulations with multiple peptides are carried out with a single pre-inserted transmembrane peptide, spontaneous pore formation occurs with a peptide-to-lipid ratio (P/L) as low as P/L=1:42. Inter-peptide salt bridges among the transmembrane peptides seem to play a role in creating compact pores with very low level of hydration. More importantly, the transmembrane peptides making up the pore are constantly pushed to the opposite side of the membrane when the mass imbalance between the two sides of membrane is significant. Thus, the pore is very dynamic, allowing multiple peptides to translocate across the membrane simultaneously.

  2. Unchanged acetylation of isoniazid by alcohol intake

    DEFF Research Database (Denmark)

    Wilcke, J T R; Døssing, M; Angelo, H R

    2004-01-01

    SETTING: In 10 healthy subjects, the influence of acute alcohol intake on the pharmacokinetics of isoniazid (INH) was studied. OBJECTIVE: To test the hypothesis that alcohol increases the conversion of INH by acetylation into its metabolite acetylisoniazid. DESIGN: In a crossover design, an oral...

  3. Acetylated flavonol triglycosides from Ammi majus L.

    Science.gov (United States)

    Singab, A N

    1998-12-01

    Two new acetylated flavonol triglycosides: kaempferol and isorhamnetin 3-O-[2"-(4"'-acetylrhamnosyl)-6"-glucosyl] glucosides, were isolated and identified from the aerial parts of Ammi majus L. In addition, three known flavonol glycosides namely; isorhamnetin-3-O-rutinoside, kaempferol-3-O-glucoside and isorhamnetin-3-O-glucoside were detected.

  4. Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed in COS-1 cells.

    Science.gov (United States)

    Maruyama, K; MacLennan, D H

    1988-01-01

    Full-length cDNAs encoding neonatal and adult isoforms of the Ca2+-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum were expressed transiently in COS-1 cells. The microsomal fraction isolated from transfected COS-1 cells contained immunoreactive Ca2+-ATPase and catalyzed Ca2+ transport at rates at least 15-fold above controls. No differences were observed in either the rates or Ca2+ dependency of Ca2+ transport catalyzed by the two isoforms. Aspartic acid-351, the site of formation of the catalytic acyl phosphate in the enzyme, was mutated to asparagine, glutamic acid, serine, threonine, histidine, or alanine. In every case, Ca2+ transport activity and Ca2+-dependent phosphorylation were eliminated. Ca2+ transport was also eliminated by mutation of lysine-352 to arginine, glutamine, or glutamic acid or by mutation of Asp351-Lys352 to Lys351-Asp352. Mutation of lysine-515, the site of fluorescein isothiocyanate modification in the enzyme, resulted in diminished Ca2+ transport activity as follows: arginine, 60%; glutamine, 25%; glutamic acid, 5%. These results demonstrate the absolute requirement of acylphosphate formation for the Ca2+ transport function and define a residue important for ATP binding. They also demonstrate the feasibility of a thorough analysis of active sites in the Ca2+-ATPase by expression and site-specific mutagenesis. Images PMID:2966962

  5. Two New Acetyl Cimicifugosides from the Rhizomes of Cimicifuga Racemosa

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Two new acetyl cimicifugosides were isolated from the rhizomes of Cimicifuga racemosa. Their structures were elucidated as 2'-O-acetyl cimicifugoside H-1 1 and 3'-O-acetyl cimicifugoside H-1 2 by the spectroscopic evidence and chemical methods.

  6. Acetylation of Chromatin-Associated Histone H3 Lysine 56 Inhibits the Development of Encysted Artemia Embryos.

    Directory of Open Access Journals (Sweden)

    Rong Zhou

    Full Text Available As a response to harsh environments, the crustacean artemia produces diapause gastrula embryos (cysts, in which cell division and embryonic development are totally arrested. This dormant state can last for very long periods but be terminated by specific environmental stimuli. Thus, artemia is an ideal model organism in which to study cell cycle arrest and embryonic development.Our study focuses on the roles of H3K56ac in the arrest of cell cycle and development during artemia diapause formation and termination. We found that the level of H3K56ac on chromatin increased during diapause formation, and decreased upon diapause termination, remaining basal level throughout subsequent embryonic development. In both HeLa cells and artemia, blocking the deacetylation with nicotinamide, a histone deacetylase inhibitor, increased the level of H3K56ac on chromatin and induced an artificial cell cycle arrest. Furthermore, we found that this arrest of the cell cycle and development was induced by H3K56ac and dephosphorylation of the checkpoint protein, retinoblastoma protein.These results have revealed the dynamic change in H3K56ac on chromatin during artemia diapause formation and termination. Thus, our findings provide insight into the regulation of cell division during arrest of artemia embryonic development and provide further insight into the functions of H3K56ac.

  7. Probing structural differences between PrPC and PrPSc by surface nitration and acetylation: evidence of conformational change in the C-terminus

    Science.gov (United States)

    We used two chemical modifiers, tetranitromethane (TNM) and acetic anhydride, which specifically target accessible tyrosine and lysine residues, respectively, to modify Syrian hamster recombinant PrP(90-231) (rPrP) and PrP27-30, aiming at finding locations of conformational change. Modified proteins...

  8. Dysregulated Lysine Acetyltransferase 2B Promotes Inflammatory Bowel Disease Pathogenesis Through Transcriptional Repression of Interleukin-10.

    Science.gov (United States)

    Bai, Alfa H C; Wu, William K K; Xu, Liangliang; Wong, Sunny H; Go, Minnie Y; Chan, Anthony W H; Harbord, Marcus; Zhang, Shenghong; Chen, Minhu; Wu, Justin C Y; Chan, Michael W Y; Chan, Matthew T V; Chan, Francis K L; Sung, Joseph J Y; Yu, Jun; Cheng, Alfred S L; Ng, Siew C

    2016-06-01

    Accumulating evidence supports epigenetic modifications in mediating intestinal immunity in inflammatory bowel disease [IBD] pathogenesis. This study aimed to identify key dysregulated epigenetic modulators and the molecular downstream pathways in IBD. Expression of 116 well-defined epigenetic modulators was profiled and validated in 96 intestinal tissues from patients with Crohn's disease [CD], ulcerative colitis [UC], and healthy controls using quantitative reverse transcriptase polymerase chain reaction [QRT-PCR], western blot, and immunohistochemistry. Dysregulation of histone modifications and IBD-related cytokines were examined by chromatin immunoprecipitation, luciferase activity, and gene expression analyses in normal colonic epithelial cell line, NCM460, upon small-molecule inhibition or RNA interference, followed by validation in primary colonic tissues. Targeted expression profiling uncovered seven differentially expressed epigenetic modulators, of which the down-regulation of lysine acetyltransferase 2B [KAT2B] mRNA and protein was the most significant and was consequently validated in inflamed CD and UC compared with healthy colonic tissues. KAT2B protein localised abundantly in nuclei of normal colonic epithelium but diminished in paired inflamed CD and UC tissues. Pharmacological inhibition of KAT2B by anacardic acid in NCM460 cells reduced the levels of histone H4 lysine 5 acetylation [H4K5ac] and interleukin-10 [IL-10] in a dose-dependent manner. Knockdown of KAT2B reduced the IL-10 promoter occupancy of KAT2B and H4K5ac, resulting in transcriptional silencing. IL-10 level was also diminished in inflamed IBD tissues. Our findings demonstrated a novel epigenetic mechanism of IL-10 dysregulation in IBD. Down-regulation of KAT2B may disrupt the innate and adaptive inflammatory responses due to the suppression of this crucial anti-inflammatory cytokine. Copyright © 2016 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University

  9. Acetylation of FoxO1 Activates Bim Expression to Induce Apoptosis in Response to Histone Deacetylase Inhibitor Depsipeptide Treatment

    Directory of Open Access Journals (Sweden)

    Yang Yang

    2009-04-01

    Full Text Available Histone deacetylase (HDAC inhibitors have been shown to induce cell cycle arrest and apoptosis in cancer cells. However, the mechanisms of HDAC inhibitor induced apoptosis are incompletely understood. In this study, depsipeptide, a novel HDAC inhibitor, was shown to be able to induce significant apoptotic cell death in human lung cancer cells. Further study showed that Bim, a BH3-only proapoptotic protein, was significantly upregulated by depsipeptide in cancer cells, and Bim's function in depsipeptide-induced apoptosis was confirmed by knockdown of Bim with RNAi. In addition, we found that depsipeptide-induced expression of Bim was directly dependent on acetylation of forkhead box class O1 (FoxO1 that is catalyzed by cyclic adenosine monophosphate-responsive element-binding protein-binding protein, and indirectly induced by a decreased four-and-a-half LIM-domain protein 2. Moreover, our results demonstrated that FoxO1 acetylation is required for the depsipeptide-induced activation of Bim and apoptosis, using transfection with a plasmid containing FoxO1 mutated at lysine sites and a luciferase reporter assay. These data show for the first time that an HDAC inhibitor induces apoptosis through the FoxO1 acetylation-Bim pathway.

  10. Peptide based DNA nanocarriers incorporating a cell-penetrating peptide derived from neurturin protein and poly-L-lysine dendrons.

    Science.gov (United States)

    Rosli, Nurlina; Christie, Michelle P; Moyle, Peter M; Toth, Istvan

    2015-05-15

    Multicomponent gene delivery systems incorporating cell-penetrating peptides (CPP) from the human neurturin protein (NRTN-30, NRTN(132-161); NRTN-17, NRTN(145-161)) and a poly-l-lysine (PLL) dendron, were synthesized and characterized for plasmid DNA (pDNA) delivery. Acetylated NRTN peptides (Ac-CPP) and peptides conjugated to a PLL dendron (DEN-CPP) efficiently condensed and stabilized pDNA. Complexes between pDNA and DEN-CPP formed smaller and more stable nanoparticles. Flow cytometry experiments showed that pDNA-DEN-CPPs were taken up more efficiently into HeLa cells. There was also no significant difference between NRTN-30 and NRTN-17 for pDNA uptake, indicating that the truncated peptide alone is sufficient as a CPP for pDNA delivery.

  11. Duplicate abalone egg coat proteins bind sperm lysin similarly, but evolve oppositely, consistent with molecular mimicry at fertilization.

    Directory of Open Access Journals (Sweden)

    Jan E Aagaard

    Full Text Available Sperm and egg proteins constitute a remarkable paradigm in evolutionary biology: despite their fundamental role in mediating fertilization (suggesting stasis, some of these molecules are among the most rapidly evolving ones known, and their divergence can lead to reproductive isolation. Because of strong selection to maintain function among interbreeding individuals, interacting fertilization proteins should also exhibit a strong signal of correlated divergence among closely related species. We use evidence of such molecular co-evolution to target biochemical studies of fertilization in North Pacific abalone (Haliotis spp., a model system of reproductive protein evolution. We test the evolutionary rates (d(N/d(S of abalone sperm lysin and two duplicated egg coat proteins (VERL and VEZP14, and find a signal of co-evolution specific to ZP-N, a putative sperm binding motif previously identified by homology modeling. Positively selected residues in VERL and VEZP14 occur on the same face of the structural model, suggesting a common mode of interaction with sperm lysin. We test this computational prediction biochemically, confirming that the ZP-N motif is sufficient to bind lysin and that the affinities of VERL and VEZP14 are comparable. However, we also find that on phylogenetic lineages where lysin and VERL evolve rapidly, VEZP14 evolves slowly, and vice versa. We describe a model of sexual conflict that can recreate this pattern of anti-correlated evolution by assuming that VEZP14 acts as a VERL mimic, reducing the intensity of sexual conflict and slowing the co-evolution of lysin and VERL.

  12. protein, tryptophan and lysine contents in quality protien maize ...

    African Journals Online (AJOL)

    owner

    for human nutrition recommended by Food and Agriculture Organization in ... METHODS: The protein, tryptophan and lysine contents of improved ... This study revealed the fact that genetic factor influences the protein, ... Ethiop J Health Sci.

  13. Digestible lysine levels in diets for laying Japanese quails

    Directory of Open Access Journals (Sweden)

    Cleverson Luís Nascimento Ribeiro

    2013-07-01

    Full Text Available The objective of this study was to estimate the digestible lysine requirement of Japanese quails in the egg-laying phase. A total of 336 female Japanese quails (Coturnix coturnix japonica of average initial age of 207 days were distributed in a completely randomized experimental design, composed of 6 treatments (lysine levels with 7 replicates and 8 birds per experimental unit, with duration of 84 days. Experimental diets were formulated from a basal diet, with corn and soybean meal, with 2.800 kcal ME/kg and 203.70 g/kg crude protein, showing levels of 9.50; 10.00; 10.50; 11.00; 11.50; and 12.00 g/kg digestible lysine; diets remained isoprotein and isocaloric. The following variables were studied: feed intake (FI; lysine intake (LI; egg production per bird per day (EPBD; egg production per bird housed (EPBH; production of marketable eggs (PME; egg weight (EW; egg mass (EM; utilization efficiency of lysine for egg mass production (UELEM; feed conversion per mass (FCEM; feed conversion per dozen eggs (FCDZ; bird availability (BA; percentages of yolk (Y, albumen (A and shell (S; specific egg weight (SW; nitrogen ingested (NI; nitrogen excreted (NE; and nitrogen balance (NB. Significant effect was only observed for LI, EW, EM, UELEM, FCEM, Y, A and SW. The digestible lysine level estimated in diets for laying Japanese quails is 11.20 g digestible lysine/kg diet, corresponding to an average daily intake of 272.23 mg lysine.

  14. A lysine-to-arginine mutation on NEDD8 markedly reduces the activity of cullin RING E3 ligase through the impairment of neddylation cascades

    Energy Technology Data Exchange (ETDEWEB)

    Sui, Yiyan; Liu, Yaobin; Xu, Guoqiang, E-mail: gux2002@suda.edu.cn

    2015-06-12

    Neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) is a ubiquitin-like modifier, which forms covalent conjugates on lysines of its substrates. This post-translational modification, neddylation, plays important roles in tumor cell proliferation and viability. Ubiquitin can form diverse polyubiquitin chains, on its seven lysines, which play important functions in various biological processes. However, the roles of lysines in NEDD8 have not been explored. Here, we generated nine NEDD8 point mutants, each with one lysine replaced by an arginine, to study the putative function of lysines in NEDD8. Our experiments discover that Lys27 in NEDD8 is a critical residue for protein neddylation. Replacement of this residue with arginine almost completely eliminates the conjugation of NEDD8 to its substrates. Furthermore, we find that the K27R mutant impairs NEDD8 conjugation to the E2 enzyme, which normally forms thioester bonds for further transferring NEDD8 to its ligases and substrates. Therefore, this mutation completely inhibits global protein neddylation, including neddylation of cullin family proteins, resulting in decreased activity of cullin-RING E3 ligases. This work sheds new light on the roles of NEDD8 lysines on neddylation cascades and provides a dominant negative mutant for the study of neddylation and its biological functions. - Highlights: • Lys27 in NEDD8 is critical for protein neddylation. • NEDD8 K27R mutant impairs the NEDD8 conjugation. • NEDD8 K27R mutant significantly reduces the activity of cullin-RING E3 ligases.

  15. Maintenance requirement and deposition efficiency of lysine in pigs

    Directory of Open Access Journals (Sweden)

    Marcos Speroni Ceron

    2013-09-01

    Full Text Available The objective of this work was to determine the maintenance requirement and the deposition efficiency of lysine in growing pigs. It was used the incomplete changeover experimental design, with replicates over time. Twelve castrated pigs with average body weight (BW of 52±2 kg were kept in metabolism crates with a controlled temperature of 22ºC. The diets were formulated to supply 30, 50, 60, and 70% of the expected requirements of standardized lysine, and provided at 2.6 times the energy requirements for maintenance. The trial lasted 24 days and was divided into two periods of 12 days: seven days for animal adaptation to the diet and five days for sample collection. The increasing content of lysine in the diet did not affect dry matter intake of the pigs. The amount of nitrogen excreted was 47% of the nitrogen intake, of which 35% was excreted through feces and 65% through urine. The estimated endogenous losses of lysine were 36.4 mg kg-1 BW0.75. The maintenance requirement of lysine for pigs weighing around 50 kg is 40.4 mg kg-1 BW0.75, and the deposition efficiency of lysine is 90%.

  16. Targeting protein lysine methylation and demethylation in cancers

    Institute of Scientific and Technical Information of China (English)

    Yunlong He; Ilia Korboukh; Jian Jin; Jing Huang

    2012-01-01

    During the last decade,we saw an explosion of studies investigating the role of lysine methylation/demethylation of histones and non-histone proteins,such as p53,NF-kappaB,and E2F1.These ‘Ying-Yang' post-translational modifications are important to fine-tuning the activity of these proteins. Lysine methylation and demethylation are catalyzed by protein lysine methyltransferases (PKMTs) and protein lysine demethylases (PKDMs).PKMTs,PKDMs,and their substrates have been shown to play important roles in cancers.Although the underlying mechanisms of tumorigenesis are still largely unknown,growing evidence is starting to link aberrant regulation of methylation to tumorigenesis.This review focuses on summarizing the recent progress in understanding of the function of protein lysine methylation,and in the discovery of small molecule inhibitors for PKMTs and PKDMs.We also discuss the potential and the caveats of targeting protein lysine methylation for the treatment of cancer.

  17. Enzymic and chemical synthesis of epilson-N-(L-propionyl-2)-L-lysine.

    Science.gov (United States)

    Fujioka, M; Tanaka, M

    1978-10-01

    Pyruvate was shown to act as an oxo acid substrate in the reverse direction of saccharopine dehydrogenase [epsilon N-(L-glutaryl-2)-L-lysine: NAD oxidoreductase (L-lysine-forming)] reaction. The enzymic condensation product of lysine and pyruvate was isolated and identified as epsilon-N-(L-propionyl-2)-L-lysine by comparison with the synthetic compound. A method for the chemical preparation of diastereoisomers of epsilon-N-(propionyl-2)-L-lysine is also described.

  18. p53 Acetylation: Regulation and Consequences

    OpenAIRE

    2014-01-01

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo ev...

  19. Preparation and characterization of acetylated starch nanoparticles as drug carrier: Ciprofloxacin as a model.

    Science.gov (United States)

    Mahmoudi Najafi, Seyed Heydar; Baghaie, Maryam; Ashori, Alireza

    2016-06-01

    The objective of this study was to characterize in-vitro the potential of acetylated corn starch (ACS) particles as a matrix for the delivery of ciprofloxacin (CFx). ACS was successfully synthesized and optimized by the reaction of native corn starch using acetic anhydride and acetic acid with low and high degrees of substitution (DS). The nanoprecipitation method was applied for the formation of the ACS-based nanoparticles, by the dropwise addition of water to acetone solution of ACS under stirring. The effects of acetylation and nanoprecipitation on the morphology and granular structure of ACS samples were examined by the FT-IR, XRD, DSL and SEM techniques. The efficiency of CFx loading was also evaluated via encapsulation efficiency (EE) in ACS nanoparticles. The average degree of acetyl substitution per glucose residue of corn starch was 0.33, 2.00, and 2.66. The nanoparticles size of the ACS and ACS-loaded with CFx were measured and analyzed relative to the solvent:non-solvent ratio. Based on the results, ACS nanoparticles with DS of 2.00 and water:acetone of 3:1 had 312nm diameter. Increasing DS in starch acetate led to increase in the EE from 67.7 to 89.1% and with increasing ratio of water/acetone from 1:1 to 3:1, the EE raised from 48.5 to 89.1%. X-ray diffraction indicated that A-type pattern of native starch was completely transformed into the V-type pattern of acetylated starch. The scanning electron microscopy showed that the different sizes of pores formed on the acetylated starch granules were utterly converted into the uniform-sized spherical nanoparticles after the nanoprecipitation. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Metabolism of Monoterpenes: Acetylation of (-)-Menthol by a Soluble Enzyme Preparation from Peppermint (Mentha piperita) Leaves.

    Science.gov (United States)

    Croteau, R; Hooper, C L

    1978-05-01

    The essential oil from mature leaves of flowering peppermint (Mentha piperita L.) contains up to 15% (-)-menthyl acetate, and leaf discs converted exogenous (-)-[G-(3)H]menthol into this ester in approximately 15% yield of the incorporated precursor. Leaf extracts catalyzed the acetyl coenzyme A-dependent acetylation of (-)-[G-(3)H]menthol and the product of this transacetylase reaction was identified by radiochromatographic techniques. Transacetylase activity was located mainly in the 100,000g supernatant fraction, and the preparation was partially purified by combination of Sephadex G-100 gel filtration and chromatography on O-diethylaminoethyl-cellulose. The transacetylase had a molecular weight of about 37,000 as judged by Sephadex G-150 gel filtration, and a pH optimum near 9. The apparent K(m) and velocity for (-)-menthol were 0.3 mm and 16 nmol/hr. mg of protein, respectively. The saturation curve for acetyl coenzyme A was sigmoidal, showing apparent saturation near 0.1 mm. Dithioerythritol was required for maximum activity and stability of the enzyme, and the enzyme was inhibited by thiol directed reagents such as p-hydroxymercuribenzoate. Diisopropylfluorophosphate also inhibited transacylation suggesting the involvement of a serine residue in catalysis. The transacylase was highly specific for acetyl coenzyme A; propionyl coenzyme A and butyryl coenzyme A were not nearly as efficient as acyl donors (11% and 2%, respectively). However, the enzyme was much less selective with regard to the alcohol substrate, suggesting that the nature of the acetate ester synthesized in mint is more dependent on the type of alcohol available than on the specificity of the transacetylase. This is the first report on an enzyme involved in monoterpenol acetylation in plants. A very similar enzyme, catalyzing this key reaction in the metabolism of menthol, was also isolated from the flowers of peppermint.