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Sample records for global protein acetylation

  1. Global Analysis of Lysine Acetylation Suggests the Involvement of Protein Acetylation in Diverse Biological Processes in Rice (Oryza sativa)

    PubMed Central

    Zhong, Xiaoxian; Tan, Feng; Mujahid, Hana; Zhang, Jian; Nanduri, Bindu; Peng, Zhaohua

    2014-01-01

    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. PMID:24586658

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

    PubMed

    Nallamilli, Babi Ramesh Reddy; Edelmann, Mariola J; Zhong, Xiaoxian; Tan, Feng; Mujahid, Hana; Zhang, Jian; Nanduri, Bindu; Peng, Zhaohua

    2014-01-01

    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.

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

    PubMed

    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.

  4. Genetic inhibition of hepatic acetyl-CoA carboxylase activity increases liver fat and alters global protein acetylationa

    PubMed Central

    Chow, Jenny D.Y.; Lawrence, Robert T.; Healy, Marin E.; Dominy, John E.; Liao, Jason A.; Breen, David S.; Byrne, Frances L.; Kenwood, Brandon M.; Lackner, Carolin; Okutsu, Saeko; Mas, Valeria R.; Caldwell, Stephen H.; Tomsig, Jose L.; Cooney, Gregory J.; Puigserver, Pere B.; Turner, Nigel; James, David E.; Villén, Judit; Hoehn, Kyle L.

    2014-01-01

    Lipid deposition in the liver is associated with metabolic disorders including fatty liver disease, type II diabetes, and hepatocellular cancer. The enzymes acetyl-CoA carboxylase 1 (ACC1) and ACC2 are powerful regulators of hepatic fat storage; therefore, their inhibition is expected to prevent the development of fatty liver. In this study we generated liver-specific ACC1 and ACC2 double knockout (LDKO) mice to determine how the loss of ACC activity affects liver fat metabolism and whole-body physiology. Characterization of LDKO mice revealed unexpected phenotypes of increased hepatic triglyceride and decreased fat oxidation. We also observed that chronic ACC inhibition led to hyper-acetylation of proteins in the extra-mitochondrial space. In sum, these data reveal the existence of a compensatory pathway that protects hepatic fat stores when ACC enzymes are inhibited. Furthermore, we identified an important role for ACC enzymes in the regulation of protein acetylation in the extra-mitochondrial space. PMID:24944901

  5. Protein acetylation in archaea, bacteria, and eukaryotes.

    PubMed

    Soppa, Jörg

    2010-09-16

    Proteins can be acetylated at the alpha-amino group of the N-terminal amino acid (methionine or the penultimate amino acid after methionine removal) or at the epsilon-amino group of internal lysines. In eukaryotes the majority of proteins are N-terminally acetylated, while this is extremely rare in bacteria. A variety of studies about N-terminal acetylation in archaea have been reported recently, and it was revealed that a considerable fraction of proteins is N-terminally acetylated in haloarchaea and Sulfolobus, while this does not seem to apply for methanogenic archaea. Many eukaryotic proteins are modified by differential internal acetylation, which is important for a variety of processes. Until very recently, only two bacterial proteins were known to be acetylation targets, but now 125 acetylation sites are known for E. coli. Knowledge about internal acetylation in archaea is extremely limited; only two target proteins are known, only one of which--Alba--was used to study differential acetylation. However, indications accumulate that the degree of internal acetylation of archaeal proteins might be underestimated, and differential acetylation has been shown to be essential for the viability of haloarchaea. Focused proteomic approaches are needed to get an overview of the extent of internal protein acetylation in archaea.

  6. Bacterial protein acetylation: new discoveries unanswered questions.

    PubMed

    Wolfe, Alan J

    2016-05-01

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

  7. Structural, Kinetic and Proteomic Characterization of Acetyl Phosphate-Dependent Bacterial Protein Acetylation

    PubMed Central

    Sahu, Alexandria; Sorensen, Dylan; Minasov, George; Lima, Bruno P.; Scholle, Michael; Mrksich, Milan; Anderson, Wayne F.; Gibson, Bradford W.; Schilling, Birgit; Wolfe, Alan J.

    2014-01-01

    The emerging view of Nε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA) to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that Nε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of Escherichia coli proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP)-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial Nε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase. PMID:24756028

  8. Nonhistone protein acetylation as cancer therapy targets

    PubMed Central

    Singh, Brahma N; Zhang, Guanghua; Hwa, Yi L; Li, Jinping; Dowdy, Sean C; Jiang, Shi-Wen

    2012-01-01

    Acetylation and deacetylation are counteracting, post-translational modifications that affect a large number of histone and nonhistone proteins. The significance of histone acetylation in the modification of chromatin structure and dynamics, and thereby gene transcription regulation, has been well recognized. A steadily growing number of nonhistone proteins have been identified as acetylation targets and reversible lysine acetylation in these proteins plays an important role(s) in the regulation of mRNA stability, protein localization and degradation, and protein–protein and protein–DNA interactions. The recruitment of histone acetyltransferases (HATs) and histone deacetylases (HDACs) to the transcriptional machinery is a key element in the dynamic regulation of genes controlling cellular proliferation, differentiation and apoptosis. Many nonhistone proteins targeted by acetylation are the products of oncogenes or tumor-suppressor genes and are directly involved in tumorigenesis, tumor progression and metastasis. Aberrant activity of HDACs has been documented in several types of cancers and HDAC inhibitors (HDACi) have been employed for therapeutic purposes. Here we review the published literature in this field and provide updated information on the regulation and function of nonhistone protein acetylation. While concentrating on the molecular mechanism and pathways involved in the addition and removal of the acetyl moiety, therapeutic modalities of HDACi are also discussed. PMID:20553216

  9. Nε- and O-Acetylation in Mycobacterium tuberculosis Lineage 7 and Lineage 4 strains: Proteins Involved in Bioenergetics, Virulence and Antimicrobial Resistance are Acetylated.

    PubMed

    Birhanu, Alemayehu Godana; Yimer, Solomon Abebe; Holm-Hansen, Carol; Norheim, Gunnstein; Aseffa, Abraham; Abebe, Markos; Tønjum, Tone

    2017-09-18

    Increasing evidence demonstrates that lysine acetylation is involved in Mycobacterium tuberculosis (Mtb) virulence and pathogenesis. However, previous investigations in Mtb have only monitored acetylation at lysine residues using selected reference strains. We analyzed the global Nε- and O-acetylation of 3 Mtb isolates; 2 lineage 7 clinical isolates and the lineage 4 H37Rv reference strain. Quantitative acetylome analysis resulted in identification of 2490 class-I acetylation sites, among them 2349 O-acetylation and 141 Nε-acetylation sites, derived from 953 unique proteins. Mtb O-acetylation was thereby significantly more abundant than Nε-acetylation. The acetylated proteins were found to be involved in central metabolism, translation, stress responses and antimicrobial drug resistance. Notably, 261 acetylation sites on 165 proteins were differentially regulated between lineage 7 and lineage 4 strains. A total of 257 acetylation sites on 161 proteins were hypoacetylated in lineage 7 strains. These proteins are involved in Mtb growth, virulence, bioenergetics, host-pathogen interaction and stress responses. This study provides the first global analysis of O-acetylated proteins in Mtb. This quantitative acetylome data expand the current understanding regarding the nature and diversity of acetylated proteins in Mtb, and opens a new avenue of research for exploring the role of protein acetylation in Mtb physiology.

  10. Protein Acetylation in Procaryotes Increases Stress Resistance

    PubMed Central

    Ma, Qun; Wood, Thomas K.

    2011-01-01

    Acetylation of lysine residues is conserved in all three kingdoms; however, its role in prokaryotes is unknown. Here we demonstrate that acetylation enables the reference bacterium Escherichia coli to withstand environmental stress. Specifically, the bacterium reaches higher cell densities and becomes more resistant to heat and oxidative stress when its proteins are acetylated as shown by deletion of the gene encoding acetyltransferase YfiQ and the gene encoding deacetylase CobB as well as by overproducing YfiQ and CobB. Furthermore, we show that the increase in oxidative stress resistance with acetylation is due to the induction of catalase activity through enhanced katG expression. We also found that two-component system proteins CpxA, PhoP, UvrY, and BasR are associated with cell catalase activity and may be responsible as the connection between bacterial acetylation and the stress response. This is the first demonstration of a specific environmental role of acetylation in prokaryotes. PMID:21703240

  11. Protein acetylation in prokaryotes increases stress resistance.

    PubMed

    Ma, Qun; Wood, Thomas K

    2011-07-15

    Acetylation of lysine residues is conserved in all three kingdoms; however, its role in prokaryotes is unknown. Here we demonstrate that acetylation enables the reference bacterium Escherichia coli to withstand environmental stress. Specifically, the bacterium reaches higher cell densities and becomes more resistant to heat and oxidative stress when its proteins are acetylated as shown by deletion of the gene encoding acetyltransferase YfiQ and the gene encoding deacetylase CobB as well as by overproducing YfiQ and CobB. Furthermore, we show that the increase in oxidative stress resistance with acetylation is due to the induction of catalase activity through enhanced katG expression. We also found that two-component system proteins CpxA, PhoP, UvrY, and BasR are associated with cell catalase activity and may be responsible as the connection between bacterial acetylation and the stress response. This is the first demonstration of a specific environmental role of acetylation in prokaryotes. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Mechanisms and Dynamics of Protein Acetylation in Mitochondria

    PubMed Central

    Baeza, Josue; Smallegan, Michael J.; Denu, John M.

    2016-01-01

    Reversible protein acetylation is a major regulatory mechanism for controlling protein function. Through genetic manipulations, dietary perturbations, and new proteomic technologies, the diverse functions of protein acetylation are coming into focus. Protein acetylation in mitochondria has taken center stage, revealing that 63% of mitochondrially localized proteins contain lysine acetylation sites. Here we summarize the field, and discuss salient topics that cover spurious versus targeted acetylation, the role of SIRT3 deacetylation, nonenzymatic acetylation, and molecular models for regulatory acetylations that display high and low stoichiometry. PMID:26822488

  13. Global proteomic analysis of lysine acetylation in zebrafish (Danio rerio) embryos.

    PubMed

    Kwon, Oh Kwang; Kim, Sunjoo; Lee, Sangkyu

    2016-12-01

    Lysine acetylation is an important post-translational modification (PTM). Since the development of MS-based proteomics technology, important roles of lysine acetylation beyond histones have focused on chromatin remodeling during the cell cycle and regulation of nuclear transport, metabolism, and translation. Zebrafish (Danio rerio) is a widely used vertebrate model in genetics and biologic studies. Although studies in several mammalian species have been performed, the mechanism of lysine acetylation in D. rerio embryos is incompletely understood. Here, we investigated the global acetylome in D. rerio embryos by using an MS-based proteomics approach. We identified 351 acetylated peptides and 377 nonredundant acetylation sites on 189 lysine-acetylated proteins in 5-day postfertilization (hpf) embryos of D. rerio. Among lysine-acetylated peptides, 40.2% indicated three motifs: (ac)KxxxK, (ac)KxxxxK, and Lx(ac)K. Of 190 acetylated proteins, 81 (42.6%) were mainly distributed in the cytoplasm. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that lysine acetylation in D. rerio was enriched in metabolic pathways. Additionally, 17 of 30 acetylated ribosomal proteins were evolutionarily conserved between zebrafish and humans. Our results indicate that acetyllysine might have regulatory effects on ribosomal proteins involved in protein biosynthesis. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

    Choudhary, Chunaram; Kumar, Chanchal; Gnad, Florian; Nielsen, Michael L; Rehman, Michael; Walther, Tobias C; Olsen, Jesper V; Mann, Matthias

    2009-08-14

    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 lysine acetylation sites on 1750 proteins and quantified acetylation changes in response to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275. Lysine acetylation preferentially targets large macromolecular complexes involved in diverse cellular processes, such as chromatin remodeling, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other major posttranslational modifications.

  15. Identification of lysine-acetylated mitochondrial proteins and their acetylation sites.

    PubMed

    Hartl, Markus; König, Ann-Christine; Finkemeier, Iris

    2015-01-01

    The (ε)N-acetylation of lysine side chains is a highly conserved posttranslational modification of both prokaryotic and eukaryotic proteins. Lysine acetylation not only occurs on histones in the nucleus but also on many mitochondrial proteins in plants and animals. As the transfer of the acetyl group to lysine eliminates its positive charge, lysine acetylation can affect the biological function of proteins. This chapter describes two methods for the identification of lysine-acetylated proteins in plant mitochondria using an anti-acetyllysine antibody. We describe the Western blot analysis of a two-dimensional blue native-polyacrylamide gel electrophoresis with an anti-acetyllysine antibody as well as the immuno-enrichment of lysine-acetylated peptides followed by liquid chromatography-tandem mass spectrometry data acquisition and analysis.

  16. Global analysis of lysine acetylation in strawberry leaves

    PubMed Central

    Fang, Xianping; Chen, Wenyue; Zhao, Yun; Ruan, Songlin; Zhang, Hengmu; Yan, Chengqi; Jin, Liang; Cao, Lingling; Zhu, Jun; Ma, Huasheng; Cheng, Zhongyi

    2015-01-01

    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. PMID:26442052

  17. Comparison of the acetylation of proteins and nucleic acids

    PubMed Central

    Paik, Woon Ki; Kim, Sangduk

    1970-01-01

    The possibility of acetylation of nucleic acids was examined. Although protein is actively acetylated with [1-14C]acetic acid in rat liver systems in vivo and in vitro and in a frog liver system in vivo, nucleic acids are not acetylated under these conditions; nucleic acids purified from these sources are without radioactivity. Requirements for acetylation in vitro of protein in rat liver are different from those in frog liver; GSH has no effect in the rat liver system and is inhibitory in the frog liver system. Among various acetylated proteins, proteins insoluble in 0.1m-sulphuric acid have the highest radioactivity. PMID:5435491

  18. Acetylation unleashes protein demons of dementia.

    PubMed

    Mattson, Mark P

    2010-09-23

    Aberrant posttranslational modifications of proteins can impair synaptic plasticity and may render neurons vulnerable to degeneration during aging. In this issue of Neuron, Min et al. show that acetylation of the amino acid lysine in the microtubule-associated protein tau prevents its ubiquitin-mediated degradation, resulting in "tau tangles" similar to those of dementias. Other recent studies suggest that lysine hyperacetylation contributes to the accumulation of amyloid β-peptide in Alzheimer's disease and to impaired cognitive function resulting from a trophic factor deficit. Copyright © 2010 Elsevier Inc. All rights reserved.

  19. Dynamic Protein Acetylation in Plant–Pathogen Interactions

    PubMed Central

    Song, Gaoyuan; Walley, Justin W.

    2016-01-01

    Pathogen infection triggers complex molecular perturbations within host cells that results in either resistance or susceptibility. Protein acetylation is an emerging biochemical modification that appears to play central roles during host–pathogen interactions. To date, research in this area has focused on two main themes linking protein acetylation to plant immune signaling. Firstly, it has been established that proper gene expression during defense responses requires modulation of histone acetylation within target gene promoter regions. Second, some pathogens can deliver effector molecules that encode acetyltransferases directly within the host cell to modify acetylation of specific host proteins. Collectively these findings suggest that the acetylation level for a range of host proteins may be modulated to alter the outcome of pathogen infection. This review will focus on summarizing our current understanding of the roles of protein acetylation in plant defense and highlight the utility of proteomics approaches to uncover the complete repertoire of acetylation changes triggered by pathogen infection. PMID:27066055

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

    PubMed

    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.

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

    PubMed Central

    Miller, Kyle M.

    2016-01-01

    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. PMID:27631103

  2. Protein acetylation affects acetate metabolism, motility and acid stress response in Escherichia coli

    PubMed Central

    Castaño-Cerezo, Sara; Bernal, Vicente; Post, Harm; Fuhrer, Tobias; Cappadona, Salvatore; Sánchez-Díaz, Nerea C; Sauer, Uwe; Heck, Albert JR; Altelaar, AF Maarten; Cánovas, Manuel

    2014-01-01

    Although protein acetylation is widely observed, it has been associated with few specific regulatory functions making it poorly understood. To interrogate its functionality, we analyzed the acetylome in Escherichia coli knockout mutants of cobB, the only known sirtuin-like deacetylase, and patZ, the best-known protein acetyltransferase. For four growth conditions, more than 2,000 unique acetylated peptides, belonging to 809 proteins, were identified and differentially quantified. Nearly 65% of these proteins are related to metabolism. The global activity of CobB contributes to the deacetylation of a large number of substrates and has a major impact on physiology. Apart from the regulation of acetyl-CoA synthetase, we found that CobB-controlled acetylation of isocitrate lyase contributes to the fine-tuning of the glyoxylate shunt. Acetylation of the transcription factor RcsB prevents DNA binding, activating flagella biosynthesis and motility, and increases acid stress susceptibility. Surprisingly, deletion of patZ increased acetylation in acetate cultures, which suggests that it regulates the levels of acetylating agents. The results presented offer new insights into functional roles of protein acetylation in metabolic fitness and global cell regulation. PMID:25518064

  3. Regulation, Function, and Detection of Protein Acetylation in Bacteria.

    PubMed

    Carabetta, Valerie J; Cristea, Ileana M

    2017-08-15

    N(ε)-Lysine acetylation is now recognized as an abundant posttranslational modification (PTM) that influences many essential biological pathways. Advancements in mass spectrometry-based proteomics have led to the discovery that bacteria contain hundreds of acetylated proteins, contrary to the prior notion of acetylation events being rare in bacteria. Although the mechanisms that regulate protein acetylation are still not fully defined, it is understood that this modification is finely tuned via both enzymatic and nonenzymatic mechanisms. The opposing actions of Gcn5-related N-acetyltransferases (GNATs) and deacetylases, including sirtuins, provide the enzymatic control of lysine acetylation. A nonenzymatic mechanism of acetylation has also been demonstrated and proven to be prominent in bacteria, as well as in mitochondria. The functional consequences of the vast majority of the identified acetylation sites remain unknown. From studies in mammalian systems, acetylation of critical lysine residues was shown to impact protein function by altering its structure, subcellular localization, and interactions. It is becoming apparent that the same diversity of functions can be found in bacteria. Here, we review current knowledge of the mechanisms and the functional consequences of acetylation in bacteria. Additionally, we discuss the methods available for detecting acetylation sites, including quantitative mass spectrometry-based methods, which promise to promote this field of research. We conclude with possible future directions and broader implications of the study of protein acetylation in bacteria. Copyright © 2017 American Society for Microbiology.

  4. Antemortem stress regulates protein acetylation and glycolysis in postmortem muscle.

    PubMed

    Li, Zhongwen; Li, Xin; Wang, Zhenyu; Shen, Qingwu W; Zhang, Dequan

    2016-07-01

    Although exhaustive research has established that preslaughter stress is a major factor contributing to pale, soft, exudative (PSE) meat, questions remain regarding the biochemistry of postmortem glycolysis. In this study, the influence of preslaughter stress on protein acetylation in relationship to glycolysis was studied. The data show that antemortem swimming significantly enhanced glycolysis and the total acetylated proteins in postmortem longissimus dorsi (LD) muscle of mice. Inhibition of protein acetylation by histone acetyltransferase (HAT) inhibitors eliminated stress induced increase in glycolysis. Inversely, antemortem injection of histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and nicotinamide (NAM), further increased protein acetylation early postmortem and the glycolysis. These data provide new insight into the biochemistry of postmortem glycolysis by showing that protein acetylation regulates glycolysis, which may participate in the regulation of preslaughter stress on glycolysis in postmortem muscle. Copyright © 2016. Published by Elsevier Ltd.

  5. Acetylation of prostaglandin synthetase by aspirin. Purification and properties of the acetylated protein from sheep vesicular gland.

    PubMed

    Roth, G J; Stanford, N; Jacobs, J W; Majerus, P W

    1977-09-20

    We previously presented evidence that aspirin (acetylsalicylic acid) inhibits prostaglandin synthetase by acetylating and active site of the enzyme. In the current work, we have labeled the enzyme from an aceton-pentane powder of sheep vesicular gland using [acetyl-3H]aspirin and purified the [3H]acetyl-protein to near homogeneity. The final preparation contains protein of a single molecular weight (85 000) and an amino-terminal sequence of Asp-Ala-Gly-Arg-Ala. The [3H]acetyl-protein contained 0.5 mol of acetyl residues per mol of protein based on amino acid composition but only a single sequence was found.

  6. Mitochondrial protein acetylation mediates nutrient sensing of mitochondrial protein synthesis and mitonuclear protein balance.

    PubMed

    Di Domenico, Antonella; Hofer, Annette; Tundo, Federica; Wenz, Tina

    2014-11-01

    Changes in nutrient supply require global metabolic reprogramming to optimize the utilization of the nutrients. Mitochondria as a central component of the cellular metabolism play a key role in this adaptive process. Since mitochondria harbor their own genome, which encodes essential enzymes, mitochondrial protein synthesis is a determinant of metabolic adaptation. While regulation of cytoplasmic protein synthesis in response to metabolic challenges has been studied in great detail, mechanisms which adapt mitochondrial translation in response to metabolic challenges remain elusive. Our results suggest that the mitochondrial acetylation status controlled by Sirt3 and its proposed opponent GCN5L1 is an important regulator of the metabolic adaptation of mitochondrial translation. Moreover, both proteins modulate regulators of cytoplasmic protein synthesis as well as the mitonuclear protein balance making Sirt3 and GCN5L1 key players in synchronizing mitochondrial and cytoplasmic translation. Our results thereby highlight regulation of mitochondrial translation as a novel component in the cellular nutrient sensing scheme and identify mitochondrial acetylation as a new regulatory principle for the metabolic competence of mitochondrial protein synthesis. © 2014 International Union of Biochemistry and Molecular Biology.

  7. Protein kinase C coordinates histone H3 phosphorylation and acetylation

    PubMed Central

    Darieva, Zoulfia; Webber, Aaron; Warwood, Stacey; Sharrocks, Andrew D

    2015-01-01

    The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at T45 are two important chromatin modifications that accompany chromatin assembly. Here we have identified the protein kinase Pkc1 as a key regulator that coordinates the deposition of these modifications in S. cerevisiae under conditions of replicative stress. Pkc1 phosphorylates the histone acetyl transferase Rtt109 and promotes its ability to acetylate H3K56. Our data also reveal novel cross-talk between two different histone modifications as Pkc1 also enhances H3T45 phosphorylation and this modification is required for H3K56 acetylation. Our data therefore uncover an important role for Pkc1 in coordinating the deposition of two different histone modifications that are important for chromatin assembly. DOI: http://dx.doi.org/10.7554/eLife.09886.001 PMID:26468616

  8. Changes in Protein Expression and Lysine Acetylation Induced by Decreased Glutathione Levels in Astrocytes.

    PubMed

    Pehar, Mariana; Ball, Lauren E; Sharma, Deep R; Harlan, Benjamin A; Comte-Walters, Susana; Neely, Benjamin A; Vargas, Marcelo R

    2016-02-01

    Astrocytes and neurons form a highly specialized functional unit, and the loss or gain of astrocytic functions can influence the initiation and progression of different neurodegenerative diseases. Neurons depend on the antioxidant protection provided by neighboring astrocytes. Glutathione (γ-l-glutamyl-l-cysteinyl-glycine) is a major component of the antioxidant system that defends cells against the toxic effects of reactive oxygen/nitrogen species. A decline in glutathione levels has been observed in aging and neurodegenerative diseases, and it aggravates the pathology in an amyotrophic lateral sclerosis-mouse model. Using a SILAC-based quantitative proteomic approach, we analyzed changes in global protein expression and lysine acetylation in primary astrocyte cultures obtained from wild-type mice or those deficient in the glutamate-cysteine ligase modifier subunit (GCLM). GCLM knockout astrocytes display an ∼80% reduction in total glutathione levels. We identified potential molecular targets and novel sites of acetylation that are affected by the chronic decrease in glutathione levels and observed a response mediated by Nrf2 activation. In addition, sequence analysis of peptides displaying increased acetylation in GCLM knockout astrocytes revealed an enrichment of cysteine residues in the vicinity of the acetylation site, which suggests potential crosstalk between lysine-acetylation and cysteine modification. Regulation of several metabolic and antioxidant pathways was observed at the level of protein expression and lysine acetylation, revealing a coordinated response involving transcriptional and posttranslational regulation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Comprehensive profiling of lysine acetylation suggests the widespread function is regulated by protein acetylation in the silkworm, Bombyx mori.

    PubMed

    Nie, Zuoming; Zhu, Honglin; Zhou, Yong; Wu, Chengcheng; Liu, Yue; Sheng, Qing; Lv, Zhengbing; Zhang, Wenping; Yu, Wei; Jiang, Caiying; Xie, Longfei; Zhang, Yaozhou; Yao, Juming

    2015-09-01

    Lysine acetylation in proteins is a dynamic and reversible PTM and plays an important role in diverse cellular processes. In this study, using lysine-acetylation (Kac) peptide enrichment coupled with nano HPLC/MS/MS, we initially identified the acetylome in the silkworms. Overall, a total of 342 acetylated proteins with 667 Kac sites were identified in silkworm. Sequence motifs analysis around Kac sites revealed an enrichment of Y, F, and H in the +1 position, and F was also enriched in the +2 and -2 positions, indicating the presences of preferred amino acids around Kac sites in the silkworm. Functional analysis showed the acetylated proteins were primarily involved in some specific biological processes. Furthermore, lots of nutrient-storage proteins, such as apolipophorin, vitellogenin, storage proteins, and 30 K proteins, were highly acetylated, indicating lysine acetylation may represent a common regulatory mechanism of nutrient utilization in the silkworm. Interestingly, Ser2 proteins, the coating proteins of larval silk, were found to contain many Kac sites, suggesting lysine acetylation may be involved in the regulation of larval silk synthesis. This study is the first to identify the acetylome in a lepidoptera insect, and expands greatly the catalog of lysine acetylation substrates and sites in insects.

  10. Histone Deacetylase Inhibitors Globally Enhance H3/H4 Tail Acetylation Without Affecting H3 Lysine 56 Acetylation

    PubMed Central

    Drogaris, Paul; Villeneuve, Valérie; Pomiès, Christelle; Lee, Eun-Hye; Bourdeau, Véronique; Bonneil, Éric; Ferbeyre, Gerardo; Verreault, Alain; Thibault, Pierre

    2012-01-01

    Histone deacetylase inhibitors (HDACi) represent a promising avenue for cancer therapy. We applied mass spectrometry (MS) to determine the impact of clinically relevant HDACi on global levels of histone acetylation. Intact histone profiling revealed that the HDACi SAHA and MS-275 globally increased histone H3 and H4 acetylation in both normal diploid fibroblasts and transformed human cells. Histone H3 lysine 56 acetylation (H3K56ac) recently elicited much interest and controversy due to its potential as a diagnostic and prognostic marker for a broad diversity of cancers. Using quantitative MS, we demonstrate that H3K56ac is much less abundant than previously reported in human cells. Unexpectedly, in contrast to H3/H4 N-terminal tail acetylation, H3K56ac did not increase in response to inhibitors of each class of HDACs. In addition, we demonstrate that antibodies raised against H3K56ac peptides cross-react against H3 N-terminal tail acetylation sites that carry sequence similarity to residues flanking H3K56. PMID:22355734

  11. N(α)-Acetylation of yeast ribosomal proteins and its effect on protein synthesis.

    PubMed

    Kamita, Masahiro; Kimura, Yayoi; Ino, Yoko; Kamp, Roza M; Polevoda, Bogdan; Sherman, Fred; Hirano, Hisashi

    2011-04-01

    N(α)-Acetyltransferases (NATs) cause the N(α)-acetylation of the majority of eukaryotic proteins during their translation, although the functions of this modification have been largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA, NatB, NatC, and NatD. In this study, the N(α)-acetylation status of ribosomal protein was analyzed using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A total of 60 ribosomal proteins were identified, of which 17 were N(α)-acetylated by NatA, and two by NatB. The N(α)-acetylation of two of these, S17 and L23, by NatA was not previously observed. Furthermore, we tested the effect of ribosomal protein N(α)-acetylation on protein synthesis using the purified ribosomes from each NAT mutant. It was found that the protein synthesis activities of ribosomes from NatA and NatB mutants were decreased by 27% and 23%, respectively, as compared to that of the normal strain. Furthermore, we have shown that ribosomal protein N(α)-acetylation by NatA influences translational fidelity in the presence of paromomycin. These results suggest that ribosomal protein N(α)-acetylation is necessary to maintain the ribosome's protein synthesis function. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. Acetylation of RNA processing proteins and cell cycle proteins in mitosis.

    PubMed

    Chuang, Carol; Lin, Sue-Hwa; Huang, Feilei; Pan, Jing; Josic, Djuro; Yu-Lee, Li-yuan

    2010-09-03

    Mitosis is a highly regulated process in which errors can lead to genomic instability, a hallmark of cancer. During this phase of the cell cycle, transcription is silent and RNA translation is inhibited. Thus, mitosis is largely driven by post-translational modification of proteins, including phosphorylation, methylation, ubiquitination, and sumoylation. Here, we show that protein acetylation is prevalent during mitosis. To identify proteins that are acetylated, we synchronized HeLa cells in early prometaphase and immunoprecipitated lysine-acetylated proteins with antiacetyl-lysine antibody. The immunoprecipitated proteins were identified by LC-ESI-MS/MS analysis. These include proteins involved in RNA translation, RNA processing, cell cycle regulation, transcription, chaperone function, DNA damage repair, metabolism, immune response, and cell structure. Immunoprecipitation followed by Western blot analyses confirmed that two RNA processing proteins, eIF4G and RNA helicase A, and several cell cycle proteins, including APC1, anillin, and NudC, were acetylated in mitosis. We further showed that acetylation of APC1 and NudC was enhanced by apicidin treatment, suggesting that their acetylation was regulated by histone deacetylase. Moreover, treating mitotic cells with apicidin or trichostatin A induced spindle abnormalities and cytokinesis failure. These studies suggest that protein acetylation/deacetylation is likely an important regulatory mechanism in mitosis.

  13. Protein acetylation sites mediated by Schistosoma mansoni GCN5

    SciTech Connect

    Moraes Maciel, Renata de; Furtado Madeiro da Costa, Rodrigo; Meirelles Bastosde Oliveira, Francisco; Rumjanek, Franklin David; Fantappie, Marcelo Rosado

    2008-05-23

    The transcriptional co-activator GCN5, a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcription activation. As in other eukaryotes, the DNA from the parasite Schistosome mansoni is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Using a series of synthetic peptides we determined that Lys-14 of histone H3 was acetylated by the recombinant SmGCN5-HAT domain. SmGCN5 was also able to acetylate schistosome non-histone proteins, such as the nuclear receptors SmRXR1 and SmNR1, and the co-activator SmNCoA-62. Electron microscopy revealed the presence of SmGCN5 protein in the nuclei of vitelline cells. Within the nucleus, SmGCN5 was found to be located in interchromatin granule clusters (IGCs), which are transcriptionally active structures. The data suggest that SmGCN5 is involved in transcription activation.

  14. Two Arabidopsis Proteins Synthesize Acetylated Xylan in Vitro

    PubMed Central

    Urbanowicz, Breeanna R.; Peña, Maria J.; Moniz, Heather A.; Moremen, Kelley W.; York, William S.

    2014-01-01

    SUMMARY Xylan is the third most abundant glycopolymer on earth after cellulose and chitin. As a major component of wood, grain and forage, this natural biopolymer has far-reaching impacts on human life. This highly acetylated cell wall polysaccharide is a vital component of the plant cell wall, which functions as a molecular scaffold, providing plants with mechanical strength and flexibility. Mutations that impair synthesis of the xylan backbone give rise to plants that fail to grow normally due to collapsed xylem cells in the vascular system. Phenotypic analysis of these mutants has implicated many proteins in xylan biosynthesis. However, the enzymes directly responsible for elongation and acetylation of the xylan backbone have not been unambiguously identified. Here we provide direct biochemical evidence that two Arabidopsis thaliana proteins, IRREGULAR XYLEM 10-L (IRX10-L) and ESKIMO1/ TRICOME BIREFRINGENCE 29 (ESK1/TBL29), catalyze these respective processes in vitro. By identifying the elusive xylan synthase and establishing ESK1/TBL29 as the archetypal plant polysaccharide O-acetyltransferase, we have resolved two long-standing questions in plant cell wall biochemistry. These findings shed light on integral steps in the molecular pathways utilized by plants to synthesize a major component of the world's biomass and expand our toolkit for producing glycopolymers with valuable properties. PMID:25141999

  15. Lysine Acetylation Is a Widespread Protein Modification for Diverse Proteins in Arabidopsis

    USDA-ARS?s Scientific Manuscript database

    Lysine acetylation (LysAc), a form of reversible protein post translational modification previously known only for histone proteins in plants, is shown to be wide spread in Arabidopsis. Sixty five lysine modification sites were identified on 58 proteins, which operate in a wide variety of pathways/...

  16. In silico analysis of protein Lys-N𝜀-acetylation in plants

    PubMed Central

    Rao, R. Shyama Prasad; Thelen, Jay J.; Miernyk, Ján A.

    2014-01-01

    Among post-translational modifications, there are some conceptual similarities between Lys-N𝜀-acetylation and Ser/Thr/Tyr O-phosphorylation. Herein we present a bioinformatics-based overview of reversible protein Lys-acetylation, including some comparisons with reversible protein phosphorylation. The study of Lys-acetylation of plant proteins has lagged behind studies of mammalian and microbial cells; 1000s of acetylation sites have been identified in mammalian proteins compared with only hundreds of sites in plant proteins. While most previous emphasis was focused on post-translational modifications of histones, more recent studies have addressed metabolic regulation. Being directly coupled with cellular CoA/acetyl-CoA and NAD/NADH, reversible Lys-N𝜀-acetylation has the potential to control, or contribute to control, of primary metabolism, signaling, and growth and development. PMID:25136347

  17. mChIP-KAT-MS, a method to map protein interactions and acetylation sites for lysine acetyltransferases

    PubMed Central

    Mitchell, Leslie; Huard, Sylvain; Cotrut, Michael; Pourhanifeh-Lemeri, Roghayeh; Steunou, Anne-Lise; Hamza, Akil; Lambert, Jean-Philippe; Zhou, Hu; Ning, Zhibin; Basu, Amrita; Côté, Jacques; Figeys, Daniel A.; Baetz, Kristin

    2013-01-01

    Recent global proteomic and genomic studies have determined that lysine acetylation is a highly abundant posttranslational modification. The next challenge is connecting lysine acetyltransferases (KATs) to their cellular targets. We hypothesize that proteins that physically interact with KATs may not only predict the cellular function of the KATs but may be acetylation targets. We have developed a mass spectrometry-based method that generates a KAT protein interaction network from which we simultaneously identify both in vivo acetylation sites and in vitro acetylation sites. This modified chromatin-immunopurification coupled to an in vitro KAT assay with mass spectrometry (mChIP-KAT-MS) was applied to the Saccharomyces cerevisiae KAT nucleosome acetyltransferase of histone H4 (NuA4). Using mChIP-KAT-MS, we define the NuA4 interactome and in vitro-enriched acetylome, identifying over 70 previously undescribed physical interaction partners for the complex and over 150 acetyl lysine residues, of which 108 are NuA4-specific in vitro sites. Through this method we determine NuA4 acetylation of its own subunit Epl1 is a means of self-regulation and identify a unique link between NuA4 and the spindle pole body. Our work demonstrates that this methodology may serve as a valuable tool in connecting KATs with their cellular targets. PMID:23572591

  18. Proteome-wide analysis reveals widespread lysine acetylation of major protein complexes in the malaria parasite

    PubMed Central

    Cobbold, Simon A.; Santos, Joana M.; Ochoa, Alejandro; Perlman, David H.; Llinás, Manuel

    2016-01-01

    Lysine acetylation is a ubiquitous post-translational modification in many organisms including the malaria parasite Plasmodium falciparum, yet the full extent of acetylation across the parasite proteome remains unresolved. Moreover, the functional significance of acetylation or how specific acetyl-lysine sites are regulated is largely unknown. Here we report a seven-fold expansion of the known parasite ‘acetylome’, characterizing 2,876 acetylation sites on 1,146 proteins. We observe that lysine acetylation targets a diverse range of protein complexes and is particularly enriched within the Apicomplexan AP2 (ApiAP2) DNA-binding protein family. Using quantitative proteomics we determined that artificial perturbation of the acetate/acetyl-CoA balance alters the acetyl-lysine occupancy of several ApiAP2 DNA-binding proteins and related transcriptional proteins. This metabolic signaling could mediate significant downstream transcriptional responses, as we show that acetylation of an ApiAP2 DNA-binding domain ablates its DNA-binding propensity. Lastly, we investigated the acetyl-lysine targets of each class of lysine deacetylase in order to begin to explore how each class of enzyme contributes to regulating the P. falciparum acetylome. PMID:26813983

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

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

  1. BET Acetyl-Lysine Binding Proteins Control Pathological Cardiac Hypertrophy

    PubMed Central

    Spiltoir, Jessica I.; Stratton, Matthew S.; Cavasin, Maria A.; Demos-Davies, Kim; Reid, Brian G.; Qi, Jun; Bradner, James E.; McKinsey, Timothy A.

    2014-01-01

    Cardiac hypertrophy is an independent predictor of adverse outcomes in patients with heart failure, and thus represents an attractive target for novel therapeutic intervention. JQ1, a small molecule inhibitor of bromodomain and extraterminal (BET) acetyl-lysine reader proteins, was identified in a high throughput screen designed to discover novel small molecule regulators of cardiomyocyte hypertrophy. JQ1 dose-dependently blocked agonist-dependent hypertrophy of cultured neonatal rat ventricular myocytes (NRVMs) and reversed the prototypical gene program associated with pathological cardiac hypertrophy. JQ1 also blocked left ventricular hypertrophy (LVH) and improved cardiac function in adult mice subjected to transverse aortic constriction (TAC). The BET family consists of BRD2, BRD3, BRD4 and BRDT. BRD4 protein expression was increased during cardiac hypertrophy, and hypertrophic stimuli promoted recruitment of BRD4 to the transcriptional start site (TSS) of the gene encoding atrial natriuretic factor (ANF). Binding of BRD4 to the ANF TSS was associated with increased phosphorylation of local RNA polymerase II. These findings define a novel function for BET proteins as signal-responsive regulators of cardiac hypertrophy, and suggest that small molecule inhibitors of these epigenetic reader proteins have potential as therapeutics for heart failure. PMID:23939492

  2. A bioinformatics-based overview of protein Lys-Ne-acetylation

    USDA-ARS?s Scientific Manuscript database

    Among posttranslational modifications, there are some conceptual similarities between Lys-N'-acetylation and Ser/Thr/Tyr O-phosphorylation. Herein we present a bioinformatics-based overview of reversible protein Lys-acetylation, including some comparisons with reversible protein phosphorylation. T...

  3. Retraction Statement: Mitochondrial protein acetylation mediates nutrient sensing of mitochondrial protein synthesis and mitonuclear protein balance.

    PubMed

    2017-07-01

    IUBMB Life (2014) 66:793-802. DOI: 10.1002/iub.1328 The above article, published online on November 15, 2014 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal's Editors-in-Chief, Angelo Azzi and William J. Whelan, Corresponding Author Tina Wenz, the University of Cologne, and Wiley Periodicals, Inc. The article has been retracted on request of the University of Cologne that, after an investigation, established that the data reported in it are not reproducible. Note from the Corresponding Author: "The paper reports on the influence of mitochondrial acetylation on protein synthesis. After publication, several irregularities appeared and have been thoroughly investigated by the lab of the Corresponding Author in cooperation with the commission of Research integrity of the University of Cologne. Both came to the conclusion that data used for the publication are erroneous and that the presented data are not reproducible by the lab of the Corresponding Author and other labs. The Corresponding Author takes responsibility and regrets not having detected these issues before publication. The appropriate corrective action is retraction of the paper. The Corresponding Author apologizes to the scientific community." Antonella Di Domenico, Annette Hofer, Federica Tundo, Tina Wenz (2014), Mitochondrial protein acetylation mediates nutrient sensing of mitochondrial protein synthesis and mitonuclear protein balance, IUBMB Life. 66: 793-802, 2014. DOI: 10.1002/iub.1328 © 2017 IUBMB Life, 69(7):553-553, 2017. © 2016 International Union of Biochemistry and Molecular Biology.

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

    SciTech Connect

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

    2012-01-01

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

  5. Acetylation of Werner syndrome protein (WRN): relationships with DNA damage, DNA replication and DNA metabolic activities.

    PubMed

    Lozada, Enerlyn; Yi, Jingjie; Luo, Jianyuan; Orren, David K

    2014-08-01

    Loss of Werner syndrome protein function causes Werner syndrome, characterized by increased genomic instability, elevated cancer susceptibility and premature aging. Although WRN is subject to acetylation, phosphorylation and sumoylation, the impact of these modifications on WRN's DNA metabolic function remains unclear. Here, we examined in further depth the relationship between WRN acetylation and its role in DNA metabolism, particularly in response to induced DNA damage. Our results demonstrate that endogenous WRN is acetylated somewhat under unperturbed conditions. However, levels of acetylated WRN significantly increase after treatment with certain DNA damaging agents or the replication inhibitor HU. Use of DNA repair-deficient cells or repair pathway inhibitors further increase levels of acetylated WRN, indicating that induced DNA lesions and their persistence are at least partly responsible for increased acetylation. Notably, acetylation of WRN correlates with inhibition of DNA synthesis, suggesting that replication blockage might underlie this effect. Moreover, WRN acetylation modulates its affinity for and activity on certain DNA structures, in a manner that may enhance its relative specificity for physiological substrates. Our results also show that acetylation and deacetylation of endogenous WRN is a dynamic process, with sirtuins and other histone deacetylases contributing to WRN deacetylation. These findings advance our understanding of the dynamics of WRN acetylation under unperturbed conditions and following DNA damage induction, linking this modification not only to DNA damage persistence but also potentially to replication stalling caused by specific DNA lesions. Our results are consistent with proposed metabolic roles for WRN and genomic instability phenotypes associated with WRN deficiency.

  6. Identification and Functional Characterization of N-Terminally Acetylated Proteins in Drosophila melanogaster

    PubMed Central

    Gerrits, Bertran; Roschitzki, Bernd; Mohanty, Sonali; Niederer, Eva M.; Laczko, Endre; Timmerman, Evy; Lange, Vinzenz; Hafen, Ernst; Aebersold, Ruedi; Vandekerckhove, Joël; Basler, Konrad; Ahrens, Christian H.; Gevaert, Kris; Brunner, Erich

    2009-01-01

    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 (X)PX 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 (X)PX 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. PMID:19885390

  7. Global Histone H4 Acetylation in the Olfactory Bulb of Lactating Rats with Different Patterns of Maternal Behavior.

    PubMed

    de Moura, Ana Carolina; da Silva, Ivy Reichert Vital; Reinaldo, Gustavo; Dani, Caroline; Elsner, Viviane Rostirola; Giovenardi, Márcia

    2016-10-01

    In rats, variations in the levels of neuromodulatory molecules and in the expression of their receptors are observed during pregnancy and postpartum. These changes may contribute to the development and management of maternal behavior. The frequency of licking the pups is used to evaluate maternal care, having mothers with low licking (LL) and high licking (HL) frequencies. Previously, we found that HL had increased levels of transcriptional expression of the receptors for serotonin (HTR1a, HTR1b), estrogen (Erα), dopamine (D1a), and prolactin (Prlr) than LL in the olfactory bulb (OB); however, the molecular mechanisms behind this phenomenon are unknown. Since evidences pointed out that epigenetic marks, which may alter gene expression, are modulated by environmental factors such as exercise, diet, maternal care, and xenobiotic exposure, our objective was to verify the acetylation levels of histone-H4 in the OB of LL and HL rats. Maternal behavior was studied for the first 7 postpartum days. LL (n = 4) and HL (n = 5) mothers were selected according to the behavior of licking their pups. Acetylation levels of histone-H4 were determined using the Global Histone-H4 Acetylation Assay Kit and expressed as ng/mg protein (mean ± SD). Analysis revealed that HL (278.36 ± 68.95) had increased H4 acetylation levels than LL (183.24 ± 73.05; p = 0.045). The enhanced expression of the previously studied receptors in the OB could be related, at least in part, to the hyperacetylation status of histone-H4 here observed. Afterward, the modulation of histone acetylation levels could exert a pivotal role through molecular mechanisms involved in the different patterns of maternal behavior.

  8. An Artificial Reaction Promoter Modulates Mitochondrial Functions via Chemically Promoting Protein Acetylation

    PubMed Central

    Shindo, Yutaka; Komatsu, Hirokazu; Hotta, Kohji; Ariga, Katsuhiko; Oka, Kotaro

    2016-01-01

    Acetylation, which modulates protein function, is an important process in intracellular signalling. In mitochondria, protein acetylation regulates a number of enzymatic activities and, therefore, modulates mitochondrial functions. Our previous report showed that tributylphosphine (PBu3), an artificial reaction promoter that promotes acetylransfer reactions in vitro, also promotes the reaction between acetyl-CoA and an exogenously introduced fluorescent probe in mitochondria. In this study, we demonstrate that PBu3 induces the acetylation of mitochondrial proteins and a decrease in acetyl-CoA concentration in PBu3-treated HeLa cells. This indicates that PBu3 can promote the acetyltransfer reaction between acetyl-CoA and mitochondrial proteins in living cells. PBu3-induced acetylation gradually reduced mitochondrial ATP concentrations in HeLa cells without changing the cytoplasmic ATP concentration, suggesting that PBu3 mainly affects mitochondrial functions. In addition, pyruvate, which is converted into acetyl-CoA in mitochondria and transiently increases ATP concentrations in the absence of PBu3, elicited a further decrease in mitochondrial ATP concentrations in the presence of PBu3. Moreover, the application and removal of PBu3 reversibly alternated mitochondrial fragmentation and elongation. These results indicate that PBu3 enhances acetyltransfer reactions in mitochondria and modulates mitochondrial functions in living cells. PMID:27374857

  9. Protein acetylation mechanisms in the regulation of insulin and insulin-like growth factor 1 signalling.

    PubMed

    Pirola, Luciano; Zerzaihi, Ouafa; Vidal, Hubert; Solari, Florence

    2012-10-15

    Lysine acetylation is a protein post-translational modification (PTM) initially discovered in abundant proteins such as tubulin, whose acetylated form confers microtubule stability, and histones, where it promotes the transcriptionally active chromatin state. Other individual reports identified lysine acetylation as a PTM regulating transcription factors and co-activators including p53, c-Myc, PGC1α and Ku70. The subsequent employment of proteomics-based approaches revealed that lysine acetylation is a widespread PTM, contributing to cellular regulation as much as protein-phosphorylation based mechanisms. In particular, most of the enzymes of central metabolic processes - glycolysis, tricarboxylic acid and urea cycles, fatty acid and glycogen metabolism - have been shown to be regulated by lysine acetylation, through the opposite actions of protein acetyltransferases and deacetylases, making protein acetylation a PTM that connects the cell's energetic state and its consequent metabolic response. In multicellular organisms, insulin/insulin-like signalling (IIS) is a major hormonal regulator of metabolism and cell growth, and very recent research indicates that most of the enzymes participating in IIS are likewise subjected to acetylation-based regulatory mechanisms, that integrate the classical phosphorylation mechanisms. Here, we review the current knowledge on acetylation/deacetylation regulatory phenomena within the IIS cascade, with emphasis on the enzymatic machinery linking the acetylation/deacetylation switch to the metabolic state. We cover this recent area of investigation because pharmacological modulation of protein acetylation/deacetylation has been shown to be a promising target for the amelioration of the metabolic abnormalities occurring in the metabolic syndrome.

  10. Resolving Acetylated and Phosphorylated Proteins by Neutral Urea Triton-Polyacrylamide Gel Electrophoresis, NUT-PAGE

    PubMed Central

    Buehl, Christopher J.; Deng, Xiexiong; Liu, Mengyu; Hovde, Stacy; Xu, Xinjing; Kuo, Min-Hao

    2014-01-01

    Protein acetylation and phosphorylation can be key modifications that regulate both normal and pathological protein functions. Current gel systems used to analyze modified proteins require either expensive reagents or time–consuming second dimension electrophoresis. In this manuscript, we present a neutral pH gel system that allows the analysis of acetylated and phosphorylated proteins. This neutral pH urea Triton-polyacrylamide gel electrophoresis system, or NUT-PAGE, separates proteins based on their charge at pH 7 and generates discrete bands from each acetylated and phosphorylated species. In addition, the gel is composed of common and inexpensive laboratory reagents, and requires only a single dimension of electrophoresis. We are able to demonstrate the effectiveness of this system by analyzing phosphorylated species of an acidic protein, α-synuclein, and both acetylated and phosphorylated species of a basic protein, histone H3. NUT-PAGE thus provides a cost-effective alternative to resolving acetylated and phosphorylated proteins, and potentially proteins with other post-translational modifications that alter net charge. Method Summary Here we present a single-dimension neutral pH urea Triton-polyacrylamide gel electrophoresis (NUT-PAGE) system affording high-resolution separation of acetylated and phosphorylated proteins. PMID:25109292

  11. Blocking an N-terminal acetylation-dependent protein interaction inhibits an E3 ligase.

    PubMed

    Scott, Daniel C; Hammill, Jared T; Min, Jaeki; Rhee, David Y; Connelly, Michele; Sviderskiy, Vladislav O; Bhasin, Deepak; Chen, Yizhe; Ong, Su-Sien; Chai, Sergio C; Goktug, Asli N; Huang, Guochang; Monda, Julie K; Low, Jonathan; Kim, Ho Shin; Paulo, Joao A; Cannon, Joe R; Shelat, Anang A; Chen, Taosheng; Kelsall, Ian R; Alpi, Arno F; Pagala, Vishwajeeth; Wang, Xusheng; Peng, Junmin; Singh, Bhuvanesh; Harper, J Wade; Schulman, Brenda A; Guy, R Kip

    2017-08-01

    N-terminal acetylation is an abundant modification influencing protein functions. Because ∼80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation-dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide-binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2-E3 ligases.

  12. Mitochondrial Matrix Ca2+ Accumulation Regulates Cytosolic NAD+/NADH Metabolism, Protein Acetylation, and Sirtuin Expression

    PubMed Central

    Marcu, Raluca; Wiczer, Brian M.; Neeley, Christopher K.

    2014-01-01

    Mitochondrial calcium uptake stimulates bioenergetics and drives energy production in metabolic tissue. It is unknown how a calcium-mediated acceleration in matrix bioenergetics would influence cellular metabolism in glycolytic cells that do not require mitochondria for ATP production. Using primary human endothelial cells (ECs), we discovered that repetitive cytosolic calcium signals (oscillations) chronically loaded into the mitochondrial matrix. Mitochondrial calcium loading in turn stimulated bioenergetics and a persistent elevation in NADH. Rather than serving as an impetus for mitochondrial ATP generation, matrix NADH rapidly transmitted to the cytosol to influence the activity and expression of cytosolic sirtuins, resulting in global changes in protein acetylation. In endothelial cells, the mitochondrion-driven reduction in both the cytosolic and mitochondrial NAD+/NADH ratio stimulated a compensatory increase in SIRT1 protein levels that had an anti-inflammatory effect. Our studies reveal the physiologic importance of mitochondrial bioenergetics in the metabolic regulation of sirtuins and cytosolic signaling cascades. PMID:24865966

  13. Identification of the sequence determinants of protein N-terminal acetylation through a decision tree approach.

    PubMed

    Yamada, Kazunori D; Omori, Satoshi; Nishi, Hafumi; Miyagi, Masaru

    2017-06-02

    N-terminal acetylation is one of the most common protein modifications in eukaryotes and occurs co-translationally when the N-terminus of the nascent polypeptide is still attached to the ribosome. This modification has been shown to be involved in a wide range of biological phenomena such as protein half-life regulation, protein-protein and protein-membrane interactions, and protein subcellular localization. Thus, accurately predicting which proteins receive an acetyl group based on their protein sequence is expected to facilitate the functional study of this modification. As the occurrence of N-terminal acetylation strongly depends on the context of protein sequences, attempts to understand the sequence determinants of N-terminal acetylation were conducted initially by simply examining the N-terminal sequences of many acetylated and unacetylated proteins and more recently by machine learning approaches. However, a complete understanding of the sequence determinants of this modification remains to be elucidated. We obtained curated N-terminally acetylated and unacetylated sequences from the UniProt database and employed a decision tree algorithm to identify the sequence determinants of N-terminal acetylation for proteins whose initiator methionine ((i)Met) residues have been removed. The results suggested that the main determinants of N-terminal acetylation are contained within the first five residues following (i)Met and that the first and second positions are the most important discriminator for the occurrence of this phenomenon. The results also indicated the existence of position-specific preferred and inhibitory residues that determine the occurrence of N-terminal acetylation. The developed predictor software, termed NT-AcPredictor, accurately predicted the N-terminal acetylation, with an overall performance comparable or superior to those of preceding predictors incorporating machine learning algorithms. Our machine learning approach based on a decision tree

  14. Cell shape regulates global histone acetylation in human mammaryepithelial cells

    SciTech Connect

    Le Beyec, Johanne; Xu, Ren; Lee, Sun-Young; Nelson, Celeste M.; Rizki, Aylin; Alcaraz, Jordi; Bissell, Mina J.

    2007-02-28

    Extracellular matrix (ECM) regulates cell morphology and gene expression in vivo; these relationships are maintained in three-dimensional (3D) cultures of mammary epithelial cells. In the presence of laminin-rich ECM (lrECM), mammary epithelial cells round up and undergo global histone deacetylation, a process critical for their functional differentiation. However, it remains unclear whether lrECM-dependent cell rounding and global histone deacetylation are indeed part of a common physical-biochemical pathway. Using 3D cultures as well as nonadhesive and micropatterned substrata, here we showed that the cell 'rounding' caused by lrECM was sufficient to induce deacetylation of histones H3 and H4 in the absence of biochemical cues. Microarray and confocal analysis demonstrated that this deacetylation in 3D culture is associated with a global increase in chromatin condensation and a reduction in gene expression. Whereas cells cultured on plastic substrata formed prominent stress fibers, cells grown in 3D lrECM or on micropatterns lacked these structures. Disruption of the actin cytoskeleton with cytochalasin D phenocopied the lrECM-induced cell rounding and histone deacetylation. These results reveal a novel link between ECM-controlled cell shape and chromatin structure, and suggest that this link is mediated by changes in the actin cytoskeleton.

  15. Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for {beta}-lactam acetylation.

    SciTech Connect

    He, H.; Ding, Y.; Bartlam, M.; Sun, F.; Le, Y.; Qin, X.; Tang, H.; Zhang, R.; Joachimiak, A.; Liu, J.; Zhao, N.; Rao, Z.; Biosciences Division; Tsinghua Univ.; Chinese Academy of Science

    2003-01-31

    Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55 {angstrom} resolution. The binary complex forms a characteristic 'V' shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.

  16. Identification and characterization of AckA-dependent protein acetylation in Neisseria gonorrhoeae

    PubMed Central

    Post, Deborah M. B.; Schilling, Birgit; Reinders, Lorri M.; D’Souza, Alexandria K.; Ketterer, Margaret R.; Kiel, Steven J.; Chande, Aroon T.; Apicella, Michael A.; Gibson, Bradford W.

    2017-01-01

    Neisseria gonorrhoeae, the causative agent of gonorrhea, has a number of factors known to contribute to pathogenesis; however, a full understanding of these processes and their regulation has proven to be elusive. Post-translational modifications (PTMs) of bacterial proteins are now recognized as one mechanism of protein regulation. In the present study, Western blot analyses, with an anti-acetyl-lysine antibody, indicated that a large number of gonococcal proteins are post-translationally modified. Previous work has shown that Nε-lysine acetylation can occur non-enzymatically with acetyl-phosphate (AcP) as the acetyl donor. In the current study, an acetate kinase mutant (1291ackA), which accumulates AcP, was generated in N. gonorrhoeae. Broth cultures of N. gonorrhoeae 1291wt and 1291ackA were grown, proteins extracted and digested, and peptides containing acetylated-lysines (K-acetyl) were affinity-enriched from both strains. Mass spectrometric analyses of these samples identified a total of 2686 unique acetylation sites. Label-free relative quantitation of the K-acetyl peptides derived from the ackA and wild-type (wt) strains demonstrated that 109 acetylation sites had an ackA/wt ratio>2 and p-values <0.05 in at least 2/3 of the biological replicates and were designated as “AckA-dependent”. Regulated K-acetyl sites were found in ribosomal proteins, central metabolism proteins, iron acquisition and regulation proteins, pilus assembly and regulation proteins, and a two-component response regulator. Since AckA is part of a metabolic pathway, comparative growth studies of the ackA mutant and wt strains were performed. The mutant showed a growth defect under aerobic conditions, an inability to grow anaerobically, and a defect in biofilm maturation. In conclusion, the current study identified AckA-dependent acetylation sites in N. gonorrhoeae and determined that these sites are found in a diverse group of proteins. This work lays the foundation for future studies

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

    PubMed

    Bennetzen, Martin V; Larsen, Dorthe Helena; Dinant, Christoffel; Watanabe, Sugiko; Bartek, Jiri; Lukas, Jiri; Andersen, Jens S

    2013-06-01

    Genotoxic insults, such as ionizing radiation (IR), cause DNA damage that evokes a multifaceted cellular DNA damage response (DDR). DNA damage signaling events that control protein activity, subcellular localization, DNA binding, protein-protein interactions, etc. rely heavily on time-dependent posttranslational modifications (PTMs). 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 EP300 and CREBBP, are dynamically acetylated; (2) that nuclear acetyltransferases themselves are regulated, not on the protein abundance level, but by (de)acetylation; and (3) that the recently reported p53 co-activator and methyltransferase MLL3 is acetylated on five lysines during the DDR. For selected examples, protein immunoprecipitation and immunoblotting were used 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 to genotoxic insults. Overall, these results present a resource of temporal profiles of a spectrum of protein acetylation sites during DDR and provide further insights into the highly dynamic nature of regulatory PTMs that help orchestrate the maintenance of genome integrity.

  18. A novel protein from mung bean hypocotyl cell walls with acetyl esterase activity.

    PubMed

    Bordenave, M; Goldberg, R; Huet, J C; Pernollet, J C

    1995-01-01

    An acetyl esterase was purified from cell walls isolated from mung bean hypocotyls. The purified enzyme had an apparent Mr of 43,300 and an apparent pI > 9. It rapidly deesterified triacetin and p-nitrophenylacetate and slowly released acetate from beet and flax pectins, the deesterification rate being increased by previous demethylation of the pectins. No significant peptide sequence identity between the acetyl esterase and any known protein could be found in protein data bases.

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

    PubMed

    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.

  20. Identification and analysis of the acetylated status of poplar proteins reveals analogous N-terminal protein processing mechanisms with other eukaryotes.

    PubMed

    Liu, Chang-Cai; Zhu, Hang-Yong; Dong, Xiu-Mei; Ning, De-Li; Wang, Hong-Xia; Li, Wei-Hua; Yang, Chuan-Ping; Wang, Bai-Chen

    2013-01-01

    The N-terminal protein processing mechanism (NPM) including N-terminal Met excision (NME) and N-terminal acetylation (N(α)-acetylation) represents a common protein co-translational process of some eukaryotes. However, this NPM occurred in woody plants yet remains unknown. To reveal the NPM in poplar, we investigated the N(α)-acetylation status of poplar proteins during dormancy by combining tandem mass spectrometry with TiO2 enrichment of acetylated peptides. We identified 58 N-terminally acetylated (N(α)-acetylated) proteins. Most proteins (47, >81%) are subjected to N(α)-acetylation following the N-terminal removal of Met, indicating that N(α)-acetylation and NME represent a common NPM of poplar proteins. Furthermore, we confirm that poplar shares the analogous NME and N(α)-acetylation (NPM) to other eukaryotes according to analysis of N-terminal features of these acetylated proteins combined with genome-wide identification of the involving methionine aminopeptidases (MAPs) and N-terminal acetyltransferase (Nat) enzymes in poplar. The N(α)-acetylated reactions and the involving enzymes of these poplar proteins are also identified based on those of yeast and human, as well as the subcellular location information of these poplar proteins. This study represents the first extensive investigation of N(α)-acetylation events in woody plants, the results of which will provide useful resources for future unraveling the regulatory mechanisms of N(α)-acetylation of proteins in poplar.

  1. The mechanism of the effect of aspirin on human platelets. I. Acetylation of a particulate fraction protein.

    PubMed Central

    Roth, G J; Majerus, P W

    1975-01-01

    Aspirin (acetylsalicylic acid) inhibits platelet prostaglandin synthesis and the ADP- and collagen-induced platelet release reaction. The mechanism of the inhibitory effect is unknown but may involve protein acetylation, since aspirin acetylates a variety of substrates, including platelet protein. We have examined the relationship between protein acetylation and aspirin's physiologic effect on platelets. Suspensions of washed human platelets were incubated at 37 degrees C with (3H)aspirin, and incorporation of radioactivity into protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Exposure to (acetyl-3H)aspirin but not (aromatic ring-3H)aspirin resulted in radioactive labeling of three platelet proteins, suggesting that the drug acetylates these three proteins. The acetylation of two of the proteins (located in the supernatant fraction) was not saturable, implying that these reactions may not be physiologically significant. Acetylation of the third protein, approximate mol wt 85,000 (located in the particulate fraction), saturated at an aspirin concentration of 30 muM and was complete within 20 min. Platelets prepared from aspirin-treated donors did not incorporate any (acetyl-3H)aspirin radioactivity into the particulate protein for 2 days after drug treatment and did not show full pretreatment uptake of radioactivity for 12 days thereafter. The course of increasing incorporation of (acetyl-3H)aspirin radioactivity parralleled that of platelet turnover. Therefore, in addition to its saturability, acetylation of the particulate fraction protein by aspirin was permanent. In two respects, the inhibition of platelet function by aspirin correlates well with the aspirin-mediated acetylation of the particulate fraction protein. Both persist for the life-span of the aspirin-treated platelet, and both occur at a similar saturating aspirin concentration. The evidence suggests that the physiologic effect of aspirin on human platelets is produced

  2. Acetyl-lysine analog peptides as mechanistic probes of protein deacetylases.

    PubMed

    Smith, Brian C; Denu, John M

    2007-12-21

    Class III histone deacetylases (Sir2 or sirtuins) catalyze the NAD+-dependent conversion of acetyl-lysine residues to nicotinamide, 2'-O-acetyl-ADP-ribose (OAADPr), and deacetylated lysine. Class I and II HDACs utilize a different deacetylation mechanism, utilizing an active site zinc to direct hydrolysis of acetyl-lysine residues to lysine and acetate. Here, using ten acetyl-lysine analog peptides, we have probed the substrate binding pockets of sirtuins and investigated the catalytic differences among sirtuins and class I and II deacetylases. For the sirtuin Hst2, acetyl-lysine analog peptide binding correlated with the hydrophobic substituent parameter pi with a slope of -0.35 from a plot of log Kd versus pi. Interestingly, propionyl- and butyryl-lysine peptides were found to bind tighter to Hst2 compared with acetyl-lysine peptide and showed measurable rates of catalysis with Hst2, Sirt1, Sirt2, and Sirt3, suggesting propionyl- and butyryl-lysine proteins may be sirtuin substrates in vivo. Unique among the acetyl-lysine analog peptides examined, homocitrulline peptide produced ADP-ribose instead of the corresponding OAADPr analog. The electron-withdrawing nature of each acetyl analog had a profound impact on the deacylation rate between deacetylase classes. The rate of catalysis with the acetyl-lysine analog peptides varied over five orders of magnitude with the class III deacetylase Hst2, revealing a linear free energy relationship with a slope of -1.57 when plotted versus the Taft constant, sigma*. HDAC8, a class I deacetylase, displayed the opposite trend with a slope of +0.79. These results are applicable toward the development of selective substrates and other mechanistic probes of protein deacetylases.

  3. Reduced Wall Acetylation Proteins Play Vital and Distinct Roles in Cell Wall O-Acetylation in Arabidopsis1[C][W][OPEN

    PubMed Central

    Manabe, Yuzuki; Verhertbruggen, Yves; Gille, Sascha; Harholt, Jesper; Chong, Sun-Li; Pawar, Prashant Mohan-Anupama; Mellerowicz, Ewa J.; Tenkanen, Maija; Cheng, Kun; Pauly, Markus; Scheller, Henrik Vibe

    2013-01-01

    The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double, triple, and quadruple loss-of-function mutants of all four members of the RWA family in Arabidopsis (Arabidopsis thaliana). In contrast to rwa2, the triple and quadruple rwa mutants display severe growth phenotypes revealing the importance of wall acetylation for plant growth and development. The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco)mannan, and xyloglucan as well as overall cell wall acetylation is affected differently in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell differentiation of cell types with secondary cell walls. PMID:24019426

  4. ARD1-mediated Hsp70 acetylation balances stress-induced protein refolding and degradation

    PubMed Central

    Seo, Ji Hae; Park, Ji-Hyeon; Lee, Eun Ji; Vo, Tam Thuy Lu; Choi, Hoon; Kim, Jun Yong; Jang, Jae Kyung; Wee, Hee-Jun; Lee, Hye Shin; Jang, Se Hwan; Park, Zee Yong; Jeong, Jaeho; Lee, Kong-Joo; Seok, Seung-Hyeon; Park, Jin Young; Lee, Bong Jin; Lee, Mi-Ni; Oh, Goo Taeg; Kim, Kyu-Won

    2016-01-01

    Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. This switch is required for the maintenance of protein homoeostasis and ultimately rescues cells from stress-induced cell death in vitro and in vivo. Therefore, ARD1-mediated Hsp70 acetylation is a regulatory mechanism that temporally balances protein refolding/degradation in response to stress. PMID:27708256

  5. ARD1-mediated Hsp70 acetylation balances stress-induced protein refolding and degradation.

    PubMed

    Seo, Ji Hae; Park, Ji-Hyeon; Lee, Eun Ji; Vo, Tam Thuy Lu; Choi, Hoon; Kim, Jun Yong; Jang, Jae Kyung; Wee, Hee-Jun; Lee, Hye Shin; Jang, Se Hwan; Park, Zee Yong; Jeong, Jaeho; Lee, Kong-Joo; Seok, Seung-Hyeon; Park, Jin Young; Lee, Bong Jin; Lee, Mi-Ni; Oh, Goo Taeg; Kim, Kyu-Won

    2016-10-06

    Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. This switch is required for the maintenance of protein homoeostasis and ultimately rescues cells from stress-induced cell death in vitro and in vivo. Therefore, ARD1-mediated Hsp70 acetylation is a regulatory mechanism that temporally balances protein refolding/degradation in response to stress.

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

    PubMed

    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.

  7. Lysine acetylation is widespread on proteins of diverse function and localization in the protozoan parasite Toxoplasma gondii.

    PubMed

    Jeffers, Victoria; Sullivan, William J

    2012-06-01

    While histone proteins are the founding members of lysine acetylation substrates, it is now clear that hundreds of other proteins can be acetylated in multiple compartments of the cell. Our knowledge of the scope of this modification throughout the kingdom of life is beginning to emerge, as proteome-wide lysine acetylation has been documented in prokaryotes, Arabidopsis thaliana, Drosophila melanogaster, and human cells. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify parasite peptides enriched by immunopurification with acetyl-lysine antibody, we produced the first proteome-wide analysis of acetylation for a protozoan organism, the opportunistic apicomplexan parasite Toxoplasma gondii. The results show that lysine acetylation is abundant in the actively proliferating tachyzoite form of the parasite, which causes acute toxoplasmosis. Our approach successfully identified known acetylation marks on Toxoplasma histones and α-tubulin and detected over 400 novel acetylation sites on a wide variety of additional proteins, including those with roles in transcription, translation, metabolism, and stress responses. Importantly, an extensive set of parasite-specific proteins, including those found in organelles unique to Apicomplexa, is acetylated in the parasite. Our data provide a wealth of new information that improves our understanding of the evolution of this vital regulatory modification while potentially revealing novel therapeutic avenues. We conclude from this study that lysine acetylation was prevalent in the early stages of eukaryotic cell evolution and occurs on proteins involved in a remarkably diverse array of cellular functions, including those that are specific to parasites.

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

    PubMed

    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.

  9. Acetylation of glucokinase regulatory protein decreases glucose metabolism by suppressing glucokinase activity

    PubMed Central

    Park, Joo-Man; Kim, Tae-Hyun; Jo, Seong-Ho; Kim, Mi-Young; Ahn, Yong-Ho

    2015-01-01

    Glucokinase (GK), mainly expressed in the liver and pancreatic β-cells, is critical for maintaining glucose homeostasis. GK expression and kinase activity, respectively, are both modulated at the transcriptional and post-translational levels. Post-translationally, GK is regulated by binding the glucokinase regulatory protein (GKRP), resulting in GK retention in the nucleus and its inability to participate in cytosolic glycolysis. Although hepatic GKRP is known to be regulated by allosteric mechanisms, the precise details of modulation of GKRP activity, by post-translational modification, are not well known. Here, we demonstrate that GKRP is acetylated at Lys5 by the acetyltransferase p300. Acetylated GKRP is resistant to degradation by the ubiquitin-dependent proteasome pathway, suggesting that acetylation increases GKRP stability and binding to GK, further inhibiting GK nuclear export. Deacetylation of GKRP is effected by the NAD+-dependent, class III histone deacetylase SIRT2, which is inhibited by nicotinamide. Moreover, the livers of db/db obese, diabetic mice also show elevated GKRP acetylation, suggesting a broader, critical role in regulating blood glucose. Given that acetylated GKRP may affiliate with type-2 diabetes mellitus (T2DM), understanding the mechanism of GKRP acetylation in the liver could reveal novel targets within the GK-GKRP pathway, for treating T2DM and other metabolic pathologies. PMID:26620281

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

    PubMed Central

    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

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

    PubMed Central

    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 acetylated by both p300 and PCAF. We further quantified the level of acetylation by analyzing, with LC-MS/MS, the proteolytic peptides of the in-vitro or in-vivo acetylated HMGA1 proteins where the unmodified lysine residues were chemically derivatized with a perdeuterated acetyl group. Quantification results revealed that p300 and PCAF exhibited different site preferences for the acetylation; the preference of p300 acetylation followed the order of Lys-64~Lys-70 > Lys-66 > Lys-14~Lys73, whereas the selectivity of PCAF acetylation followed the sequence of Lys-70~Lys-73 > Lys-64~Lys-66 > Lys-14. HMGA1b was acetylated in a very similar fashion as HMGA1a. We also demonstrated that C-terminal phosphorylation of HMGA1 proteins did not affect the in-vitro acetylation of the two proteins by either p300 or PCAF. Moreover, we examined the acetylation of lysine residues in HMGA1a and HMGA1b isolated from PC-3 human prostate cancer cells. Our results showed that all the above five lysine residues were also acetylated in vivo, with Lys-64, Lys-66 and Lys-70 in HMGA1a exhibiting higher levels of acetylation than Lys-14 and Lys-73. PMID:17627840

  12. Physical and Functional HAT/HDAC Interplay Regulates Protein Acetylation Balance

    PubMed Central

    Peserico, Alessia; Simone, Cristiano

    2011-01-01

    The balance between protein acetylation and deacetylation controls several physiological and pathological cellular processes, and the enzymes involved in the maintenance of this equilibrium—acetyltransferases (HATs) and deacetylases (HDACs)—have been widely studied. Presently, the evidences obtained in this field suggest that the dynamic acetylation equilibrium is mostly maintained through the physical and functional interplay between HAT and HDAC activities. This model overcomes the classical vision in which the epigenetic marks of acetylation have only an activating function whereas deacetylation marks have a repressing activity. Given the existence of several players involved in the preservation of this equilibrium, the identification of these complex networks of interacting proteins will likely foster our understanding of how cells regulate intracellular processes and respond to the extracellular environment and will offer the rationale for new therapeutic approaches based on epigenetic drugs in human diseases. PMID:21151613

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

    PubMed

    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.

  14. A Method to determine lysine acetylation stoichiometries

    SciTech Connect

    Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; Shukla, Anil K.; Weitz, Karl K.; Moore, Ronald J.; Hixson, Kim K.; Kim, Jong Seo; Petyuk, Vladislav A.; Monroe, Matthew E.; Pasa-Tolic, Ljiljana; Qian, Weijun; Smith, Richard D.; Adkins, Joshua N.; Ansong, Charles

    2014-07-21

    A major bottleneck to fully understanding the functional aspects of lysine acetylation is the lack of stoichiometry information. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of lysine acetylation on proteins globally. Using this technique, we determined the modification occupancy on hundreds of acetylated peptides from cell lysates and cross-validated the measurements via immunoblotting.

  15. Tat acetylation modulates assembly of a viral-host RNA–protein transcription complex

    PubMed Central

    D'Orso, Iván; Frankel, Alan D.

    2009-01-01

    HIV-1 Tat enhances viral transcription elongation by forming a ribonucleoprotein complex with transactivating responsive (TAR) RNA and P-TEFb, an elongation factor composed of cyclin T1 (CycT1) and Cdk9 that phosphorylates the C-terminal domain of RNA polymerase II. Previous studies have shown that Lys-28 in the activation domain (AD) of Tat is essential for HIV-1 transcription and replication and is acetylated by p300/CBP-associated factor (PCAF), but the mechanistic basis of the Lys-28 requirement is unknown. Here, we show that Lys-28 acetylation modulates the affinity and stability of HIV-1 Tat–CycT1–TAR complexes by enhancing an interaction with the CycT1 Tat–TAR recognition motif. High-affinity assembly correlates strongly with stimulation of transcription elongation in vitro and Tat activation in vivo. In marked contrast, bovine lentiviral Tat proteins have evolved a high-affinity TAR interaction that does not require PCAF-mediated acetylation of the Tat AD or CycT1 for RNA binding, whereas HIV-2 Tat has evolved an intermediate mechanism that uses a duplicated TAR element and CycT1 to enhance RNA affinity and consequently transcription activation. The coevolution of Tat acetylation, CycT1 dependence, and TAR binding affinity is seen in viral replication assays using Tat proteins that rely on CycT1 for TAR binding but are acetylation deficient, where compensatory mutations rapidly accrue in TAR to generate high-affinity, CycT1-independent complexes reminiscent of the bovine viruses. Thus, lysine acetylation can be used to modulate and evolve the strength of a viral-host RNA–protein complex, thereby tuning the levels of transcription elongation. PMID:19223581

  16. Evidence for lysine acetylation in the coat protein of a Polerovirus

    USDA-ARS?s Scientific Manuscript database

    Virions of the RPV strain of Cereal yellow dwarf virus (CYDV-RPV) were purified from infected oat tissue and analyzed by mass spectrometry. Two conserved residues, K147 and K181, residing in the virus coat protein, were confidently identified to contain epsilon-N-acetyl groups. While no functional ...

  17. Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

    SciTech Connect

    Biswas, Arunima; Pasquel, Danielle; Tyagi, Rakesh Kumar; Mani, Sridhar

    2011-03-18

    Research highlights: {yields} Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. {yields} PXR undergoes dynamic deacetylation upon ligand-mediated activation. {yields} SIRT1 partially mediates PXR deacetylation. {yields} PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.

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

    PubMed Central

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

    2012-01-01

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

  19. The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation

    SciTech Connect

    Coutinho Carneiro, Vitor; Moraes Maciel, Renata de; Caetano de Abreu da Silva, Isabel; Furtado Madeira da Costa, Rodrigo; Neto Paiva, Claudia; Torres Bozza, Marcelo; Rosado Fantappie, Marcelo

    2009-12-25

    Schistosoma mansoni HMGB1 (SmHMGB1) was revealed to be a substrate for the parasite histone acetyltransferases SmGCN5 and SmCBP1. We found that full-length SmHMGB1, as well as its HMG-box B (but not HMG-box A) were acetylated in vitro by SmGCN5 and SmCBP1. However, SmCBP1 was able to acetylate both substrates more efficiently than SmGCN5. Interestingly, the removal of the C-terminal acidic tail of SmHMGB1 (SmHMGB1{Delta}C) resulted in increased acetylation of the protein. We showed by mammalian cell transfection assays that SmHMGB1 and SmHMGB1{Delta}C were transported from the nucleus to the cytoplasm after sodium butyrate (NaB) treatment. Importantly, after NaB treatment, SmHMGB1 was also present outside the cell. Together, our data suggest that acetylation of SmHMGB1 plays a role in cellular trafficking, culminating with its secretion to the extracellular milieu. The possible role of SmHMGB1 acetylation in the pathogenesis of schistosomiasis is discussed.

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

    PubMed

    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.

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

    PubMed Central

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

    2014-01-01

    Hormone systems evolved over 500 million years of animal evolution 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. PMID:24050258

  2. Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses

    DOE PAGES

    Nafisi, Majse; Stranne, Maria; Fimognari, Lorenzo; ...

    2015-07-22

    The epidermis on leaves protects plants from pathogen invasion and provides a waterproof barrier. It consists of a layer of cells that is surrounded by thick cell walls, which are partially impregnated by highly hydrophobic cuticular components. We show that the Arabidopsis T-DNA insertion mutants of REDUCED WALL ACETYLATION 2 (rwa2), previously identified as having reduced O-acetylation of both pectins and hemicelluloses, exhibit pleiotrophic phenotype on the leaf surface. The cuticle layer appeared diffused and was significantly thicker and underneath cell wall layer was interspersed with electron-dense deposits. A large number of trichomes were collapsed and surface permeability of themore » leaves was enhanced in rwa2 as compared to the wild type. A massive reprogramming of the transcriptome was observed in rwa2 as compared to the wild type, including a coordinated up-regulation of genes involved in responses to abiotic stress, particularly detoxification of reactive oxygen species and defense against microbial pathogens (e.g., lipid transfer proteins, peroxidases). In accordance, peroxidase activities were found to be elevated in rwa2 as compared to the wild type. In conclusion, these results indicate that cell wall acetylation is essential for maintaining the structural integrity of leaf epidermis, and that reduction of cell wall acetylation leads to global stress responses in Arabidopsis.« less

  3. Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses

    SciTech Connect

    Nafisi, Majse; Stranne, Maria; Fimognari, Lorenzo; Atwell, Susanna; Martens, Helle J.; Pedas, Pai R.; Hansen, Sara F.; Nawrath, Christiane; Scheller, Henrik V.; Kliebenstein, Daniel J.; Sakuragi, Yumiko

    2015-07-22

    Here we report that the epidermis on leaves protects plants from pathogen invasion and provides a waterproof barrier. It consists of a layer of cells that is surrounded by thick cell walls, which are partially impregnated by highly hydrophobic cuticular components. We show that the Arabidopsis T-DNA insertion mutants of REDUCED WALL ACETYLATION 2 (rwa2), previously identified as having reduced O-acetylation of both pectins and hemicelluloses, exhibit pleiotrophic phenotype on the leaf surface. The cuticle layer appeared diffused and was significantly thicker and underneath cell wall layer was interspersed with electron-dense deposits. A large number of trichomes were collapsed and surface permeability of the leaves was enhanced in rwa2 as compared to the wild type. A massive reprogramming of the transcriptome was observed in rwa2 as compared to the wild type, including a coordinated up-regulation of genes involved in responses to abiotic stress, particularly detoxification of reactive oxygen species and defense against microbial pathogens (e.g., lipid transfer proteins, peroxidases). In accordance, peroxidase activities were found to be elevated in rwa2 as compared to the wild type. These results indicate that cell wall acetylation is essential for maintaining the structural integrity of leaf epidermis, and that reduction of cell wall acetylation leads to global stress responses in Arabidopsis.

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

    PubMed

    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.

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

    PubMed

    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. Keap1-Independent Regulation of Nrf2 Activity by Protein Acetylation and a BET Bromodomain Protein

    PubMed Central

    Chatterjee, Nirmalya; Tian, Min; Spirohn, Kerstin; Boutros, Michael; Bohmann, Dirk

    2016-01-01

    Mammalian BET proteins comprise a family of bromodomain-containing epigenetic regulators with complex functions in chromatin organization and gene regulation. We identified the sole member of the BET protein family in Drosophila, Fs(1)h, as an inhibitor of the stress responsive transcription factor CncC, the fly ortholog of Nrf2. Fs(1)h physically interacts with CncC in a manner that requires the function of its bromodomains and the acetylation of CncC. Treatment of cultured Drosophila cells or adult flies with fs(1)h RNAi or with the BET protein inhibitor JQ1 de-represses CncC transcriptional activity and engages protective gene expression programs. The mechanism by which Fs(1)h inhibits CncC function is distinct from the canonical mechanism that stimulates Nrf2 function by abrogating Keap1-dependent proteasomal degradation. Consistent with the independent modes of CncC regulation by Keap1 and Fs(1)h, combinations of drugs that can specifically target these pathways cause a strong synergistic and specific activation of protective CncC- dependent gene expression and boosts oxidative stress resistance. This synergism might be exploitable for the design of combinatorial therapies to target diseases associated with oxidative stress or inflammation. PMID:27233051

  7. Molecular basis for the autoregulation of the protein acetyl transferase Rtt109

    PubMed Central

    Stavropoulos, Pete; Nagy, Vivien; Blobel, Günter; Hoelz, André

    2008-01-01

    Rtt109 is a protein acetyltransferase (PAT) that is responsible for the acetylation of lysine-56 of histone 3 (H3K56) in yeast. H3K56 acetylation has been implicated in the weakening of the interaction between the histone core and the surrounding DNA in the nucleosomal particle. Rtt109, in cooperation with various histone chaperones, promotes genomic stability and is required for resistance to DNA damaging agents. Here, we present the crystal structure of Rtt109 in complex with acetyl-CoA at a 2.0-Å resolution. Rtt109 consists of a core PAT domain, which binds the acetyl-CoA cofactor. A second domain, the activation domain, is tightly associated with the PAT domain. Autoacetylation of lysine-290 within the activation domain is required for stabilizing the interaction between the two domains and is essential for catalysis. Biochemical analysis demonstrates the requirement of a loop within the PAT domain for the binding of the histone chaperone Vps75, and mutational analysis identifies key residues for catalysis. We propose a model in which the autoacetylation of Rtt109 is crucial for the regulation of its catalytic activity. PMID:18719104

  8. Modulation of myelin basic protein gene expression by acetyl-L-carnitine.

    PubMed

    Traina, Giovanna; Federighi, Giuseppe; Macchi, Monica; Bernardi, Rodolfo; Durante, Mauro; Brunelli, Marcello

    2011-08-01

    Acetyl-L-carnitine (ALC), the acetyl ester of L-carnitine, is a naturally occurring molecule which plays an essential role in intermediary and mitochondrial metabolism. It has also neurotrophic and antioxidant actions, demonstrating efficacy and high tolerability in the treatment of neuropathies of various etiologies. ALC is a molecule of considerable interest for its clinical application in various neural disorders, although little is known regarding its effects on gene expression. Suppression subtractive hybridization methodology was used for the generation of subtracted complementary DNA libraries and the subsequent identification of differentially expressed transcripts in the rat brain after chronic ALC treatments. We provided evidence for a downregulation of the expression of all of the isoforms of myelin basic protein gene following prolonged ALC treatment, indicating a possible role in the modulation of myelin basic protein turnover, stabilizing and maintaining myelin integrity.

  9. Calorie restriction and SIRT3 trigger global reprogramming of the mitochondrial protein acetylome.

    PubMed

    Hebert, Alexander S; Dittenhafer-Reed, Kristin E; Yu, Wei; Bailey, Derek J; Selen, Ebru Selin; Boersma, Melissa D; Carson, Joshua J; Tonelli, Marco; Balloon, Allison J; Higbee, Alan J; Westphall, Michael S; Pagliarini, David J; Prolla, Tomas A; Assadi-Porter, Fariba; Roy, Sushmita; Denu, John M; Coon, Joshua J

    2013-01-10

    Calorie restriction (CR) extends life span in diverse species. Mitochondria play a key role in CR adaptation; however, the molecular details remain elusive. We developed and applied a quantitative mass spectrometry method to probe the liver mitochondrial acetyl-proteome during CR versus control diet in mice that were wild-type or lacked the protein deacetylase SIRT3. Quantification of 3,285 acetylation sites-2,193 from mitochondrial proteins-rendered a comprehensive atlas of the acetyl-proteome and enabled global site-specific, relative acetyl occupancy measurements between all four experimental conditions. Bioinformatic and biochemical analyses provided additional support for the effects of specific acetylation on mitochondrial protein function. Our results (1) reveal widespread reprogramming of mitochondrial protein acetylation in response to CR and SIRT3, (2) identify three biochemically distinct classes of acetylation sites, and (3) provide evidence that SIRT3 is a prominent regulator in CR adaptation by coordinately deacetylating proteins involved in diverse pathways of metabolism and mitochondrial maintenance.

  10. Acetylation of Surface Lysine Groups of a Protein Alters the Organization and Composition of Its Crystal Contacts.

    PubMed

    Kang, Kyungtae; Choi, Jeong-Mo; Fox, Jerome M; Snyder, Phillip W; Moustakas, Demetri T; Whitesides, George M

    2016-07-14

    This paper uses crystals of bovine carbonic anhydrase (CA) and its acetylated variant to examine (i) how a large negative formal charge can be accommodated in protein-protein interfaces, (ii) why lysine residues are often excluded from them, and (iii) how changes in the surface charge of a protein can alter the structure and organization of protein-protein interfaces. It demonstrates that acetylation of lysine residues on the surface of CA increases the participation of polar residues (particularly acetylated lysine) in protein-protein interfaces, and decreases the participation of nonpolar residues in those interfaces. Negatively charged residues are accommodated in protein-protein interfaces via (i) hydrogen bonds or van der Waals interactions with polar residues or (ii) salt bridges with other charged residues. The participation of acetylated lysine in protein-protein interfaces suggests that unacetylated lysine tends to be excluded from interfaces because of its positive charge, and not because of a loss in conformational entropy. Results also indicate that crystal contacts in acetylated CA become less constrained geometrically and, as a result, more closely packed (i.e., more tightly clustered spatially) than those of native CA. This study demonstrates a physical-organic approach-and a well-defined model system-for studying the role of charges in protein-protein interactions.

  11. Discovery of protein acetylation patterns by deconvolution of peptide isomer mass spectra.

    PubMed

    Abshiru, Nebiyu; Caron-Lizotte, Olivier; Rajan, Roshan Elizabeth; Jamai, Adil; Pomies, Christelle; Verreault, Alain; Thibault, Pierre

    2015-10-15

    Protein post-translational modifications (PTMs) play important roles in the control of various biological processes including protein-protein interactions, epigenetics and cell cycle regulation. Mass spectrometry-based proteomics approaches enable comprehensive identification and quantitation of numerous types of PTMs. However, the analysis of PTMs is complicated by the presence of indistinguishable co-eluting isomeric peptides that result in composite spectra with overlapping features that prevent the identification of individual components. In this study, we present Iso-PeptidAce, a novel software tool that enables deconvolution of composite MS/MS spectra of isomeric peptides based on features associated with their characteristic fragment ion patterns. We benchmark Iso-PeptidAce using dilution series prepared from mixtures of known amounts of synthetic acetylated isomers. We also demonstrate its applicability to different biological problems such as the identification of site-specific acetylation patterns in histones bound to chromatin assembly factor-1 and profiling of histone acetylation in cells treated with different classes of HDAC inhibitors.

  12. Modification of the conductance, selectivity and concentration-dependent saturation of Pseudomonas aeruginosa protein P channels by chemical acetylation.

    PubMed

    Hancock, R E; Poole, K; Gimple, M; Benz, R

    1983-10-26

    Protein P, an anion-specific channel-forming protein from the outer membrane of Pseudomonas aeruginosa was chemically modified by acetylation and syccinylation of its accessible amino groups. The chemically modified protein retained its ability to form oligomers on sodium dodecyl sulfate polyacrylamide gels, whereas only the acetylated protein formed channels in reconstitution experiments with lipid bilayers. Acetylated protein P demonstrated a substantially reduced mean single channel conductance (25 pS at 1 M KCl) compared to the native protein P channels (250 pS at 1 M KCl) when reconstituted into black lipid bilayer membranes. The homogeneous size distribution of single-channel conductances suggested that all of the protein P molecules had been acetylated. Zero-current potential measurements demonstrated that the acetylated protein P channel was only weakly selective for anions and allowed the permeation of cations, in contrast to the native protein P channels, which were more than 100-fold selective for anions over cations. The dependence of conductance on salt concentration was changed upon acetylation, in that acetylated protein P demonstrated a linear concentration-conductance relationship, whereas native protein P channels became saturated at high salt concentrations. These data strongly suggested that the basis of anion selectivity for native protein P channels is fixed amino groups. In agreement with this, we could demonstrate a 2.5-fold decrease in single-channel conductance between pH 7 and pH 9, between which pH values the epsilon-amino groups of amino acids would start to become deprotonated. Two alternative schemes for the topography of the protein P channel and localization of the fixed amino groups are presented and discussed.

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

    PubMed

    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.

  14. Acetylation of the SUN protein Mps3 by Eco1 regulates its function in nuclear organization

    PubMed Central

    Ghosh, Suman; Gardner, Jennifer M.; Smoyer, Christine J.; Friederichs, Jennifer M.; Unruh, Jay R.; Slaughter, Brian D.; Alexander, Richard; Chisholm, Robert D.; Lee, Kenneth K.; Workman, Jerry L.; Jaspersen, Sue L.

    2012-01-01

    The Saccharomyces cerevisiae SUN-domain protein Mps3 is required for duplication of the yeast centrosome-equivalent organelle, the spindle pole body (SPB), and it is involved in multiple aspects of nuclear organization, including telomere tethering and gene silencing at the nuclear membrane, establishment of sister chromatid cohesion, and repair of certain types of persistent DNA double-stranded breaks. How these diverse SUN protein functions are regulated is unknown. Here we show that the Mps3 N-terminus is a substrate for the acetyltransferase Eco1/Ctf7 in vitro and in vivo and map the sites of acetylation to three lysine residues adjacent to the Mps3 transmembrane domain. Mutation of these residues shows that acetylation is not essential for growth, SPB duplication, or distribution in the nuclear membrane. However, analysis of nonacetylatable mps3 mutants shows that this modification is required for accurate sister chromatid cohesion and for chromosome recruitment to the nuclear membrane. Acetylation of Mps3 by Eco1 is one of the few regulatory mechanisms known to control nuclear organization. PMID:22593213

  15. Discovery of protein acetylation patterns by deconvolution of peptide isomer mass spectra

    PubMed Central

    Abshiru, Nebiyu; Caron-Lizotte, Olivier; Rajan, Roshan Elizabeth; Jamai, Adil; Pomies, Christelle; Verreault, Alain; Thibault, Pierre

    2015-01-01

    Protein post-translational modifications (PTMs) play important roles in the control of various biological processes including protein–protein interactions, epigenetics and cell cycle regulation. Mass spectrometry-based proteomics approaches enable comprehensive identification and quantitation of numerous types of PTMs. However, the analysis of PTMs is complicated by the presence of indistinguishable co-eluting isomeric peptides that result in composite spectra with overlapping features that prevent the identification of individual components. In this study, we present Iso-PeptidAce, a novel software tool that enables deconvolution of composite MS/MS spectra of isomeric peptides based on features associated with their characteristic fragment ion patterns. We benchmark Iso-PeptidAce using dilution series prepared from mixtures of known amounts of synthetic acetylated isomers. We also demonstrate its applicability to different biological problems such as the identification of site-specific acetylation patterns in histones bound to chromatin assembly factor-1 and profiling of histone acetylation in cells treated with different classes of HDAC inhibitors. PMID:26468920

  16. Acetylation-dependent function of human single-stranded DNA binding protein 1

    PubMed Central

    Wu, Yuanzhong; Chen, Hongxia; Lu, Jinping; Zhang, Meifang; Zhang, Ruhua; Duan, Tingmei; Wang, Xin; Huang, Jun; Kang, Tiebang

    2015-01-01

    Human single-stranded DNA binding protein 1 (hSSB1) plays a critical role in responding to DNA damage and maintaining genome stability. However, the regulation of hSSB1 remains poorly studied. Here, we determined that hSSB1 acetylation at K94 mediated by the acetyltransferase p300 and the deacetylases SIRT4 and HDAC10 impaired its ubiquitin-mediated degradation by proteasomes. Moreover, we demonstrated that the hSSB1-K94R mutant had reduced cell survival in response to DNA damage by radiation or chemotherapy drugs. Furthermore, the p300/CBP inhibitor C646 significantly enhanced the sensitivity of cancer cells to chemotherapy drugs, and a positive correlation between hSSB1 and p300 level was observed in clinical colorectal cancer samples. Acetylation, a novel regulatory modification of hSSB1, is crucial for its function under both physiological and pathological conditions. This finding supports the notion that the combination of chemotherapy drugs with acetylation inhibitors may benefit cancer patients. PMID:26170237

  17. Global market for dairy proteins.

    PubMed

    Lagrange, Veronique; Whitsett, Dacia; Burris, Cameron

    2015-03-01

    This review examines the global market for dairy ingredients by assessing the global demand for dairy products in relation to major dairy ingredient categories. Each broad category of dairy ingredients is reviewed including its definition, production and trade status, key applications, and future trends. Ingredient categories examined include whole and skim milk powders (WMPs, SMPs), whey protein concentrates (WPCs) and whey protein isolates (WPIs), milk protein concentrates (MPCs) and milk protein isolates (MPIs), caseins, and caseinates. Increases in world population and improvements in socioeconomic conditions will continue to drive the demand for dairy products and ingredients in the future. Dairy proteins are increasingly recognized to have nutritional and functional advantages compared to many protein sources, and the variety of ingredients with different protein concentrations, functionality, and flavor can meet the needs of the increasingly global dairy consumption. A thorough understanding of the variety of ingredients, how the ingredients are derived from milk, and how the demand from particular markets affects the supply situation are critical elements in understanding the current ingredient marketplace.

  18. The viral protein gp120 decreases the acetylation of neuronal tubulin: potential mechanism of neurotoxicity.

    PubMed

    Avdoshina, Valeria; Caragher, Seamus P; Wenzel, Erin D; Taraballi, Francesca; Mocchetti, Italo; Harry, Gaylia Jean

    2017-05-01

    The human immunodeficiency virus (HIV) envelope protein gp120 promotes axonal damage and neurite pruning, similar to that observed in HIV-positive subjects with neurocognitive disorders. Thus, gp120 has been used to examine molecular and cellular pathways underlying HIV-mediated neuronal dysfunction. Gp120 binds to tubulin beta III, a component of neuronal microtubules. Microtubule function, which modulates the homeostasis of neurons, is regulated by polymerization and post-translational modifications. Based on these considerations, we tested the hypothesis that gp120 induces dynamic instability of neuronal microtubules. We first observed that gp120 prevents the normal polymerization of tubulin in vitro. We then tested whether gp120 alters the post-translational modifications in tubulin by examining the ability of gp120 to change the levels of acetylated tubulin in primary rat neuronal cultures. Gp120 elicited a time-dependent decrease in tubulin acetylation that was reversed by Helix-A peptide, a compound that competitively displaces the binding of gp120 to neuronal microtubules. To determine whether post-translational modifications in tubulin also occur in vivo, we measured acetylated tubulin in the cerebral cortex of HIV transgenic rats (HIV-tg). We observed a decrease in tubulin acetylation in 5- and 9-month-old HIV-tg rats when compared to age-matched wild type. Neither changes in microglia morphology nor alterations in mRNA levels for interleukin-1β and tumor necrosis factor α were detected in 5-month-old animals. Our findings propose neuronal microtubule instability as a novel mechanism of HIV neurotoxicity, without evidence of enhanced inflammation. © 2017 International Society for Neurochemistry.

  19. Modulation of Protein Phosphorylation, N-Glycosylation and Lys-Acetylation in Grape (Vitis vinifera) Mesocarp and Exocarp Owing to Lobesia botrana Infection*

    PubMed Central

    Melo-Braga, Marcella N.; Verano-Braga, Thiago; León, Ileana R.; Antonacci, Donato; Nogueira, Fábio C. S.; Thelen, Jay J.; Larsen, Martin R.; Palmisano, Giuseppe

    2012-01-01

    Grapevine (Vitis vinifera) is an economically important fruit crop that is subject to many types of insect and pathogen attack. To better elucidate the plant response to Lobesia botrana pathogen infection, we initiated a global comparative proteomic study monitoring steady-state protein expression as well as changes in N-glycosylation, phosphorylation, and Lys-acetylation in control and infected mesocarp and exocarp from V. vinifera cv Italia. A multi-parallel, large-scale proteomic approach employing iTRAQ labeling prior to three peptide enrichment techniques followed by tandem mass spectrometry led to the identification of a total of 3059 proteins, 1135 phosphorylation sites, 323 N-linked glycosylation sites and 138 Lys-acetylation sites. Of these, we could identify changes in abundance of 899 proteins. The occupancy of 110 phosphorylation sites, 10 N-glycosylation sites and 20 Lys-acetylation sites differentially changed during L. botrana infection. Sequence consensus analysis for phosphorylation sites showed eight significant motifs, two of which containing up-regulated phosphopeptides (X-G-S-X and S-X-X-D) and two containing down-regulated phosphopeptides (R-X-X-S and S-D-X-E) in response to pathogen infection. Topographical distribution of phosphorylation sites within primary sequences reveal preferential phosphorylation at both the N- and C termini, and a clear preference for C-terminal phosphorylation in response to pathogen infection suggesting induction of region-specific kinase(s). Lys-acetylation analysis confirmed the consensus X-K-Y-X motif previously detected in mammals and revealed the importance of this modification in plant defense. The importance of N-linked protein glycosylation in plant response to biotic stimulus was evident by an up-regulated glycopeptide belonging to the disease resistance response protein 206. This study represents a substantial step toward the understanding of protein and PTMs-mediated plant-pathogen interaction shedding

  20. Modulation of protein phosphorylation, N-glycosylation and Lys-acetylation in grape (Vitis vinifera) mesocarp and exocarp owing to Lobesia botrana infection.

    PubMed

    Melo-Braga, Marcella N; Verano-Braga, Thiago; León, Ileana R; Antonacci, Donato; Nogueira, Fábio C S; Thelen, Jay J; Larsen, Martin R; Palmisano, Giuseppe

    2012-10-01

    Grapevine (Vitis vinifera) is an economically important fruit crop that is subject to many types of insect and pathogen attack. To better elucidate the plant response to Lobesia botrana pathogen infection, we initiated a global comparative proteomic study monitoring steady-state protein expression as well as changes in N-glycosylation, phosphorylation, and Lys-acetylation in control and infected mesocarp and exocarp from V. vinifera cv Italia. A multi-parallel, large-scale proteomic approach employing iTRAQ labeling prior to three peptide enrichment techniques followed by tandem mass spectrometry led to the identification of a total of 3059 proteins, 1135 phosphorylation sites, 323 N-linked glycosylation sites and 138 Lys-acetylation sites. Of these, we could identify changes in abundance of 899 proteins. The occupancy of 110 phosphorylation sites, 10 N-glycosylation sites and 20 Lys-acetylation sites differentially changed during L. botrana infection. Sequence consensus analysis for phosphorylation sites showed eight significant motifs, two of which containing up-regulated phosphopeptides (X-G-S-X and S-X-X-D) and two containing down-regulated phosphopeptides (R-X-X-S and S-D-X-E) in response to pathogen infection. Topographical distribution of phosphorylation sites within primary sequences reveal preferential phosphorylation at both the N- and C termini, and a clear preference for C-terminal phosphorylation in response to pathogen infection suggesting induction of region-specific kinase(s). Lys-acetylation analysis confirmed the consensus X-K-Y-X motif previously detected in mammals and revealed the importance of this modification in plant defense. The importance of N-linked protein glycosylation in plant response to biotic stimulus was evident by an up-regulated glycopeptide belonging to the disease resistance response protein 206. This study represents a substantial step toward the understanding of protein and PTMs-mediated plant-pathogen interaction shedding

  1. Mitochondrial Complex I Deficiency Increases Protein Acetylation and Accelerates Heart Failure

    PubMed Central

    Karamanlidis, Georgios; Lee, Chi Fung; Garcia-Menendez, Lorena; Kolwicz, Stephen C.; Suthammarak, Wichit; Gong, Guohua; Sedensky, Margaret M.; Morgan, Philip G.; Wang, Wang; Tian, Rong

    2013-01-01

    Summary Mitochondrial respiratory dysfunction is linked to the pathogenesis of multiple diseases including heart failure but the specific mechanisms for this link remain largely elusive. We modeled the impairment of mitochondrial respiration by inactivation of the Ndufs4 gene, a protein critical for Complex I (C-I) assembly, in the mouse heart (cKO). While C-I supported respiration decreased by >40%, the cKO mice maintained normal cardiac function in vivo and high-energy phosphate content in isolated perfused hearts. However, the cKO mice developed accelerated heart failure after pressure overload or repeated pregnancy. Decreased NAD+/NADH ratio by C-I deficiency inhibited Sirt3 activity, leading to increase in protein acetylation, and sensitization of the permeability transition in mitochondria (mPTP). NAD+ precursor supplementation to cKO mice partially normalized the NAD+/NADH ratio, protein acetylation and mPTP sensitivity. These findings describe a mechanism connecting mitochondrial dysfunction to the susceptibility to diseases and propose a potential therapeutic target. PMID:23931755

  2. Long-term exposure to a ‘safe’ dose of bisphenol A reduced protein acetylation in adult rat testes

    NASA Astrophysics Data System (ADS)

    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.

  3. Long-term exposure to a ‘safe’ dose of bisphenol A reduced protein acetylation in adult rat testes

    PubMed Central

    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

  4. Acetaminophen analog N-acetyl-m-aminophenol, but not its reactive metabolite, N-acetyl-p-benzoquinone imine induces CYP3A activity via inhibition of protein degradation.

    PubMed

    Santoh, Masataka; Sanoh, Seigo; Ohtsuki, Yuya; Ejiri, Yoko; Kotake, Yaichiro; Ohta, Shigeru

    2017-05-06

    Cytochrome P450 (CYP) 3A subfamily members are known to metabolize various types of drugs, highlighting the importance of understanding drug-drug interactions (DDI) depending on CYP3A induction or inhibition. While transcriptional regulation of CYP3A members is widely understood, post-translational regulation needs to be elucidated. We previously reported that acetaminophen (APAP) induces CYP3A activity via inhibition of protein degradation and proposed a novel DDI concept. N-Acetyl-p-benzoquinone imine (NAPQI), the reactive metabolite of APAP formed by CYP, is known to cause adverse events related to depletion of intracellular reduced glutathione (GSH). We aimed to inspect whether NAPQI rather than APAP itself could cause the inhibitory effects on protein degradation. We found that N-acetyl-l-cysteine, the precursor of GSH, and 1-aminobenzotriazole, a nonselective CYP inhibitor, had no effect on CYP3A1/23 protein levels affected by APAP. Thus, we used APAP analogs to test CYP3A1/23 mRNA levels, protein levels, and CYP3A activity. We found N-acetyl-m-aminophenol (AMAP), a regioisomer of APAP, has the same inhibitory effects of CYP3A1/23 protein degradation, while p-acetamidobenzoic acid (PAcBA), a carboxy-substituted form of APAP, shows no inhibitory effects. AMAP and PAcBA cannot be oxidized to quinone imine forms such as NAPQI, so the inhibitory effects could depend on the specific chemical structure of APAP. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. N-Acetylaspartate reductions in brain injury: impact on post-injury neuroenergetics, lipid synthesis, and protein acetylation

    PubMed Central

    Moffett, John R.; Arun, Peethambaran; Ariyannur, Prasanth S.; Namboodiri, Aryan M. A.

    2013-01-01

    N-Acetylaspartate (NAA) is employed as a non-invasive marker for neuronal health using proton magnetic resonance spectroscopy (MRS). This utility is afforded by the fact that NAA is one of the most concentrated brain metabolites and that it produces the largest peak in MRS scans of the healthy human brain. NAA levels in the brain are reduced proportionately to the degree of tissue damage after traumatic brain injury (TBI) and the reductions parallel the reductions in ATP levels. Because NAA is the most concentrated acetylated metabolite in the brain, we have hypothesized that NAA acts in part as an extensive reservoir of acetate for acetyl coenzyme A synthesis. Therefore, the loss of NAA after TBI impairs acetyl coenzyme A dependent functions including energy derivation, lipid synthesis, and protein acetylation reactions in distinct ways in different cell populations. The enzymes involved in synthesizing and metabolizing NAA are predominantly expressed in neurons and oligodendrocytes, respectively, and therefore some proportion of NAA must be transferred between cell types before the acetate can be liberated, converted to acetyl coenzyme A and utilized. Studies have indicated that glucose metabolism in neurons is reduced, but that acetate metabolism in astrocytes is increased following TBI, possibly reflecting an increased role for non-glucose energy sources in response to injury. NAA can provide additional acetate for intercellular metabolite trafficking to maintain acetyl CoA levels after injury. Here we explore changes in NAA, acetate, and acetyl coenzyme A metabolism in response to brain injury. PMID:24421768

  6. Acetylation of TUG protein promotes the accumulation of GLUT4 glucose transporters in an insulin-responsive intracellular compartment.

    PubMed

    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.

  7. MOF and H4 K16 Acetylation Play Important Roles in DNA Damage Repair by Modulating Recruitment of DNA Damage Repair Protein Mdc1 ▿

    PubMed Central

    Li, Xiangzhi; Corsa, Callie Ann Sprunger; Pan, Patricia W.; Wu, Lipeng; Ferguson, David; Yu, Xiaochun; Min, Jinrong; Dou, Yali

    2010-01-01

    MOF (MYST1) is the major enzyme to catalyze acetylation of histone H4 lysine 16 (K16) and is highly conserved through evolution. Using a conditional knockout mouse model and the derived mouse embryonic fibroblast cell lines, we showed that loss of Mof led to a global reduction of H4 K16 acetylation, severe G2/M cell cycle arrest, massive chromosome aberration, and defects in ionizing radiation-induced DNA damage repair. We further showed that although early DNA damage sensing and signaling by ATM were normal in Mof-null cells, the recruitment of repair mediator protein Mdc1 and its downstream signaling proteins 53bp1 and Brca1 to DNA damage foci was completely abolished. Mechanistic studies suggested that Mof-mediated H4 K16 acetylation and an intact acidic pocket on H2A.X were essential for the recruitment of Mdc1. Removal of Mof and its associated proteins phenocopied a charge-neutralizing mutant of H2A.X. Given the well-characterized H4-H2A trans interactions in regulating higher-order chromatin structure, our study revealed a novel chromatin-based mechanism that regulates the DNA damage repair process. PMID:20837706

  8. MOF and H4 K16 acetylation play important roles in DNA damage repair by modulating recruitment of DNA damage repair protein Mdc1.

    PubMed

    Li, Xiangzhi; Corsa, Callie Ann Sprunger; Pan, Patricia W; Wu, Lipeng; Ferguson, David; Yu, Xiaochun; Min, Jinrong; Dou, Yali

    2010-11-01

    MOF (MYST1) is the major enzyme to catalyze acetylation of histone H4 lysine 16 (K16) and is highly conserved through evolution. Using a conditional knockout mouse model and the derived mouse embryonic fibroblast cell lines, we showed that loss of Mof led to a global reduction of H4 K16 acetylation, severe G(2)/M cell cycle arrest, massive chromosome aberration, and defects in ionizing radiation-induced DNA damage repair. We further showed that although early DNA damage sensing and signaling by ATM were normal in Mof-null cells, the recruitment of repair mediator protein Mdc1 and its downstream signaling proteins 53bp1 and Brca1 to DNA damage foci was completely abolished. Mechanistic studies suggested that Mof-mediated H4 K16 acetylation and an intact acidic pocket on H2A.X were essential for the recruitment of Mdc1. Removal of Mof and its associated proteins phenocopied a charge-neutralizing mutant of H2A.X. Given the well-characterized H4-H2A trans interactions in regulating higher-order chromatin structure, our study revealed a novel chromatin-based mechanism that regulates the DNA damage repair process.

  9. A bromodomain–DNA interaction facilitates acetylation-dependent bivalent nucleosome recognition by the BET protein BRDT

    PubMed Central

    Miller, Thomas C. R.; Simon, Bernd; Rybin, Vladimir; Grötsch, Helga; Curtet, Sandrine; Khochbin, Saadi; Carlomagno, Teresa; Müller, Christoph W.

    2016-01-01

    Bromodomains are critical components of many chromatin modifying/remodelling proteins and are emerging therapeutic targets, yet how they interact with nucleosomes, rather than acetylated peptides, remains unclear. Using BRDT as a model, we characterized how the BET family of bromodomains interacts with site-specifically acetylated nucleosomes. Here we report that BRDT interacts with nucleosomes through its first (BD1), but not second (BD2) bromodomain, and that acetylated histone recognition by BD1 is complemented by a bromodomain–DNA interaction. Simultaneous DNA and histone recognition enhances BRDT's nucleosome binding affinity and specificity, and its ability to localize to acetylated chromatin in cells. Conservation of DNA binding in bromodomains of BRD2, BRD3 and BRD4, indicates that bivalent nucleosome recognition is a key feature of these bromodomains and possibly others. Our results elucidate the molecular mechanism of BRDT association with nucleosomes and identify structural features of the BET bromodomains that may be targeted for therapeutic inhibition. PMID:27991587

  10. Quantitative assessment of the impact of the gut microbiota on lysine epsilon-acetylation of host proteins using gnotobiotic mice.

    PubMed

    Simon, Gabriel M; Cheng, Jiye; Gordon, Jeffrey I

    2012-07-10

    The gut microbiota influences numerous aspects of human biology. One facet that has not been thoroughly explored is its impact on the host proteome. We hypothesized that the microbiota may produce certain of its effects through covalent modification of host proteins. We focused on protein lysine ε-acetylation because of its recently discovered roles in regulation of cell metabolism, and the potential for products of microbial fermentation to interact with the lysine acetylation machinery of host cells. Germ-free mice, fed a (15)N-labeled diet for two generations, were colonized as adults with a microbiota harvested from conventionally raised mouse donors. Using high-resolution mass spectrometry, we quantified 3,891 liver and proximal colonic proteins, 558 of which contained 1,602 sites of lysine acetylation, 43% not previously described. Multiple proteins from multiple subcellular compartments underwent microbiota-associated increases in their levels of lysine acetylation at one or more residues, in one or both tissues. Acetylated proteins were enriched in functions related to energy production, respiration, and primary metabolism. A number of the acetylation events affect lysine residues at or near the active sites of enzymes, whereas others occur at locations that may affect other facets of protein function. One of these modifications, affecting Lys292 in mouse α-1-antitrypsin, was detected in the corresponding lysine of the human serum protein. Methods described in this report can be applied to other co- or posttranslational modifications, and add quantitation of protein expression and covalent modification to the arsenal of techniques for characterizing the dynamic, important interactions between gut symbionts and their hosts.

  11. Acetyl-phosphate is a critical determinant of lysine acetylation in E. coli.

    PubMed

    Weinert, Brian T; Iesmantavicius, Vytautas; Wagner, Sebastian A; Schölz, Christian; Gummesson, Bertil; Beli, Petra; Nyström, Thomas; Choudhary, Chunaram

    2013-07-25

    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 in a manner that depended on the formation of acetyl-phosphate (AcP) through glycolysis. Mutant cells unable to produce AcP had significantly reduced acetylation levels, while mutant cells unable to convert AcP to acetate had significantly elevated acetylation levels. We showed that AcP can chemically 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 level and is dynamically affected by metabolism and cell proliferation in a global, uniform manner.

  12. Aging and exercise affect the level of protein acetylation and SIRT1 activity in cerebellum of male rats.

    PubMed

    Marton, Orsolya; Koltai, Erika; Nyakas, Csaba; Bakonyi, Tibor; Zenteno-Savin, Tania; Kumagai, Shuzo; Goto, Sataro; Radak, Zsolt

    2010-12-01

    Aging is associated with a gradual decline in cognitive and motor functions, the result of complex biochemical processes including pre- and posttranslational modifications of proteins. Sirtuins are NAD(+) dependent protein deacetylases. These enzymes modulate the aging process by lysine deacetylation, which alters the activity and stability of proteins. Exercise can increase mean life-span and improve quality of life. Data from our laboratories revealed that 4 weeks of treadmill running improves performance in the Morris Maze test for young (4 months, old) but not old (30 months, old) male rats, and the exercise could not prevent the age-associated loss in muscle strength assessed by a gripping test. The positive correlation between protein acetylation and the gripping test suggests that the age-dependent decrease in relative activity of SIRT1 in the cerebellum impairs motor function. Similarly to the acetylation level of total proteins, the acetylation of ά -tubulin is also increased with aging, while the effect of exercise training was not found to be significant. Moreover, the protein content of nicotinamide phosphoribosyltransferase, one of the key enzymes of NAD biosynthesis, decreased in the young exercise group. These data suggest that aging results in decreased specific activity of SIRT1 in cerebellum, which could lead to increased acetylation of protein residues, including ά-tubulin, that interfere with motor function.

  13. NAT10, a nucleolar protein, localizes to the midbody and regulates cytokinesis and acetylation of microtubules

    SciTech Connect

    Shen, Qi; Zheng, Xingzheng; McNutt, Michael A.; Guang, Lizhao; Sun, Ying; Wang, Jiaochen; Gong, Yilei; Hou, Lin; Zhang, Bo

    2009-06-10

    The midbody is a structural organelle formed in late phase mitosis which is responsible for completion of cytokinesis. Although various kinds of proteins have been found to distribute or immigrate to this organelle, their functions have still not been completely worked out. In this study, we demonstrated that NAT10 (N-acetyltransferase 10, NAT10) is not only predominantly distributed in the nucleolus in interphase, but is also concentrated in the mitotic midbody during telophase. The domain in N-terminal residues 549-834 of NAT10 specifically mediated its subcellular localization. Treatment with genotoxic agents or irradiation increased concentration of NAT10 in both the nucleolus and midbody. Moreover, DNA damage induced increase of NAT10 in the midbody apparently accompanied by in situ elevation of the level of acetylated {alpha}-tubulin, suggesting that it plays a role in maintaining or enhancing stability of {alpha}-tubulin. The depletion of NAT10 induced defects in nucleolar assembly, cytokinesis and decreased acetylated {alpha}-tubulin, leading to G2/M cell cycle arrest or delay of mitotic exit. In addition, over-expression of NAT10 was found in a variety of soft tissue sarcomas, and correlated with tumor histological grading. These results indicate that NAT10 may play an important role in cell division through facilitating reformation of the nucleolus and midbody in the late phase of cell mitosis, and stabilization of microtubules.

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

    PubMed

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

    2012-07-27

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

  15. Global Proteome Analyses of Lysine Acetylation and Succinylation Reveal the Widespread Involvement of both Modification in Metabolism in the Embryo of Germinating Rice Seed.

    PubMed

    He, Dongli; Wang, Qiong; Li, Ming; Damaris, Rebecca Njeri; Yi, Xingling; Cheng, Zhongyi; Yang, Pingfang

    2016-03-04

    Regulation of rice seed germination has been shown to mainly occur at post-transcriptional levels, of which the changes on proteome status is a major one. Lysine acetylation and succinylation are two prevalent protein post-translational modifications (PTMs) involved in multiple biological processes, especially for metabolism regulation. To investigate the potential mechanism controlling metabolism regulation in rice seed germination, we performed the lysine acetylation and succinylation analyses simultaneously. Using high-accuracy nano-LC-MS/MS in combination with the enrichment of lysine acetylated or succinylated peptides from digested embryonic proteins of 24 h after imbibition (HAI) rice seed, a total of 699 acetylated sites from 389 proteins and 665 succinylated sites from 261 proteins were identified. Among these modified lysine sites, 133 sites on 78 proteins were commonly modified by two PTMs. The overlapped PTM sites were more likely to be in polar acidic/basic amino acid regions and exposed on the protein surface. Both of the acetylated and succinylated proteins cover nearly all aspects of cellular functions. Ribosome complex and glycolysis/gluconeogenesis-related proteins were significantly enriched in both acetylated and succinylated protein profiles through KEGG enrichment and protein-protein interaction network analyses. The acetyl-CoA and succinyl-CoA metabolism-related enzymes were found to be extensively modified by both modifications, implying the functional interaction between the two PTMs. This study provides a rich resource to examine the modulation of the two PTMs on the metabolism pathway and other biological processes in germinating rice seed.

  16. Acetylation and phosphorylation control both local and global stability of the chloroplast F1 ATP synthase

    PubMed Central

    Schmidt, Carla; Beilsten-Edmands, Victoria; Mohammed, Shabaz; Robinson, Carol V.

    2017-01-01

    ATP synthases (ATPases) are enzymes that produce ATP and control the pH in the cell or cellular compartments. While highly conserved over different species, ATPases are structurally well-characterised but the existence and functional significance of many post-translational modifications (PTMs) is not well understood. We combined a range of mass spectrometric techniques to unravel the location and extent of PTMs in the chloroplast ATP synthase (cATPase) purified from spinach leaves. We identified multiple phosphorylation and acetylation sites and found that both modifications stabilise binding of ε and δ subunits. Comparing cross-linking of naturally modified cATPase with the in vitro deacetylated enzyme revealed a major conformational change in the ε subunit in accord with extended and folded forms of the subunit. Locating modified residues within the catalytic head we found that phosphorylated and acetylated residues are primarily on α/β and β/α interfaces respectively. By aligning along different interfaces the higher abundance acetylated residues are proximal to the regulatory sites while the lower abundance phosphorylation sites are more densely populated at the catalytic sites. We propose that modifications in the catalytic head, together with the conformational change in subunit ε, work in synergy to fine-tune the enzyme during adverse conditions. PMID:28276484

  17. Acetylation of Werner syndrome protein (WRN): relationships with DNA damage, DNA replication and DNA metabolic activities

    PubMed Central

    Lozada, Enerlyn; Yi, Jingjie; Luo, Jianyuan; Orren, David K.

    2014-01-01

    Loss of WRN function causes Werner Syndrome, characterized by increased genomic instability, elevated cancer susceptibility and premature aging. Although WRN is subject to acetylation, phosphorylation and sumoylation, the impact of these modifications on WRN’s DNA metabolic function remains unclear. Here, we examined in further depth the relationship between WRN acetylation and its role in DNA metabolism, particularly in response to induced DNA damage. Our results demonstrate that endogenous WRN is acetylated somewhat under unperturbed conditions. However, levels of acetylated WRN significantly increase after treatment with certain DNA damaging agents or the replication inhibitor hydroxyurea. Use of DNA repair-deficient cells or repair pathway inhibitors further increase levels of acetylated WRN, indicating that induced DNA lesions and their persistence are at least partly responsible for increased acetylation. Notably, acetylation of WRN correlates with inhibition of DNA synthesis, suggesting that replication blockage might underlie this effect. Moreover, WRN acetylation modulates its affinity for and activity on certain DNA structures, in a manner that may enhance its relative specificity for physiological substrates. Our results also show that acetylation and deacetylation of endogenous WRN is a dynamic process, with sirtuins and other histone deacetylases contributing to WRN deacetylation. These findings advance our understanding of the dynamics of WRN acetylation under unperturbed conditions and following DNA damage induction, linking this modification not only to DNA damage persistence but also potentially to replication stalling caused by specific DNA lesions. Our results are consistent with proposed metabolic roles for WRN and genomic instability phenotypes associated with WRN deficiency. PMID:24965941

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

    PubMed

    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.

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

    PubMed Central

    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

  20. Arsenic Trioxide Reduces Global Histone H4 Acetylation at Lysine 16 through Direct Binding to Histone Acetyltransferase hMOF in Human Cells

    PubMed Central

    Liu, Da; Wu, Donglu; Zhao, Linhong; Yang, Yang; Ding, Jian; Dong, Liguo; Hu, Lianghai; Wang, Fei; Zhao, Xiaoming; Cai, Yong; Jin, Jingji

    2015-01-01

    Histone post-translational modification heritably regulates gene expression involved in most cellular biological processes. Experimental studies suggest that alteration of histone modifications affects gene expression by changing chromatin structure, causing various cellular responses to environmental influences. Arsenic (As), a naturally occurring element and environmental pollutant, is an established human carcinogen. Recently, increasing evidence suggests that As-mediated epigenetic mechanisms may be involved in its toxicity and carcinogenicity, but how this occurs is still unclear. Here we present evidence that suggests As-induced global histone H4K16 acetylation (H4K16ac) partly due to the direct physical interaction between As and histone acetyltransferase (HAT) hMOF (human male absent on first) protein, leading to the loss of hMOF HAT activity. Our data show that decreased global H4K16ac and increased deacetyltransferase HDAC4 expression occurred in arsenic trioxide (As2O3)-exposed HeLa or HEK293T cells. However, depletion of HDAC4 did not affect global H4K16ac, and it could not raise H4K16ac in cells exposed to As2O3, suggesting that HDAC4 might not directly be involved in histone H4K16 de-acetylation. Using As-immobilized agarose, we confirmed that As binds directly to hMOF, and that this interaction was competitively inhibited by free As2O3. Also, the direct interaction of As and C2CH zinc finger peptide was verified by MAIDI-TOF mass and UV absorption. In an in vitro HAT assay, As2O3 directly inhibited hMOF activity. hMOF over-expression not only increased resistance to As and caused less toxicity, but also effectively reversed reduced H4K16ac caused by As exposure. These data suggest a theoretical basis for elucidating the mechanism of As toxicity. PMID:26473953

  1. Two Trichome Birefringence-Like Proteins Mediate Xylan Acetylation, Which Is Essential for Leaf Blight Resistance in Rice.

    PubMed

    Gao, Yaping; He, Congwu; Zhang, Dongmei; Liu, Xiangling; Xu, Zuopeng; Tian, Yanbao; Liu, Xue-Hui; Zang, Shanshan; Pauly, Markus; Zhou, Yihua; Zhang, Baocai

    2017-01-01

    Acetylation is a ubiquitous modification on cell wall polymers, which play a structural role in plant growth and stress defenses. However, the mechanisms for how crop plants accomplish cell wall polymer O-acetylation are largely unknown. Here, we report on the isolation and characterization of two trichome birefringence-like (tbl) mutants in rice (Oryza sativa), which are affected in xylan O-acetylation. ostbl1 and ostbl2 single mutant and the tbl1 tbl2 double mutant displayed a stunted growth phenotype with varied degree of dwarfism. As shown by chemical assays, the wall acetylation level is affected in the mutants and the knock-down and overexpression transgenic plants. Furthermore, NMR spectroscopy analyses showed that all those mutants have varied decreases in xylan monoacetylation. The divergent expression levels of OsTBL1 and OsTBL2 explained the chemotype difference and indicated that OsTBL1 is a functionally dominant gene. OsTBL1 was found to be Golgi-localized. The recombinant OsTBL1 protein incorporates acetyl groups onto xylan. By using xylopentaose, a preferred acceptor substrate, OsTBL1 can transfer up to four acetyl residues onto xylopentaose, and this activity showed saturable kinetics. 2D-NMR spectroscopy showed that OsTBL1 transfers acetate to both 2-O and 3-O sites of xylosyl residues. In addition, ostbl1 and tbl1 tbl2 displayed susceptibility to rice blight disease, indicating that this xylan modification is required for pathogen resistance. This study identifies the major genes responsible for xylan acetylation in rice plants.

  2. Two Trichome Birefringence-Like Proteins Mediate Xylan Acetylation, Which Is Essential for Leaf Blight Resistance in Rice1[OPEN

    PubMed Central

    He, Congwu; Liu, Xiangling; Tian, Yanbao; Liu, Xue-Hui; Pauly, Markus

    2017-01-01

    Acetylation is a ubiquitous modification on cell wall polymers, which play a structural role in plant growth and stress defenses. However, the mechanisms for how crop plants accomplish cell wall polymer O-acetylation are largely unknown. Here, we report on the isolation and characterization of two trichome birefringence-like (tbl) mutants in rice (Oryza sativa), which are affected in xylan O-acetylation. ostbl1 and ostbl2 single mutant and the tbl1 tbl2 double mutant displayed a stunted growth phenotype with varied degree of dwarfism. As shown by chemical assays, the wall acetylation level is affected in the mutants and the knock-down and overexpression transgenic plants. Furthermore, NMR spectroscopy analyses showed that all those mutants have varied decreases in xylan monoacetylation. The divergent expression levels of OsTBL1 and OsTBL2 explained the chemotype difference and indicated that OsTBL1 is a functionally dominant gene. OsTBL1 was found to be Golgi-localized. The recombinant OsTBL1 protein incorporates acetyl groups onto xylan. By using xylopentaose, a preferred acceptor substrate, OsTBL1 can transfer up to four acetyl residues onto xylopentaose, and this activity showed saturable kinetics. 2D-NMR spectroscopy showed that OsTBL1 transfers acetate to both 2-O and 3-O sites of xylosyl residues. In addition, ostbl1 and tbl1 tbl2 displayed susceptibility to rice blight disease, indicating that this xylan modification is required for pathogen resistance. This study identifies the major genes responsible for xylan acetylation in rice plants. PMID:27864442

  3. The Role of the Plant-Specific ALTERED XYLOGLUCAN9 Protein in Arabidopsis Cell Wall Polysaccharide O-Acetylation1[OPEN

    PubMed Central

    Schultink, Alex; Naylor, Dan; Dama, Murali; Pauly, Markus

    2015-01-01

    A mutation in the ALTERED XYLOGLUCAN9 (AXY9) gene was found to be causative for the decreased xyloglucan acetylation phenotype of the axy9.1 mutant, which was identified in a forward genetic screen for Arabidopsis (Arabidopsis thaliana) mutants. The axy9.1 mutant also exhibits decreased O-acetylation of xylan, implying that the AXY9 protein has a broad role in polysaccharide acetylation. An axy9 insertional mutant exhibits severe growth defects and collapsed xylem, demonstrating the importance of wall polysaccharide O-acetylation for normal plant growth and development. Localization and topological experiments indicate that the active site of the AXY9 protein resides within the Golgi lumen. The AXY9 protein appears to be a component of the plant cell wall polysaccharide acetylation pathway, which also includes the REDUCED WALL ACETYLATION and TRICHOME BIREFRINGENCE-LIKE proteins. The AXY9 protein is distinct from the TRICHOME BIREFRINGENCE-LIKE proteins, reported to be polysaccharide acetyltransferases, but does share homology with them and other acetyltransferases, suggesting that the AXY9 protein may act to produce an acetylated intermediate that is part of the O-acetylation pathway. PMID:25681330

  4. PCAF-mediated acetylation of transcriptional factor HOXB9 suppresses lung adenocarcinoma progression by targeting oncogenic protein JMJD6

    PubMed Central

    Wan, Junhu; Xu, Weizhi; Zhan, Jun; Ma, Ji; Li, Xueying; Xie, Yuping; Wang, Jiadong; Zhu, Wei-guo; Luo, Jianyuan; Zhang, Hongquan

    2016-01-01

    HOXB9 is a homeobox domain-containing transcription factor, playing an important role in embryonic development and cancer progression. However, the precise post-translational modifications (PTMs) of HOXB9 and the corresponding roles are unclear. Here, we report that acetyltransferase p300/CBP-associated factor (PCAF) interacts with and acetylates HOXB9 both in vivo and in vitro. Conversely, the acetylation of HOXB9 can be reversed by deacetylase SIRT1. Furthermore, we found that HOXB9 is acetylated at lysine 27 (AcK27). Functionally, in contrast to the wild type HOXB9, AcK27-HOXB9 decreased its capacity in promoting lung cancer cell migration and tumor growth in mice. Mechanistically, AcK27-HOXB9 suppresses the transcription of its target gene Jumonji domain-containing protein 6 (JMJD6) by direct occupying the promoter of JMJD6 gene. For clinical relevance, elevated HOXB9 acetylation at K27 predicts a better prognosis in lung adenocarcinoma patients. Taken together, we identified the first PTM of HOXB9 by demonstrating that HOXB9 can be acetylated and AcK27-HOXB9 counteracts the role of the wild-type HOXB9 in regulating lung adenocarcinoma progression. PMID:27613418

  5. The chromatin-binding protein HMGN3 stimulates histone acetylation and transcription across the Glyt1 gene

    PubMed Central

    Barkess, Gráinne; Postnikov, Yuri; Campos, Chrisanne D.; Mishra, Shivam; Mohan, Gokula; Verma, Sakshi; Bustin, Michael; West, Katherine L.

    2013-01-01

    HMGNs are nucleosome-binding proteins that alter the pattern of histone modifications and modulate the binding of linker histones to chromatin. The HMGN3 family member exists as two splice forms, HMGN3a which is full-length and HMGN3b which lacks the C-terminal RD (regulatory domain). In the present study, we have used the Glyt1 (glycine transporter 1) gene as a model system to investigate where HMGN proteins are bound across the locus in vivo, and to study how the two HMGN3 splice variants affect histone modifications and gene expression. We demonstrate that HMGN1, HMGN2, HMGN3a and HMGN3b are bound across the Glyt1 gene locus and surrounding regions, and are not enriched more highly at the promoter or putative enhancer. We conclude that the peaks of H3K4me3 (trimethylated Lys4 of histone H3) and H3K9ac (acetylated Lys9 of histone H3) at the active Glyt1a promoter do not play a major role in recruiting HMGN proteins. HMGN3a/b binding leads to increased H3K14 (Lys14 of histone H3) acetylation and stimulates Glyt1a expression, but does not alter the levels of H3K4me3 or H3K9ac enrichment. Acetylation assays show that HMGN3a stimulates the ability of PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] to acetylate nucleosomal H3 in vitro, whereas HMGN3b does not. We propose a model where HMGN3a/b-stimulated H3K14 acetylation across the bodies of large genes such as Glyt1 can lead to more efficient transcription elongation and increased mRNA production. PMID:22150271

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  8. O-Acetylation of Arabidopsis Hemicellulose Xyloglucan Requires AXY4 or AXY4L, Proteins with a TBL and DUF231 Domain[W][OA

    PubMed Central

    Gille, Sascha; de Souza, Amancio; Xiong, Guangyan; Benz, Monique; Cheng, Kun; Schultink, Alex; Reca, Ida-Barbara; Pauly, Markus

    2011-01-01

    In an Arabidopsis thaliana forward genetic screen aimed at identifying mutants with altered structures of their hemicellulose xyloglucan (axy mutants) using oligosaccharide mass profiling, two nonallelic mutants (axy4-1 and axy4-2) that have a 20 to 35% reduction in xyloglucan O-acetylation were identified. Mapping of the mutation in axy4-1 identified AXY4, a type II transmembrane protein with a Trichome Birefringence-Like domain and a domain of unknown function (DUF231). Loss of AXY4 transcript results in a complete lack of O-acetyl substituents on xyloglucan in several tissues, except seeds. Seed xyloglucan is instead O-acetylated by the paralog AXY4like, as demonstrated by the analysis of the corresponding T-DNA insertional lines. Wall fractionation analysis of axy4 knockout mutants indicated that only a fraction containing xyloglucan is non-O-acetylated. Hence, AXY4/AXY4L is required for the O-acetylation of xyloglucan, and we propose that these proteins represent xyloglucan-specific O-acetyltransferases, although their donor and acceptor substrates have yet to be identified. An Arabidopsis ecotype, Ty-0, has reduced xyloglucan O-acetylation due to mutations in AXY4, demonstrating that O-acetylation of xyloglucan does not impact the plant’s fitness in its natural environment. The relationship of AXY4 with another previously identified group of Arabidopsis proteins involved in general wall O-acetylation, reduced wall acetylation, is discussed. PMID:22086088

  9. Acetylation of Transition Protein 2 (TP2) by KAT3B (p300) Alters Its DNA Condensation Property and Interaction with Putative Histone Chaperone NPM3*

    PubMed Central

    Pradeepa, Madapura M.; Nikhil, Gupta; Hari Kishore, Annavarapu; Bharath, Giriyapura N.; Kundu, Tapas K.; Rao, Manchanahalli R. Satyanarayana

    2009-01-01

    The hallmark of mammalian spermiogenesis is the dramatic chromatin remodeling process wherein the nucleosomal histones are replaced by the transition proteins TP1, TP2, and TP4. Subsequently these transition proteins are replaced by the protamines P1 and P2. Hyperacetylation of histone H4 is linked to their replacement by transition proteins. Here we report that TP2 is acetylated in vivo as detected by anti-acetylated lysine antibody and mass spectrometric analysis. Further, recombinant TP2 is acetylated in vitro by acetyltransferase KAT3B (p300) more efficiently than by KAT2B (PCAF). In vivo p300 was demonstrated to acetylate TP2. p300 acetylates TP2 in its C-terminal domain, which is highly basic in nature and possesses chromatin-condensing properties. Mass spectrometric analysis showed that p300 acetylates four lysine residues in the C-terminal domain of TP2. Acetylation of TP2 by p300 leads to significant reduction in its DNA condensation property as studied by circular dichroism and atomic force microscopy analysis. TP2 also interacts with a putative histone chaperone, NPM3, wherein expression is elevated in haploid spermatids. Interestingly, acetylation of TP2 impedes its interaction with NPM3. Thus, acetylation of TP2 adds a new dimension to its role in the dynamic reorganization of chromatin during mammalian spermiogenesis. PMID:19710011

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

    PubMed

    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.

  11. The Fasted/Fed Mouse Metabolic Acetylome: N6-Acetylation Differences Suggest Acetylation Coordinates Organ-Specific Fuel Switching

    PubMed Central

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

    2011-01-01

    The elucidation of extra-nuclear lysine acetylation has been of growing interest, as the co-substrate 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-MSMS 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. PMID:21728379

  12. Characterization of N-Acetyl-Tryptophan Degradation in Protein Therapeutic Formulations.

    PubMed

    Hogan, Kyle L; Leiske, Danielle; Salisbury, Cleo M

    2017-08-24

    N-Acetyl-tryptophan (NAT) is used as a stabilizer for preparations of human serum albumin and has more recently been demonstrated to provide oxidative protection for labile Trp residues in monoclonal antibodies. As a component in the formulations of protein therapeutics, NAT is sacrificially degraded; therefore understanding the identity and quantity of NAT degradants potentially formed in these drug products is essential to understanding the potential patient impact of this additive. Here we report a simple reversed phase chromatography approach that allows systematic investigation of NAT degradation in relevant formulations under stressed conditions. Screening a panel of NAT-containing samples following a variety of forced stress conditions led to a range of NAT degradation from minimal (3%) to significant (83%). NAT degradants were observed to be largely conserved between oxidative and thermal stress conditions. Online mass spectrometry and standard compound synthesis allowed for identification of the major degradants in the stressed sample panel. NAT degradation was minimal under recommended storage conditions and in relevant thermal stress conditions for a representative protein therapeutic drug product, suggesting that NAT is stable under normal manufacturing, storage, and handling conditions. This work supports the use of NAT as an antioxidant in liquid drug product formulations. Copyright © 2017. Published by Elsevier Inc.

  13. Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis.

    PubMed

    Xie, Longxiang; Wang, Xiaobo; Zeng, Jie; Zhou, Mingliang; Duan, Xiangke; Li, Qiming; Zhang, Zhen; Luo, Hongping; Pang, Lei; Li, Wu; Liao, Guojian; Yu, Xia; Li, Yunxu; Huang, Hairong; Xie, Jianping

    2015-02-01

    N(ɛ)-Acetylation of lysine residues represents a pivotal post-translational modification used by both eukaryotes and prokaryotes to modulate diverse biological processes. Mycobacterium tuberculosis is the causative agent of tuberculosis, one of the most formidable public health threats. Many aspects of the biology of M. tuberculosis remain elusive, in particular the extent and function of N(ɛ)-lysine acetylation. With a combination of anti-acetyllysine antibody-based immunoaffinity enrichment with high-resolution mass spectrometry, we identified 1128 acetylation sites on 658 acetylated M. tuberculosis proteins. GO analysis of the acetylome showed that acetylated proteins are involved in the regulation of diverse cellular processes including metabolism and protein synthesis. Six types of acetylated peptide sequence motif were revealed from the acetylome. Twenty lysine-acetylated proteins showed homology with acetylated proteins previously identified from Escherichia coli, Salmonella enterica, Bacillus subtilis and Streptomyces roseosporus, with several acetylation sites highly conserved among four or five bacteria, suggesting that acetylated proteins are more conserved. Notably, several proteins including isocitrate lyase involved in the persistence, virulence and antibiotic resistance are acetylated, and site-directed mutagenesis of isocitrate lyase acetylation site to glutamine led to a decrease of the enzyme activity, indicating major roles of KAc in these proteins engaged cellular processes. Our data firstly provides a global survey of M. tuberculosis acetylation, and implicates extensive regulatory role of acetylation in this pathogen. This may serve as an important basis to address the roles of lysine acetylation in M. tuberculosis metabolism, persistence and virulence. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Maximal oxidative capacity during exercise is associated with skeletal muscle fuel selection and dynamic changes in mitochondrial protein acetylation

    PubMed Central

    Overmyer, Katherine A.; Evans, Charles R.; Qi, Nathan R.; Minogue, Catherine E.; Carson, Joshua J.; Chermside-Scabbo, Christopher J.; Koch, Lauren G.; Britton, Steven L.; Pagliarini, David J.; Coon, Joshua J.; Burant, Charles F.

    2015-01-01

    Summary Maximal exercise-associated oxidative capacity is strongly correlated with health and longevity in humans. Rats selectively bred for high running capacity (HCR) have improved metabolic health and are longer-lived than their low capacity counterparts (LCR). Using metabolomic and proteomic profiling, we show that HCR efficiently oxidize fatty acids (FA) and branched-chain amino acid (BCAA), sparing glycogen and reducing accumulation of short- and medium-chain acylcarnitines. HCR mitochondria have reduced acetylation of mitochondrial proteins within oxidative pathways at rest, and there is rapid protein deacetylation with exercise, which is greater in HCR than LCR. Fluxomic analysis of valine degradation with exercise demonstrates a functional role of differential protein acetylation in HCR and LCR. Our data suggest efficient FA and BCAA utilization contribute to high intrinsic exercise capacity and the health and longevity benefits associated with enhanced fitness. PMID:25738461

  15. Acyl hydrolases from trans-AT polyketide synthases target acetyl units on acyl carrier proteins.

    PubMed

    Jenner, Matthew; Afonso, Jose P; Kohlhaas, Christoph; Karbaum, Petra; Frank, Sarah; Piel, Jörn; Oldham, Neil J

    2016-04-18

    Acyl hydrolase (AH) domains are a common feature of trans-AT PKSs. They have been hypothesised to perform a proofreading function by removing acyl chains from stalled sites. This study determines the substrate tolerance of the AH PedC for a range of acyl-ACPs. Clear preference towards short, linear acyl-ACPs is shown, with acetyl-ACP the best substrate. These results imply a more targeted housekeeping role for PedC: namely the removal of unwanted acetyl groups from ACP domains caused by erroneous transfer of acetyl-CoA, or possibly by decarboxylation of malonyl-ACP.

  16. Repetitive transcranial magnetic stimulation induces long-lasting changes in protein expression and histone acetylation

    PubMed Central

    Etiévant, Adeline; Manta, Stella; Latapy, Camille; Magno, Luiz Alexandre V.; Fecteau, Shirley; Beaulieu, Jean-Martin

    2015-01-01

    The use of non-invasive brain stimulation like repetitive transcranial magnetic stimulation (rTMS) is an increasingly popular set of methods with promising results for the treatment of neurological and psychiatric disorders. Despite great enthusiasm, the impact of non-invasive brain stimulation on its neuronal substrates remains largely unknown. Here we show that rTMS applied over the frontal cortex of awaken mice induces dopamine D2 receptor dependent persistent changes of CDK5 and PSD-95 protein levels specifically within the stimulated brain area. Importantly, these modifications were associated with changes of histone acetylation at the promoter of these genes and prevented by administration of the histone deacetylase inhibitor MS-275. These findings show that, like several other psychoactive treatments, repeated rTMS sessions can exert long-lasting effects on neuronal substrates. This underscores the need of understanding these effects in the development of future clinical applications as well as in the establishment of improved guidelines to use rTMS in non-medical settings. PMID:26585834

  17. Evidence that acetyl phosphate functions as a global signal during biofilm development.

    PubMed

    Wolfe, Alan J; Chang, Dong-Eun; Walker, Jason D; Seitz-Partridge, Jeanine E; Vidaurri, Michael D; Lange, Charles F; Prüss, Birgit M; Henk, Margaret C; Larkin, John C; Conway, Tyrrell

    2003-05-01

    We used DNA macroarray analysis to identify genes that respond to the status of the intracellular acetyl phosphate (acP) pool. Genes whose expression correlated negatively with the ability to synthesize acP (i.e. negatively regulated genes) function primarily in flagella biosynthesis, a result consistent with observations that we published previously (Prüss and Wolfe, 1994, Mol Microbiol 12: 973-984). In contrast, genes whose expression correlated positively with the ability to synthesize acP (i.e. positively regulated genes) include those for type 1 pilus assembly, colanic acid (capsule) biosynthesis and certain stress effectors. To our knowledge, this constitutes the first report that these genes may respond to the status of the intracellular acP pool. Previously, other researchers have implicated flagella, type 1 pili, capsule and diverse stress effectors in the formation of biofilms. We therefore tested whether cells altered in their ability to metabolize acP could construct normal biofilms, and found that they could not. Cells defective for the production of acP and cells defective for the degradation of acP could both form biofilms, but these biofilms exhibited characteristics substantially different from each other and from biofilms formed by their wild-type parent. We confirmed the role of individual cell surface structures, the expression of which appears to correlate with acP levels, in fim or fli mutants that cannot assemble type 1 pili or flagella respectively. Thus, the information gained by expression profiling of cells with altered acP metabolism indicates that acP may help to co-ordinate the expression of surface structures and cellular processes involved in the initial stages of wild-type biofilm development.

  18. The S protein of bovine coronavirus is a hemagglutinin recognizing 9-O-acetylated sialic acid as a receptor determinant.

    PubMed Central

    Schultze, B; Gross, H J; Brossmer, R; Herrler, G

    1991-01-01

    The S protein of bovine coronavirus (BCV) has been isolated from the viral membrane and purified by gradient centrifugation. Purified S protein was identified as a viral hemagglutinin. Inactivation of the cellular receptors by sialate 9-O-acetylesterase and generation of receptors by sialylation of erythrocytes with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicate that S protein recognizes 9-O-acetylated sialic acid as a receptor determinant as has been shown previously for intact virions. The second glycoprotein of BCV, HE, which has been thought previously to be responsible for the hemagglutinating activity of BCV, is a less efficient hemagglutinin; it agglutinates mouse and rat erythrocytes, but in contrast to S protein, it is unable to agglutinate chicken erythrocytes, which contain a lower level of Neu5,9Ac2 on their surface. S protein is proposed to be responsible for the primary attachment of virus to cell surface. S protein is proposed to be responsible for the primary attachement of virus to cell surface receptors. The potential of S protein as a probe for the detection of Neu5,9Ac2-containing glycoconjugates is demonstrated. Images PMID:1920630

  19. P. aeruginosa SGNH Hydrolase-Like Proteins AlgJ and AlgX Have Similar Topology but Separate and Distinct Roles in Alginate Acetylation

    PubMed Central

    Moynihan, Patrick J.; Kitova, Elena N.; Walvoort, Marthe T. C.; Little, Dustin J.; Whitney, John C.; Dawson, Karen; Weadge, Joel T.; Robinson, Howard; Ohman, Dennis E.; Codée, Jeroen D. C.; Klassen, John S.; Clarke, Anthony J.; Howell, P. Lynne

    2014-01-01

    The O-acetylation of polysaccharides is a common modification used by pathogenic organisms to protect against external forces. Pseudomonas aeruginosa secretes the anionic, O-acetylated exopolysaccharide alginate during chronic infection in the lungs of cystic fibrosis patients to form the major constituent of a protective biofilm matrix. Four proteins have been implicated in the O-acetylation of alginate, AlgIJF and AlgX. To probe the biological function of AlgJ, we determined its structure to 1.83 Å resolution. AlgJ is a SGNH hydrolase-like protein, which while structurally similar to the N-terminal domain of AlgX exhibits a distinctly different electrostatic surface potential. Consistent with other SGNH hydrolases, we identified a conserved catalytic triad composed of D190, H192 and S288 and demonstrated that AlgJ exhibits acetylesterase activity in vitro. Residues in the AlgJ signature motifs were found to form an extensive network of interactions that are critical for O-acetylation of alginate in vivo. Using two different electrospray ionization mass spectrometry (ESI-MS) assays we compared the abilities of AlgJ and AlgX to bind and acetylate alginate. Binding studies using defined length polymannuronic acid revealed that AlgJ exhibits either weak or no detectable polymer binding while AlgX binds polymannuronic acid specifically in a length-dependent manner. Additionally, AlgX was capable of utilizing the surrogate acetyl-donor 4-nitrophenyl acetate to catalyze the O-acetylation of polymannuronic acid. Our results, combined with previously published in vivo data, suggest that the annotated O-acetyltransferases AlgJ and AlgX have separate and distinct roles in O-acetylation. Our refined model for alginate acetylation places AlgX as the terminal acetlytransferase and provides a rationale for the variability in the number of proteins required for polysaccharide O-acetylation. PMID:25165982

  20. Global Insight into Lysine Acetylation Events and Their Links to Biological Aspects in Beauveria bassiana, a Fungal Insect Pathogen

    PubMed Central

    Wang, Zhi-Kang; Cai, Qing; Liu, Jin; Ying, Sheng-Hua; Feng, Ming-Guang

    2017-01-01

    Lysine acetylation (Kac) events in filamentous fungi are poorly explored. Here we show a lysine acetylome generated by LC-MS/MS analysis of immunoaffinity-based Kac peptides from normal hyphal cells of Beauveria bassiana, a fungal entomopathogen. The acetylome comprised 283 Kac proteins and 464 Kac sites. These proteins were enriched to eight molecular functions, 20 cellular components, 27 biological processes, 20 KEGG pathways and 12 subcellular localizations. All Kac sites were characterized as six Kac motifs, including a novel motif (KacW) for 26 Kac sites of 17 unknown proteins. Many Kac sites were predicted to be multifunctional, largely expanding the fungal Kac events. Biological importance of identified Kac sites was confirmed through functional analysis of Kac sites on Pmt1 and Pmt4, two O-mannosyltransferases. Singular site mutations (K88R and K482R) of Pmt1 resulted in impaired conidiation, attenuated virulence and decreased tolerance to oxidation and cell wall perturbation. These defects were close to or more severe than those caused by the deletion of pmt1. The Pmt4 K360R mutation facilitated colony growth under normal and stressful conditions and enhanced the fungal virulence. Our findings provide the first insight into the Kac events of B. bassiana and their links to the fungal potential against insect pests. PMID:28295016

  1. Lysine acetylation in mitochondria: From inventory to function.

    PubMed

    Hosp, Fabian; Lassowskat, Ines; Santoro, Valeria; De Vleesschauwer, David; Fliegner, Daniela; Redestig, Henning; Mann, Matthias; Christian, Sven; Hannah, Matthew A; Finkemeier, Iris

    2017-03-01

    Cellular signaling pathways are regulated in a highly dynamic fashion in order to quickly adapt to distinct environmental conditions. Acetylation of lysine residues represents a central process that orchestrates cellular metabolism and signaling. In mitochondria, acetylation seems to be the most prevalent post-translational modification, presumably linked to the compartmentation and high turnover of acetyl-CoA in this organelle. Similarly, the elevated pH and the higher concentration of metabolites in mitochondria seem to favor non-enzymatic lysine modifications, as well as other acylations. Hence, elucidating the mechanisms for metabolic control of protein acetylation is crucial for our understanding of cellular processes. Recent advances in mass spectrometry-based proteomics have considerably increased our knowledge of the regulatory scope of acetylation. Here, we review the current knowledge and functional impact of mitochondrial protein acetylation across species. We first cover the experimental approaches to identify and analyze lysine acetylation on a global scale, we then explore both commonalities and specific differences of plant and animal acetylomes and the evolutionary conservation of protein acetylation, as well as its particular impact on metabolism and diseases. Important future directions and technical challenges are discussed, and it is pointed out that the transfer of knowledge between species and diseases, both in technology and biology, is of particular importance for further advancements in this field. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  2. Global Conformation of Tau Protein Mapped by Raman Spectroscopy.

    PubMed

    Gorantla, Nalini Vijay; Khandelwal, Puneet; Poddar, Pankaj; Chinnathambi, Subashchandrabose

    2017-01-01

    Alzheimer's disease (AD) is one of the neurodegenerative disease characterized by progressive neuronal loss in the brain. Its two major hallmarks are extracellular senile plaques and intracellular neurofibrillary tangles (NFTs), formed by aggregation of amyloid β-42 (Aβ-42) and Tau protein respectively. Aβ-42 is a transmembrane protein, which is produced after the sequential action of β- and γ-secretases, thus obtained peptide is released extracellularly and gets deposited on the neuron forming senile plaques. NFTs are composed of microtubule-associated protein-Tau (MAPT). Tau protein's major function is to stabilize the microtubule that provides a track on which the cargo proteins are shuttled and the stabilized microtubule also maintains shape and integrity of the neuronal cell. Tau protein is subjected to various modifications such as phosphorylation, ubiquitination, glycation, acetylation, truncation, glycosylation, deamination, and oxidation; these modifications ultimately lead to its aggregation. Phosphorylation is the major modification and is extensively studied with respect to Tau protein. Tau protein, however, undergoes certain level of phosphorylation and dephosphorylation, which regulates its affinity for microtubule and ultimately leading to microtubule assembly and disassembly. Our main aim was to study the native state of longest isoform of Tau (hTau40WT-4R2N) and its shortest isoform, (hTau23WT-3R0N), at various temperatures such as 10, 25, and 37 °C. Raman spectroscopic results suggested that the proportion of random coils or unordered structure depends on the temperature of the protein environment. Upon increase in the temperature from 10 to 37 °C, the proportion of random coils or unordered structures increased in the case of hTau40WT. However, we did not find a significant effect of temperature on the structure of hTau23WT. This current approach enables one to analyze the global conformation of soluble Tau in solution.

  3. SILProNAQ: A Convenient Approach for Proteome-Wide Analysis of Protein N-Termini and N-Terminal Acetylation Quantitation.

    PubMed

    Bienvenut, Willy V; Giglione, Carmela; Meinnel, Thierry

    2017-01-01

    Protein N-terminal modifications have recently been involved in overall proteostasis through their impact on cell fate and protein life time. This explains the development of new approaches to characterize more precisely the N-terminal end of mature proteins. Although few approaches are available to perform N-terminal enrichment based on positive or negative discriminations, these methods are usually restricted to the enrichment in N-terminal peptides and their characterization by mass spectrometry. Recent investigation highlights both (1) the knowledge of the N-terminal acetylation status of most cytosolic proteins and (2) post-translational addition of this modification on the N-terminus of nuclear coded chloroplast proteins imported in the plastid and after the cleavage of the transit peptide. The workflow involves stable isotope labeling to assess N-acetylation rates followed by Strong Cation eXchange (SCX ) fractionation of the samples to provide protein N-terminal enriched fractions. Combined with mass spectrometry analyses, the technology finally requires extensive data processing. This last step aims first at discriminating the most relevant mature N-termini from the characterized peptides, next at determining its experimental position and then at calculating the N-terminal acetylation yield. Stable-Isotope Protein N-terminal Acetylation Quantification (SILProNAQ) is a complete workflow combining wet-lab techniques together with dry-lab processing to determine the N-terminal acetylation yield of mature proteins for a clearly defined localization.

  4. Global histone modification fingerprinting in human cells using epigenetic reverse phase protein array

    PubMed Central

    Partolina, Marina; Thoms, Hazel C; MacLeod, Kenneth G; Rodriguez-Blanco, Giovanny; Clarke, Matthew N; Venkatasubramani, Anuroop V; Beesoo, Rima; Larionov, Vladimir; Neergheen-Bhujun, Vidushi S; Serrels, Bryan; Kimura, Hiroshi; Carragher, Neil O; Kagansky, Alexander

    2017-01-01

    The balance between acetylation and deacetylation of histone proteins plays a critical role in the regulation of genomic functions. Aberrations in global levels of histone modifications are linked to carcinogenesis and are currently the focus of intense scrutiny and translational research investments to develop new therapies, which can modify complex disease pathophysiology through epigenetic control. However, despite significant progress in our understanding of the molecular mechanisms of epigenetic machinery in various genomic contexts and cell types, the links between epigenetic modifications and cellular phenotypes are far from being clear. For example, enzymes controlling histone modifications utilize key cellular metabolites associated with intra- and extracellular feedback loops, adding a further layer of complexity to this process. Meanwhile, it has become increasingly evident that new assay technologies which provide robust and precise measurement of global histone modifications are required, for at least two pressing reasons: firstly, many approved drugs are known to influence histone modifications and new cancer therapies are increasingly being developed towards targeting histone deacetylases (HDACs) and other epigenetic readers and writers. Therefore, robust assays for fingerprinting the global effects of such drugs on preclinical cell, organoid and in vivo models is required; and secondly, robust histone-fingerprinting assays applicable to patient samples may afford the development of next-generation diagnostic and prognostic tools. In our study, we have used a panel of monoclonal antibodies to determine the relative changes in the global abundance of post-translational modifications on histones purified from cancer cell lines treated with HDAC inhibitors using a novel technique, called epigenetic reverse phase protein array. We observed a robust increase in acetylation levels within 2–24 h after inhibition of HDACs in different cancer cell lines

  5. Acetyl-Phosphate Is Not a Global Regulatory Bridge between Virulence and Central Metabolism in Borrelia burgdorferi

    PubMed Central

    Richards, Crystal L.; Lawrence, Kevin A.; Su, Hua; Yang, Youyun; Yang, X. Frank; Dulebohn, Daniel P.; Gherardini, Frank C.

    2015-01-01

    In B. burgdorferi, the Rrp2-RpoN-RpoS signaling cascade is a distinctive system that coordinates the expression of virulence factors required for successful transition between its arthropod vector and mammalian hosts. Rrp2 (BB0763), an RpoN specific response regulator, is essential to activate this regulatory pathway. Previous investigations have attempted to identify the phosphate donor of Rrp2, including the cognate histidine kinase, Hk2 (BB0764), non-cognate histidine kinases such as Hk1, CheA1, and CheA2, and small molecular weight P-donors such as carbamoyl-phosphate and acetyl-phosphate (AcP). In a report by Xu et al., exogenous sodium acetate led to increased expression of RpoS and OspC and it was hypothesized this effect was due to increased levels of AcP via the enzyme AckA (BB0622). Genome analyses identified only one pathway that could generate AcP in B. burgdorferi: the acetate/mevalonate pathway that synthesizes the lipid, undecaprenyl phosphate (C55-P, lipid I), which is essential for cell wall biogenesis. To assess the role of AcP in Rrp2–dependent regulation of RpoS and OspC, we used a unique selection strategy to generate mutants that lacked ackA (bb0622: acetate to AcP) or pta (bb0589: AcP to acetyl-CoA). These mutants have an absolute requirement for mevalonate and demonstrate that ackA and pta are required for cell viability. When the ΔackA or Δpta mutant was exposed to conditions (i.e., increased temperature or cell density) that up-regulate the expression of RpoS and OspC, normal induction of those proteins was observed. In addition, adding 20mM acetate or 20mM benzoate to the growth media of B. burgdorferi strain B31 ΔackA induced the expression of RpoS and OspC. These data suggest that AcP (generated by AckA) is not directly involved in modulating the Rrp2-RpoN-RpoS regulatory pathway and that exogenous acetate or benzoate are triggering an acid stress response in B. burgdorferi. PMID:26681317

  6. Acetylproteomic analysis reveals functional implications of lysine acetylation in human spermatozoa (sperm).

    PubMed

    Yu, Heguo; Diao, Hua; Wang, Chunmei; Lin, Yan; Yu, Fudong; Lu, Hui; Xu, Wei; Li, Zheng; Shi, Huijuan; Zhao, Shimin; Zhou, Yuchuan; Zhang, Yonglian

    2015-04-01

    Male infertility is a medical condition that has been on the rise globally. Lysine acetylation of human sperm, an essential posttranslational modification involved in the etiology of sperm abnormality, is not fully understood. Therefore, we first generated a qualified pan-anti-acetyllysine monoclonal antibody to characterize the global lysine acetylation of uncapacitated normal human sperm with a proteomics approach. With high enrichment ratios that were up to 31%, 973 lysine-acetylated sites that matched to 456 human sperm proteins, including 671 novel lysine acetylation sites and 205 novel lysine-acetylated proteins, were identified. These proteins exhibited conserved motifs XXXKYXXX, XXXKFXXX, and XXXKHXXX, were annotated to function in multiple metabolic processes, and were localized predominantly in the mitochondrion and cytoplasmic fractions. Between the uncapacitated and capacitated sperm, different acetylation profiles in regard to functional proteins involved in sperm capacitation, sperm-egg recognition, sperm-egg plasma fusion, and fertilization were observed, indicating that acetylation of functional proteins may be required during sperm capacitation. Bioinformatics analysis revealed association of acetylated proteins with diseases and drugs. Novel acetylation of voltage-dependent anion channel proteins was also found. With clinical sperm samples, we observed differed lysine acetyltransferases and lysine deacetylases expression between normal sperm and abnormal sperm of asthenospermia or necrospermia. Furthermore, with sperm samples impaired by epigallocatechin gallate to mimic asthenospermia, we observed that inhibition of sperm motility was partly through the blockade of voltage-dependent anion channel 2 Lys-74 acetylation combined with reduced ATP levels and mitochondrial membrane potential. Taken together, we obtained a qualified pan-anti-acetyllysine monoclonal antibody, analyzed the acetylproteome of uncapacitated human sperm, and revealed

  7. Acetylproteomic Analysis Reveals Functional Implications of Lysine Acetylation in Human Spermatozoa (sperm)*

    PubMed Central

    Yu, Heguo; Diao, Hua; Wang, Chunmei; Lin, Yan; Yu, Fudong; Lu, Hui; Xu, Wei; Li, Zheng; Shi, Huijuan; Zhao, Shimin; Zhou, Yuchuan; Zhang, Yonglian

    2015-01-01

    Male infertility is a medical condition that has been on the rise globally. Lysine acetylation of human sperm, an essential posttranslational modification involved in the etiology of sperm abnormality, is not fully understood. Therefore, we first generated a qualified pan-anti-acetyllysine monoclonal antibody to characterize the global lysine acetylation of uncapacitated normal human sperm with a proteomics approach. With high enrichment ratios that were up to 31%, 973 lysine-acetylated sites that matched to 456 human sperm proteins, including 671 novel lysine acetylation sites and 205 novel lysine-acetylated proteins, were identified. These proteins exhibited conserved motifs XXXKYXXX, XXXKFXXX, and XXXKHXXX, were annotated to function in multiple metabolic processes, and were localized predominantly in the mitochondrion and cytoplasmic fractions. Between the uncapacitated and capacitated sperm, different acetylation profiles in regard to functional proteins involved in sperm capacitation, sperm-egg recognition, sperm-egg plasma fusion, and fertilization were observed, indicating that acetylation of functional proteins may be required during sperm capacitation. Bioinformatics analysis revealed association of acetylated proteins with diseases and drugs. Novel acetylation of voltage-dependent anion channel proteins was also found. With clinical sperm samples, we observed differed lysine acetyltransferases and lysine deacetylases expression between normal sperm and abnormal sperm of asthenospermia or necrospermia. Furthermore, with sperm samples impaired by epigallocatechin gallate to mimic asthenospermia, we observed that inhibition of sperm motility was partly through the blockade of voltage-dependent anion channel 2 Lys-74 acetylation combined with reduced ATP levels and mitochondrial membrane potential. Taken together, we obtained a qualified pan-anti-acetyllysine monoclonal antibody, analyzed the acetylproteome of uncapacitated human sperm, and revealed

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

    SciTech Connect

    Goupille, Olivier; Penglong, Tipparat; Kadri, Zahra; Granger-Locatelli, Marine; Fucharoen, Suthat; Maouche-Chrétien, Leila; Prost, Stéphane; Leboulch, Philippe; Chrétien, Stany

    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.

  9. Identification of an extended N-acetylated sequence adjacent to the protein-linkage region of fibroblast heparan sulphate.

    PubMed Central

    Lyon, M; Steward, W P; Hampson, I N; Gallagher, J T

    1987-01-01

    The distribution of N-sulphate groups within fibroblast heparan sulphate chains was investigated. The detergent-extractable heparan sulphate proteoglycan from adult human skin fibroblasts, radiolabelled with [3H]glucosamine and [35S]sulphate, was coupled to CNBr-activated Sepharose 4B. After partial depolymerization of the heparan sulphate with nitrous acid, the remaining Sepharose-bound fragments were removed by treatment with alkali. These fragments, of various sizes, but all containing an intact reducing xylose residue, were fractionated on Sephacryl S-300 and the distribution of the 3H and 35S radiolabels was analysed. A decreased degree of sulphation was observed towards the reducing termini of the chains. After complete nitrous acid hydrolysis of the Sepharose-bound proteoglycan, analysis of the proximity of N-sulphation to the reducing end revealed the existence of an extended N-acetylated sequence directly adjacent to the protein-linkage sequence. The size of this N-acetylated domain was estimated by gel filtration to be approximately eight disaccharide units. This domain appears to be highly conserved, being present in virtually all the chains derived from this proteoglycan, implying the existence of a mechanism capable of generating such a non-random sequence during the post-polymeric modification of heparan sulphate. Comparison with the corresponding situation in heparin suggests that different mechanisms regulate polymer N-sulphation in the vicinity of the protein-linkage region of these chemically related glycosaminoglycans. PMID:2954540

  10. Global Proteomics Analysis of Protein Lysine Methylation.

    PubMed

    Cao, Xing-Jun; Garcia, Benjamin A

    2016-11-01

    Lysine methylation is a common protein post-translational modification dynamically mediated by protein lysine methyltransferases (PKMTs) and protein lysine demethylases (PKDMs). Beyond histone proteins, lysine methylation on non-histone proteins plays a substantial role in a variety of functions in cells and is closely associated with diseases such as cancer. A large body of evidence indicates that the dysregulation of some PKMTs leads to tumorigenesis via their non-histone substrates. However, most studies on other PKMTs have made slow progress owing to the lack of approaches for extensive screening of lysine methylation sites. However, recently, there has been a series of publications to perform large-scale analysis of protein lysine methylation. In this unit, we introduce a protocol for the global analysis of protein lysine methylation in cells by means of immunoaffinity enrichment and mass spectrometry. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  11. Acetylome analysis reveals diverse functions of lysine acetylation in Mycobacterium tuberculosis.

    PubMed

    Liu, Fengying; Yang, Mingkun; Wang, Xude; Yang, Shanshan; Gu, Jing; Zhou, Jie; Zhang, Xian-En; Deng, Jiaoyu; Ge, Feng

    2014-12-01

    The lysine acetylation of proteins is a reversible post-translational modification that plays a critical regulatory role in both eukaryotes and prokaryotes. Mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. Increasing evidence shows that lysine acetylation may play an important role in the pathogenesis of M. tuberculosis. However, only a few acetylated proteins of M. tuberculosis are known, presenting a major obstacle to understanding the functional roles of reversible lysine acetylation in this pathogen. We performed a global acetylome analysis of M. tuberculosis H37Ra by combining protein/peptide prefractionation, antibody enrichment, and LC-MS/MS. In total, we identified 226 acetylation sites in 137 proteins of M. tuberculosis H37Ra. The identified acetylated proteins were functionally categorized into an interaction map and shown to be involved in various biological processes. Consistent with previous reports, a large proportion of the acetylation sites were present on proteins involved in glycolysis/gluconeogenesis, the citrate cycle, and fatty acid metabolism. A NAD(+)-dependent deacetylase (MRA_1161) deletion mutant of M. tuberculosis H37Ra was constructed and its characterization showed a different colony morphology, reduced biofilm formation, and increased tolerance of heat stress. Interestingly, lysine acetylation was found, for the first time, to block the immunogenicity of a peptide derived from a known immunogen, HspX, suggesting that lysine acetylation plays a regulatory role in immunogenicity. Our data provide the first global survey of lysine acetylation in M. tuberculosis. The dataset should be an important resource for the functional analysis of lysine acetylation in M. tuberculosis and facilitate the clarification of the entire metabolic networks of this life-threatening pathogen. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Acetylome Analysis Reveals Diverse Functions of Lysine Acetylation in Mycobacterium tuberculosis*

    PubMed Central

    Liu, Fengying; Yang, Mingkun; Wang, Xude; Yang, Shanshan; Gu, Jing; Zhou, Jie; Zhang, Xian-En; Deng, Jiaoyu; Ge, Feng

    2014-01-01

    The lysine acetylation of proteins is a reversible post-translational modification that plays a critical regulatory role in both eukaryotes and prokaryotes. Mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. Increasing evidence shows that lysine acetylation may play an important role in the pathogenesis of M. tuberculosis. However, only a few acetylated proteins of M. tuberculosis are known, presenting a major obstacle to understanding the functional roles of reversible lysine acetylation in this pathogen. We performed a global acetylome analysis of M. tuberculosis H37Ra by combining protein/peptide prefractionation, antibody enrichment, and LC-MS/MS. In total, we identified 226 acetylation sites in 137 proteins of M. tuberculosis H37Ra. The identified acetylated proteins were functionally categorized into an interaction map and shown to be involved in various biological processes. Consistent with previous reports, a large proportion of the acetylation sites were present on proteins involved in glycolysis/gluconeogenesis, the citrate cycle, and fatty acid metabolism. A NAD+-dependent deacetylase (MRA_1161) deletion mutant of M. tuberculosis H37Ra was constructed and its characterization showed a different colony morphology, reduced biofilm formation, and increased tolerance of heat stress. Interestingly, lysine acetylation was found, for the first time, to block the immunogenicity of a peptide derived from a known immunogen, HspX, suggesting that lysine acetylation plays a regulatory role in immunogenicity. Our data provide the first global survey of lysine acetylation in M. tuberculosis. The dataset should be an important resource for the functional analysis of lysine acetylation in M. tuberculosis and facilitate the clarification of the entire metabolic networks of this life-threatening pathogen. PMID:25180227

  13. Human papillomavirus E6 proteins mediate resistance to interferon-induced growth arrest through inhibition of p53 acetylation.

    PubMed

    Hebner, Christy; Beglin, Melanie; Laimins, Laimonis A

    2007-12-01

    The high-risk human papillomavirus (HPV) E6 and E7 proteins act cooperatively to mediate multiple activities in viral pathogenesis. For instance, E7 acts to increase p53 levels while E6 accelerates its rate of turnover through the binding of the cellular ubiquitin ligase E6AP. Interferons are important antiviral agents that modulate both the initial and persistent phases of viral infection. The expression of HPV type 16 E7 was found to sensitize keratinocytes to the growth-inhibitory effects of interferon, while coexpression of E6 abrogates this inhibition. Treatment of E7-expressing cells with interferon ultimately resulted in cellular senescence through a process that is dependent upon acetylation of p53 by p300/CBP at lysine 382. Cells expressing mutant forms of E6 that are unable to bind p300/CBP or bind p53 failed to block acetylation of p53 at lysine 382 and were sensitive to growth arrest by interferon. In contrast, mutant forms of E6 that are unable to bind E6AP remain resistant to the effects of interferon, demonstrating that the absolute levels of p53 are not the major determinants of this activity. Finally, p53 acetylation at lysine 382 was found not to be an essential determinant of other types of senescence such as that induced by overexpression of Ras in human fibroblasts. This study identifies an important physiological role for E6 binding to p300/CBP in blocking growth arrest of human keratinocytes in the presence of interferon and so contributes to the persistence of HPV-infected cells.

  14. Chaperone-mediated acetylation of histones by Rtt109 identified by quantitative proteomics.

    PubMed

    Abshiru, Nebiyu; Ippersiel, Kevin; Tang, Yong; Yuan, Hua; Marmorstein, Ronen; Verreault, Alain; Thibault, Pierre

    2013-04-09

    Rtt109 is a fungal-specific histone acetyltransferase (HAT) that associates with either Vps75 or Asf1 to acetylate histone H3. Recent biochemical and structural studies suggest that site-specific acetylation of H3 by Rtt109 is dictated by the binding chaperone where Rtt109-Asf1 acetylates K56, while Rtt109-Vps75 acetylates K9 and K27. To gain further insights into the roles of Vps75 and Asf1 in directing site-specific acetylation of H3, we used quantitative proteomics to profile the global and site-specific changes in H3 and H4 during in vitro acetylation assays with Rtt109 and its chaperones. Our analyses showed that Rtt109-Vps75 preferentially acetylates H3 K9 and K23, the former residue being the major acetylation site. At high enzyme-to-substrate ratio, Rtt109 also acetylated K14, K18, K27 and to a lower extent K56 of histone H3. Importantly, this study revealed that in contrast to Rtt109-Vps75, Rtt109-Asf1 displayed a far greater site-specificity, with K56 being the primary site of acetylation. For the first time, we also report the acetylation of histone H4 K12 by Rtt109-Vps75, whereas Rtt109-Asf1 showed no detectable activity toward H4. This article is part of a Special Issue entitled: From protein structures to clinical applications.

  15. Differential Effect of Endurance Training on Mitochondrial Protein Damage, Degradation, and Acetylation in the Context of Aging

    PubMed Central

    Johnson, Matthew L.; Irving, Brian A.; Lanza, Ian R.; Vendelbo, Mikkel H.; Konopka, Adam R.; Robinson, Matthew M.; Henderson, Gregory C.; Klaus, Katherine A.; Morse, Dawn M.; Heppelmann, Carrie; Bergen, H. Robert; Dasari, Surendra; Schimke, Jill M.; Jakaitis, Daniel R.

    2015-01-01

    Acute aerobic exercise increases reactive oxygen species and could potentially damage proteins, but exercise training (ET) enhances mitochondrial respiration irrespective of age. Here, we report a differential impact of ET on protein quality in young and older participants. Using mass spectrometry we measured oxidative damage to skeletal muscle proteins before and after 8 weeks of ET and find that young but not older participants reduced oxidative damage to both total skeletal muscle and mitochondrial proteins. Young participants showed higher total and mitochondrial derived semitryptic peptides and 26S proteasome activity indicating increased protein degradation. ET however, increased the activity of the endogenous antioxidants in older participants. ET also increased skeletal muscle content of the mitochondrial deacetylase SIRT3 in both groups. A reduction in the acetylation of isocitrate dehydrogenase 2 was observed following ET that may counteract the effect of acute oxidative stress. In conclusion aging is associated with an inability to improve skeletal muscle and mitochondrial protein quality in response to ET by increasing degradation of damaged proteins. ET does however increase muscle and mitochondrial antioxidant capacity in older individuals, which provides increased buffering from the acute oxidative effects of exercise. PMID:25504576

  16. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae.

    PubMed

    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.

  17. Global Proteomics Analysis of Protein Lysine Methylation

    PubMed Central

    Cao, Xing-Jun; Garcia, Benjamin A.

    2017-01-01

    Lysine methylation is a common protein post-translational modification dynamically mediated by protein lysine methyltransferases (PKMTs) and demethylases (PKDMs). Beyond histone proteins, lysine methylation on non-histone proteins play substantial roles in a variety of functions in cells, and is closely associated with diseases such as cancer. A large body of evidence indicates that the dysregulation of some PKMTs lead to tumorigenesis via their non-histone substrates. However, more studies on other PKMTs have made slow progress owing to the lack of the approaches for extensive screening of lysine methylation sites. Recently a series of publications to perform large-scale analysis of protein lysine methylation have emerged. In this unit, we introduce a protocol for the global analysis of protein lysine methylation in cells by means of immunoaffinity enrichment and mass spectrometry. PMID:27801517

  18. Identification and quantification of N alpha-acetylated Y. pestis fusion protein F1-V expressed in Escherichia coli using LCMS E.

    PubMed

    Bariola, Pauline A; Russell, Brett A; Monahan, Steven J; Stroop, Steven D

    2007-05-31

    N-terminal acetylation in E coli is a rare event catalyzed by three known N-acetyl-transferases (NATs), each having a specific ribosomal protein substrate. Multiple, gram-scale lots of recombinant F1-V, a fusion protein constructed from Y. Pestis antigens, were expressed and purified from a single stably transformed E. coli cell bank. A variant form of F1-V with mass increased by 42-43 Da was detected in all purified lots by electrospray orthogonal acceleration time-of-flight mass spectrometry (MS). Peptide mapping LCMS localized the increased mass to an N-terminal Lys-C peptide, residues 1-24, and defined it as +42.0308+/-0.0231 Da using a LockSpray exact mass feature and a leucine enkaphalin mass standard. Sequencing of the variant 1-24 peptide by LCMS and high-energy collision induced dissociation (LCMS(E)) further localized the modification to the amino terminal tri-peptide ADL and identified the modification as N(alpha)-acetylation. The average content of N(alpha)-acetylated F1-V in five lots was 24.7+/-2.6% indicating that a stable acetylation activity for F1-V was established in the E. coli expression system. Alignment of the F1-V N-terminal sequence with those of other known N(alpha)-acetylated ectopic proteins expressed in E. coli reveals a substrate motif analogous to the eukaryote NatA' acetylation pathway and distinct from endogenous E. coli NAT substrates.

  19. The effect of a high mobility group protein (HMG 17) on the structure of acetylated and control core HeLa cell chromatin.

    PubMed

    Sasi, R; Fasman, G D

    1984-05-15

    The effect of binding a high mobility group protein (HMG 17) on the stability and conformation of acetylated and control HeLa high molecular weight core chromatin (stripped of H1 and non-histone chromosomal proteins) was studied by circular dichroism and thermal-denaturation measurements. Previously it had been shown that conformational differences exist between native whole chromatin derived from butyrate-treated (acetylated) and control HeLa cells and that these conformational differences disappear by removing H1 and non-histone chromosomal proteins ( Reczek , P.R., Weissman , D., Huvos , P.E. and Fasman, G.D. (1982) Biochemistry 21, 993-1002). The circular dichroism spectra and the thermal denaturation profiles of control and acetylated core chromatin were found to be similar. The circular dichroism properties of HMG 17 reconstituted highly acetylated and control core chromatin indicated the same alteration of chromatin structure at low ionic strength (1 mM sodium phosphate/0.25 mM EDTA, pH 7.0). The magnitudes of the decrease in ellipticity were proportional to the amount of HMG 17 bound and were found to be the same for both the acetylated and control core chromatin. Thermal denaturation profiles confirmed this change in structure induced by HMG 17 on control and highly acetylated core chromatin. The thermal denaturation profiles, which were resolved into three component transitions, exhibited a shifting of hyperchromicity from the lower melting transitions to the higher melting transitions, with a concomitant rise in Tm, on HMG 17 binding to both control and acetylated chromatin. The natures of the interactions of HMG 17 at higher ionic strength (50 mM NaCl/0.25 mM EDTA/1 mM sodium phosphate, pH 7.0) with acetylated and control core chromatin were slightly different, as measured by circular dichroism; however, a decrease in ellipticity was observed for both samples upon binding of HMG 17. These observations suggest that acetylation coupled with HMG 17 binding

  20. Regulation of S-Adenosylhomocysteine Hydrolase by Lysine Acetylation*

    PubMed Central

    Wang, Yun; Kavran, Jennifer M.; Chen, Zan; Karukurichi, Kannan R.; Leahy, Daniel J.; Cole, Philip A.

    2014-01-01

    S-Adenosylhomocysteine hydrolase (SAHH) is an NAD+-dependent tetrameric enzyme that catalyzes the breakdown of S-adenosylhomocysteine to adenosine and homocysteine and is important in cell growth and the regulation of gene expression. Loss of SAHH function can result in global inhibition of cellular methyltransferase enzymes because of high levels of S-adenosylhomocysteine. Prior proteomics studies have identified two SAHH acetylation sites at Lys401 and Lys408 but the impact of these post-translational modifications has not yet been determined. Here we use expressed protein ligation to produce semisynthetic SAHH acetylated at Lys401 and Lys408 and show that modification of either position negatively impacts the catalytic activity of SAHH. X-ray crystal structures of 408-acetylated SAHH and dually acetylated SAHH have been determined and reveal perturbations in the C-terminal hydrogen bonding patterns, a region of the protein important for NAD+ binding. These crystal structures along with mutagenesis data suggest that such hydrogen bond perturbations are responsible for SAHH catalytic inhibition by acetylation. These results suggest how increased acetylation of SAHH may globally influence cellular methylation patterns. PMID:25248746

  1. Hexavalent Chromium (Cr(VI)) Down-Regulates Acetylation of Histone H4 at Lysine 16 through Induction of Stressor Protein Nupr1

    PubMed Central

    Chen, Danqi; Kluz, Thomas; Fang, Lei; Zhang, Xiaoru; Sun, Hong; Jin, Chunyuan; Costa, Max

    2016-01-01

    The environmental and occupational carcinogen Hexavalent Chromium (Cr(VI)) has been shown to cause lung cancer in humans when inhaled. In spite of a considerable research effort, the mechanisms of Cr(VI)-induced carcinogenesis remain largely unknown. Nupr1 (nuclear protein 1) is a small, highly basic, and unfolded protein with molecular weight of 8,800 daltons and is induced by a variety of stressors. Studies in animal models have suggested that Nupr1 is a key factor in the development of lung and pancreatic cancers, with little known about the underlying molecular mechanisms. Here we report that the level of Nupr1 is significantly increased in human bronchial epithelial BEAS2B cells following exposure to Cr(VI) through epigenetic mechanisms. Interestingly, Cr(VI) exposure also results in the loss of acetylation at histone H4K16, which is considered a ‘hallmark’ of human cancer. Cr(VI)-induced reduction of H4K16 acetylation appears to be caused by the induction of Nupr1, since (a) overexpression of Nupr1 decreased the levels of both H4K16 acetylation and the histone acetyltransferase MOF (male absent on the first; also known as Kat8, Myst 1), which specifically acetylates H4K16; (b) the loss of acetylation of H4K16 upon Cr(VI) exposure is greatly compromised by knockdown of Nupr1. Moreover, Nupr1-induced reduction of H4K16 acetylation correlates with the transcriptional down-regulation at several genomic loci. Notably, overexpression of Nupr1 induces anchorage-independent cell growth and knockdown of Nupr1 expression prevents Cr(VI)-induced cell transformation. We propose that Cr(VI) induces Nupr1 and rapidly perturbs gene expression by downregulating H4K16 acetylation, thereby contributing to Cr(VI)-induced carcinogenesis. PMID:27285315

  2. The catalytic requirements for reduction and acetylation of protein X and the related regulation of various forms of resolved pyruvate dehydrogenase kinase.

    PubMed

    Rahmatullah, M; Roche, T E

    1987-07-25

    The pyruvate dehydrogenase kinase consists of a catalytic subunit (Kc) and a basic subunit (Kb) which appear to be anchored to the dihydrolipoyl transacetylase core component (E2) by another subunit, referred to as protein X (Rahmatullah, M., Jilka, J. M., Radke, G. A., and Roche, T. E. (1986) J. Biol. Chem. 261, 6515-6523). We determined the catalytic requirements for reduction and acetylation of the lipoyl moiety in protein X and linked those changes in protein X to regulatory effects on kinase activity. Using fractions prepared by resolution and proteolytic treatments, we evaluated which subunits are required for regulatory effects on kinase activity. With X-KcKb fraction (treated to remove the mercurial agent used in its preparation), we found that the resolved pyruvate dehydrogenase component, the isolated inner domain of E2 (lacking the lipoyl-bearing region of E2), and the dihydrolipoyl dehydrogenase component directly utilize protein X as a substrate. The resulting reduction and acetylation of protein X occurs in association with enhancement of kinase activity. Following tryptic cleavage of E2 and protein X into subdomains, full acetylation of the lipoyl-bearing subdomains of these proteins is retained along with the capacity of acetylating substrates to stimulate kinase activity. All kinase-containing fractions, including those in which the Kb subunit was digested, were inhibited by pyruvate or ADP, alone, and synergistically by the combination suggesting that pyruvate and ADP bind to Kc. Our results suggest that the Kb subunit of the kinase does not contribute to the observed regulatory effects. A dynamic role of protein X in attenuating kinase activity based on changes in the mitochondrial redox and acetylating potentials is considered.

  3. N-Acetyl-L-Cystein downregulates beta-amyloid precursor protein gene transcription in human neuroblastoma cells.

    PubMed

    Studer, R; Baysang, G; Brack, C

    2001-01-01

    The causes for the sporadic form of Alzheimer's disease (AD) are still poorly understood, except from the fact that age is an important risk factor. The main component of the characteristic amyloid plaques in brains of AD patients are Abeta peptides, derivatives of the amyloid precursor protein APP. Oxidative stress may contribute to the aetiology of AD by dysregulation of APP metabolism. Overexpression of the APP gene could result in an increased secretion of neurotoxic Abeta peptides, while preventing the overexpression might be protective. We here report that the antioxidant N-Acetyl-L-Cystein (NAC) downregulates APP gene transcription in human neuroblastoma cells. The effect is reversible when cells are returned to NAC free medium. These results open up new possibilities for the development of therapeutic agents that intervene at the transcriptional level.

  4. An acetylated 120-kDa lysosomal transmembrane protein is absent from mucopolysaccharidosis IIIC fibroblasts: a candidate molecule for MPS IIIC.

    PubMed

    Ausseil, Jérôme; Landry, Karine; Seyrantepe, Volkan; Trudel, Stéphanie; Mazur, Alexander; Lapointe, Fanny; Pshezhetsky, Alexey V

    2006-01-01

    Genetic deficiency of the lysosomal enzyme, acetyl-CoA: alpha-glucosaminide N-acetyltransferase (N-acetyltransferase), which catalyses the transmembrane acetylation of heparan sulfate results in severe neurodegenerative disease, mucopolysaccharidosis IIIC. N-Acetyltransferase has never been characterized structurally and its gene has never been identified. We combined traditional methods of enzyme purification with organellar proteomics, isolating lysosomal membranes from mouse liver using differential centrifugation and osmolysis, followed by detergent extraction and purification of N-acetyltransferase by liquid chromatography. Partially purified enzyme had a molecular mass of 240 kDa and pI of 7.4 by gel filtration and chromatofocusing. Its specific activity varied with protein concentration typical of oligomeric enzymes or multienzyme complexes. Incubation of N-acetyltransferase with acetyl[14C]CoA in the absence of the acceptor of the acetyl group resulted in radioactive labeling of a 120-kDa polypeptide, suggesting that it represents a subunit containing the enzyme active site. Furthermore, following acetyl[14C]-labeling, the 120-kDa protein was present in the lysosomal membranes purified from the normal human skin fibroblasts but absent in those from the skin fibroblasts of MPS IIIC patients.

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

    PubMed Central

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

    2005-01-01

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

  6. Protein oxidation under extremely low frequency electric field in guinea pigs. Effect of N-acetyl-L-cysteine treatment.

    PubMed

    Güler, Göknur; Türközer, Zerrin; Ozgur, Elcin; Tomruk, Arin; Seyhan, Nesrin; Karasu, Cimen

    2009-03-01

    Modern age exposes humans to an increasing level of electromagnetic activity in their environment due to overhead power lines and transformers around residential areas. Studies have shown that treatment with antioxidants can suppress the oxidative damage induced by electromagnetic fields in various frequencies of the non-ionizing radiation band. In this study, we detected protein carbonyl content (PCO), advanced oxidation protein products (AOPP) in liver and 3-nitrotyrosine (3-NT) levels in plasma of guinea pigs in order to investigate the effects of N-acetyl-L-cysteine (NAC) administration on oxidative protein damage induced by power frequency electric (E) field (50 Hz, 12 kV/m, 7 days/8 h/day). We also analyzed hepatic hydroxyproline level to study protein synthesis. According to the findings of the present study, no statistically significant changes occurred in PCO, AOPP and 3-NT levels of the guinea pigs that were exposed to the E field with respect to the control group. However, liver hydroxyproline level was significantly diminished in the E field exposure group compared to the control and PCO, hydroxyproline and 3-NT levels changed significantly in the NAC-administrated groups.

  7. Optimization of proteins and minerals removal from shrimp shells to produce highly acetylated chitin.

    PubMed

    Younes, Islem; Hajji, Sawssen; Rinaudo, Marguerite; Chaabouni, Moncef; Jellouli, Kemel; Nasri, Moncef

    2016-03-01

    Chitin and derivatives used for biomedical or pharmaceutical applications require a high level of purity and quality that are difficult to achieve. In this study, we propose to optimize the extraction of chitin in order to obtain pure product keeping a structure as close as possible to the native form. Thus, demineralization step was firstly optimized using response surface methodology. In the optimized conditions predicted by the model, the obtained chitin has an acetylation degree (DA) and a demineralization degree (DM) equal to 99% and 100%, respectively. Then, different microbial and fish crude alkaline proteases were tested for their efficiency in deproteinization. Crude alkaline proteases giving the highest deproteinization degrees (DP), Bacillus mojavensis A21 and Scorpaena scrofa, were selected for chitin extraction. The obtained DP was 88±2% and 83±1%, respectively. At the end, effect of the use of mixed enzymatic treatment with the two selected crude enzymes and the order of demineralization/deproteinization steps were tested. The results demonstrated that two separated steps in enzymatic treatments realized on demineralized sample give the best DP (96%) preserving the DA (99%).

  8. Wheat acetyl-coenzyme A carboxylase: cDNA and protein structure.

    PubMed Central

    Gornicki, P; Podkowinski, J; Scappino, L A; DiMaio, J; Ward, E; Haselkorn, R

    1994-01-01

    cDNA fragments encoding part of wheat (Triticum aestivum) acetyl-CoA carboxylase (ACC; EC 6.4.1.2) were cloned by PCR using primers based on the alignment of several biotin-dependent carboxylases. A set of overlapping clones encoding the entire wheat ACC was then isolated by using these fragments as probes. The cDNA sequence contains a 2257-amino acid reading frame encoding a 251-kDa polypeptide. The amino acid sequence of the most highly conserved domain, corresponding to the biotin carboxylases of prokaryotes, is 52-55% identical to ACC of yeast, rat, and diatom. Identity with the available C-terminal amino acid sequence of maize ACC is 66%. The biotin attachment site has the typical eukaryotic EVMKM sequence. The cDNA does not encode an obvious chloroplast targeting sequence. Various cDNA fragments hybridize in Northern blots to a 7.9-kb mRNA. Southern analysis with cDNA probes revealed multiple hybridizing fragments in hexaploid wheat DNA. Some of the wheat cDNA probes also hybridize with ACC-specific DNA from other plants, indicating significant conservation among plant ACCs. Images PMID:7913745

  9. Quaternion maps of global protein structure.

    PubMed

    Hanson, Andrew J; Thakur, Sidharth

    2012-09-01

    The geometric structures of proteins are vital to the understanding of biochemical interactions. However, there is much yet to be understood about the spatial arrangements of the chains of amino acids making up any given protein. In particular, while conventional analysis tools like the Ramachandran plot supply some insight into the local relative orientation of pairs of amino acid residues, they provide little information about the global relative orientations of large groups of residues. We apply quaternion maps to families of coordinate frames defined naturally by amino acid residue structures as a way to expose global spatial relationships among residues within proteins. The resulting visualizations enable comparisons of absolute orientations as well as relative orientations, and thus generalize the framework of the Ramachandran plot. There are a variety of possible quaternion frames and visual representation strategies that can be chosen, and very complex quaternion maps can result. Just as Ramachandran plots are useful for addressing particular questions and not others, quaternion tools have characteristic domains of relevance. In particular, quaternion maps show great potential for answering specific questions about global residue alignment in crystallographic data and statistical orientation properties in Nuclear Magnetic Resonance (NMR) data that are very difficult to treat by other methods. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Vaccinia virus K1 ankyrin repeat protein inhibits NF-κB activation by preventing RelA acetylation.

    PubMed

    Bravo Cruz, Ariana G; Shisler, Joanna L

    2016-10-01

    The vaccinia virus (VACV) K1 protein inhibits dsRNA-dependent protein kinase (PKR) activation. A consequence of this function is that K1 inhibits PKR-induced NF-κB activation during VACV infection. However, transient expression of K1 also inhibits Toll-like receptor (TLR)-induced NF-κB activation. This suggests that K1 has a second NF-κB inhibitory mechanism that is PKR-independent. This possibility was explored by expressing K1 independently of infection and stimulating NF-κB under conditions that minimized or excluded PKR activation. K1 inhibited both TNF- and phorbol 12-myristate 13-acetate (PMA)-induced NF-κB activation, as detected by transcription of synthetic (e.g. luciferase) and natural (e.g. CXCL8) genes controlled by NF-κB. K1 also inhibited NF-κB activity in PKRkd cells, cells that have greatly decreased amounts of PKR. K1 no longer prevented IκBα degradation or NF-κB nuclear translocation in the absence of PKR, suggesting that K1 acted on a nuclear event. Indeed, K1 was present in the nucleus and cytoplasm of stimulated and unstimulated cells. K1 inhibited acetylation of the RelA (p65) subunit of NF-κB, a nuclear event known to be required for NF-κB activation. Moreover, p65-CBP (CREB-binding protein) interactions were blocked in the presence of K1. However, K1 did not preclude NF-κB binding to oligonucleotides containing κB-binding sites. The current interpretation of these data is that NF-κB-promoter interactions still occur in the presence of K1, but NF-κB cannot properly trigger transcriptional activation because K1 antagonizes acetylation of RelA. Thus, in comparison to all known VACV NF-κB inhibitory proteins, K1 acts at one of the most downstream events of NF-κB activation.

  11. Tissue-specific regulation of acetyl-CoA carboxylase gene expression by dietary soya protein isolate in rats.

    PubMed

    Xiao, Chao Wu; Wood, Carla; Huang, Wenxin; L'Abbé, Mary R; Gilani, G Sarwar; Cooke, Gerard M; Curran, Ivan

    2006-06-01

    We have recently reported that intake of soya protein isolate (SPI) inhibited the DNA-binding activities of hepatic thyroid hormone receptor (TR). The genes for acetyl-CoA carboxylase (ACC), a rate-limiting enzyme in fatty acid synthesis, contain the thyroid hormone response element in their promoters and are regulated by TR. The present study has examined the effect of long-term feeding of SPI and soya isoflavones (ISF) on the gene expression and protein phosphorylation of different ACC isoforms in different tissues and plasma triacylglycerol (TAG) levels in rats. Sprague-Dawley female rats were fed diets containing 20 % casein or alcohol-washed SPI with or without supplemental ISF for 70, 190 and 310 d. SPI intake significantly reduced plasma TAG concentrations compared with casein, whereas supplemental ISF had no effect. Hepatic ACCalpha and ACCbeta mRNA abundance and protein content were markedly lower in the rats fed SPI than in those fed casein. The protein contents of ACCalpha in the kidney and ACCbeta, the predominant isoform in the heart and kidney, were unchanged by dietary SPI. The ratios of phospho-ACCalpha/ACCalpha and phospho-ACCbeta/ACCbeta were not different among dietary groups in all tissues measured. The present study demonstrates that ingestion of SPI decreases plasma TAG level and down-regulates ACCalpha and ACCbeta gene expression in the liver but not in the heart and kidney. The results indicate that the effect of SPI is tissue-specific and that alteration of ACC gene expression rather than phosphorylation status may play a major role in the regulation of ACC activities by soya proteins.

  12. Acetyl-lysine Binding Site of Bromodomain-Containing Protein 4 (BRD4) Interacts with Diverse Kinase Inhibitors

    PubMed Central

    2014-01-01

    Members of the bromodomain and extra terminal (BET) family of proteins are essential for the recognition of acetylated lysine (KAc) residues in histones and have emerged as promising drug targets in cancer, inflammation, and contraception research. In co-crystallization screening campaigns using the first bromodomain of BRD4 (BRD4-1) against kinase inhibitor libraries, we identified and characterized 14 kinase inhibitors (10 distinct chemical scaffolds) as ligands of the KAc binding site. Among these, the PLK1 inhibitor BI2536 and the JAK2 inhibitor TG101209 displayed strongest inhibitory potential against BRD4 (IC50 = 25 nM and 130 nM, respectively) and high selectivity for BET bromodomains. Comparative structural analysis revealed markedly different binding modes of kinase hinge-binding scaffolds in the KAc binding site, suggesting that BET proteins are potential off-targets of diverse kinase inhibitors. Combined, these findings provide a new structural framework for the rational design of next-generation BET-selective and dual-activity BET-kinase inhibitors. PMID:24568369

  13. Glucose-regulated protein 78 inhibits scavenger receptor A-mediated internalization of acetylated low density lipoprotein.

    PubMed

    Ben, Jingjing; Gao, Song; Zhu, Xudong; Zheng, Yuan; Zhuang, Yan; Bai, Hui; Xu, Yong; Ji, Yong; Sha, Jiahao; He, Zhigang; Chen, Qi

    2009-11-01

    Class A scavenger receptor (SR-A) plays an important role in foam cell formation. However, the mechanism underlying the internalization of the receptor-ligand complexes remains unclear. The aim of the present study was to investigate the molecular mechanism to regulate SR-A-mediated intracellular lipid accumulation in macrophages. A pull-down assay was performed and glucose-regulated protein 78 (GRP78) was identified to bind with the cytoplasmic domain of SR-A (CSR-A). Immunoprecipitation and artificially expressed protein binding assay demonstrated the direct specific binding of GRP78 with SR-A in cells. Indirect immunofluorescence assay and western blot analysis showed their co-localization in membrane and cytoplasm. Over-expression of GRP78 specifically inhibited SR-A-mediated uptake of fluorescent acetylated low-density lipoprotein, a specific ligand for SR-A, without altering cellular SR-A expression and binding ability, and significantly inhibited cholesterol ester accumulation in cells, which can be partly attributed to the suppression of c-Jun-NH2-terminal kinase signaling pathway. These results suggest that GRP78 may act as an inhibitor of SR-A-mediated internalization of modified low-density lipoprotein into macrophages.

  14. GENERAL CONTROL NONREPRESSED PROTEIN5-Mediated Histone Acetylation of FERRIC REDUCTASE DEFECTIVE3 Contributes to Iron Homeostasis in Arabidopsis1

    PubMed Central

    Wang, Tianya; Liu, Zhenshan; Xu, Jianqin; Yao, Yingyin; Peng, Huiru; Xin, Mingming; Ni, Zhongfu

    2015-01-01

    Iron homeostasis is essential for plant growth and development. Here, we report that a mutation in GENERAL CONTROL NONREPRESSED PROTEIN5 (GCN5) impaired iron translocation from the root to the shoot in Arabidopsis (Arabidopsis thaliana). Illumina high-throughput sequencing revealed 879 GCN5-regulated candidate genes potentially involved in iron homeostasis. Chromatin immunoprecipitation assays indicated that five genes (At3G08040, At2G01530, At2G39380, At2G47160, and At4G05200) are direct targets of GCN5 in iron homeostasis regulation. Notably, GCN5-mediated acetylation of histone 3 lysine 9 and histone 3 lysine 14 of FERRIC REDUCTASE DEFECTIVE3 (FRD3) determined the dynamic expression of FRD3. Consistent with the function of FRD3 as a citrate efflux protein, the iron retention defect in gcn5 was rescued and fertility was partly restored by overexpressing FRD3. Moreover, iron retention in gcn5 roots was significantly reduced by the exogenous application of citrate. Collectively, these data suggest that GCN5 plays a critical role in FRD3-mediated iron homeostasis. Our results provide novel insight into the chromatin-based regulation of iron homeostasis in Arabidopsis. PMID:26002909

  15. Comparative covalent protein binding of 2,5-hexanedione and 3-acetyl-2,5-hexanedione in the rat.

    PubMed

    DeCaprio, Anthony P; Kinney, Elizabeth A; LoPachin, Richard M

    2009-01-01

    2,5-Hexanedione (HD) is the metabolite implicated in n-hexane neurotoxicity. This gamma-diketone reacts with protein lysine amines to form 2,5-dimethylpyrrole adducts. Pyrrole adduction of neurofilaments (NF) and/or other axonal proteins was proposed as a critical step in the neuropathy. While pyrrole adduction is widely accepted as necessary, subsequent pyrrole oxidation, which may result in protein cross-linking, was alternatively postulated as the critical mechanistic step. Previous studies have indicated that 3-acetyl-2,5-HD (AcHD), an analogue that forms pyrroles that do not oxidize, was not neurotoxic in rats. However, relative levels of pyrrole adduction of NF or other axonal proteins were not reported. In the present study, groups of 6 male Wistar rats were given saline, [1,6-(14)C]-HD (3 mmol/kg/d), or [5-(14)C]-AcHD (0.1 mmol/kg/d), i.p. for 21 d. HD- and AcHD-treated rats lost 10% and gained 14% body weight, respectively, compared to a 22% gain for control rats. At termination, HD- and AcHD-treated rats exhibited mean scores of 3.5 and 1.4, respectively, for hindlimb weakness (0-5 scale). Incorporation of radiolabel from HD was 27.8 +/- 3.9, 13.9 +/- 2.6, and 7.8 +/- 0.6 nmol/mg in plasma protein, purified globin, and axonal cytoskeletal proteins, respectively, compared to 0.6 +/- 0.1, 1.6 +/- 0.5, and 1.0 +/- 0.1 for AcHD. Binding of HD to the NF-L, -M, and -H subunit proteins from treated animals was 4-, 24-, and 13-fold higher, respectively, that that of AcHD, indicating differing stoichiometry and patterns of NF adduction for the two diketones. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of globin and NF proteins did not demonstrate protein cross-linking for either diketone at the dose levels and time period examined. These results indicate that that the lack of neurotoxicity previously reported for AcHD may reflect differences in adduct levels at critical axonal target sites rather than an inability to form cross

  16. Acetyl chloride

    Integrated Risk Information System (IRIS)

    Acetyl chloride ; CASRN 75 - 36 - 5 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  17. Global view of the protein universe.

    PubMed

    Nepomnyachiy, Sergey; Ben-Tal, Nir; Kolodny, Rachel

    2014-08-12

    To explore protein space from a global perspective, we consider 9,710 SCOP (Structural Classification of Proteins) domains with up to 70% sequence identity and present all similarities among them as networks: In the "domain network," nodes represent domains, and edges connect domains that share "motifs," i.e., significantly sized segments of similar sequence and structure. We explore the dependence of the network on the thresholds that define the evolutionary relatedness of the domains. At excessively strict thresholds the network falls apart completely; for very lax thresholds, there are network paths between virtually all domains. Interestingly, at intermediate thresholds the network constitutes two regions that can be described as "continuous" versus "discrete." The continuous region comprises a large connected component, dominated by domains with alternating alpha and beta elements, and the discrete region includes the rest of the domains in isolated islands, each generally corresponding to a fold. We also construct the "motif network," in which nodes represent recurring motifs, and edges connect motifs that appear in the same domain. This network also features a large and highly connected component of motifs that originate from domains with alternating alpha/beta elements (and some all-alpha domains), and smaller isolated islands. Indeed, the motif network suggests that nature reuses such motifs extensively. The networks suggest evolutionary paths between domains and give hints about protein evolution and the underlying biophysics. They provide natural means of organizing protein space, and could be useful for the development of strategies for protein search and design.

  18. Molecular characterization of the cysJIH promoters of Salmonella typhimurium and Escherichia coli: regulation by cysB protein and N-acetyl-L-serine.

    PubMed Central

    Ostrowski, J; Kredich, N M

    1989-01-01

    The cysJIH promoter regions from Salmonella typhimurium LT7 and Escherichia coli B were cloned and sequenced. Primer extension analyses showed that the major in vivo transcription initiation site in S. typhimurium is located 171 nucleotides upstream of the cysJ start codon. Minor start sites were found 8 and 9 nucleotides downstream of the major site. In vivo transcription initiation in E. coli was found to occur at a single site 66 nucleotides upstream of the cysJ start codon. Primer extension studies also indicated that chromosomal cysJIH transcription is stimulated by sulfur limitation and repressed by growth on L-cystine. Paradoxically, in strains carrying plasmids containing the S. typhimurium cysJIH region, the highest levels of primer extension products were found with RNA from cells grown on L-cystine, even though levels of the proteins encoded by cysJ and cysI were normally repressed. In vitro transcription runoff studies with DNA template from the S. typhimurium cysJIH promoter region showed synthesis of a product originating at the major in vivo start site, which was dependent on the presence of purified cysB protein and either O-acetyl-L-serine or N-acetyl-L-serine. N-Acetyl-L-serine was 10- to 30-fold more active than O-acetyl-L-serine as an in vitro inducer of cysJIH transcription. Images PMID:2701932

  19. High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation[S

    PubMed Central

    Currie, Erin; Guo, Xiuling; Christiano, Romain; Chitraju, Chandramohan; Kory, Nora; Harrison, Kenneth; Haas, Joel; Walther, Tobias C.; Farese, Robert V.

    2014-01-01

    Accurate protein inventories are essential for understanding an organelle’s functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae. Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism. PMID:24868093

  20. Systematic analysis of the lysine acetylome reveals diverse functions of lysine acetylation in the oleaginous yeast Yarrowia lipolytica.

    PubMed

    Wang, Guangyuan; Guo, Lizhong; Liang, Wenxing; Chi, Zhenming; Liu, Lin

    2017-12-01

    Lysine acetylation of proteins, a major post-translational modification, plays a critical regulatory role in almost every aspects in both eukaryotes and prokaryotes. Yarrowia lipolytica, an oleaginous yeast, is considered as a model for bio-oil production due to its ability to accumulate a large amount of lipids. However, the function of lysine acetylation in this organism is elusive. Here, we performed a global acetylproteome analysis of Y. lipolytica ACA-DC 50109. In total, 3163 lysine acetylation sites were identified in 1428 proteins, which account for 22.1% of the total proteins in the cell. Fifteen conserved acetylation motifs were detected. The acetylated proteins participate in a wide variety of biological processes. Notably, a total of 65 enzymes involved in lipid biosynthesis were found to be acetylated. The acetylation sites are distributed in almost every type of conserved domains in the multi-enzymatic complexes of fatty acid synthetases. The provided dataset probably illuminates the crucial role of reversible acetylation in oleaginous microorganisms, and serves as an important resource for exploring the physiological role of lysine acetylation in eukaryotes.

  1. Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling

    PubMed Central

    Evans, Eric G.B.; Millhauser, Glenn L.

    2016-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain “K1,” and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron–electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite

  2. Local vs global motions in protein folding

    PubMed Central

    Maisuradze, Gia G.; Liwo, Adam; Senet, Patrick; Scheraga, Harold A.

    2013-01-01

    It is of interest to know whether local fluctuations in a polypeptide chain play any role in the mechanism by which the chain folds to the native structure of a protein. This question is addressed by analyzing folding and non-folding trajectories of a protein; as an example, the analysis is applied to the 37-residue triple β-strand WW domain from the Formin binding protein 28 (FBP28) (PDB ID: 1E0L). Molecular dynamics (MD) trajectories were generated with the coarse-grained united-residue force field, and one- and two-dimensional free-energy landscapes (FELs) along the backbone virtual-bond angle θ and backbone virtual-bond-dihedral angle γ of each residue, and principal components, respectively, were analyzed. The key residues involved in the folding of the FBP28 WW domain are elucidated by this analysis. The correlations between local and global motions are found. It is shown that most of the residues in the folding trajectories of the system studied here move in a concerted fashion, following the dynamics of the whole system. This demonstrates how the choice of a pathway has to involve concerted movements in order for this protein to fold. This finding also sheds light on the effectiveness of principal component analysis (PCA) for the description of the folding dynamics of the system studied. It is demonstrated that the FEL along the PCs, computed by considering only several critically-placed residues, can correctly describe the folding dynamics. PMID:23914144

  3. Post-translational Serine/Threonine Phosphorylation and Lysine Acetylation: A Novel Regulatory Aspect of the Global Nitrogen Response Regulator GlnR in S. coelicolor M145

    PubMed Central

    Amin, Rafat; Franz-Wachtel, Mirita; Tiffert, Yvonne; Heberer, Martin; Meky, Mohamed; Ahmed, Yousra; Matthews, Arne; Krysenko, Sergii; Jakobi, Marco; Hinder, Markus; Moore, Jane; Okoniewski, Nicole; Maček, Boris; Wohlleben, Wolfgang; Bera, Agnieszka

    2016-01-01

    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. This study clearly

  4. Widespread and Enzyme-independent Nϵ-Acetylation and Nϵ-Succinylation of Proteins in the Chemical Conditions of the Mitochondrial Matrix*♦

    PubMed Central

    Wagner, Gregory R.; Payne, R. Mark

    2013-01-01

    Alterations in mitochondrial protein acetylation are implicated in the pathophysiology of diabetes, the metabolic syndrome, mitochondrial disorders, and cancer. However, a viable mechanism responsible for the widespread acetylation in mitochondria remains unknown. Here, we demonstrate that the physiologic pH and acyl-CoA concentrations of the mitochondrial matrix are sufficient to cause dose- and time-dependent, but enzyme-independent acetylation and succinylation of mitochondrial and nonmitochondrial proteins in vitro. These data suggest that protein acylation in mitochondria may be a chemical event facilitated by the alkaline pH and high concentrations of reactive acyl-CoAs present in the mitochondrial matrix. Although these results do not exclude the possibility of enzyme-mediated protein acylation in mitochondria, they demonstrate that such a mechanism may not be required in its unique chemical environment. These findings may have implications for the evolutionary roles that the mitochondria-localized SIRT3 deacetylase and SIRT5 desuccinylase have in the maintenance of metabolic health. PMID:23946487

  5. Global view of the protein universe

    PubMed Central

    Nepomnyachiy, Sergey; Ben-Tal, Nir; Kolodny, Rachel

    2014-01-01

    To explore protein space from a global perspective, we consider 9,710 SCOP (Structural Classification of Proteins) domains with up to 70% sequence identity and present all similarities among them as networks: In the “domain network,” nodes represent domains, and edges connect domains that share “motifs,” i.e., significantly sized segments of similar sequence and structure. We explore the dependence of the network on the thresholds that define the evolutionary relatedness of the domains. At excessively strict thresholds the network falls apart completely; for very lax thresholds, there are network paths between virtually all domains. Interestingly, at intermediate thresholds the network constitutes two regions that can be described as “continuous” versus “discrete.” The continuous region comprises a large connected component, dominated by domains with alternating alpha and beta elements, and the discrete region includes the rest of the domains in isolated islands, each generally corresponding to a fold. We also construct the “motif network,” in which nodes represent recurring motifs, and edges connect motifs that appear in the same domain. This network also features a large and highly connected component of motifs that originate from domains with alternating alpha/beta elements (and some all-alpha domains), and smaller isolated islands. Indeed, the motif network suggests that nature reuses such motifs extensively. The networks suggest evolutionary paths between domains and give hints about protein evolution and the underlying biophysics. They provide natural means of organizing protein space, and could be useful for the development of strategies for protein search and design. PMID:25071170

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

    NASA Astrophysics Data System (ADS)

    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.

  7. Characterization and Prediction of Lysine (K)-Acetyl-Transferase Specific Acetylation Sites*

    PubMed Central

    Li, Tingting; Du, Yipeng; Wang, Likun; Huang, Lei; Li, Wenlin; Lu, Ming; Zhang, Xuegong; Zhu, Wei-Guo

    2012-01-01

    Lysine acetylation is a well-studied post-translational modification on both histone and nonhistone proteins. More than 2000 acetylated proteins and 4000 lysine acetylation sites have been identified by large scale mass spectrometry or traditional experimental methods. Although over 20 lysine (K)-acetyl-transferases (KATs) have been characterized, which KAT is responsible for a given protein or lysine site acetylation is mostly unknown. In this work, we collected KAT-specific acetylation sites manually and analyzed sequence features surrounding the acetylated lysine of substrates from three main KAT families (CBP/p300, GCN5/PCAF, and the MYST family). We found that each of the three KAT families acetylates lysines with different sequence features. Based on these differences, we developed a computer program, Acetylation Set Enrichment Based method to predict which KAT-families are responsible for acetylation of a given protein or lysine site. Finally, we evaluated the efficiency of our method, and experimentally detected four proteins that were predicted to be acetylated by two KAT families when one representative member of the KAT family is over expressed. We conclude that our approach, combined with more traditional experimental methods, may be useful for identifying KAT families responsible for acetylated substrates proteome-wide. PMID:21964354

  8. Mapping sites of aspirin-induced acetylations in live cells by quantitative acid-cleavable activity-based protein profiling (QA-ABPP).

    PubMed

    Wang, Jigang; Zhang, Chong-Jing; Zhang, Jianbin; He, Yingke; Lee, Yew Mun; Chen, Songbi; Lim, Teck Kwang; Ng, Shukie; Shen, Han-Ming; Lin, Qingsong

    2015-01-20

    Target-identification and understanding of mechanism-of-action (MOA) are challenging for development of small-molecule probes and their application in biology and drug discovery. For example, although aspirin has been widely used for more than 100 years, its molecular targets have not been fully characterized. To cope with this challenge, we developed a novel technique called quantitative acid-cleavable activity-based protein profiling (QA-ABPP) with combination of the following two parts: (i) activity-based protein profiling (ABPP) and iTRAQ™ quantitative proteomics for identification of target proteins and (ii) acid-cleavable linker-based ABPP for identification of peptides with specific binding sites. It is known that reaction of aspirin with its target proteins leads to acetylation. We thus applied the above technique using aspirin-based probes in human cancer HCT116 cells. We identified 1110 target proteins and 2775 peptides with exact acetylation sites. By correlating these two sets of data, 523 proteins were identified as targets of aspirin. We used various biological assays to validate the effects of aspirin on inhibition of protein synthesis and induction of autophagy which were elicited from the pathway analysis of Aspirin target profile. This technique is widely applicable for target identification in the field of drug discovery and biology, especially for the covalent drugs.

  9. Mapping sites of aspirin-induced acetylations in live cells by quantitative acid-cleavable activity-based protein profiling (QA-ABPP)

    PubMed Central

    Wang, Jigang; Zhang, Chong-Jing; Zhang, Jianbin; He, Yingke; Lee, Yew Mun; Chen, Songbi; Lim, Teck Kwang; Ng, Shukie; Shen, Han-Ming; Lin, Qingsong

    2015-01-01

    Target-identification and understanding of mechanism-of-action (MOA) are challenging for development of small-molecule probes and their application in biology and drug discovery. For example, although aspirin has been widely used for more than 100 years, its molecular targets have not been fully characterized. To cope with this challenge, we developed a novel technique called quantitative acid-cleavable activity-based protein profiling (QA-ABPP) with combination of the following two parts: (i) activity-based protein profiling (ABPP) and iTRAQ™ quantitative proteomics for identification of target proteins and (ii) acid-cleavable linker-based ABPP for identification of peptides with specific binding sites. It is known that reaction of aspirin with its target proteins leads to acetylation. We thus applied the above technique using aspirin-based probes in human cancer HCT116 cells. We identified 1110 target proteins and 2775 peptides with exact acetylation sites. By correlating these two sets of data, 523 proteins were identified as targets of aspirin. We used various biological assays to validate the effects of aspirin on inhibition of protein synthesis and induction of autophagy which were elicited from the pathway analysis of Aspirin target profile. This technique is widely applicable for target identification in the field of drug discovery and biology, especially for the covalent drugs. PMID:25600173

  10. Feedback regulation of plastidic acetyl-CoA carboxylase by 18:1-acyl carrier protein in Brassica napus

    PubMed Central

    Andre, Carl; Haslam, Richard P.; Shanklin, John

    2012-01-01

    Plant seed oil represents a major renewable source of reduced carbon, but little is known about the biochemical regulation of its synthesis. The goal of this research was to identify potential feedback regulation of fatty acid biosynthesis in Brassica napus embryo-derived cell cultures and to characterize both the feedback signals and enzymatic targets of the inhibition. Fatty acids delivered via Tween esters rapidly reduced the rate of fatty acid synthesis in a dose-dependent and reversible manner, demonstrating the existence of feedback inhibition in an oil-accumulating tissue. Tween feeding did not affect fatty acid elongation in the cytosol or the incorporation of radiolabeled malonate into nascent fatty acids, which together pinpoint plastidic acetyl-CoA carboxylase (ACCase) as the enzymatic target of feedback inhibition. To identify the signal responsible for feedback, a variety of Tween esters were tested for their effects on the rate of fatty acid synthesis. Maximum inhibition was achieved upon feeding oleic acid (18:1) Tween esters that resulted in the intracellular accumulation of 18:1 free fatty acid, 18:1-CoA, and 18:1-acyl-carrier protein (ACP). Direct, saturable inhibition of ACCase enzyme activity was observed in culture extracts and in extracts of developing canola seeds supplemented with 18:1-ACP at physiological concentrations. A mechanism for feedback inhibition is proposed in which reduced demand for de novo fatty acids results in the accumulation of 18:1-ACP, which directly inhibits plastidic ACCase, leading to reduced fatty acid synthesis. Defining this mechanism presents an opportunity for mitigating feedback inhibition of fatty acid synthesis in crop plants to increase oil yield. PMID:22665812

  11. Interaction of the Nitrogen Regulatory Protein GlnB (PII) with Biotin Carboxyl Carrier Protein (BCCP) Controls Acetyl-CoA Levels in the Cyanobacterium Synechocystis sp. PCC 6803

    PubMed Central

    Hauf, Waldemar; Schmid, Katharina; Gerhardt, Edileusa C. M.; Huergo, Luciano F.; Forchhammer, Karl

    2016-01-01

    The family of PII signal transduction proteins (members GlnB, GlnK, NifI) plays key roles in various cellular processes related to nitrogen metabolism at different functional levels. Recent studies implied that PII proteins may also be involved in the regulation of fatty acid metabolism, since GlnB proteins from Proteobacteria and from Arabidopsis thaliana were shown to interact with biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase (ACC). In case of Escherichia coli ACCase, this interaction reduces the kcat of acetyl-CoA carboxylation, which should have a marked impact on the acetyl-CoA metabolism. In this study we show that the PII protein of a unicellular cyanobacterium inhibits the biosynthetic activity of E. coli ACC and also interacts with cyanobacterial BCCP in an ATP and 2-oxoglutarate dependent manner. In a PII mutant strain of Synechocystis strain PCC 6803, the lacking control leads to reduced acetyl-CoA levels, slightly increased levels of fatty acids and formation of lipid bodies as well as an altered fatty acid composition. PMID:27833596

  12. Interaction of the Nitrogen Regulatory Protein GlnB (PII) with Biotin Carboxyl Carrier Protein (BCCP) Controls Acetyl-CoA Levels in the Cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Hauf, Waldemar; Schmid, Katharina; Gerhardt, Edileusa C M; Huergo, Luciano F; Forchhammer, Karl

    2016-01-01

    The family of PII signal transduction proteins (members GlnB, GlnK, NifI) plays key roles in various cellular processes related to nitrogen metabolism at different functional levels. Recent studies implied that PII proteins may also be involved in the regulation of fatty acid metabolism, since GlnB proteins from Proteobacteria and from Arabidopsis thaliana were shown to interact with biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase (ACC). In case of Escherichia coli ACCase, this interaction reduces the kcat of acetyl-CoA carboxylation, which should have a marked impact on the acetyl-CoA metabolism. In this study we show that the PII protein of a unicellular cyanobacterium inhibits the biosynthetic activity of E. coli ACC and also interacts with cyanobacterial BCCP in an ATP and 2-oxoglutarate dependent manner. In a PII mutant strain of Synechocystis strain PCC 6803, the lacking control leads to reduced acetyl-CoA levels, slightly increased levels of fatty acids and formation of lipid bodies as well as an altered fatty acid composition.

  13. Histone acetylation in heterochromatin assembly

    PubMed Central

    Kim, Jeong-Hoon; Workman, Jerry L.

    2010-01-01

    Histone acetylation is generally considered a mark involved in activating gene expression by making chromatin structures less compact. In the April 1, 2010, issue of Genes & Development, Xhemalce and Kouzarides (pp. 647–652) demonstrate that the acetylation of histone H3 at Lys 4 (H3K4) plays a role in the formation of repressive heterochromatin in Schizosaccharomyces pombe. H3K4 acetylation mediates a switch of chromodomain proteins associated with methylated H3K9 during heterochromatin assembly. PMID:20395362

  14. The age-associated loss of ischemic preconditioning in the kidney is accompanied by mitochondrial dysfunction, increased protein acetylation and decreased autophagy.

    PubMed

    Jankauskas, Stanislovas S; Pevzner, Irina B; Andrianova, Nadezda V; Zorova, Ljubava D; Popkov, Vasily A; Silachev, Denis N; Kolosova, Nataliya G; Plotnikov, Egor Y; Zorov, Dmitry B

    2017-03-15

    In young rats, ischemic preconditioning (IPC), which consists of 4 cycles of ischemia and reperfusion alleviated kidney injury caused by 40-min ischemia. However,old rats lost their ability to protect the ischemic kidney by IPC. A similar aged phenotype was demonstrated in 6-month-old OXYS rats having signs of premature aging. In the kidney of old and OXYS rats, the levels of acetylated nuclear proteins were higher than in young rats, however, unlike in young rats, acetylation levels in old and OXYS rats were further increased after IPC. In contrast to Wistar rats, age-matched OXYS demonstrated no increase in lysosome abundance and LC3 content in the kidney after ischemia/reperfusion. The kidney LC3 levels were also lower in OXYS, even under basal conditions, and mitochondrial PINK1 and ubiquitin levels were higher, suggesting impaired mitophagy. The kidney mitochondria from old rats contained a population with diminished membrane potential and this fraction was expanded by IPC. Apparently, oxidative changes with aging result in the appearance of malfunctioning renal mitochondria due to a low efficiency of autophagy. Elevated protein acetylation might be a hallmark of aging which is associated with a decreased autophagy, accumulation of dysfunctional mitochondria, and loss of protection against ischemia by IPC.

  15. Functional Organization of the Yeast SAGA Complex: Distinct Components Involved in Structural Integrity, Nucleosome Acetylation, and TATA-Binding Protein Interaction

    PubMed Central

    Sterner, David E.; Grant, Patrick A.; Roberts, Shannon M.; Duggan, Laura J.; Belotserkovskaya, Rimma; Pacella, Lisa A.; Winston, Fred; Workman, Jerry L.; Berger, Shelley L.

    1999-01-01

    SAGA, a recently described protein complex in Saccharomyces cerevisiae, is important for transcription in vivo and possesses histone acetylation function. Here we report both biochemical and genetic analyses of members of three classes of transcription regulatory factors contained within the SAGA complex. We demonstrate a correlation between the phenotypic severity of SAGA mutants and SAGA structural integrity. Specifically, null mutations in the Gcn5/Ada2/Ada3 or Spt3/Spt8 classes cause moderate phenotypes and subtle structural alterations, while mutations in a third subgroup, Spt7/Spt20, as well as Ada1, disrupt the complex and cause severe phenotypes. Interestingly, double mutants (gcn5Δ spt3Δ and gcn5Δ spt8Δ) causing loss of a member of each of the moderate classes have severe phenotypes, similar to spt7Δ, spt20Δ, or ada1Δ mutants. In addition, we have investigated biochemical functions suggested by the moderate phenotypic classes and find that first, normal nucleosomal acetylation by SAGA requires a specific domain of Gcn5, termed the bromodomain. Deletion of this domain also causes specific transcriptional defects at the HIS3 promoter in vivo. Second, SAGA interacts with TBP, the TATA-binding protein, and this interaction requires Spt8 in vitro. Overall, our data demonstrate that SAGA harbors multiple, distinct transcription-related functions, including direct TBP interaction and nucleosomal histone acetylation. Loss of either of these causes slight impairment in vivo, but loss of both is highly detrimental to growth and transcription. PMID:9858534

  16. Acetylation of Mitochondrial Trifunctional Protein α-Subunit Enhances Its Stability To Promote Fatty Acid Oxidation and Is Decreased in Nonalcoholic Fatty Liver Disease

    PubMed Central

    Guo, Liang; Zhou, Shui-Rong; Wei, Xiang-Bo; Liu, Yuan; Chang, Xin-Xia; Liu, Yang; Ge, Xin; Dou, Xin; Huang, Hai-Yan; Qian, Shu-Wen; Li, Xi; Lei, Qun-Ying

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD) has become the most common liver disease, and decreased fatty acid oxidation is one of the important contributors to NAFLD. Mitochondrial trifunctional protein α-subunit (MTPα) functions as a critical enzyme for fatty acid β-oxidation, but whether dysregulation of MTPα is pathogenically connected to NAFLD is poorly understood. We show that MTPα is acetylated at lysine residues 350, 383, and 406 (MTPα-3K), which promotes its protein stability by antagonizing its ubiquitylation on the same three lysines (MTPα-3K) and blocking its subsequent degradation. Sirtuin 4 (SIRT4) has been identified as the deacetylase, deacetylating and destabilizing MTPα. Replacement of MTPα-3K with either MTPα-3KR or MTPα-3KQ inhibits cellular lipid accumulation both in free fatty acid (FFA)-treated alpha mouse liver 12 (AML12) cells and primary hepatocytes and in the livers of high-fat/high-sucrose (HF/HS) diet-fed mice. Moreover, knockdown of SIRT4 could phenocopy the effects of MTPα-3K mutant expression in mouse livers, and MTPα-3K mutants more efficiently attenuate SIRT4-mediated hepatic steatosis in HF/HS diet-fed mice. Importantly, acetylation of both MTPα and MTPα-3K is decreased while SIRT4 is increased in the livers of mice and humans with NAFLD. Our study reveals a novel mechanism of MTPα regulation by acetylation and ubiquitylation and a direct functional link of this regulation to NAFLD. PMID:27457618

  17. The age-associated loss of ischemic preconditioning in the kidney is accompanied by mitochondrial dysfunction, increased protein acetylation and decreased autophagy

    PubMed Central

    Jankauskas, Stanislovas S.; Pevzner, Irina B.; Andrianova, Nadezda V.; Zorova, Ljubava D.; Popkov, Vasily A.; Silachev, Denis N.; Kolosova, Nataliya G.; Plotnikov, Egor Y.; Zorov, Dmitry B.

    2017-01-01

    In young rats, ischemic preconditioning (IPC), which consists of 4 cycles of ischemia and reperfusion alleviated kidney injury caused by 40-min ischemia. However,old rats lost their ability to protect the ischemic kidney by IPC. A similar aged phenotype was demonstrated in 6-month-old OXYS rats having signs of premature aging. In the kidney of old and OXYS rats, the levels of acetylated nuclear proteins were higher than in young rats, however, unlike in young rats, acetylation levels in old and OXYS rats were further increased after IPC. In contrast to Wistar rats, age-matched OXYS demonstrated no increase in lysosome abundance and LC3 content in the kidney after ischemia/reperfusion. The kidney LC3 levels were also lower in OXYS, even under basal conditions, and mitochondrial PINK1 and ubiquitin levels were higher, suggesting impaired mitophagy. The kidney mitochondria from old rats contained a population with diminished membrane potential and this fraction was expanded by IPC. Apparently, oxidative changes with aging result in the appearance of malfunctioning renal mitochondria due to a low efficiency of autophagy. Elevated protein acetylation might be a hallmark of aging which is associated with a decreased autophagy, accumulation of dysfunctional mitochondria, and loss of protection against ischemia by IPC. PMID:28294175

  18. Pseudo-acetylation of multiple sites on human Tau proteins alters Tau phosphorylation and microtubule binding, and ameliorates amyloid beta toxicity.

    PubMed

    Gorsky, Marianna Karina; Burnouf, Sylvie; Sofola-Adesakin, Oyinkan; Dols, Jacqueline; Augustin, Hrvoje; Weigelt, Carina Marianne; Grönke, Sebastian; Partridge, Linda

    2017-08-30

    Tau is a microtubule-associated protein that is highly soluble and natively unfolded. Its dysfunction is involved in the pathogenesis of several neurodegenerative disorders including Alzheimer's disease (AD), where it aggregates within neurons. Deciphering the physiological and pathogenic roles of human Tau (hTau) is crucial to further understand the mechanisms leading to its dysfunction in vivo. We have used a knock-out/knock-in strategy in Drosophila to generate a strain with hTau inserted into the endogenous fly tau locus and expressed under the control of the endogenous fly tau promoter, thus avoiding potential toxicity due to genetic over-expression. hTau knock-in (KI) proteins were expressed at normal, endogenous levels, bound to fly microtubules and were post-translationally modified, hence displaying physiological properties. We used this new model to investigate the effects of acetylation on hTau toxicity in vivo. The simultaneous pseudo-acetylation of hTau at lysines 163, 280, 281 and 369 drastically decreased hTau phosphorylation and significantly reduced its binding to microtubules in vivo. These molecular alterations were associated with ameliorated amyloid beta toxicity. Our results indicate acetylation of hTau on multiple sites regulates its biology and ameliorates amyloid beta toxicity in vivo.

  19. Comprehensive profiling of lysine acetylproteome analysis reveals diverse functions of lysine acetylation in common wheat.

    PubMed

    Zhang, Yumei; Song, Limin; Liang, Wenxing; Mu, Ping; Wang, Shu; Lin, Qi

    2016-02-15

    Lysine acetylation of proteins, a dynamic and reversible post-translational modification, plays a critical regulatory role in both eukaryotes and prokaryotes. Several researches have been carried out on acetylproteome in plants. However, until now, there have been no data on common wheat, the major cereal crop in the world. In this study, we performed a global acetylproteome analysis of common wheat variety (Triticum aestivum L.), Chinese Spring. In total, 416 lysine modification sites were identified on 277 proteins, which are involved in a wide variety of biological processes. Consistent with previous studies, a large proportion of the acetylated proteins are involved in metabolic process. Interestingly, according to the functional enrichment analysis, 26 acetylated proteins are involved in photosynthesis and Calvin cycle, suggesting an important role of lysine acetylation in these processes. Moreover, protein interaction network analysis reveals that diverse interactions are modulated by protein acetylation. These data represent the first report of acetylome in common wheat and serve as an important resource for exploring the physiological role of lysine acetylation in this organism and likely in all plants.

  20. Comprehensive profiling of lysine acetylproteome analysis reveals diverse functions of lysine acetylation in common wheat

    PubMed Central

    Zhang, Yumei; Song, Limin; Liang, Wenxing; Mu, Ping; Wang, Shu; Lin, Qi

    2016-01-01

    Lysine acetylation of proteins, a dynamic and reversible post-translational modification, plays a critical regulatory role in both eukaryotes and prokaryotes. Several researches have been carried out on acetylproteome in plants. However, until now, there have been no data on common wheat, the major cereal crop in the world. In this study, we performed a global acetylproteome analysis of common wheat variety (Triticum aestivum L.), Chinese Spring. In total, 416 lysine modification sites were identified on 277 proteins, which are involved in a wide variety of biological processes. Consistent with previous studies, a large proportion of the acetylated proteins are involved in metabolic process. Interestingly, according to the functional enrichment analysis, 26 acetylated proteins are involved in photosynthesis and Calvin cycle, suggesting an important role of lysine acetylation in these processes. Moreover, protein interaction network analysis reveals that diverse interactions are modulated by protein acetylation. These data represent the first report of acetylome in common wheat and serve as an important resource for exploring the physiological role of lysine acetylation in this organism and likely in all plants. PMID:26875666

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

    PubMed Central

    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

  2. Time-resolved luminescence biosensor for continuous activity detection of protein acetylation-related enzymes based on DNA-sensitized terbium(III) probes.

    PubMed

    Han, Yitao; Li, Hao; Hu, Yufang; Li, Pei; Wang, Huixia; Nie, Zhou; Yao, Shouzhuo

    2015-09-15

    Protein acetylation of histone is an essential post-translational modification (PTM) mechanism in epigenetic gene regulation, and its status is reversibly controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Herein, we have developed a sensitive and label-free time-resolved luminescence (TRL) biosensor for continuous detection of enzymatic activity of HATs and HDACs, respectively, based on acetylation-mediated peptide/DNA interaction and Tb(3+)/DNA luminescent probes. Using guanine (G)-rich DNA-sensitized Tb(3+) luminescence as the output signal, the polycationic substrate peptides interact with DNA with high affinity and subsequently replace Tb(3+), eliminating the luminescent signal. HAT-catalyzed acetylation remarkably reduces the positive charge of the peptides and diminishes the peptide/DNA interaction, resulting in the signal on detection via recovery of DNA-sensitized Tb(3+) luminescence. With this TRL sensor, HAT (p300) can be sensitively detected with a wide linear range from 0.2 to 100 nM and a low detection limit of 0.05 nM. The proposed sensor was further used to continuously monitor the HAT activity in real time. Additionally, the TRL biosensor was successfully applied to evaluating HAT inhibition by two specific inhibitors, anacardic acid and C464, and satisfactory Z'-factors above 0.73 were obtained. Moreover, this sensor is feasible to continuously monitor the HDAC (Sirt1)-catalyzed deacetylation with a linear range from 0.5 to 500 nM and a detection limit of 0.5 nM. The proposed sensor is a convenient, sensitive, and mix-and-read assay, presenting a promising platform for protein acetylation-targeted epigenetic research and drug discovery.

  3. Resveratrol and acetyl-resveratrol modulate activity of VEGF and IL-8 in ovarian cancer cell aggregates via attenuation of the NF-κB protein.

    PubMed

    Tino, Alexandria B; Chitcholtan, Kenny; Sykes, Peter H; Garrill, Ashley

    2016-12-01

    Key features of advanced ovarian cancer include metastasis via cell clusters in the abdominal cavity and increased chemoresistance. Resveratrol and derivatives of resveratrol have been shown to have antitumour properties. The purpose of this study was to investigate the effect of resveratrol and acetyl-resveratrol on 3D cell aggregates of ovarian cancer, and establish if NF-κB signalling may be a potential target. Poly-HEMA coated wells were used to produce 3D aggregates of two ovarian cancer cell lines, SKOV-3 and OVCAR-5. The aggregates were exposed to 10, 20 or 30 μM resveratrol or acetyl-resveratrol for 2, 4 or 6 days. Cell growth and metabolism were measured then ELISA, western blot and immunofluorescence were utilised to evaluate VEGF, IL-8 and NF-κB levels. Resveratrol and acetyl-resveratrol reduced cell growth and metabolism of SKOV-3 aggregates in a dose- and time-dependent manner. After 6 days all three doses of both compounds inhibited cell growth. This growth inhibition correlated with the attenuated secretion of VEGF and a decrease of NF-κB protein levels. Conversely, the secretion of IL-8 increased with treatment. The effects of the compounds were limited in OVCAR-5 cell clusters. The results suggest that resveratrol and its derivative acetyl-resveratrol may inhibit in vitro 3D cell growth of certain subtypes of ovarian cancer, and growth restriction may be associated with the secretion of VEGF under the control of the NF-κB protein.

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

    PubMed Central

    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

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

    PubMed

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

    2017-03-02

    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

  6. NatB Domain-Containing CRA-1 Antagonizes Hydrolase ACER-1 Linking Acetyl-CoA Metabolism to the Initiation of Recombination during C. elegans Meiosis

    PubMed Central

    Gao, Jinmin; Kim, Hyun-Min; Elia, Andrew E.; Elledge, Stephen J.; Colaiácovo, Monica P.

    2015-01-01

    The formation of DNA double-strand breaks (DSBs) must take place during meiosis to ensure the formation of crossovers, which are required for accurate chromosome segregation, therefore avoiding aneuploidy. However, DSB formation must be tightly regulated to maintain genomic integrity. How this regulation operates in the context of different chromatin architectures and accessibility, and how it is linked to metabolic pathways, is not understood. We show here that global histone acetylation levels undergo changes throughout meiotic progression. Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans. We provide evidence that the regulation of histone acetylation requires CRA-1, a NatB domain-containing protein homologous to human NAA25, which controls the levels of acetyl-Coenzyme A (acetyl-CoA) by antagonizing ACER-1, a previously unknown and conserved acetyl-CoA hydrolase. CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein. We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation. PMID:25768301

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

    PubMed

    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.

  8. A. baumannii histone acetyl transferase Hpa2: optimization of homology modeling, analysis of protein-protein interaction and virtual screening.

    PubMed

    Tomar, Jyoti Singh; Peddinti, Rama Krishna

    2017-04-01

    In the current scenario, widespread multidrug resistivity in ESKAPE pathogens demands identification of novel drug targets to keep their infections at bay. For this purpose, we have identified a novel target Hpa2 of A. baumannii, a member of GNAT superfamily of HATs. But due to sequence identity of equal or less than 35%, the correct sequence alignment and construction of 3D monomeric and dimeric models of Hpa2 having optimal structural parameters is a troublesome task. To circumvent these problems, we have designed an easy and optimized protocol for Hpa2 monomer modeling, and for generation of dimeric Hpa2 model using data-driven protein-protein docking experiment. Improvement in the structural features of generated model is an onerous process and generally achieved by paying time and computational cost. Herein, it is achieved by reconciliation of FoldX commands which takes less time in execution. Evaluations performed to validate structural parameters and stability of monomeric and dimeric Hpa2 attests to its quality. Analysis of interfacial residues, energy terms and RMSD values indicated a clear correlation between experimental and theoretical interface properties of the dimers, corroborating to the regime used for Hpa2 dimer generation. Structural information from the refined models was used for virtual screening of substrate-derived library and polyamines to achieve a new platform for developing A. baumannii inhibitory molecules. Molecules showing preferential binding at the dimer interface could be used as allosteric inhibitors. Binding of polyamines with model illustrated the same binding pattern as described experimentally in case of yeast Hpa2.

  9. Acetylation of prostaglandin synthase by aspirin.

    PubMed Central

    Roth, G J; Stanford, N; Majerus, P W

    1975-01-01

    When microsomes of sheep or bovine seminal vesicles are incubated with [acetyl-3H]aspirin (acetyl salicylic acid), 200 Ci/mol, we observe acetylation of a single protein, as measured by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The protein has a molecular weight of 85,000 and corresponds to a similar acetylated protein found in the particulate fraction of aspirin-treated human platelets. The aspirin-mediated acetylation reaction proceeds with the same time course and at the same concentration as does the inhibition of prostaglandin synthase (cyclo-oxygenase) (EC 1.14.99.1; 8,11,14-eicosatrienoate, hydrogen-donor:oxygen oxidoreductase) by the drug. At 100 muM aspirin, 50% inhibition of prostaglandin synthase and 50% of maximal acetylation are observed after 15 min at 37 degrees. Furthermore, the substrate for cyclo-oxygenase, arachidonic acid, inhibits protein acetylation by aspirin at concentrations (50% inhibition at 10-30 muM) which correlate with the Michaelis constant of arachidonic acid as a substrate for cyclooxygenase. Arachidonic acid analogues and indomethacin inhibit the acetylation reaction in proportion to their effectiveness as cyclo-oxygenase inhibitors. The results suggest that aspirin acts as an active-site acetylating agent for the enzyme cyclo-oxygenase. This action of aspirin may account for its anti-inflammatory and anti-platelet action. PMID:810797

  10. Lysine acetylation and cancer: A proteomics perspective.

    PubMed

    Gil, Jeovanis; Ramírez-Torres, Alberto; Encarnación-Guevara, Sergio

    2017-01-06

    Lysine acetylation is a reversible modification controlled by two groups of enzymes: lysine acetyltransferases (KATs) and lysine deacetylases (KDACs). Acetylated lysine residues are recognized by bromodomains, a family of evolutionarily conserved domains. The use of high-resolution mass spectrometry-based proteomics, in combination with the enrichment of acetylated peptides through immunoprecipitation with anti-acetyl-lysine antibodies, has expanded the number of acetylated proteins from histones and a few nuclear proteins to more than 2000 human proteins. Because acetylation targets almost all cellular processes, this modification has been associated with cancer. Several KATs, KDACs and bromodomain-containing proteins have been linked to cancer development. Many small molecules targeting some of these proteins have been or are being tested as potential cancer therapies. The stoichiometry of lysine acetylation has not been explored in cancer, representing a promising field in which to increase our knowledge of how this modification is affected in cancer. In this review, we will focus on the strategies that can be used to go deeper in the characterization of the protein lysine acetylation emphasizing in cancer research.

  11. A strategy to quantitate global phosphorylation of bone matrix proteins.

    PubMed

    Sroga, Grażyna E; Vashishth, Deepak

    2016-04-15

    Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and their landscapes of phosphorylation in the regulation of different cellular functions. However, global changes in phosphorylation of extracellular matrix phosphoproteins measured "in bulk" are equally important. For example, correct global phosphorylation of different bone matrix proteins is critical to healthy tissue biomineralization. To study changes of bone matrix global phosphorylation, we developed a strategy that combines a procedure for in vitro phosphorylation/dephosphorylation of fully mineralized bone in addition to quantitation of the global phosphorylation levels of bone matrix proteins. For the first time, we show that it is possible to enzymatically phosphorylate/dephosphorylate fully mineralized bone originating from either cadaveric human donors or laboratory animals (mice). Using our strategy, we detected the difference in the global phosphorylation levels of matrix proteins isolated from wild-type and osteopontin knockout mice. We also observed that the global phosphorylation levels of matrix proteins isolated from human cortical bone were lower than those isolated from trabecular bone. The developed strategy has the potential to open new avenues for studies on the global phosphorylation of bone matrix proteins and their role in biomineralization as well for other tissues/cells and protein-based materials.

  12. The E3 ubiquitin ligase Itch regulates tumor suppressor protein RASSF5/NORE1 stability in an acetylation-dependent manner.

    PubMed

    Suryaraja, R; Anitha, M; Anbarasu, K; Kumari, G; Mahalingam, S

    2013-03-28

    Ras association (RalGDS/AF-6) domain family member RASSF5 is a non-enzymatic RAS effector super family protein, known to be involved in cell growth regulation. Expression of RASSF5 is found to be extinguished by promoter hypermethylation in different human cancers, and its ectopic expression suppresses cell proliferation and tumorigenicity. Interestingly, this role in tumorigenesis has been confounded by the fact that regulation at molecular level remains unclear and many transformed cells actually display elevated RASSF5 expression. Here, we demonstrate that E3 ubiquitin ligase Itch is a unique binding partner of RASSF5. Itch can interact with PPxY motif in RASSF5 both in vivo and in vitro through its WW domains. Importantly, the overexpression of Itch induces RASSF5 degradation by poly-ubiquitination via 26S proteasome pathway. In addition, our results indicate that the elevated levels of RASSF5 found in tumor cells due to acetylation, which restricts its binding to Itch and results in a more stable inert protein. Inhibition of RASSF5 acetylation permits its interaction with Itch and provokes proteasomal degradation. These data suggest that apart from promoter methylation, hyperacetylation could also be downregulating RASSF5 function in different human cancer. Finally, results from functional assays suggest that the overexpression of wild type, not the ligase activity defective Itch negatively regulate RASSF5-mediated G1 phase transition of cell cycle as well as apoptosis, suggesting that Itch alone is sufficient to alter RASSF5 function. Collectively, the present investigation identifies a HECT class E3 ubiquitin ligase Itch as a unique negative regulator of RASSF5, and suggests the possibility that acetylation as a potential therapeutic target for human cancer.

  13. A Method to Determine Lysine Acetylation Stoichiometries

    DOE PAGES

    Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; ...

    2014-01-01

    Lysine acetylation is a common protein posttranslational modification that regulates a variety of biological processes. A major bottleneck to fully understanding the functional aspects of lysine acetylation is the difficulty in measuring the proportion of lysine residues that are acetylated. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of protein lysine acetylation. Using this technique, we determined the modification occupancy for ~750 acetylated peptides from mammalian cell lysates. Furthermore, the acetylation on N-terminal tail of histone H4 was cross-validated by treating cells with sodiummore » butyrate, a potent deacetylase inhibitor, and comparing changes in stoichiometry levels measured by our method with immunoblotting measurements. Of note we observe that acetylation stoichiometry is high in nuclear proteins, but very low in mitochondrial and cytosolic proteins. In summary, our method opens new opportunities to study in detail the relationship of lysine acetylation levels of proteins with their biological functions.« less

  14. Transcription and protein synthesis inhibitors influence long-term effects of acetyl-l-carnitine on non-associative learning in the leech.

    PubMed

    Traina, Giovanna; Scuri, Rossana

    2015-01-01

    Acetyl-l-carnitine (ALC) is the principal acetyl ester of L-carnitine and it plays an essential role in intermediary metabolism. ALC affects several targets in the nervous system. Along this line of investigation, we analyzed the long-term effects of ALC on elementary nonassociative learning in the swimming induction model of the leech Hirudo medicinalis, in which nociceptive stimulation of the dorsal skin produces a more rapid swim response to a test stimulus (sensitization). In this simplified model a single ALC administration blocked the sensitizing effects of nociceptive stimulation in swim induction showing increasingly long lasting effects. Herein, we have analyzed the long-term effects of ALC on sensitization and dishabituation. Leeches were treated with inhibitors of either transcription or protein synthesis 30 min after the administration of ALC and, subsequently, subjected to noxious stimuli: the animals exhibited a sensitized swimming response 6 days after ALC treatment but not after 2 hours indicating that the long-term suppressive effects of ALC on sensitization/dishabituation needed mRNA and protein synthesis.

  15. Detection of methylation, acetylation and glycosylation of protein residues by monitoring (13)C chemical-shift changes: A quantum-chemical study.

    PubMed

    Garay, Pablo G; Martin, Osvaldo A; Scheraga, Harold A; Vila, Jorge A

    2016-01-01

    Post-translational modifications of proteins expand the diversity of the proteome by several orders of magnitude and have a profound effect on several biological processes. Their detection by experimental methods is not free of limitations such as the amount of sample needed or the use of destructive procedures to obtain the sample. Certainly, new approaches are needed and, therefore, we explore here the feasibility of using (13)C chemical shifts of different nuclei to detect methylation, acetylation and glycosylation of protein residues by monitoring the deviation of the (13)C chemical shifts from the expected (mean) experimental value of the non-modified residue. As a proof-of-concept, we used (13)C chemical shifts, computed at the DFT-level of theory, to test this hypothesis. Moreover, as a validation test of this approach, we compare our theoretical computations of the (13)Cε chemical-shift values against existing experimental data, obtained from NMR spectroscopy, for methylated and acetylated lysine residues with good agreement within ∼1 ppm. Then, further use of this approach to select the most suitable (13)C-nucleus, with which to determine other modifications commonly seen, such as methylation of arginine and glycosylation of serine, asparagine and threonine, shows encouraging results.

  16. Major cancer protein amplifies global gene expression

    Cancer.gov

    Scientists may have discovered why a protein called MYC can provoke a variety of cancers. Like many proteins associated with cancer, MYC helps regulate cell growth. A new study carried out by researchers at the National Institutes of Health and colleagues

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

    PubMed

    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.

  18. N-acetyl ornithine deacetylase is a moonlighting protein and is involved in the adaptation of Entamoeba histolytica to nitrosative stress

    PubMed Central

    Shahi, Preeti; Trebicz-Geffen, Meirav; Nagaraja, Shruti; Hertz, Rivka; Alterzon-Baumel, Sharon; Methling, Karen; Lalk, Michael; Mazumder, Mohit; Samudrala, Gourinath; Ankri, Serge

    2016-01-01

    Adaptation of the Entamoeba histolytica parasite to toxic levels of nitric oxide (NO) that are produced by phagocytes may be essential for the establishment of chronic amebiasis and the parasite’s survival in its host. In order to obtain insight into the mechanism of E. histolytica’s adaptation to NO, E. histolytica trophozoites were progressively adapted to increasing concentrations of the NO donor drug, S-nitrosoglutathione (GSNO) up to a concentration of 110 μM. The transcriptome of NO adapted trophozoites (NAT) was investigated by RNA sequencing (RNA-seq). N-acetyl ornithine deacetylase (NAOD) was among the 208 genes that were upregulated in NAT. NAOD catalyzes the deacetylation of N-acetyl-L-ornithine to yield ornithine and acetate. Here, we report that NAOD contributes to the better adaptation of the parasite to nitrosative stress (NS) and that this function does not depend on NAOD catalytic activity. We also demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is detrimental to E. histolytica exposed to NS and that this detrimental effect is neutralized by NAOD or by a catalytically inactive NAOD (mNAOD). These results establish NAOD as a moonlighting protein, and highlight the unexpected role of this metabolic enzyme in the adaptation of the parasite to NS. PMID:27808157

  19. Protein kinase CK2-mediated phosphorylation of HDAC2 regulates co-repressor formation, deacetylase activity and acetylation of HDAC2 by cigarette smoke and aldehydes.

    PubMed

    Adenuga, David; Rahman, Irfan

    2010-06-01

    Histone deacetylase 2 (HDAC2) mediates the repression of pro-inflammatory genes by deacetylating core histones, RelA/p65 and the glucocorticoid receptor. Reduced level of HDAC2 is associated with steroid resistant inflammation caused by cigarette smoke (CS)-derived oxidants and aldehydes. However, the molecular mechanisms regulating HDAC2 in response to CS and aldehydes is not known. Here, we report that CS extract, and aldehyde acrolein induced phosphorylation of HDAC2 which was abolished by mutations at serine sites S(394), S(411), S(422) and S(424). HDAC2 phosphorylation required direct interaction with serine-phosphorylated protein kinase CK2alpha and involved reduced HDAC2 deacetylase activity. Furthermore, HDAC2 phosphorylation was required for HDAC2 interaction with transcription factors, co-repressor complex formation, CBP recruitment, acetylation on lysine residues and modulates transrepression activity. Thus, phospho-acetylation of HDAC2 negatively regulates its deacetylase activity which has implications in steroid resistance in chronic inflammatory conditions.

  20. Gene expression in macrophage-rich human atherosclerotic lesions. 15-lipoxygenase and acetyl low density lipoprotein receptor messenger RNA colocalize with oxidation specific lipid-protein adducts.

    PubMed Central

    Ylä-Herttuala, S; Rosenfeld, M E; Parthasarathy, S; Sigal, E; Särkioja, T; Witztum, J L; Steinberg, D

    1991-01-01

    Oxidatively modified low density lipoprotein (LDL) exhibits several potentially atherogenic properties, and inhibition of LDL oxidation in rabbits decreases the rate of the development of atherosclerotic lesions. In vitro studies have suggested that cellular lipoxygenases may be involved in LDL oxidation, and we have shown previously that 15-lipoxygenase and oxidized LDL are present in rabbit atherosclerotic lesions. We now report that epitopes of oxidized LDL are also found in macrophage-rich areas of human fatty streaks as well as in more advanced human atherosclerotic lesions. Using in situ hybridization and immunostaining techniques, we also report that 15-lipoxygenase mRNA and protein colocalize to the same macrophage-rich areas. Moreover, these same lesions express abundant mRNA for the acetyl LDL receptor but no detectable mRNA for the LDL receptor. We suggest that atherogenesis in human arteries may be linked to macrophage-induced oxidative modification of LDL mediated by 15-lipoxygenase, leading to subsequent enhanced macrophage uptake, partly by way of the acetyl LDL receptor. Images PMID:2010531

  1. N-acetyl ornithine deacetylase is a moonlighting protein and is involved in the adaptation of Entamoeba histolytica to nitrosative stress.

    PubMed

    Shahi, Preeti; Trebicz-Geffen, Meirav; Nagaraja, Shruti; Hertz, Rivka; Alterzon-Baumel, Sharon; Methling, Karen; Lalk, Michael; Mazumder, Mohit; Samudrala, Gourinath; Ankri, Serge

    2016-11-03

    Adaptation of the Entamoeba histolytica parasite to toxic levels of nitric oxide (NO) that are produced by phagocytes may be essential for the establishment of chronic amebiasis and the parasite's survival in its host. In order to obtain insight into the mechanism of E. histolytica's adaptation to NO, E. histolytica trophozoites were progressively adapted to increasing concentrations of the NO donor drug, S-nitrosoglutathione (GSNO) up to a concentration of 110 μM. The transcriptome of NO adapted trophozoites (NAT) was investigated by RNA sequencing (RNA-seq). N-acetyl ornithine deacetylase (NAOD) was among the 208 genes that were upregulated in NAT. NAOD catalyzes the deacetylation of N-acetyl-L-ornithine to yield ornithine and acetate. Here, we report that NAOD contributes to the better adaptation of the parasite to nitrosative stress (NS) and that this function does not depend on NAOD catalytic activity. We also demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is detrimental to E. histolytica exposed to NS and that this detrimental effect is neutralized by NAOD or by a catalytically inactive NAOD (mNAOD). These results establish NAOD as a moonlighting protein, and highlight the unexpected role of this metabolic enzyme in the adaptation of the parasite to NS.

  2. Quantitative measurement of histone tail acetylation reveals stage-specific regulation and response to environmental changes during Drosophila development

    PubMed Central

    Henry, Ryan A.; Singh, Tanu; Kuo, Yin-Ming; Biester, Alison; O’Keefe, Abigail; Lee, Sandy; Andrews, Andrew J.; O’Reilly, Alana M.

    2016-01-01

    Histone modification plays a major role in regulating gene transcription and ensuring the healthy development of an organism. Numerous studies have suggested that histones are dynamically modified during developmental events to control gene expression levels in a temporal and spatial manner. However, the study of histone acetylation dynamics using currently available techniques is hindered by the difficulty of simultaneously measuring acetylation of the numerous potential sites of modification present in histones. Here, we present a methodology that allows us to combine mass spectrometry-based histone analysis with Drosophila developmental genetics. Using this system, we characterized histone acetylation patterns during multiple developmental stages of the fly. Additionally, we utilized this analysis to characterize how treatments with pharmacological agents or environmental changes such as gamma-irradiation altered histone acetylation patterns. Strikingly, gamma-irradiation dramatically increased acetylation at H3K18, a site linked to DNA repair via non-homologous end joining. In mutant fly strains deficient in DNA repair proteins, however, this increase in H3K18 acetylation was lost. These results demonstrate the efficacy of our combined mass spectrometry system with a Drosophila model system, and provide interesting insight into the changes in histone acetylation during development, as well as the effects of both pharmacological and environmental agents on global histone acetylation. PMID:26836402

  3. Metabolic control of methylation and acetylation.

    PubMed

    Su, Xiaoyang; Wellen, Kathryn E; Rabinowitz, Joshua D

    2016-02-01

    Methylation and acetylation of DNA and histone proteins are the chemical basis for epigenetics. From bacteria to humans, methylation and acetylation are sensitive to cellular metabolic status. Modification rates depend on the availability of one-carbon and two-carbon substrates (S-adenosylmethionine, acetyl-CoA, and in bacteria also acetyl-phosphate). In addition, they are sensitive to demodification enzyme cofactors (α-ketoglutarate, NAD(+)) and structural analog metabolites that function as epigenetic enzyme inhibitors (e.g., S-adenosylhomocysteine, 2-hydroxyglutarate). Methylation and acetylation likely initially evolved to tailor protein activities in microbes to their metabolic milieu. While the extracellular environment of mammals is more tightly controlled, the combined impact of nutrient abundance and metabolic enzyme expression impacts epigenetics in mammals sufficiently to drive important biological outcomes such as stem cell fate and cancer.

  4. SPOTing Acetyl-Lysine Dependent Interactions

    PubMed Central

    Picaud, Sarah; Filippakopoulos, Panagis

    2015-01-01

    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. PMID:27600229

  5. Metabolic control of methylation and acetylation

    PubMed Central

    Su, Xiaoyang; Wellen, Kathryn E.; Rabinowitz, Joshua D

    2015-01-01

    Methylation and acetylation of DNA and histone proteins are the chemical basis for epigenetics. From bacteria to humans, methylation and acetylation are sensitive to cellular metabolic status. Modification rates depend on the availability of one-carbon and two-carbon substrates (S-adenosylmethionine, acetyl-CoA, and in bacteria also acetyl-phosphate). In addition, they are sensitive to demodification enzyme cofactors (α-ketoglutarate, NAD+) and structural analog metabolites that function as epigenetic enzyme inhibitors (e.g., S-adenosylhomocysteine, 2-hydroxyglutarate). Methylation and acetylation likely initially evolved to tailor protein activities in microbes to their metabolic milieu. While the extracellular environment of mammals is more tightly controlled, the combined impact of nutrient abundance and metabolic enzyme expression impacts epigenetics in mammals sufficiently to drive important biological outcomes such as stem cell fate and cancer. PMID:26629854

  6. Acetylation of woody lignocellulose: significance and regulation

    PubMed Central

    Pawar, Prashant Mohan-Anupama; Koutaniemi, Sanna; Tenkanen, Maija; Mellerowicz, Ewa J.

    2013-01-01

    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 toward 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. PMID:23734153

  7. Acetylation of histones in neocortex and hippocampus of rats exposed to different modes of hypobaric hypoxia: Implications for brain hypoxic injury and tolerance.

    PubMed

    Samoilov, Mikhail; Churilova, Anna; Gluschenko, Tatjana; Vetrovoy, Oleg; Dyuzhikova, Natalia; Rybnikova, Elena

    2016-03-01

    Acetylation of nucleosome histones results in relaxation of DNA and its availability for the transcriptional regulators, and is generally associated with the enhancement of gene expression. Although it is well known that activation of a variety of pro-adaptive genes represents a key event in the development of brain hypoxic/ischemic tolerance, the role of epigenetic mechanisms, in particular histone acetylation, in this process is still unexplored. The aim of the present study was to investigate changes in acetylation of histones in vulnerable brain neurons using original well-standardized model of hypobaric hypoxia and preconditioning-induced tolerance of the brain. Using quantitative immunohistochemistry and Western blot, effects of severe injurious hypobaric hypoxia (SH, 180mm Hg, 3h) and neuroprotective preconditioning mode (three episodes of 360mm Hg for 2h spaced at 24h) on the levels of the acetylated proteins and acetylated H3 Lys24 (H3K24ac) in the neocortex and hippocampus of rats were studied. SH caused global repression of the acetylation processes in the neocortex (layers II-III, V) and hippocampus (CA1, CA3) by 3-24h, and this effect was prevented by the preconditioning. Moreover, hypoxic preconditioning remarkably increased the acetylation of H3K24 in response to SH in the brain areas examined. The preconditioning hypoxia without subsequent SH also stimulated acetylation processes in the neocortex and hippocampus. The moderately enhanced expression of the acetylated proteins in the preconditioned rats was maintained for 24h, whereas acetylation of H3K24 was intense but transient, peaked at 3h. The novel data obtained in the present study indicate that large activation of the acetylation processes, in particular acetylation of histones might be essential for the development of brain hypoxic tolerance.

  8. Early postnatal feed restriction reduces liver connective tissue levels and affects H3K9 acetylation state of regulated genes associated with protein metabolism in low birth weight pigs.

    PubMed

    Nebendahl, Constance; Görs, Solvig; Albrecht, Elke; Krüger, Ricarda; Martens, Karen; Giller, Katrin; Hammon, Harald M; Rimbach, Gerald; Metges, Cornelia C

    2016-03-01

    Intrauterine growth retardation is associated with metabolic consequences in adulthood. Since our previous data indicate birth weight-dependent effects of feed restriction (R) on protein degradation processes in the liver, it should be investigated whether effects on connective tissue turnover are obvious and could be explained by global changes of histone H3K9me3 and H3K9ac states in regulated genes. For this purpose, female littermate pigs with low (U) or normal (N) birth weight were subjected to 3-week R (60% of ad libitum fed controls) with subsequent refeeding (REF) for further 5 weeks. The 3-week R-period induced a significant reduction of connective tissue area by 43% in the liver of U animals at 98 d of age, which was not found in age-matched N animals. Of note, after REF at 131 d of age, in previously feed-restricted U animals (UR), the percentage of mean connective tissue was only 53% of ad libitum fed controls (UK), indicating a persistent effect. In U animals, R induced H3K9 acetylation of regulated genes (e.g. XBP1, ERLEC1, GALNT2, PTRH2), which were inter alia associated with protein metabolism. In contrast, REF was mostly accompanied by deacetylation in U and N animals. Thus, our epigenetic data may give a first explanation for the observed birth weight-dependent differences in this connective tissue phenotype.

  9. Global Geometric Affinity for Revealing High Fidelity Protein Interaction Network

    PubMed Central

    Fang, Yi; Benjamin, William; Sun, Mengtian; Ramani, Karthik

    2011-01-01

    Protein-protein interaction (PPI) network analysis presents an essential role in understanding the functional relationship among proteins in a living biological system. Despite the success of current approaches for understanding the PPI network, the large fraction of missing and spurious PPIs and a low coverage of complete PPI network are the sources of major concern. In this paper, based on the diffusion process, we propose a new concept of global geometric affinity and an accompanying computational scheme to filter the uncertain PPIs, namely, reduce the spurious PPIs and recover the missing PPIs in the network. The main concept defines a diffusion process in which all proteins simultaneously participate to define a similarity metric (global geometric affinity (GGA)) to robustly reflect the internal connectivity among proteins. The robustness of the GGA is attributed to propagating the local connectivity to a global representation of similarity among proteins in a diffusion process. The propagation process is extremely fast as only simple matrix products are required in this computation process and thus our method is geared toward applications in high-throughput PPI networks. Furthermore, we proposed two new approaches that determine the optimal geometric scale of the PPI network and the optimal threshold for assigning the PPI from the GGA matrix. Our approach is tested with three protein-protein interaction networks and performs well with significant random noises of deletions and insertions in true PPIs. Our approach has the potential to benefit biological experiments, to better characterize network data sets, and to drive new discoveries. PMID:21559288

  10. The DAF-16 FOXO Transcription Factor Regulates natc-1 to Modulate Stress Resistance in Caenorhabditis elegans, Linking Insulin/IGF-1 Signaling to Protein N-Terminal Acetylation

    PubMed Central

    Warnhoff, Kurt; Murphy, John T.; Kumar, Sandeep; Schneider, Daniel L.; Peterson, Michelle; Hsu, Simon; Guthrie, James; Robertson, J. David; Kornfeld, Kerry

    2014-01-01

    The insulin/IGF-1 signaling pathway plays a critical role in stress resistance and longevity, but the mechanisms are not fully characterized. To identify genes that mediate stress resistance, we screened for C. elegans mutants that can tolerate high levels of dietary zinc. We identified natc-1, which encodes an evolutionarily conserved subunit of the N-terminal acetyltransferase C (NAT) complex. N-terminal acetylation is a widespread modification of eukaryotic proteins; however, relatively little is known about the biological functions of NATs. We demonstrated that loss-of-function mutations in natc-1 cause resistance to a broad-spectrum of physiologic stressors, including multiple metals, heat, and oxidation. The C. elegans FOXO transcription factor DAF-16 is a critical target of the insulin/IGF-1 signaling pathway that mediates stress resistance, and DAF-16 is predicted to directly bind the natc-1 promoter. To characterize the regulation of natc-1 by DAF-16 and the function of natc-1 in insulin/IGF-1 signaling, we analyzed molecular and genetic interactions with key components of the insulin/IGF-1 pathway. natc-1 mRNA levels were repressed by DAF-16 activity, indicating natc-1 is a physiological target of DAF-16. Genetic studies suggested that natc-1 functions downstream of daf-16 to mediate stress resistance and dauer formation. Based on these findings, we hypothesize that natc-1 is directly regulated by the DAF-16 transcription factor, and natc-1 is a physiologically significant effector of the insulin/IGF-1 signaling pathway that mediates stress resistance and dauer formation. These studies identify a novel biological function for natc-1 as a modulator of stress resistance and dauer formation and define a functionally significant downstream effector of the insulin/IGF-1 signaling pathway. Protein N-terminal acetylation mediated by the NatC complex may play an evolutionarily conserved role in regulating stress resistance. PMID:25330323

  11. Site-Specific Acetyl Lysine Antibodies Reveal Differential Regulation of Histone Acetylation upon Kinase Inhibition.

    PubMed

    Chen, Shi; Chen, Suping; Duan, Qianqian; Xu, Guoqiang

    2017-03-01

    Lysine acetylation regulates diverse biological functions for the modified proteins. Mass spectrometry-based proteomic approaches have identified thousands of lysine acetylation sites in cells and tissues. However, functional studies of these acetylation sites were limited by the lack of antibodies recognizing the specific modification sites. Here, we generated 55 site-specific acetyl lysine antibodies for the detection of this modification in cell lysates and evaluated the quality of these antibodies. Based on the immunoblotting analyses, we found that the nature of amino acid sequences adjacent to the modification sites affected the specificity of the site-specific acetyl lysine antibodies. Amino acids with charged, hydrophilic, small, or flexible side chains adjacent to the modification sites increase the likelihood of obtaining high quality site-specific acetyl lysine antibodies. This result may provide valuable insights in fine-tuning the amino acid sequences of the epitopes for the generation of site-specific acetyl lysine antibodies. Using the site-specific acetyl lysine antibodies, we further discovered that acetylation of histone 3 at four lysine residues was differentially regulated by kinase inhibitors. This result demonstrates the potential application of these antibodies in the study of new signaling pathways that lysine acetylation may participate in.

  12. A proteomic strategy for global analysis of plant protein complexes.

    PubMed

    Aryal, Uma K; Xiong, Yi; McBride, Zachary; Kihara, Daisuke; Xie, Jun; Hall, Mark C; Szymanski, Daniel B

    2014-10-01

    Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions.

  13. Acetylation modulates the STAT signaling code.

    PubMed

    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.

  14. Replication-independent nucleosome exchange is enhanced by local and specific acetylation of histone H4

    PubMed Central

    Elliott, Giles O.; Murphy, Kevin J.; Hayes, Jeffrey J.; Thiriet, Christophe

    2013-01-01

    We used a novel single-cell strategy to examine the fate of histones during G2-phase. Consistent with previous results, we find that in G2-phase, the majority of nuclear histones are assembled into chromatin, whereas a small fraction comprises an unassembled pool. Small increases in the amount of histones within the free pool affect the extent of exchange, suggesting that the free pool is in dynamic equilibrium with chromatin proteins. Unexpectedly, acetylated H4 is preferentially partitioned to the unassembled pool. Although an increase in global histone acetylation did not affect overall nucleosome dynamics, an H4 containing lysine to glutamine substitutions as mimics of acetylation significantly increased the rate of exchange, but did not affect the acetylation state of neighbouring nucleosomes. Interestingly, transcribed regions are particularly predisposed to exchange on incorporation of H4 acetylation mimics compared with surrounding regions. Our results support a model whereby histone acetylation on K8 and K16 specifically marks nucleosomes for eviction, with histones being rapidly deacetylated on reassembly. PMID:23303778

  15. Mitochondrial protein adducts formation and mitochondrial dysfunction during N-acetyl-m-aminophenol (AMAP)-induced hepatotoxicity in primary human hepatocytes

    PubMed Central

    Xie, Yuchao; McGill, Mitchell R.; Du, Kuo; Dorko, Kenneth; Kumer, Sean C.; Schmitt, Timothy M.; Ding, Wen-Xing; Jaeschke, Hartmut

    2015-01-01

    3′-Hydroxyacetanilide or N-acetyl-meta-aminophenol (AMAP) is generally regarded as a non-hepatotoxic analog of acetaminophen (APAP). Previous studies demonstrated absence of toxicity after AMAP in mice, hamsters, primary mouse hepatocytes and several cell lines. In contrast, experiments with liver slices suggested that it may be toxic to human hepatocytes; however, the mechanism of toxicity is unclear. To explore this, we treated primary human hepatocytes (PHH) with AMAP or APAP for up to 48 h and measured several parameters to assess metabolism and injury. Although less toxic than APAP, AMAP dose-dependently triggered cell death in PHH as indicated by alanine aminotransferase (ALT) release and propidium iodide (PI) staining. Similar to APAP, AMAP also significantly depleted glutathione (GSH) in PHH and caused mitochondrial damage as indicated by glutamate dehydrogenase (GDH) release and the JC-1 assay. However, unlike APAP, AMAP treatment did not cause relevant c-jun-N-terminal kinase (JNK) activation in the cytosol or phospho-JNK translocation to mitochondria. To compare, AMAP toxicity was assessed in primary mouse hepatocytes (PMH). No cytotoxicity was observed as indicated by the lack of lactate dehydrogenase release and no PI staining. Furthermore, there was no GSH depletion or mitochondrial dysfunction after AMAP treatment in PMH. Immunoblotting for arylated proteins suggested that AMAP treatment caused extensive mitochondrial protein adducts formation in PHH but not in PMH. In conclusion, AMAP is hepatotoxic in PHH and the mechanism involves formation of mitochondrial protein adducts and mitochondrial dysfunction. PMID:26431796

  16. Acetylation of the c-MYC oncoprotein is required for cooperation with the HTLV-1 p30{sup II} accessory protein and the induction of oncogenic cellular transformation by p30{sup II}/c-MYC

    SciTech Connect

    Romeo, Megan M.; Ko, Bookyung; Kim, Janice; Brady, Rebecca; Heatley, Hayley C.; He, Jeffrey; Harrod, Carolyn K.; Barnett, Braden; Ratner, Lee; Lairmore, Michael D.; Martinez, Ernest; Lüscher, Bernhard; Robson, Craig N.; Henriksson, Marie; Harrod, Robert

    2015-02-15

    The human T-cell leukemia retrovirus type-1 (HTLV-1) p30{sup II} protein is a multifunctional latency-maintenance factor that negatively regulates viral gene expression and deregulates host signaling pathways involved in aberrant T-cell growth and proliferation. We have previously demonstrated that p30{sup II} interacts with the c-MYC oncoprotein and enhances c-MYC-dependent transcriptional and oncogenic functions. However, the molecular and biochemical events that mediate the cooperation between p30{sup II} and c-MYC remain to be completely understood. Herein we demonstrate that p30{sup II} induces lysine-acetylation of the c-MYC oncoprotein. Acetylation-defective c-MYC Lys→Arg substitution mutants are impaired for oncogenic transformation with p30{sup II} in c-myc{sup −/−} HO15.19 fibroblasts. Using dual-chromatin-immunoprecipitations (dual-ChIPs), we further demonstrate that p30{sup II} is present in c-MYC-containing nucleoprotein complexes in HTLV-1-transformed HuT-102 T-lymphocytes. Moreover, p30{sup II} inhibits apoptosis in proliferating cells expressing c-MYC under conditions of genotoxic stress. These findings suggest that c-MYC-acetylation is required for the cooperation between p30{sup II}/c-MYC which could promote proviral replication and contribute to HTLV-1-induced carcinogenesis. - Highlights: • Acetylation of c-MYC is required for oncogenic transformation by HTLV-1 p30{sup II}/c-MYC. • Acetylation-defective c-MYC mutants are impaired for foci-formation by p30{sup II}/c-MYC. • The HTLV-1 p30{sup II} protein induces lysine-acetylation of c-MYC. • p30{sup II} is present in c-MYC nucleoprotein complexes in HTLV-1-transformed T-cells. • HTLV-1 p30{sup II} inhibits apoptosis in c-MYC-expressing proliferating cells.

  17. The protein folding problem: global optimization of the force fields.

    PubMed

    Scheraga, H A; Liwo, A; Oldziej, S; Czaplewski, C; Pillardy, J; Ripoll, D R; Vila, J A; Kazmierkiewicz, R; Saunders, J A; Arnautova, Y A; Jagielska, A; Chinchio, M; Nanias, M

    2004-09-01

    The evolutionary development of a theoretical approach to the protein folding problem, in our laboratory, is traced. The theoretical foundations and the development of a suitable empirical all-atom potential energy function and a global optimization search are examined. Whereas the all-atom approach has thus far succeeded for relatively small molecules and for alpha-helical proteins containing up to 46 residues, it has been necessary to develop a hierarchical approach to treat larger proteins. In the hierarchical approach to single- and multiple-chain proteins, global optimization is carried out for a simplified united residue (UNRES) description of a polypeptide chain to locate the region in which the global minimum lies. Conversion of the UNRES structures in this region to all-atom structures is followed by a local search in this region. The performance of this approach in successive CASP blind tests for predicting protein structure by an ab initio physics-based method is described. Finally, a recent attempt to compute a folding pathway is discussed.

  18. Short-term supplementation with acetyl-L-carnitine and lipoic acid alters plasma protein carbonyl levels but does not improve cognition in aged beagles

    PubMed Central

    Christie, Lori-Ann; Opii, Wycliffe O.; Head, Elizabeth; Araujo, Joseph A.; De Rivera, Christina; Milgram, Norton W.; Cotman, Carl W.

    2009-01-01

    Previous work has shown that a diet enriched with antioxidants and mitochondrial co-factors improves cognition in aged dogs, which was accompanied by a reduction oxidative damage in the brain. The objective of the present study was to assess the effects of supplementation with mitochondrial co-factors on cognition and plasma protein carbonyl levels in aged dogs. Specifically, we aimed to test whether the individual or combined action of lipoic acid (LA) and acetyl-L-carnitine (ALCAR) could account for the beneficial effects of the enriched diet that contained both plus antioxidants. Dogs were given LA or ALCAR, alone and then in combination and cognition was assessed using a spatial learning task and two discrimination and reversal paradigms. Dogs receiving the ALCAR supplement showed an increase in protein carbonyl levels that was associated with increased error scores on the spatial task, and which was reduced upon additional supplementation with LA. We did not observe significant positive effects on cognition. The present findings suggest that short-term supplementation with LA and ALCAR is insufficient to improve cognition in aged dogs, and that the beneficial effects of the full spectrum diet arose from either the cellular antioxidants alone or their interaction with LA and ALCAR. PMID:19735717

  19. The tep1 gene of Sinorhizobium meliloti coding for a putative transmembrane efflux protein and N-acetyl glucosamine affect nod gene expression and nodulation of alfalfa plants.

    PubMed

    van Dillewijn, Pieter; Sanjuán, Juan; Olivares, José; Soto, María José

    2009-01-27

    Soil bacteria collectively known as Rhizobium, characterized by their ability to establish beneficial symbiosis with legumes, share several common characteristics with pathogenic bacteria when infecting the host plant. Recently, it was demonstrated that a fadD mutant of Sinorhizobium meliloti is altered in the control of swarming, a type of co-ordinated movement previously associated with pathogenicity, and is also impaired in nodulation efficiency on alfalfa roots. In the phytopathogen Xanthomonas campestris, a fadD homolog (rpfB) forms part of a cluster of genes involved in the regulation of pathogenicity factors. In this work, we have investigated the role in swarming and symbiosis of SMc02161, a S. meliloti fadD-linked gene. The SMc02161 locus in S. meliloti shows similarities with members of the Major Facilitator Superfamily (MFS) of transporters. A S. meliloti null-mutant shows increased sensitivity to chloramphenicol. This indication led us to rename the locus tep1 for transmembrane efflux protein. The lack of tep1 does not affect the appearance of swarming motility. Interestingly, nodule formation efficiency on alfalfa plants is improved in the tep1 mutant during the first days of the interaction though nod gene expression is lower than in the wild type strain. Curiously, a nodC mutation or the addition of N-acetyl glucosamine to the wild type strain lead to similar reductions in nod gene expression as in the tep1 mutant. Moreover, aminosugar precursors of Nod factors inhibit nodulation. tep1 putatively encodes a transmembrane protein which can confer chloramphenicol resistance in S. meliloti by expelling the antibiotic outside the bacteria. The improved nodulation of alfalfa but reduced nod gene expression observed in the tep1 mutant suggests that Tep1 transports compounds which influence nodulation. In contrast to Bradyrhizobium japonicum, we show that in S. meliloti there is no feedback regulation of nodulation genes. Moreover, the Nod factor precursor

  20. The tep1 gene of Sinorhizobium meliloti coding for a putative transmembrane efflux protein and N-acetyl glucosamine affect nod gene expression and nodulation of alfalfa plants

    PubMed Central

    2009-01-01

    Background Soil bacteria collectively known as Rhizobium, characterized by their ability to establish beneficial symbiosis with legumes, share several common characteristics with pathogenic bacteria when infecting the host plant. Recently, it was demonstrated that a fadD mutant of Sinorhizobium meliloti is altered in the control of swarming, a type of co-ordinated movement previously associated with pathogenicity, and is also impaired in nodulation efficiency on alfalfa roots. In the phytopathogen Xanthomonas campestris, a fadD homolog (rpfB) forms part of a cluster of genes involved in the regulation of pathogenicity factors. In this work, we have investigated the role in swarming and symbiosis of SMc02161, a S. meliloti fadD-linked gene. Results The SMc02161 locus in S. meliloti shows similarities with members of the Major Facilitator Superfamily (MFS) of transporters. A S. meliloti null-mutant shows increased sensitivity to chloramphenicol. This indication led us to rename the locus tep1 for transmembrane efflux protein. The lack of tep1 does not affect the appearance of swarming motility. Interestingly, nodule formation efficiency on alfalfa plants is improved in the tep1 mutant during the first days of the interaction though nod gene expression is lower than in the wild type strain. Curiously, a nodC mutation or the addition of N-acetyl glucosamine to the wild type strain lead to similar reductions in nod gene expression as in the tep1 mutant. Moreover, aminosugar precursors of Nod factors inhibit nodulation. Conclusion tep1 putatively encodes a transmembrane protein which can confer chloramphenicol resistance in S. meliloti by expelling the antibiotic outside the bacteria. The improved nodulation of alfalfa but reduced nod gene expression observed in the tep1 mutant suggests that Tep1 transports compounds which influence nodulation. In contrast to Bradyrhizobium japonicum, we show that in S. meliloti there is no feedback regulation of nodulation genes

  1. Shape-Dependent Global Deformation Modes of Large Protein Structures

    PubMed Central

    Miloshevsky, Gennady V.; Hassanein, Ahmed; Jordan, Peter C.

    2010-01-01

    Conformational changes are central to the functioning of pore-forming proteins that open and close their molecular gates in response to external stimuli such as pH, ionic strength, membrane voltage or ligand binding. Normal mode analysis (NMA) is used to identify and characterize the slowest motions in the gA, KcsA, ClC-ec1, LacY and LeuTAa proteins at the onset of gating. Global deformation modes of the essentially cylindrical gA, KcsA, LacY and LeuTAa biomolecules are reminiscent of global twisting, transverse and longitudinal motions in a homogeneous elastic rod. The ClC-ec1 protein executes a splaying motion in the plane perpendicular to the lipid bilayer. These global collective deformations are determined by protein shape. New methods, all-atom Monte Carlo Normal Mode Following and its simplification using a rotation-translation of protein blocks (RTB), are described and applied to gain insight into the nature of gating transitions in gA and KcsA. These studies demonstrate the severe limitations of standard NMA in characterizing the structural rearrangements associated with gating transitions. Comparison of all-atom and RTB transition pathways in gA clearly illustrates the impact of the rigid protein block approximation and the need to include all degrees of freedom and their relaxation in computational studies of protein gating. The effects of atomic level structure, pH, hydrogen bonding and charged residues on the large scale conformational changes associated with gating transitions are discussed. PMID:20526444

  2. Shape-dependent global deformation modes of large protein structures

    NASA Astrophysics Data System (ADS)

    Miloshevsky, Gennady V.; Hassanein, Ahmed; Jordan, Peter C.

    2010-05-01

    Conformational changes are central to the functioning of pore-forming proteins that open and close their molecular gates in response to external stimuli such as pH, ionic strength, membrane voltage or ligand binding. Normal mode analysis (NMA) is used to identify and characterize the slowest motions in the gA, KcsA, ClC-ec1, LacY and LeuT Aa proteins at the onset of gating. Global deformation modes of the essentially cylindrical gA, KcsA, LacY and LeuT Aa biomolecules are reminiscent of global twisting, transverse and longitudinal motions in a homogeneous elastic rod. The ClC-ec1 protein executes a splaying motion in the plane perpendicular to the lipid bilayer. These global collective deformations are determined by protein shape. New methods, all-atom Monte Carlo Normal Mode Following and its simplification using a rotation-translation of protein blocks (RTB), are described and applied to gain insight into the nature of gating transitions in gA and KcsA. These studies demonstrate the severe limitations of standard NMA in characterizing the structural rearrangements associated with gating transitions. Comparison of all-atom and RTB transition pathways in gA clearly illustrates the impact of the rigid protein block approximation and the need to include all degrees of freedom and their relaxation in computational studies of protein gating. The effects of atomic level structure, pH, hydrogen bonding and charged residues on the large-scale conformational changes associated with gating transitions are discussed.

  3. Zinc finger protein 668 interacts with Tip60 to promote H2AX acetylation after DNA damage.

    PubMed

    Hu, Ruozhen; Wang, Edward; Peng, Guang; Dai, Hui; Lin, Shiaw-Yih

    2013-07-01

    Many tumor suppressors play an important role in the DNA damage pathway. Zinc finger protein 668 (ZNF668) has recently been identified as one of the potential tumor suppressors in breast cancer, but its function in DNA damage response is unknown. Herein, we report that ZNF668 is a regulator of DNA repair. ZNF668 knockdown impairs cell survival after DNA damage without affecting the ATM/ATR DNA-damage signaling cascade. However, recruitment of repair proteins to DNA lesions is decreased. In response to IR, ZNF668 knockdown reduces Tip60-H2AX interaction and impairs IR-induced histone H2AX hyperacetylation, thus impairing chromatin relaxation. Impaired chromatin relaxation causes decreased recruitment of repair proteins to DNA lesions, defective homologous recombination (HR) repair and impaired cell survival after IR. In addition, ZNF668 knockdown decreased RPA phosphorylation and its recruitment to DNA damage foci in response to UV. In both IR and UV damage responses, chromatin relaxation counteracted the impaired loading of repair proteins and DNA repair defects in ZNF668-deficient U2OS cells, indicating that impeded chromatin accessibility at sites of DNA breaks caused the DNA repair defects observed in the absence of ZNF668. Our findings suggest that ZNF668 is a key molecule that links chromatin relaxation with DNA damage response in DNA repair control.

  4. Global Analysis of Protein Expression of Inner Ear Hair Cells.

    PubMed

    Hickox, Ann E; Wong, Ann C Y; Pak, Kwang; Strojny, Chelsee; Ramirez, Miguel; Yates, John R; Ryan, Allen F; Savas, Jeffrey N

    2017-02-01

    The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes.

  5. Identification of functionally important cysteine residues of the human renin-binding protein as the enzyme N-acetyl-D-glucosamine 2-epimerase.

    PubMed

    Takahashi, S; Takahashi, K; Kaneko, T; Ogasawara, H; Shindo, S; Saito, K; Kawamura, Y

    2001-04-01

    Renin-binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney. It was recently identified as the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353] and its active site residue was determined to be cysteine 380 by site-directed mutagenesis [Takahashi, S. et al. (1999) J. Biochem. 126, 639-642]. To further investigate the relationship between structure and function of recombinant human (rh) RnBP as a GlcNAc 2-epimerase, we have constructed several C-terminal deletion and multi-cysteine/serine mutants of rhGlcNAc 2-epimerase and expressed them in Escherichia coli cells. The expression was detected by Western blotting using anti-rhRnBP antiserum. The C-terminal deletion mutant, Delta400-417, had approximately 50% activity relative to the wild-type enzyme, but other C-terminal deletion mutants, Delta380-417, Delta386-417, and Delta390-417, had no enzymatic activity. Mutational analysis of multi-cysteine/serine mutants revealed that cysteines 41 and 390 were critical for the activity or stabilization of the enzyme, while cysteine residues in the middle of the enzyme, cysteines 125, 210, 239, and 302, had no essential function in relation to the activity.

  6. Antioxidant N-acetyl-L-cysteine ameliorates symptoms of premature aging associated with the deficiency of the circadian protein BMAL1

    PubMed Central

    Kondratov, Roman V.; Vykhovanets, Olena; Kondratova, Anna A.; Antoch, Marina P.

    2009-01-01

    Deficiency of the circadian clock protein BMAL1 leads to premature aging and increased levels of reactivate oxygen species in several tissues of mice. In order to investigate the role of oxidative stress in accelerated aging and development of age-related pathologies, we continuously administered the antioxidant N-acetyl-L-cysteine toBmal1-deficient mice through their entire lifespan by supplementing drinking water. We found that the life long treatment with antioxidant significantly increased average and maximal lifespan and reduced the rate of age-dependent weight loss and development of cataracts. At the same time, it had no effect on time of onset and severity of other age-related pathologies characteristic of Bmal1-/- mice, such as joint ossification, reduced hair regrowth and sarcopenia. We conclude that chronic oxidative stress affects longevity and contributes to the development of at least some age-associated pathology, although ROS-independent mechanisms may also play a role. Our bioinformatics analysis identified the presence of a conservative E box element in the promoter regions of several genes encoding major antioxidant enzymes. We speculate that BMAL1 controls antioxidant defense by regulating the expression of major antioxidant enzymes. PMID:20157581

  7. Transport of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2).

    PubMed

    Tsirulnikov, Kirill; Abuladze, Natalia; Koag, Myong-Chul; Newman, Debra; Scholz, Karoline; Bondar, Galyna; Zhu, Quansheng; Avliyakulov, Nuraly K; Dekant, Wolfgang; Faull, Kym; Kurtz, Ira; Pushkin, Alexander

    2010-04-15

    N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine (Ac-DCVC) and S-(1,2-dichlorovinyl)-l-cysteine (DCVC) are the glutathione conjugation pathway metabolites of a common industrial contaminant and potent nephrotoxicant trichloroethylene (TCE). Ac-DCVC and DCVC are accumulated in the renal proximal tubule where they may be secreted into the urine by an unknown apical transporter(s). In this study, we explored the hypothesis that the apical transport of Ac-DCVC and/or DCVC may be mediated by the multidrug resistance associated protein 2 (Mrp2, ABCC2), which is known to mediate proximal tubular apical ATP-dependent transport of glutathione and numerous xenobiotics and endogenous substances conjugated with glutathione. Transport experiments using membrane vesicles prepared from mouse proximal tubule derived cells expressing mouse Mrp2 utilizing ATPase assay and direct measurements of Ac-DCVC/DCVC using liquid chromatography/tandem mass-spectrometry (LC/MS/MS) demonstrated that mouse Mrp2 mediates ATP-dependent transport of Ac-DCVC. Expression of mouse Mrp2 antisense mRNA significantly inhibited the vectorial basolateral to apical transport of Ac-DCVC but not DCVC in mouse proximal tubule derived cells endogenously expressing mouse Mrp2. The results suggest that Mrp2 may be involved in the renal secretion of Ac-DCVC.

  8. Transport of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2)

    PubMed Central

    Tsirulnikov, Kirill; Abuladze, Natalia; Koag, Myong-Chul; Newman, Debra; Scholz, Karoline; Bondar, Galyna; Zhu, Quansheng; Avliyakulov, Nuraly K.; Dekant, Wolfgang; Faull, Kym; Kurtz, Ira; Pushkin, Alexander

    2010-01-01

    N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (Ac-DCVC) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) are the glutathione conjugation pathway metabolites of a common industrial contaminant and potent nephrotoxicant trichloroethylene (TCE). Ac-DCVC and DCVC are accumulated in the renal proximal tubule where they may be secreted into the urine by an unknown apical transporter(s). In this study we explored the hypothesis that the apical transport of Ac-DCVC and/or DCVC may be mediated by the multidrug resistance associated protein 2 (Mrp2, ABCC2), which is known to mediate proximal tubular apical ATP-dependent transport of glutathione and numerous xenobiotics and endogenous substances conjugated with glutathione. Transport experiments using membrane vesicles prepared from mouse proximal tubule derived cells expressing mouse Mrp2 utilizing ATPase assay and direct measurements of Ac-DCVC/DCVC using liquid chromatography/tandem mass-spectrometry (LC/MS/MS) demonstrated that mouse Mrp2 mediates ATP-dependent transport of Ac-DCVC. Expression of mouse Mrp2 antisense mRNA significantly inhibited the vectorial basolateral to apical transport of Ac-DCVC but not DCVC in mouse proximal tubule derived cells endogenously expressing mouse Mrp2. The results suggest that Mrp2 may be involved in the renal secretion of Ac-DCVC. PMID:20060011

  9. N-acetylated Proline–Glycine–Proline induced G-protein dependent chemotaxis of neutrophils is independent of CXCL8 release

    PubMed Central

    Overbeek, Saskia A.; Henricks, Paul A.J.; Srienc, Anja I.; Koelink, Pim J.; de Kruijf, Petra; Lim, Herman D.; Smit, Martine J.; Zaman, Guido J.R.; Garssen, Johan; Nijkamp, Frans P.; Kraneveld, Aletta D.; Folkerts, Gert

    2013-01-01

    Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline–Glycine–Proline (N-ac-PGP). In this study, we investigated in more detail the mechanism of action of N-ac-PGP in neutrophilic inflammation. N-ac-PGP was chemotactic for human neutrophils via pertussis toxin sensitive G protein-coupled receptors in vitro and directly activated this cell type, which led to cytosolic calcium mobilization and release of CXCL8. Furthermore, using a selective CXCR2 antagonist confirmed that N-ac-PGP-induced neutrophil chemotaxis is mediated through CXCR2 activation. To determine whether N-ac-PGP was solely responsible for the migration and activation of human neutrophils in vitro and not the released CXCL8 upon stimulation with N-ac-PGP, an antibody directed against CXCL8 was used. Performing chemotaxis and calcium influx assays in the presence of this antibody did not alter the effects of N-ac-PGP whereas effects of CXCL8 were attenuated. These experiments indicate that N-ac-PGP, in addition to the direct induction of chemotaxis, also directly activates neutrophils to release CXCL8. In vivo, this may lead in the long term to a self-maintaining situation enhanced by both N-ac-PGP and CXCL8, leading to a further increase in neutrophil infiltration and chronic inflammation. PMID:21458443

  10. N-acetylated Proline-Glycine-Proline induced G-protein dependent chemotaxis of neutrophils is independent of CXCL8 release.

    PubMed

    Overbeek, Saskia A; Henricks, Paul A J; Srienc, Anja I; Koelink, Pim J; de Kruijf, Petra; Lim, Herman D; Smit, Martine J; Zaman, Guido J R; Garssen, Johan; Nijkamp, Frans P; Kraneveld, Aletta D; Folkerts, Gert

    2011-10-15

    Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline-Glycine-Proline (N-ac-PGP). In this study, we investigated in more detail the mechanism of action of N-ac-PGP in neutrophilic inflammation. N-ac-PGP was chemotactic for human neutrophils via pertussis toxin sensitive G protein-coupled receptors in vitro and directly activated this cell type, which led to cytosolic calcium mobilization and release of CXCL8. Furthermore, using a selective CXCR2 antagonist confirmed that N-ac-PGP-induced neutrophil chemotaxis is mediated through CXCR2 activation. To determine whether N-ac-PGP was solely responsible for the migration and activation of human neutrophils in vitro and not the released CXCL8 upon stimulation with N-ac-PGP, an antibody directed against CXCL8 was used. Performing chemotaxis and calcium influx assays in the presence of this antibody did not alter the effects of N-ac-PGP whereas effects of CXCL8 were attenuated. These experiments indicate that N-ac-PGP, in addition to the direct induction of chemotaxis, also directly activates neutrophils to release CXCL8. In vivo, this may lead in the long term to a self-maintaining situation enhanced by both N-ac-PGP and CXCL8, leading to a further increase in neutrophil infiltration and chronic inflammation. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Penicillium purpurogenum produces a family 1 acetyl xylan esterase containing a carbohydrate-binding module: characterization of the protein and its gene.

    PubMed

    Gordillo, Felipe; Caputo, Valentina; Peirano, Alessandra; Chavez, Renato; Van Beeumen, Jozef; Vandenberghe, Isabel; Claeyssens, Marc; Bull, Paulina; Ravanal, María Cristina; Eyzaguirre, Jaime

    2006-10-01

    At least three acetyl xylan esterases (AXE I, II and III) are secreted by Penicillium purpurogenum. This publication describes more detailed work on AXE I and its gene. AXE I binds cellulose but not xylan; it is glycosylated and inactivated by phenylmethylsulphonyl fluoride, showing that it is a serine esterase. The axe1 gene presents an open reading frame of 1278 bp, including two introns of 68 and 61 bp; it codes for a signal peptide of 31 residues and a mature protein of 351 amino acids (molecular weight 36,693). AXE I has a modular structure: a catalytic module at the amino terminus belonging to family 1 of the carbohydrate esterases, a linker rich in serines and threonines, and a family 1 carboxy terminal carbohydrate binding module (CBM). The CBM is similar to that of AXE from Trichoderma reesei, (with a family 5 catalytic module) indicating that the genes for catalytic modules and CBMs have evolved separately, and that they have been linked by gene fusion. The promoter sequence of axe1 contains several putative sequences for binding of gene expression regulators also found in other family 1 esterase gene promoters. It is proposed that AXE I and II act in succession in xylan degradation; first, xylan is attacked by AXE I and other xylanases possessing CBMs (which facilitate binding to lignocellulose), followed by other enzymes acting mainly on soluble substrates.

  12. Transport of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2)

    SciTech Connect

    Tsirulnikov, Kirill; Abuladze, Natalia; Koag, Myong-Chul; Newman, Debra; Bondar, Galyna; Zhu Quansheng; Dekant, Wolfgang; Faull, Kym; Kurtz, Ira

    2010-04-15

    N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (Ac-DCVC) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) are the glutathione conjugation pathway metabolites of a common industrial contaminant and potent nephrotoxicant trichloroethylene (TCE). Ac-DCVC and DCVC are accumulated in the renal proximal tubule where they may be secreted into the urine by an unknown apical transporter(s). In this study, we explored the hypothesis that the apical transport of Ac-DCVC and/or DCVC may be mediated by the multidrug resistance associated protein 2 (Mrp2, ABCC2), which is known to mediate proximal tubular apical ATP-dependent transport of glutathione and numerous xenobiotics and endogenous substances conjugated with glutathione. Transport experiments using membrane vesicles prepared from mouse proximal tubule derived cells expressing mouse Mrp2 utilizing ATPase assay and direct measurements of Ac-DCVC/DCVC using liquid chromatography/tandem mass-spectrometry (LC/MS/MS) demonstrated that mouse Mrp2 mediates ATP-dependent transport of Ac-DCVC. Expression of mouse Mrp2 antisense mRNA significantly inhibited the vectorial basolateral to apical transport of Ac-DCVC but not DCVC in mouse proximal tubule derived cells endogenously expressing mouse Mrp2. The results suggest that Mrp2 may be involved in the renal secretion of Ac-DCVC.

  13. Antioxidant N-acetyl-L-cysteine ameliorates symptoms of premature aging associated with the deficiency of the circadian protein BMAL1.

    PubMed

    Kondratov, Roman V; Vykhovanets, Olena; Kondratova, Anna A; Antoch, Marina P

    2009-12-30

    Deficiency of the circadian clock protein BMAL1 leads to premature aging and increased levels of reactivate oxygen species in several tissues of mice. In order to investigate the role of oxidative stress in accelerated aging and development of age-related pathologies, we continuously administered the antioxidant N-acetyl-L-cysteine toBmal1-deficient mice through their entire lifespan by supplementing drinking water. We found that the life long treatment with antioxidant significantly increased average and maximal lifespan and reduced the rate of age-dependent weight loss and development of cataracts. At the same time, it had no effect on time of onset and severity of other age-related pathologies characteristic of Bmal1-/- mice, such as joint ossification, reduced hair regrowth and sarcopenia. We conclude that chronic oxidative stress affects longevity and contributes to the development of at least some age-associated pathology, although ROS-independent mechanisms may also play a role. Our bioinformatics analysis identified the presence of a conservative E box element in the promoter regions of several genes encoding major antioxidant enzymes. We speculate that BMAL1 controls antioxidant defense by regulating the expression of major antioxidant enzymes.

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

    PubMed

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

    2001-08-10

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

  15. Alteration of cell wall xylan acetylation triggers defense responses that counterbalance the immune deficiencies of plants impaired in the β-subunit of the heterotrimeric G-protein.

    PubMed

    Escudero, Viviana; Jordá, Lucía; Sopeña-Torres, Sara; Mélida, Hugo; Miedes, Eva; Muñoz-Barrios, Antonio; Swami, Sanjay; Alexander, Danny; McKee, Lauren S; Sánchez-Vallet, Andrea; Bulone, Vincent; Jones, Alan M; Molina, Antonio

    2017-08-09

    Arabidopsis heterotrimeric G-protein complex modulates pathogen-associated molecular pattern-triggered immunity (PTI) and disease resistance responses to different types of pathogens. It also plays a role in plant cell wall integrity as mutants impaired in the Gβ- (agb1-2) or Gγ-subunits have an altered wall composition compared with wild-type plants. Here we performed a mutant screen to identify suppressors of agb1-2 (sgb) that restore susceptibility to pathogens to wild-type levels. Out of the four sgb mutants (sgb10-sgb13) identified, sgb11 is a new mutant allele of ESKIMO1 (ESK1), which encodes a plant-specific polysaccharide O-acetyltransferase involved in xylan acetylation. Null alleles (sgb11/esk1-7) of ESK1 restore to wild-type levels the enhanced susceptibility of agb1-2 to the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM), but not to the bacterium Pseudomonas syringae pv. tomato DC3000 or to the oomycete Hyaloperonospora arabidopsidis. The enhanced resistance to PcBMM of the agb1-2 esk1-7 double mutant was not the result of the re-activation of deficient PTI responses in agb1-2. Alteration of cell wall xylan acetylation caused by ESK1 impairment was accompanied by an enhanced accumulation of abscisic acid, the constitutive expression of genes encoding antibiotic peptides and enzymes involved in the biosynthesis of tryptophan-derived metabolites, and the accumulation of disease resistance-related secondary metabolites and different osmolites. These esk1-mediated responses counterbalance the defective PTI and PcBMM susceptibility of agb1-2 plants, and explain the enhanced drought resistance of esk1 plants. These results suggest that a deficient PTI-mediated resistance is partially compensated by the activation of specific cell-wall-triggered immune responses. © 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

  16. Protein Surface Matching by Combining Local and Global Geometric Information

    PubMed Central

    Ellingson, Leif; Zhang, Jinfeng

    2012-01-01

    Comparison of the binding sites of proteins is an effective means for predicting protein functions based on their structure information. Despite the importance of this problem and much research in the past, it is still very challenging to predict the binding ligands from the atomic structures of protein binding sites. Here, we designed a new algorithm, TIPSA (Triangulation-based Iterative-closest-point for Protein Surface Alignment), based on the iterative closest point (ICP) algorithm. TIPSA aims to find the maximum number of atoms that can be superposed between two protein binding sites, where any pair of superposed atoms has a distance smaller than a given threshold. The search starts from similar tetrahedra between two binding sites obtained from 3D Delaunay triangulation and uses the Hungarian algorithm to find additional matched atoms. We found that, due to the plasticity of protein binding sites, matching the rigid body of point clouds of protein binding sites is not adequate for satisfactory binding ligand prediction. We further incorporated global geometric information, the radius of gyration of binding site atoms, and used nearest neighbor classification for binding site prediction. Tested on benchmark data, our method achieved a performance comparable to the best methods in the literature, while simultaneously providing the common atom set and atom correspondences. PMID:22815760

  17. Acetylation and characterization of banana (Musa paradisiaca) starch.

    PubMed

    Bello-Pérez, L A; Contreras-Ramos, S M; Jìmenez-Aparicio, A; Paredes-López, O

    2000-01-01

    Banana native starch was acetylated and some of its functional properties were evaluated and compared to corn starch. In general, acetylated banana starch presented higher values in ash, protein and fat than corn acetylated starch. The modified starches had minor tendency to retrogradation assessed as % transmittance of starch pastes. At high temperature acetylated starches presented a water retention capacity similar to their native counterpart. The acetylation considerably increased the solubility of starches, and a similar behavior was found for swelling power. When freeze-thaw stability was studied, acetyl banana starch drained approximately 60% of water in the first and second cycles, but in the third and fourth cycles the percentage of separated water was low. However, acetyl corn starch showed lower freeze-thaw stability than the untreated sample. The modification increased the viscosity of banana starch pastes.

  18. The convergent chemical synthesis of histone H3 protein for site-specific acetylation at Lys56 and ubiquitination at Lys122.

    PubMed

    Qi, Yun-Kun; He, Qiao-Qiao; Ai, Hua-Song; Guo, Jing; Li, Jia-Bin

    2017-03-29

    Deposition of (H3-H4)2 tetramers is believed to be the critical step in nucleosome assembly. Site-specific acetylation and ubiquitination of histone H3 have been speculated to synergistically facilitate the formation and deposition of (H3-H4)2 tetramers. Here we report our endeavors toward the first chemical synthesis of homogenous histone H3, which bears Lys56 acetylation and Lys122 ubiquitination, for in vitro biochemical and biophysical studies.

  19. Optimizing a global alignment of protein interaction networks

    PubMed Central

    Chindelevitch, Leonid; Ma, Cheng-Yu; Liao, Chung-Shou; Berger, Bonnie

    2013-01-01

    Motivation: The global alignment of protein interaction networks is a widely studied problem. It is an important first step in understanding the relationship between the proteins in different species and identifying functional orthologs. Furthermore, it can provide useful insights into the species’ evolution. Results: We propose a novel algorithm, PISwap, for optimizing global pairwise alignments of protein interaction networks, based on a local optimization heuristic that has previously demonstrated its effectiveness for a variety of other intractable problems. PISwap can begin with different types of network alignment approaches and then iteratively adjust the initial alignments by incorporating network topology information, trading it off for sequence information. In practice, our algorithm efficiently refines other well-studied alignment techniques with almost no additional time cost. We also show the robustness of the algorithm to noise in protein interaction data. In addition, the flexible nature of this algorithm makes it suitable for different applications of network alignment. This algorithm can yield interesting insights into the evolutionary dynamics of related species. Availability: Our software is freely available for non-commercial purposes from our Web site, http://piswap.csail.mit.edu/. Contact: bab@csail.mit.edu or csliao@ie.nthu.edu.tw Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24048352

  20. Poly-acetylated chromatin signatures are preferred epitopes for site-specific histone H4 acetyl antibodies.

    PubMed

    Rothbart, Scott B; Lin, Shu; Britton, Laura-Mae; Krajewski, Krzysztof; Keogh, Michael-C; Garcia, Benjamin A; Strahl, Brian D

    2012-01-01

    Antibodies specific for histone post-translational modifications (PTMs) have been central to our understanding of chromatin biology. Here, we describe an unexpected and novel property of histone H4 site-specific acetyl antibodies in that they prefer poly-acetylated histone substrates. By all current criteria, these antibodies have passed specificity standards. However, we find these site-specific histone antibodies preferentially recognize chromatin signatures containing two or more adjacent acetylated lysines. Significantly, we find that the poly-acetylated epitopes these antibodies prefer are evolutionarily conserved and are present at levels that compete for these antibodies over the intended individual acetylation sites. This alarming property of acetyl-specific antibodies has far-reaching implications for data interpretation and may present a challenge for the future study of acetylated histone and non-histone proteins.

  1. Atom-by-atom analysis of global downhill protein folding

    NASA Astrophysics Data System (ADS)

    Sadqi, Mourad; Fushman, David; Muñoz, Victor

    2006-07-01

    Protein folding is an inherently complex process involving coordination of the intricate networks of weak interactions that stabilize native three-dimensional structures. In the conventional paradigm, simple protein structures are assumed to fold in an all-or-none process that is inaccessible to experiment. Existing experimental methods therefore probe folding mechanisms indirectly. A widely used approach interprets changes in protein stability and/or folding kinetics, induced by engineered mutations, in terms of the structure of the native protein. In addition to limitations in connecting energetics with structure, mutational methods have significant experimental uncertainties and are unable to map complex networks of interactions. In contrast, analytical theory predicts small barriers to folding and the possibility of downhill folding. These theoretical predictions have been confirmed experimentally in recent years, including the observation of global downhill folding. However, a key remaining question is whether downhill folding can indeed lead to the high-resolution analysis of protein folding processes. Here we show, with the use of nuclear magnetic resonance (NMR), that the downhill protein BBL from Escherichia coli unfolds atom by atom starting from a defined three-dimensional structure. Thermal unfolding data on 158 backbone and side-chain protons out of a total of 204 provide a detailed view of the structural events during folding. This view confirms the statistical nature of folding, and exposes the interplay between hydrogen bonding, hydrophobic forces, backbone conformation and side-chain entropy. From the data we also obtain a map of the interaction network in this protein, which reveals the source of folding cooperativity. Our approach can be extended to other proteins with marginal barriers (less than 3RT), providing a new tool for the study of protein folding.

  2. Analytical strategies for the global quantification of intact proteins.

    PubMed

    Collier, Timothy S; Muddiman, David Charles

    2012-09-01

    The quantification of intact proteins is a relatively recent development in proteomics. In eukaryotic organisms, proteins are present as multiple isoforms as the result of variations in genetic code, alternative splicing, post-translational modification and other processing events. Understanding the identities and biological functions of these isoforms and how their concentrations vary across different states is the central goal of proteomics. To date, the bulk of proteomics research utilizes a "bottom-up" approach, digesting proteins into their more manageable constitutive peptides, but sacrificing information about the specific isoform and combinations of post-translational modifications present on the protein. Very specific strategies for protein quantification such as the enzyme-linked immunosorbent assay and Western blot are commonplace in laboratories and clinics, but impractical for the study of global biological changes. Herein, we describe strategies for the quantification of intact proteins, their distinct advantages, and challenges to their employment. Techniques contained in this review include the more traditional and widely employed methodology of differential gel electrophoresis and more recently developed mass spectrometry-based techniques including metabolic labeling, chemical labeling, and label-free methodologies.

  3. Global, quantitative and dynamic mapping of protein subcellular localization

    PubMed Central

    Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg HH

    2016-01-01

    Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology. DOI: http://dx.doi.org/10.7554/eLife.16950.001 PMID:27278775

  4. THE EXCHANGE REACTION OF ACETYL FLUORIDE AND ACETYL HEXAFLUOROARSENATE,

    DTIC Science & Technology

    From the temperature dependence of the exchange rate of the methyl protons between acetyl fluoride and acetyl hexafluoroarsenate an Arrhenius...the reaction was found to be one-half order in acetyl hexafluoroarsenate and zero order in acetyl fluoride. (Author)

  5. Platelet activating factor-induced expression of p21 is correlated with histone acetylation

    PubMed Central

    Damiani, Elisabetta; Puebla-Osorio, Nahum; Lege, Bree M.; Liu, Jingwei; Neelapu, Sattva S.; Ullrich, Stephen E.

    2017-01-01

    Ultraviolet (UV)-irradiated keratinocytes secrete the lipid mediator of inflammation, platelet-activating factor (PAF). PAF plays an essential role in UV-induced immune suppression and skin cancer induction. Dermal mast cell migration from the skin to the draining lymph nodes plays a prominent role in activating systemic immune suppression. UV-induced PAF activates mast cell migration by up-regulating mast cell CXCR4 surface expression. Recent findings indicate that PAF up-regulates CXCR4 expression via histone acetylation. UV-induced PAF also activates cell cycle arrest and disrupts DNA repair, in part by increasing p21 expression. Do epigenetic alterations play a role in p21 up-regulation? Here we show that PAF increases Acetyl-CREB-binding protein (CBP/p300) histone acetyltransferase expression in a time and dose-dependent fashion. Partial deletion of the HAT domain in the CBP gene, blocked these effects. Chromatin immunoprecipitation assays indicated that PAF-treatment activated the acetylation of the p21 promoter. PAF-treatment had no effect on other acetylating enzymes (GCN5L2, PCAF) indicating it is not a global activator of histone acetylation. This study provides further evidence that PAF activates epigenetic mechanisms to affect important cellular processes, and we suggest this bioactive lipid can serve as a link between the environment and the epigenome. PMID:28157211

  6. Structural Basis for p53 Lys120-Acetylation-Dependent DNA-Binding Mode.

    PubMed

    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.

  7. Platelet activating factor-induced expression of p21 is correlated with histone acetylation.

    PubMed

    Damiani, Elisabetta; Puebla-Osorio, Nahum; Lege, Bree M; Liu, Jingwei; Neelapu, Sattva S; Ullrich, Stephen E

    2017-02-03

    Ultraviolet (UV)-irradiated keratinocytes secrete the lipid mediator of inflammation, platelet-activating factor (PAF). PAF plays an essential role in UV-induced immune suppression and skin cancer induction. Dermal mast cell migration from the skin to the draining lymph nodes plays a prominent role in activating systemic immune suppression. UV-induced PAF activates mast cell migration by up-regulating mast cell CXCR4 surface expression. Recent findings indicate that PAF up-regulates CXCR4 expression via histone acetylation. UV-induced PAF also activates cell cycle arrest and disrupts DNA repair, in part by increasing p21 expression. Do epigenetic alterations play a role in p21 up-regulation? Here we show that PAF increases Acetyl-CREB-binding protein (CBP/p300) histone acetyltransferase expression in a time and dose-dependent fashion. Partial deletion of the HAT domain in the CBP gene, blocked these effects. Chromatin immunoprecipitation assays indicated that PAF-treatment activated the acetylation of the p21 promoter. PAF-treatment had no effect on other acetylating enzymes (GCN5L2, PCAF) indicating it is not a global activator of histone acetylation. This study provides further evidence that PAF activates epigenetic mechanisms to affect important cellular processes, and we suggest this bioactive lipid can serve as a link between the environment and the epigenome.

  8. Structural analysis of a type 1 ribosome inactivating protein reveals multiple L-asparagine-N-acetyl-D-glucosamine monosaccharide modifications: Implications for cytotoxicity

    PubMed Central

    HOGG, TANIS; MENDEL, JAMESON T.; LAVEZO, JONATHAN L.

    2015-01-01

    Pokeweed antiviral protein (PAP) belongs to the family of type I ribosome-inactivating proteins (RIPs): Ribotoxins, which function by depurinating the sarcin-ricin loop of ribosomal RNA. In addition to its antibacterial and antifungal properties, PAP has shown promise in antiviral and targeted tumor therapy owing to its ability to depurinate viral RNA and eukaryotic rRNA. Several PAP genes are differentially expressed across pokeweed tissues, with natively isolated seed forms of PAP exhibiting the greatest cytotoxicity. To help elucidate the molecular basis of increased cytotoxicity of PAP isoenzymes from seeds, the present study used protein sequencing, mass spectroscopy and X-ray crystallography to determine the complete covalent structure and 1.7 Å X-ray crystal structure of PAP-S1aci isolated from seeds of Asian pokeweed (Phytolacca acinosa). PAP-S1aci shares ~95% sequence identity with PAP-S1 from P. americana and contains the signature catalytic residues of the RIP superfamily, corresponding to Tyr72, Tyr122, Glu175 and Arg178 in PAP-S1aci. A rare proline substitution (Pro174) was identified in the active site of PAP-S1aci, which has no effect on catalytic Glu175 positioning or overall active-site topology, yet appears to come at the expense of strained main-chain geometry at the pre-proline residue Val173. Notably, a rare type of N-glycosylation was detected consisting of N-acetyl-D-glucosamine monosaccharide residues linked to Asn10, Asn44 and Asn255 of PAP-S1aci. Of note, our modeling studies suggested that the ribosome depurination activity of seed PAPs would be adversely affected by the N-glycosylation of Asn44 and Asn255 with larger and more typical oligosaccharide chains, as they would shield the rRNA-binding sites on the protein. These results, coupled with evidence gathered from the literature, suggest that this type of minimal N-glycosylation in seed PAPs and other type I seed RIPs may serve to enhance cytotoxicity by exploiting receptor

  9. Protein Data Bank (PDB): The Single Global Macromolecular Structure Archive.

    PubMed

    Burley, Stephen K; Berman, Helen M; Kleywegt, Gerard J; Markley, John L; Nakamura, Haruki; Velankar, Sameer

    2017-01-01

    The Protein Data Bank (PDB)--the single global repository of experimentally determined 3D structures of biological macromolecules and their complexes--was established in 1971, becoming the first open-access digital resource in the biological sciences. The PDB archive currently houses ~130,000 entries (May 2017). It is managed by the Worldwide Protein Data Bank organization (wwPDB; wwpdb.org), which includes the RCSB Protein Data Bank (RCSB PDB; rcsb.org), the Protein Data Bank Japan (PDBj; pdbj.org), the Protein Data Bank in Europe (PDBe; pdbe.org), and BioMagResBank (BMRB; www.bmrb.wisc.edu). The four wwPDB partners operate a unified global software system that enforces community-agreed data standards and supports data Deposition, Biocuration, and Validation of ~11,000 new PDB entries annually (deposit.wwpdb.org). The RCSB PDB currently acts as the archive keeper, ensuring disaster recovery of PDB data and coordinating weekly updates. wwPDB partners disseminate the same archival data from multiple FTP sites, while operating complementary websites that provide their own views of PDB data with selected value-added information and links to related data resources. At present, the PDB archives experimental data, associated metadata, and 3D-atomic level structural models derived from three well-established methods: crystallography, nuclear magnetic resonance spectroscopy (NMR), and electron microscopy (3DEM). wwPDB partners are working closely with experts in related experimental areas (small-angle scattering, chemical cross-linking/mass spectrometry, Forster energy resonance transfer or FRET, etc.) to establish a federation of data resources that will support sustainable archiving and validation of 3D structural models and experimental data derived from integrative or hybrid methods.

  10. Global Analysis of Protein Expression of Inner Ear Hair Cells

    PubMed Central

    Wong, Ann C.Y.; Pak, Kwang; Strojny, Chelsee; Ramirez, Miguel

    2017-01-01

    The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes. SIGNIFICANCE STATEMENT Hearing and balance rely on specialized sensory hair cells (HCs) in the inner ear (IE) to convey information about sound, acceleration, and orientation to the brain. Genetically and environmentally induced perturbations to HC proteins can result in deafness and severe imbalance. We used transgenic mice with GFP-expressing HCs, coupled with FACS sorting and tandem mass spectrometry, to define the most complete HC and IE proteome to date. We show that

  11. The protein BpsB is a poly-β-1,6-N-acetyl-D-glucosamine deacetylase required for biofilm formation in Bordetella bronchiseptica.

    PubMed

    Little, Dustin J; Milek, Sonja; Bamford, Natalie C; Ganguly, Tridib; DiFrancesco, Benjamin R; Nitz, Mark; Deora, Rajendar; Howell, P Lynne

    2015-09-11

    Bordetella pertussis and Bordetella bronchiseptica are the causative agents of whooping cough in humans and a variety of respiratory diseases in animals, respectively. Bordetella species produce an exopolysaccharide, known as the Bordetella polysaccharide (Bps), which is encoded by the bpsABCD operon. Bps is required for Bordetella biofilm formation, colonization of the respiratory tract, and confers protection from complement-mediated killing. In this report, we have investigated the role of BpsB in the biosynthesis of Bps and biofilm formation by B. bronchiseptica. BpsB is a two-domain protein that localizes to the periplasm and outer membrane. BpsB displays metal- and length-dependent deacetylation on poly-β-1,6-N-acetyl-d-glucosamine (PNAG) oligomers, supporting previous immunogenic data that suggests Bps is a PNAG polymer. BpsB can use a variety of divalent metal cations for deacetylase activity and showed highest activity in the presence of Ni(2+) and Co(2+). The structure of the BpsB deacetylase domain is similar to the PNAG deacetylases PgaB and IcaB and contains the same circularly permuted family four carbohydrate esterase motifs. Unlike PgaB from Escherichia coli, BpsB is not required for polymer export and has unique structural differences that allow the N-terminal deacetylase domain to be active when purified in isolation from the C-terminal domain. Our enzymatic characterizations highlight the importance of conserved active site residues in PNAG deacetylation and demonstrate that the C-terminal domain is required for maximal deacetylation of longer PNAG oligomers. Furthermore, we show that BpsB is critical for the formation and complex architecture of B. bronchiseptica biofilms.

  12. The Protein BpsB Is a Poly-β-1,6-N-acetyl-d-glucosamine Deacetylase Required for Biofilm Formation in Bordetella bronchiseptica*

    PubMed Central

    Little, Dustin J.; Milek, Sonja; Bamford, Natalie C.; Ganguly, Tridib; DiFrancesco, Benjamin R.; Nitz, Mark; Deora, Rajendar; Howell, P. Lynne

    2015-01-01

    Bordetella pertussis and Bordetella bronchiseptica are the causative agents of whooping cough in humans and a variety of respiratory diseases in animals, respectively. Bordetella species produce an exopolysaccharide, known as the Bordetella polysaccharide (Bps), which is encoded by the bpsABCD operon. Bps is required for Bordetella biofilm formation, colonization of the respiratory tract, and confers protection from complement-mediated killing. In this report, we have investigated the role of BpsB in the biosynthesis of Bps and biofilm formation by B. bronchiseptica. BpsB is a two-domain protein that localizes to the periplasm and outer membrane. BpsB displays metal- and length-dependent deacetylation on poly-β-1,6-N-acetyl-d-glucosamine (PNAG) oligomers, supporting previous immunogenic data that suggests Bps is a PNAG polymer. BpsB can use a variety of divalent metal cations for deacetylase activity and showed highest activity in the presence of Ni2+ and Co2+. The structure of the BpsB deacetylase domain is similar to the PNAG deacetylases PgaB and IcaB and contains the same circularly permuted family four carbohydrate esterase motifs. Unlike PgaB from Escherichia coli, BpsB is not required for polymer export and has unique structural differences that allow the N-terminal deacetylase domain to be active when purified in isolation from the C-terminal domain. Our enzymatic characterizations highlight the importance of conserved active site residues in PNAG deacetylation and demonstrate that the C-terminal domain is required for maximal deacetylation of longer PNAG oligomers. Furthermore, we show that BpsB is critical for the formation and complex architecture of B. bronchiseptica biofilms. PMID:26203190

  13. EGF-Induced Acetylation of Heterogeneous Nuclear Ribonucleoproteins Is Dependent on KRAS Mutational Status in Colorectal Cancer Cells.

    PubMed

    Roda, Desamparados; Castillo, Josefa; Telechea-Fernández, Marcelino; Gil, Anabel; López-Rodas, Gerardo; Franco, Luís; González-Rodríguez, Patricia; Roselló, Susana; Pérez-Fidalgo, J Alejandro; García-Trevijano, Elena R; Cervantes, Andrés; Zaragozá, Rosa

    2015-01-01

    KRAS mutational status is considered a negative predictive marker of the response to anti-EGFR therapies in colorectal cancer (CRC) patients. However, conflicting data exist regarding the variable response to EGFR-targeted therapy. The effects of oncogenic KRAS on downstream targets were studied in cell lines with different KRAS mutations. Cells harboring a single KRASG13D allele showed the most tumorigenic profile, with constitutive activation of the downstream pathway, rendering them EGF-unresponsive. Conversely, KRASA146T cells showed a full EGF-response in terms of signal transduction pathways, cell proliferation, migration or adhesion. Moreover, the global acetylome of CRC cells was also dependent on KRAS mutational status. Several hnRNP family members were identified within the 36 acetylated-proteins. Acetylation status is known to be involved in the modulation of EGF-response. In agreement with results presented herein, hnRNPA1 and L acetylation was induced in response to EGF in KRASA146T cells, whereas acetyl-hnRNPA1 and L levels remained unchanged after growth factor treatment in KRASG13D unresponsive cells. Our results showed that hnRNPs induced-acetylation is dependent on KRAS mutational status. Nevertheless hnRNPs acetylation might also be the point where different oncogenic pathways converge.

  14. EGF-Induced Acetylation of Heterogeneous Nuclear Ribonucleoproteins Is Dependent on KRAS Mutational Status in Colorectal Cancer Cells

    PubMed Central

    Telechea-Fernández, Marcelino; Gil, Anabel; López-Rodas, Gerardo; Franco, Luís; González-Rodríguez, Patricia; Roselló, Susana; Pérez-Fidalgo, J. Alejandro; García-Trevijano, Elena R.; Cervantes, Andrés; Zaragozá, Rosa

    2015-01-01

    KRAS mutational status is considered a negative predictive marker of the response to anti-EGFR therapies in colorectal cancer (CRC) patients. However, conflicting data exist regarding the variable response to EGFR-targeted therapy. The effects of oncogenic KRAS on downstream targets were studied in cell lines with different KRAS mutations. Cells harboring a single KRASG13D allele showed the most tumorigenic profile, with constitutive activation of the downstream pathway, rendering them EGF-unresponsive. Conversely, KRASA146T cells showed a full EGF-response in terms of signal transduction pathways, cell proliferation, migration or adhesion. Moreover, the global acetylome of CRC cells was also dependent on KRAS mutational status. Several hnRNP family members were identified within the 36 acetylated-proteins. Acetylation status is known to be involved in the modulation of EGF-response. In agreement with results presented herein, hnRNPA1 and L acetylation was induced in response to EGF in KRASA146T cells, whereas acetyl-hnRNPA1 and L levels remained unchanged after growth factor treatment in KRASG13D unresponsive cells. Our results showed that hnRNPs induced-acetylation is dependent on KRAS mutational status. Nevertheless hnRNPs acetylation might also be the point where different oncogenic pathways converge. PMID:26110767

  15. Acetylation of interferon regulatory factor-7 by p300/CREB-binding protein (CBP)-associated factor (PCAF) impairs its DNA binding.

    PubMed

    Caillaud, Alexandre; Prakash, Arun; Smith, Eric; Masumi, Atsuko; Hovanessian, Ara G; Levy, David E; Marié, Isabelle

    2002-12-20

    Interferon regulatory factor 7 (IRF7) is an interferon-inducible transcription factor required for induction of delayed early interferon alpha genes and the onset of a potent antiviral state. After induction of IRF7 by autocrine interferon, latent IRF7 is activated by virus-induced phosphorylation on serine residues within the C-terminal regulatory domain. Although it is likely that IRF7 is subjected to a cascade of events responsible for regulating its biological activity, to date no mechanism other than phosphorylation has been reported to modulate IRF7 activity. Here, we report that IRF7 is acetylated in vivo by the histone acetyltransferases p300/CBP-associated factor (PCAF) and GCN5. The single lysine residue target for acetylation, lysine 92, is located in the DNA-binding domain and is conserved throughout the entire IRF family. Mutation of lysine 92 resulted in complete abolition of DNA binding ability. However, a mutant that cannot be acetylated by PCAF due to a change in the surrounding amino acid context of lysine 92 showed increased DNA binding and activity compared with wild type IRF7. Conversely, we showed that acetylated IRF7 displayed impaired DNA binding capability and that over-expression of PCAF led to decreased IRF7 activity. Together, our results strongly suggest that acetylation of lysine 92 negatively modulates IRF7 DNA binding.

  16. A two-process model describes the hydrogen exchange behavior of cytochrome c in the molten globule state with various extents of acetylation.

    PubMed

    Szewczuk, Z; Konishi, Y; Goto, Y

    2001-08-14

    Acetylation of Lys residues of horse cytochrome c steadily stabilizes the molten globule state in 18 mM HCl as more Lys residues are acetylated [Goto and Nishikiori (1991) J. Mol. Biol. 222, 679-686]. The dynamic features of the molten globule state were characterized by hydrogen/deuterium exchange of amide protons, monitored by mass spectrometry as each deuteration increased the protein mass by 1 Da. Electrospray mass spectrometry enabled us to monitor simultaneously the exchange kinetics of more than seven species with a different number of acetyl groups. One to four Lys residue-acetylated cytochrome c showed almost no protection of the amide protons from rapid exchange. The transition from the unprotected to the protected state occurred between five and eight Lys residue-acetylated species. For species with more than nine acetylated Lys residues, the exchange kinetics were independent of the extent of acetylation, and 26 amide protons were protected at 60 min of exchange, indicating the formation of a rigid hydrophobic core with hydrogen-bonded secondary structures. The apparent transition to the protected state required a higher degree of acetylation than the conformational transition measured by circular dichroism, which had a midpoint at about four acetylated residues. This difference in the transitions suggested a two-process model in which the exchange occurs either from the protected folded state or from the unprotected unfolded state through global unfolding. On the basis of a two-process model and with the reported values of the exchange and stability parameters, we simulated the exchange kinetics of a series of acetylated cytochrome c species. The simulated kinetics reproduced the observed kinetics well, indicating validity of this model for hydrogen exchange of the molten globule state.

  17. Total Survivin and acetylated Survivin correlate with distinct molecular subtypes of breast cancer.

    PubMed

    Yakirevich, Evgeny; Samkari, Ayman; Holloway, Michael P; Lu, Shaolei; Singh, Kamaljeet; Yu, Jovian; Fenton, Mary Anne; Altura, Rachel A

    2012-06-01

    Global gene expression profiling studies led to the recent classification of breast cancer into 4 distinct molecular subtypes including luminal, human epidermal growth factor receptor 2 enriched, basal like, and unclassified. Here, we used immunohistochemistry to evaluate expression of the antiapoptotic protein Survivin and its recently described acetylated form, Survivin acetyl129, in normal breast tissue and in 226 primary breast tumors of different molecular subtypes. Correlation of Survivin expression with molecular markers and its impact on patient outcomes were analyzed. Eighty-four percent of basal-like tumors expressed high levels of total Survivin, whereas 52% of luminal tumors expressed high levels of acetylated Survivin (P < .001). Overall survival (91%) for tumors expressing low levels of total Survivin was better than that for tumors expressing high levels of total Survivin (72%, P = .02), whereas the reverse was true for tumors expressing acetylated Survivin. In hierarchical cluster analysis, total Survivin clustered with basal marker expression, whereas acetylated Survivin clustered with luminal marker expression. In multivariate analysis, high total Survivin expression was an independent predictor of worse overall survival in patients with breast cancer (relative risk, 11; P < .01). These data indicate that high levels of total Survivin predict poor outcome in patients with grade 3 invasive ductal carcinoma and correlate directly with a basal-like phenotype. In contrast, high expression of the acetylated form of the protein associates with a favorable outcome and preferentially correlates with luminal-type tumors. Survivin likely has different functions in distinct breast cancer subtypes, and diagnostic strategies that incorporate immunohistochemical markers that detect both Survivin forms may help better strategize patient risk and direct therapy.

  18. The p38-interacting Protein (p38IP) Regulates G2/M Progression by Promoting α-Tubulin Acetylation via Inhibiting Ubiquitination-induced Degradation of the Acetyltransferase GCN5*

    PubMed Central

    Liu, Xin; Xiao, Wei; Wang, Xu-Dong; Li, Yue-Fang; Han, Jiahuai; Li, Yingqiu

    2013-01-01

    p38-interacting protein (p38IP) is a component of the GCN5 histone acetyltransferase-containing coactivator complex (GCN5-SAGA complex). It remains unclear whether p38IP or GCN5-SAGA is involved in cell cycle regulation. Using RNA interference to knock down p38IP, we observed that cells were arrested at the G2/M phase, exhibiting accumulation of cyclins, shrunken spindles, and hypoacetylation of α-tubulin. Further analysis revealed that knockdown of p38IP led to proteasome-dependent degradation of GCN5. GCN5 associated with and acetylated α-tubulin, and recovering GCN5 protein levels in p38IP knockdown cells by ectopic expression of GCN5 efficiently reversed α-tubulin hypoacetylation and G2/M arrest. During the G2/M transition, the association of α-tubulin with GCN5 increased, and the acetylation of α-tubulin reached a peak. Biochemical analyses demonstrated that the interaction between p38IP and GCN5 depended on the p38IP N terminus (1–381 amino acids) and GCN5 histone acetyltransferase domain and bromodomain. The p38IP N terminus could effectively reverse p38IP depletion-induced GCN5 degradation, thus recovering α-tubulin acetylation and G2/M progression. p38IP-mediated suppression of GCN5 ubiquitination most likely occurs via nuclear sequestration of GCN5. Our data indicate that the GCN5-SAGA complex is required for G2/M progression, mainly because p38IP promotes the acetylation of α-tubulin by preventing the degradation of GCN5, in turn facilitating the formation of the mitotic spindle. PMID:24220028

  19. Tubulin acetylation protects long-lived microtubules against mechanical ageing.

    PubMed

    Portran, Didier; Schaedel, Laura; Xu, Zhenjie; Théry, Manuel; Nachury, Maxence V

    2017-04-01

    Long-lived microtubules endow the eukaryotic cell with long-range transport abilities. While long-lived microtubules are acetylated on Lys40 of α-tubulin (αK40), acetylation takes place after stabilization and does not protect against depolymerization. Instead, αK40 acetylation has been proposed to mechanically stabilize microtubules. Yet how modification of αK40, a residue exposed to the microtubule lumen and inaccessible to microtubule-associated proteins and motors, could affect microtubule mechanics remains an open question. Here we develop FRET-based assays that report on the lateral interactions between protofilaments and find that αK40 acetylation directly weakens inter-protofilament interactions. Congruently, αK40 acetylation affects two processes largely governed by inter-protofilament interactions, reducing the nucleation frequency and accelerating the shrinkage rate. Most relevant to the biological function of acetylation, microfluidics manipulations demonstrate that αK40 acetylation enhances flexibility and confers resilience against repeated mechanical stresses. Thus, unlike deacetylated microtubules that accumulate damage when subjected to repeated stresses, long-lived microtubules are protected from mechanical ageing through their acquisition of αK40 acetylation. In contrast to other tubulin post-translational modifications that act through microtubule-associated proteins, motors and severing enzymes, intraluminal acetylation directly tunes the compliance and resilience of microtubules.

  20. Acetyl-L-carnitine normalizes the impaired long-term potentiation and spine density in a rat model of global ischemia.

    PubMed

    Kocsis, K; Knapp, L; Gellért, L; Oláh, G; Kis, Zs; Takakuwa, H; Iwamori, N; Ono, E; Toldi, J; Farkas, T

    2014-06-06

    As a consequence of an ischemic episode, energy production is disturbed, leading to neuronal cell death. Despite intensive research, the quest for promising neuroprotective drugs has largely failed, not only because of ineffectiveness, but also because of serious side-effects and dosing difficulties. Acetyl-l-carnitine (ALC) is an essential nutrient which plays a key role in energy metabolism by transporting fatty acids into mitochondria for β-oxidation. It is an endogenous compound and can be used at high dose without toxicity in research into ischemia. Its neuroprotective properties have been reported in many studies, but its potential action on long-term potentiation (LTP) and dendritic spine density has not been described to date. The aim of the present study was an evaluation of the possible protective effect of ALC after ischemic insults inflicted on hippocampal synaptic plasticity in a 2-vessel occlusion (2VO) model in rats. For electrophysiological measurements, LTP was tested on hippocampal slices. The Golgi-Cox staining technique was used to determine spine density. 2VO resulted in a decreased, unstable LTP and a significant loss of dendritic spines. ALC administered after 2VO was not protective, but as pretreatment prior to 2VO it restored LTP nearly to the control level. This finding paralleled the histological analysis: ALC pretreatment resulted in the reappearance of dendritic spines on the CA1 pyramidal cells. Our data demonstrate that ALC administration can restore hippocampal function and spine density. ALC probably acts by enhancing the aerobic metabolic pathway, which is inhibited during and following ischemic attacks.

  1. Acetylation of the c-MYC oncoprotein is required for cooperation with the HTLV-1 p30(II) accessory protein and the induction of oncogenic cellular transformation by p30(II)/c-MYC.

    PubMed

    Romeo, Megan M; Ko, Bookyung; Kim, Janice; Brady, Rebecca; Heatley, Hayley C; He, Jeffrey; Harrod, Carolyn K; Barnett, Braden; Ratner, Lee; Lairmore, Michael D; Martinez, Ernest; Lüscher, Bernhard; Robson, Craig N; Henriksson, Marie; Harrod, Robert

    2015-02-01

    The human T-cell leukemia retrovirus type-1 (HTLV-1) p30(II) protein is a multifunctional latency-maintenance factor that negatively regulates viral gene expression and deregulates host signaling pathways involved in aberrant T-cell growth and proliferation. We have previously demonstrated that p30(II) interacts with the c-MYC oncoprotein and enhances c-MYC-dependent transcriptional and oncogenic functions. However, the molecular and biochemical events that mediate the cooperation between p30(II) and c-MYC remain to be completely understood. Herein we demonstrate that p30(II) induces lysine-acetylation of the c-MYC oncoprotein. Acetylation-defective c-MYC Lys→Arg substitution mutants are impaired for oncogenic transformation with p30(II) in c-myc(-/-) HO15.19 fibroblasts. Using dual-chromatin-immunoprecipitations (dual-ChIPs), we further demonstrate that p30(II) is present in c-MYC-containing nucleoprotein complexes in HTLV-1-transformed HuT-102 T-lymphocytes. Moreover, p30(II) inhibits apoptosis in proliferating cells expressing c-MYC under conditions of genotoxic stress. These findings suggest that c-MYC-acetylation is required for the cooperation between p30(II)/c-MYC which could promote proviral replication and contribute to HTLV-1-induced carcinogenesis.

  2. Acetylation of the c-MYC oncoprotein is required for cooperation with the HTLV-1 p30II accessory protein and the induction of oncogenic cellular transformation by p30II/c-MYC

    PubMed Central

    Romeo, Megan M.; Ko, Bookyung; Kim, Janice; Brady, Rebecca; Heatley, Hayley C.; He, Jeffrey; Harrod, Carolyn K.; Barnett, Braden; Ratner, Lee; Lairmore, Michael D.; Martinez, Ernest; Lüscher, Bernhard; Robson, Craig N.; Henriksson, Marie; Harrod, Robert

    2014-01-01

    The human T-cell leukemia retrovirus type-1 (HTLV-1) p30II protein is a multifunctional latency-maintenance factor that negatively regulates viral gene expression and deregulates host signaling pathways involved in aberrant T-cell growth and proliferation. We have previously demonstrated that p30II interacts with the c-MYC oncoprotein and enhances c-MYC-dependent transcriptional and oncogenic functions. However, the molecular and biochemical events that mediate the cooperation between p30II and c-MYC remain to be completely understood. Herein we demonstrate that p30II induces lysine-acetylation of the c-MYC oncoprotein. Acetylation-defective c-MYC Lys→Arg substitution mutants are impaired for oncogenic transformation with p30II in c-myc−/− HO15.19 fibroblasts. Using dual-chromatin-immunoprecipitations (dual-ChIPs), we further demonstrate that p30II is present in c-MYC-containing nucleoprotein complexes in HTLV-1-transformed HuT-102 T-lymphocytes. Moreover, p30II inhibits apoptosis in proliferating cells expressing c-MYC under conditions of genotoxic stress. These findings suggest that c-MYC-acetylation is required for the cooperation between p30II/c-MYC which could promote proviral replication and contribute to HTLV-1-induced carcinogenesis. PMID:25569455

  3. Roles of P-glycoprotein and multidrug resistance protein in transporting para-aminosalicylic acid and its N-acetylated metabolite in mice brain

    PubMed Central

    Hong, Lan; Xu, Cong; O'Neal, Stefanie; Bi, Hui-chang; Huang, Min; Zheng, Wei; Zeng, Su

    2014-01-01

    Aim: Para-aminosalicylic acid (PAS) is effective in the treatment of manganism-induced neurotoxicity (manganism). In this study we investigated the roles of P-glycoprotein (MDR1a) and multidrug resistance protein (MRP) in transporting PAS and its N-acetylated metabolite AcPAS through blood-brain barrier. Methods: MDR1a-null or wild-type mice were intravenously injected with PAS (200 mg/kg). Thirty minutes after the injection, blood samples and brains were collected, and the concentrations of PAS and AcPAS in brain capillaries and parenchyma were measured using HPLC. Both MDCK-MDR1 and MDCK-MRP1 cells that overexpressed P-gp and MRP1, respectively, were used in two-chamber Transwell transport studies in vitro. Results: After injection of PAS, the brain concentration of PAS was substantially higher in MDR1a-null mice than in wild-type mice, but the brain concentration of AcPAS had no significant difference between MDR1a-null mice and wild-type mice. Concomitant injection of PAS with the MRP-specific inhibitor MK-571 (50 mg/kg) further increased the brain concentration of PAS in MDR1a-null mice, and increased the brain concentration of AcPAS in both MDR1a-null mice and wild-type mice. Two-chamber Transwell studies with MDCK-MDR1 cells demonstrated that PAS was not only a substrate but also a competitive inhibitor of P-gp, while AcPAS was not a substrate of P-gp. Two-chamber Transwell studies with the MDCK-MRP1 cells showed that MRP1 had the ability to transport both PAS and AcPAS across the BBB. Conclusion: P-gp plays a major role in the efflux of PAS from brain parenchyma into blood in mice, while MRP1 is involved in both PAS and AcPAS transport in the brain. PMID:25418377

  4. Histone acetylation in neurodevelopment.

    PubMed

    Contestabile, Antonio; Sintoni, Silvia

    2013-01-01

    Post-translational modification of histones is a primary mechanism through which epigenetic regulation of DNA transcription does occur. Among these modifications, regulation of histone acetylation state is an important tool to influence gene expression. Epigenetic regulation of neurodevelopment contributes to the structural and functional shaping of the brain during neurogenesis and continues to impact on neural plasticity lifelong. Alterations of these mechanisms during neurodevelopment may result in later occurrence of neuropsychatric disorders. The present paper reviews and discusses available data on histone modifications, in particular histone acetylation, in neurogenesis considering results obtained in culture systems of neural progenitors as well as in in vivo studies. Possible teratogenic effects of altered histone acetylation state during development are also considered. The use during pregnancy of drugs such as valproic acid, which acts as a histone deacetylase inhibitor, may result during postnatal development in autistic-like symptoms. The effect of gestational administration of the drug has been, therefore, tested on adult hippocampal neurogenesis in animals showing behavioral impairment as a consequence of the drug administration at a specific stage of pregnancy. These experimental results show that adult neurogenesis in the hippocampal dentate gyrus is not quantitatively altered by gestational valproic acid administration. Future steps and goals of research on the role and mechanisms of histone acetylation in neurodevelopment are briefly discussed.

  5. Histone H4 acetylation and the epigenetic reader Brd4 are critical regulators of pluripotency in embryonic stem cells.

    PubMed

    Gonzales-Cope, Michelle; Sidoli, Simone; Bhanu, Natarajan V; Won, Kyoung-Jae; Garcia, Benjamin A

    2016-02-04

    Pluripotent cells can be differentiated into many different cell types in vitro. Successful differentiation is guided in large part by epigenetic reprogramming and regulation of critical gene expression patterns. Recent genome-wide studies have identified the distribution of different histone-post-translational modifications (PTMs) in various conditions and during cellular differentiation. However, our understanding of the abundance of histone PTMs and their regulatory mechanisms still remain unknown. Here, we present a quantitative and comprehensive study of the abundance levels of histone PTMs during the differentiation of mouse embryonic stem cells (ESCs) using mass spectrometry (MS). We observed dynamic changes of histone PTMs including increased H3K9 methylation levels in agreement with previously reported results. More importantly, we found a global decrease of multiply acetylated histone H4 peptides. Brd4 targets acetylated H4 with a strong affinity to multiply modified H4 acetylation sites. We observed that the protein levels of Brd4 decreased upon differentiation together with global histone H4 acetylation. Inhibition of Brd4:histone H4 interaction by the BET domain inhibitor (+)-JQ1 in ESCs results in enhanced differentiation to the endodermal lineage, by disrupting the protein abundance dynamics. Genome-wide ChIP-seq mapping showed that Brd4 and H4 acetylation are co-occupied in the genome, upstream of core pluripotency genes such as Oct4 and Nanog in ESCs and lineage-specific genes in embryoid bodies (EBs). Together, our data demonstrate the fundamental role of Brd4 in monitoring cell differentiation through its interaction with acetylated histone marks and disruption of Brd4 may cause aberrant differentiation.

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

    PubMed

    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

  7. Acetylation of Stat1 modulates NF-κB activity

    PubMed Central

    Krämer, Oliver H.; Baus, Daniela; Knauer, Shirley K.; Stein, Stefan; Jäger, Elke; Stauber, Roland H.; Grez, Manuel; Pfitzner, Edith; Heinzel, Thorsten

    2006-01-01

    Acetylation of signaling molecules can lead to apoptosis or differentiation of carcinoma cells. The molecular mechanisms underlying these processes and the biological role of enzymes mediating the transfer or removal of an acetyl-group are currently under intense investigation. Our study shows that Stat1 is an acetylated protein. Stat1 acetylation depends on the balance between Stat1-associated histone deacetylases (HDACs) and histone acetyltransferases (HATs) such as CBP. Remarkably both inhibitors of HDACs and the cytokine interferon α alter this equilibrium and induce Stat1 acetylation. The analysis of Stat1 mutants reveals Lys 410 and Lys 413 as acetylation sites. Experiments with Stat1 mutants mimicking either constitutively acetylated or nonacetylated states show that only acetylated Stat1 is able to interact with NF-κB p65. As a consequence, p65 DNA binding, nuclear localization, and expression of anti-apoptotic NF-κB target genes decrease. These findings show how the acetylation of Stat1 regulates NF-κB activity and thus ultimately apoptosis. PMID:16481475

  8. Identification of N-terminal protein acetylation and arginine methylation of the voltage-gated sodium channel in end-stage heart failure human heart.

    PubMed

    Beltran-Alvarez, Pedro; Tarradas, Anna; Chiva, Cristina; Pérez-Serra, Alexandra; Batlle, Montserrat; Pérez-Villa, Félix; Schulte, Uwe; Sabidó, Eduard; Brugada, Ramon; Pagans, Sara

    2014-11-01

    The α subunit of the cardiac voltage-gated sodium channel, NaV1.5, provides the rapid sodium inward current that initiates cardiomyocyte action potentials. Here, we analyzed for the first time the post-translational modifications of NaV1.5 purified from end-stage heart failure human cardiac tissue. We identified R526 methylation as the major post-translational modification of any NaV1.5 arginine or lysine residue. Unexpectedly, we found that the N terminus of NaV1.5 was: 1) devoid of the initiation methionine, and 2) acetylated at the resulting initial alanine residue. This is the first evidence for N-terminal acetylation in any member of the voltage-gated ion channel superfamily. Our results open the door to explore NaV1.5 N-terminal acetylation and arginine methylation levels as drivers or markers of end-stage heart failure. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. N-Terminal Acetylation Acts as an Avidity Enhancer Within an Interconnected Multiprotein Complex

    SciTech Connect

    Scott, Daniel C.; Monda, Julie K.; Bennett, Eric J.; Harper, J. Wade; Schulman, Brenda A.

    2012-10-25

    Although many eukaryotic proteins are amino (N)-terminally acetylated, structural mechanisms by which N-terminal acetylation mediates protein interactions are largely unknown. Here, we found that N-terminal acetylation of the E2 enzyme, Ubc12, dictates distinctive E3-dependent ligation of the ubiquitin-like protein Nedd8 to Cul1. Structural, biochemical, biophysical, and genetic analyses revealed how complete burial of Ubc12's N-acetyl-methionine in a hydrophobic pocket in the E3, Dcn1, promotes cullin neddylation. The results suggest that the N-terminal acetyl both directs Ubc12's interactions with Dcn1 and prevents repulsion of a charged N terminus. Our data provide a link between acetylation and ubiquitin-like protein conjugation and define a mechanism for N-terminal acetylation-dependent recognition.

  10. An Alternative Strategy for Pan-acetyl-lysine Antibody Generation.

    PubMed

    Kim, Sun-Yee; Sim, Choon Kiat; Zhang, Qiongyi; Tang, Hui; Brunmeir, Reinhard; Pan, Hong; Karnani, Neerja; Han, Weiping; Zhang, Kangling; Xu, Feng

    2016-01-01

    Lysine acetylation is an important post-translational modification in cell signaling. In acetylome studies, a high-quality pan-acetyl-lysine antibody is key to successful enrichment of acetylated peptides for subsequent mass spectrometry analysis. Here we show an alternative method to generate polyclonal pan-acetyl-lysine antibodies using a synthesized random library of acetylated peptides as the antigen. Our antibodies are tested to be specific for acetyl-lysine peptides/proteins via ELISA and dot blot. When pooled, five of our antibodies show broad reactivity to acetyl-lysine peptides, complementing a commercial antibody in terms of peptide coverage. The consensus sequence of peptides bound by our antibody cocktail differs slightly from that of the commercial antibody. Lastly, our antibodies are tested in a proof-of-concept to analyze the acetylome of HEK293 cells. In total we identified 1557 acetylated peptides from 416 proteins. We thus demonstrated that our antibodies are well-qualified for acetylome studies and can complement existing commercial antibodies.

  11. Kinetics of CO Insertion and Acetyl Group Transfer Steps, and a Model of the Acetyl-CoA Synthase Catalytic Mechanism

    PubMed Central

    Tan, Xiangshi; Surovtsev, Ivan V.; Lindahl, Paul A.

    2008-01-01

    Acetyl-CoA synthase/carbon monoxide dehydrogenase is a Ni-Fe-S-containing enzyme that catalyzes the synthesis of acetyl-CoA from CO, CoA and a methyl group. The methyl group is transferred onto the enzyme from a corrinoid-iron-sulfur protein (CoFeSP). The kinetics of two steps within the catalytic mechanism were studied using the stopped-flow method, including the insertion of CO into a putative Ni2+-CH3 bond and the transfer of the resulting acetyl group to CoA. Neither step had been studied previously. Reactions were monitored indirectly, starting with the methylated intermediate form of the enzyme. Resulting traces were analyzed by constructing a simple kinetic model describing the catalytic mechanism under reducing conditions. Besides methyl group transfer, CO insertion, and acetyl group transfer, fitting to experimental traces required the inclusion of an inhibitory step in which CO reversibly bound to the form of the enzyme obtained immediately after product release. Global simulation of the reported datasets afforded a consistent set of kinetic parameters. The equilibrium constant for the overall synthesis of acetyl-CoA was estimated and compared to the product of the individual equilibrium constants. Simulations obtained with the model recapitulated the essential behavior of the enzyme, in terms of the variation of activity with [CO], and the time-dependent decay of the NiFeC EPR signal upon reaction with CoFeSP. Under standard assay conditions, the model suggests that the vast majority of active enzyme molecules in a population should be in the methylated form, suggesting that the subsequent catalytic step, namely CO insertion, is rate limiting. This conclusion is further supported by a sensitivity analysis showing that the rate is most sensitively affected by a change in the rate-coefficient associated with the CO insertion step. PMID:16967985

  12. Comparative specificities of Calreticulin Transacetylase to O-acetyl, N-acetyl and S-acetyl derivative of 4-methylcoumarins and their inhibitory effect on AFB1-induced genotoxicity in vitro and in vivo.

    PubMed

    Kumar, Ajit; Ponnan, Prija; Raj, Hanumantharao G; Parmar, Virinder S; Saso, Luciano

    2013-02-01

    We have earlier conclusively established the Calreticulin Transacetylase (CRTAase) catalyzed modifications of functional proteins such as cytochrome-P450-linked mixed function oxidases (Cyt-P450-linked MFOs), NADPH cytochrome c reductase, and glutathione S-transferase by acetoxy derivatives of polyphenols. In this study, we have investigated the comparative specificities of CRTAase to N-acetyl derivative, 7-acetamido-4-methylcoumarin (7-N-AMC), O-acetyl derivative, 7-acetoxy-4-methylcoumarin (7-AMC), S-acetyl derivative, 7-thioacetyl-4-methycoumarin (7-S-AMC) and their parent compounds in the modulation of catalytic activities of aforesaid proteins. Special attention concentrated on the comparative inhibitory effect of aforesaid acetyl moiety on Cyt-P450-linked MFOs such as 7-ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-dealkylase (PROD) and aflatoxin B(1) (AFB(1))-induced genotoxicity in vitro and in vivo. The results clearly indicated that N-acetyl and O-acetyl derivatives were better substrates for CRTAase while the S-acetyl was found to be a poorer substrate. Our study involving atomic charge, charge density and molecular electrostatic potential (MEP) calculations indicated the pivotal role of electronegativity and charge distribution values of O, N and S atoms of the acetyl group at C-7 position of the 4-methylcoumarins in CRTAase activity. These facts reinforce our hypothesis that the CRTAase catalyzed modifications of the catalytic activities of aforesaid proteins by acetyl derivative of 4-methylcoumarins is probably due to acetylation of these proteins.

  13. Insulin accelerates global and mitochondrial protein synthesis rates in neonatal muscle during sepsis

    USDA-ARS?s Scientific Manuscript database

    In neonatal pigs, sepsis decreases protein synthesis in skeletal muscle by decreasing translation initiation. However, insulin stimulates muscle protein synthesis despite persistent repression of translation initiation signaling. To determine whether the insulin-induced increase in global rates of m...

  14. Data detailing the platelet acetyl-lysine proteome.

    PubMed

    Aslan, Joseph E; David, Larry L; McCarty, Owen J T

    2015-12-01

    Here we detail proteomics data that describe the acetyl-lysine proteome of blood platelets (Aslan et al., 2015 [1]). An affinity purification - mass spectrometry (AP-MS) approach was used to identify proteins modified by Nε-lysine acetylation in quiescent, washed human platelets. The data provide insights into potential regulatory mechanisms of platelet function mediated by protein lysine acetylation. Additionally, as platelets are anucleate and lack histone proteins, they offer a unique and valuable system to study the regulation of cytosolic proteins by lysine acetylation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014 [2]) via with PRIDE partner repository with the dataset identifier PXD002332.

  15. Amyloid precursor protein (APP) affects global protein synthesis in dividing human cells.

    PubMed

    Sobol, Anna; Galluzzo, Paola; Liang, Shuang; Rambo, Brittany; Skucha, Sylvia; Weber, Megan J; Alani, Sara; Bocchetta, Maurizio

    2015-05-01

    Hypoxic non-small cell lung cancer (NSCLC) is dependent on Notch-1 signaling for survival. Targeting Notch-1 by means of γ-secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post-mortem analysis of GSI-treated, NSCLC-burdened mice suggested enhanced phosphorylation of 4E-BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non-canonical 4E-BP1 phosphorylation pattern rearrangement-a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF-4F composition indicating increased recruitment of eIF-4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF-4A assembly into eIF-4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap- and IRES-dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin-1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC-1) inhibition affected 4E-BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC-1. Key phenomena described in this study were reversed by overexpression of the APP C-terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC-1 regulation of cap-dependent protein synthesis. © 2014 Wiley Periodicals, Inc.

  16. Amyloid Precursor Protein (APP) Affects Global Protein Synthesis in Dividing Human Cells

    PubMed Central

    Sobol, Anna; Galluzzo, Paola; Liang, Shuang; Rambo, Brittany; Skucha, Sylvia; Weber, Megan J.; Alani, Sara

    2015-01-01

    Hypoxic non‐small cell lung cancer (NSCLC) is dependent on Notch‐1 signaling for survival. Targeting Notch‐1 by means of γ‐secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post‐mortem analysis of GSI‐treated, NSCLC‐burdened mice suggested enhanced phosphorylation of 4E‐BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non‐canonical 4E‐BP1 phosphorylation pattern rearrangement—a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF‐4F composition indicating increased recruitment of eIF‐4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF‐4A assembly into eIF‐4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap‐ and IRES‐dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin‐1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC‐1) inhibition affected 4E‐BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC‐1. Key phenomena described in this study were reversed by overexpression of the APP C‐terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC‐1 regulation of cap‐dependent protein synthesis. J. Cell. Physiol. 230: 1064–1074, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:25283437

  17. Structural basis for regulation of the human acetyl-CoA thioesterase 12 and interactions with the steroidogenic acute regulatory protein-related lipid transfer (START) domain.

    PubMed

    Swarbrick, Crystall M D; Roman, Noelia; Cowieson, Nathan; Patterson, Edward I; Nanson, Jeffrey; Siponen, Marina I; Berglund, Helena; Lehtiö, Lari; Forwood, Jade K

    2014-08-29

    Acetyl-CoA plays a fundamental role in cell signaling and metabolic pathways, with its cellular levels tightly controlled through reciprocal regulation of enzymes that mediate its synthesis and catabolism. ACOT12, the primary acetyl-CoA thioesterase in the liver of human, mouse, and rat, is responsible for cleavage of the thioester bond within acetyl-CoA, producing acetate and coenzyme A for a range of cellular processes. The enzyme is regulated by ADP and ATP, which is believed to be mediated through the ligand-induced oligomerization of the thioesterase domains, whereby ATP induces active dimers and tetramers, whereas apo- and ADP-bound ACOT12 are monomeric and inactive. Here, using a range of structural and biophysical techniques, it is demonstrated that ACOT12 is a trimer rather than a tetramer and that neither ADP nor ATP exert their regulatory effects by altering the oligomeric status of the enzyme. Rather, the binding site and mechanism of ADP regulation have been determined to occur through two novel regulatory regions, one involving a large loop that links the thioesterase domains (Phe(154)-Thr(178)), defined here as RegLoop1, and a second region involving the C terminus of thioesterase domain 2 (Gln(304)-Gly(326)), designated RegLoop2. Mutagenesis confirmed that Arg(312) and Arg(313) are crucial for this mode of regulation, and novel interactions with the START domain are presented together with insights into domain swapping within eukaryotic thioesterases for substrate recognition. In summary, these experiments provide the first structural insights into the regulation of this enzyme family, revealing an alternate hypothesis likely to be conserved throughout evolution. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Acetylation of Mammalian ADA3 Is Required for Its Functional Roles in Histone Acetylation and Cell Proliferation.

    PubMed

    Mohibi, Shakur; Srivastava, Shashank; Bele, Aditya; Mirza, Sameer; Band, Hamid; Band, Vimla

    2016-10-01

    Alteration/deficiency in activation 3 (ADA3) is an essential component of specific histone acetyltransferase (HAT) complexes. We have previously shown that ADA3 is required for establishing global histone acetylation patterns and for normal cell cycle progression (S. Mohibi et al., J Biol Chem 287:29442-29456, 2012, http://dx.doi.org/10.1074/jbc.M112.378901). Here, we report that these functional roles of ADA3 require its acetylation. We show that ADA3 acetylation, which is dynamically regulated in a cell cycle-dependent manner, reflects a balance of coordinated actions of its associated HATs, GCN5, PCAF, and p300, and a new partner that we define, the deacetylase SIRT1. We use mass spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300. Acetylation-defective mutants are capable of interacting with HATs and other components of HAT complexes but are deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3-deleted murine embryonic fibroblasts (MEFs). Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including the cell cycle, our study presents a novel mechanism to regulate the function of these complexes through dynamic ADA3 acetylation.

  19. Acetylation of Mammalian ADA3 Is Required for Its Functional Roles in Histone Acetylation and Cell Proliferation

    PubMed Central

    Mohibi, Shakur; Srivastava, Shashank; Bele, Aditya; Mirza, Sameer; Band, Hamid

    2016-01-01

    Alteration/deficiency in activation 3 (ADA3) is an essential component of specific histone acetyltransferase (HAT) complexes. We have previously shown that ADA3 is required for establishing global histone acetylation patterns and for normal cell cycle progression (S. Mohibi et al., J Biol Chem 287:29442–29456, 2012, http://dx.doi.org/10.1074/jbc.M112.378901). Here, we report that these functional roles of ADA3 require its acetylation. We show that ADA3 acetylation, which is dynamically regulated in a cell cycle-dependent manner, reflects a balance of coordinated actions of its associated HATs, GCN5, PCAF, and p300, and a new partner that we define, the deacetylase SIRT1. We use mass spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300. Acetylation-defective mutants are capable of interacting with HATs and other components of HAT complexes but are deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3-deleted murine embryonic fibroblasts (MEFs). Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including the cell cycle, our study presents a novel mechanism to regulate the function of these complexes through dynamic ADA3 acetylation. PMID:27402865

  20. N-acetyl-D-glucosamine-asparagine structure in ribosome-inactivating proteins from the seeds of Luffa cylindrica and Phytolacca americana.

    PubMed

    Islam, M R; Kung, S S; Kimura, Y; Funatsu, G

    1991-05-01

    Glycosylation-site-containing peptides were isolated from the proteolytic digests of luffin-a, luffin-b, PAP-S and CNBr-fragments of PAP-S by reverse-phase HPLC, and their amino acid compositions and sequences were analyzed. Six peptides were obtained from luffin-a, and three each from luffin-b and PAP-S. All of these peptides were negative toward the phenol-H2SO4 reaction and gave only N-acetyl-D-glucosamine in gas chromatography after methanolysis and reacetylation. Amounts of N-acetyl-D-glucosamine in these peptides were determined as D-glucosamine to be approximately one mol per peptide by an amino acid analyzer after HCl hydrolysis. Based on these results we concluded that Asn residues at positions of 28, 33, 77, 84, 206, and 227 in luffin-a, of 2, 78, and 85 in luffin-b, and of 10, 44, and 255 in PAP-S were glycosylated with only GlcNAc, and contained one residue per site.

  1. Acetylated Rhamnogalacturonans from Immature Fruits of Abelmoschus esculentus Inhibit the Adhesion of Helicobacter pylori to Human Gastric Cells by Interaction with Outer Membrane Proteins.

    PubMed

    Thöle, Christian; Brandt, Simone; Ahmed, Niyaz; Hensel, Andreas

    2015-09-15

    Polysaccharide containing extracts from immature fruits of okra (Abelmoschus esculentus) are known to exhibit antiadhesive effects against bacterial adhesion of Helicobacter pylori (H. pylori) to stomach tissue. The present study investigates structural and functional features of polymers responsible for this inhibition of bacterial attachment to host cells. Ammonium sulfate precipitation of an aqueous extract yielded two fractions at 60% and 90% saturation with significant antiadhesive effects against H. pylori, strain J99, (FE60% 68% ± 15%; FE90% 75% ± 11% inhibition rates) after preincubation of the bacteria at 1 mg/mL. Sequential extraction of okra fruits yielded hot buffer soluble solids (HBSS) with dose dependent antiadhesive effects against strain J99 and three clinical isolates. Preincubation of H. pylori with HBSS (1 mg/mL) led to reduced binding to 3'-sialyl lactose, sialylated Le(a) and Le(x). A reduction of bacterial binding to ligands complementary to BabA and SabA was observed when bacteria were pretreated with FE90%. Structural analysis of the antiadhesive polysaccharides (molecular weight, monomer composition, linkage analysis, stereochemistry, and acetylation) indicated the presence of acetylated rhamnogalacturonan-I polymers, decorated with short galactose side chains. Deacetylation of HBSS and FE90% resulted in loss of the antiadhesive activity, indicating esterification being a prerequisite for antiadhesive activity.

  2. Methamphetamine-induced neuronal protein NAT8L is the NAA biosynthetic enzyme: implications for specialized acetyl coenzyme A metabolism in the CNS.

    PubMed

    Ariyannur, Prasanth S; Moffett, John R; Manickam, Pachiappan; Pattabiraman, Nagarajan; Arun, Peethambaran; Nitta, Atsumi; Nabeshima, Toshitaka; Madhavarao, Chikkathur N; Namboodiri, Aryan M A

    2010-06-04

    N-acetylaspartate (NAA) is a concentrated, neuron-specific brain metabolite routinely used as a magnetic resonance spectroscopy marker for brain injury and disease. Despite decades of research, the functional roles of NAA remain unclear. Biochemical investigations over several decades have associated NAA with myelin lipid synthesis and energy metabolism. However, studies have been hampered by an inability to identify the gene for the NAA biosynthetic enzyme aspartate N-acetyltransferase (Asp-NAT). A very recent report has identified Nat8l as the gene encoding Asp-NAT and confirmed that the only child diagnosed with a lack of NAA on brain magnetic resonance spectrograms has a 19-bp deletion in this gene. Based on in vitro Nat8l expression studies the researchers concluded that many previous biochemical investigations have been technically flawed and that NAA may not be associated with brain energy or lipid metabolism. In studies done concurrently in our laboratory we have demonstrated via cloning, expression, specificity for acetylation of aspartate, responsiveness to methamphetamine treatment, molecular modeling and comparative immunolocalization that NAT8L is the NAA biosynthetic enzyme Asp-NAT. We conclude that NAA is a major storage and transport form of acetyl coenzyme A specific to the nervous system, thus linking it to both lipid synthesis and energy metabolism.

  3. First Comprehensive Proteome Analyses of Lysine Acetylation and Succinylation in Seedling Leaves of Brachypodium distachyon L.

    PubMed Central

    Zhen, Shoumin; Deng, Xiong; Wang, Jian; Zhu, Gengrui; Cao, Hui; Yuan, Linlin; Yan, Yueming

    2016-01-01

    Protein acetylation and succinylation are the most crucial protein post-translational modifications (PTMs) involved in the regulation of plant growth and development. In this study, we present the first lysine-acetylation and lysine-succinylation proteome analysis of seedling leaves in Brachypodium distachyon L (Bd). Using high accuracy nano LC-MS/MS combined with affinity purification, we identified a total of 636 lysine-acetylated sites in 353 proteins and 605 lysine-succinylated sites in 262 proteins. These proteins participated in many biology processes, with various molecular functions. In particular, 119 proteins and 115 sites were found to be both acetylated and succinylated, simultaneously. Among the 353 acetylated proteins, 148 had acetylation orthologs in Oryza sativa L., Arabidopsis thaliana, Synechocystis sp. PCC 6803, and Glycine max L. Among the 262 succinylated proteins, 170 of them were found to have homologous proteins in Oryza sativa L., Escherichia coli, Sacchayromyces cerevisiae, or Homo sapiens. Motif-X analysis of the acetylated and succinylated sites identified two new acetylated motifs (K---K and K-I-K) and twelve significantly enriched succinylated motifs for the first time, which could serve as possible binding loci for future studies in plants. Our comprehensive dataset provides a promising starting point for further functional analysis of acetylation and succinylation in Bd and other plant species. PMID:27515067

  4. Nucleosome acetylation sequencing to study the establishment of chromatin acetylation.

    PubMed

    Mittal, Chitvan; Blacketer, Melissa J; Shogren-Knaak, Michael A

    2014-07-15

    The establishment of posttranslational chromatin modifications is a major mechanism for regulating how genomic DNA is utilized. However, current in vitro chromatin assays do not monitor histone modifications at individual nucleosomes. Here we describe a strategy, nucleosome acetylation sequencing, that allows us to read the amount of modification at each nucleosome. In this approach, a bead-bound trinucleosome substrate is enzymatically acetylated with radiolabeled acetyl CoA by the SAGA complex from Saccharomyces cerevisae. The product is digested by restriction enzymes that cut at unique sites between the nucleosomes and then counted to quantify the extent of acetylation at each nucleosomal site. We find that we can sensitively, specifically, and reproducibly follow enzyme-mediated nucleosome acetylation. Applying this strategy, when acetylation proceeds extensively, its distribution across nucleosomes is relatively uniform. However, when substrates are used that contain nucleosomes mutated at the major sites of SAGA-mediated acetylation, or that are studied under initial rate conditions, changes in the acetylation distribution can be observed. Nucleosome acetylation sequencing should be applicable to analyzing a wide range of modifications. Additionally, because our trinucleosomes synthesis strategy is highly modular and efficient, it can be used to generate nucleosomal systems in which nucleosome composition differs across the array.

  5. The p53-SET Interplays Reveal A New Mode of Acetylation-dependent Regulation

    PubMed Central

    Lasso, Gorka; Jiang, Le; Leng, Wenchuan; Zhu, Wei-Guo; Qin, Jun; Honig, Barry; Gu, Wei

    2016-01-01

    Summary Although lysine acetylation is now recognized as a general protein modification for both histones and non-histone proteins1-3, the mechanisms of acetylation mediated actions are not completely understood. Acetylation of the C-terminal domain (CTD) of p53 was the first example for non-histone protein acetylation4. Yet the precise role of the CTD acetylation remains elusive. Lysine acetylation often creates binding sites for bromodomain-containing “reader” proteins5,6; surprisingly, in a proteomic screen, we identified SET as a major cellular factor whose binding with p53 is totally dependent on the CTD acetylation status. SET profoundly inhibits p53 transcriptional activity in unstressed cells but SET-mediated repression is completely abolished by stress-induced p53 CTD acetylation. Moreover, loss of the interaction with SET activates p53, resulting in tumor regression in mouse xenograft models. Notably, the acidic domain of SET acts as a “reader” for unacetylated CTD of p53 and this mechanism of acetylation-dependent regulation is widespread in nature. For example, p53 acetylation also modulates its interactions with similar acidic domains found in other p53 regulators including VPRBP, DAXX and PELP1 (refs. 7-9), and computational analysis of the proteome identified numerous proteins with the potential to serve as the acidic domain readers and lysine-rich ligands. Unlike bromodomain readers, which preferentially bind the acetylated forms of their cognate ligands, the acidic domain readers specifically recognize the unacetylated forms of their ligands. Finally, the acetylation-dependent regulation of p53 was further validated in vivo by using a knockin mouse model expressing an acetylation-mimicking form of p53. These results reveal that the acidic domain-containing factors act as a new class of acetylation-dependent regulators by targeting p53 and potentially, beyond. PMID:27626385

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

    PubMed

    Weinert, Brian T; Schölz, Christian; Wagner, Sebastian A; Iesmantavicius, Vytautas; Su, Dan; Daniel, Jeremy A; Choudhary, Chunaram

    2013-08-29

    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.

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

    PubMed

    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

  8. Global Analysis of Protein Activities Using Proteome Chips

    NASA Astrophysics Data System (ADS)

    Zhu, Heng; Bilgin, Metin; Bangham, Rhonda; Hall, David; Casamayor, Antonio; Bertone, Paul; Lan, Ning; Jansen, Ronald; Bidlingmaier, Scott; Houfek, Thomas; Mitchell, Tom; Miller, Perry; Dean, Ralph A.; Gerstein, Mark; Snyder, Michael

    2001-09-01

    To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.

  9. STAT5 acetylation

    PubMed Central

    Kosan, Christian; Ginter, Torsten; Heinzel, Thorsten; Krämer, Oliver H

    2013-01-01

    The cytokine-inducible transcription factors signal transducer and activator of transcription 5A and 5B (STAT5A and STAT5B) are important for the proper development of multicellular eukaryotes. Disturbed signaling cascades evoking uncontrolled expression of STAT5 target genes are associated with cancer and immunological failure. Here, we summarize how STAT5 acetylation is integrated into posttranslational modification networks within cells. Moreover, we focus on how inhibitors of deacetylases and tyrosine kinases can correct leukemogenic signaling nodes involving STAT5. Such small molecules can be exploited in the fight against neoplastic diseases and immunological disorders. PMID:24416653

  10. Histone acetylation in insect chromosomes.

    PubMed

    Allfrey, V G; Pogo, B G; Littau, V C; Gershey, E L; Mirsky, A E

    1968-01-19

    Acetylation of histones takes place along the salivary gland chromosomes of Chironomus thummi when RNA synthesis is active. It can be observed but not measured quantitatively by autoradiography of chromosome squashes. The "fixatives" commonly used in preparing squashes of insect chromosomes preferentially extract the highly acetylated "arginine-rich" histone fractions; the use of such fixatives may explain the reported absence of histone acetylation in Drosophila melanogaster.

  11. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.

    PubMed

    Krogan, Nevan J; Cagney, Gerard; Yu, Haiyuan; Zhong, Gouqing; Guo, Xinghua; Ignatchenko, Alexandr; Li, Joyce; Pu, Shuye; Datta, Nira; Tikuisis, Aaron P; Punna, Thanuja; Peregrín-Alvarez, José M; Shales, Michael; Zhang, Xin; Davey, Michael; Robinson, Mark D; Paccanaro, Alberto; Bray, James E; Sheung, Anthony; Beattie, Bryan; Richards, Dawn P; Canadien, Veronica; Lalev, Atanas; Mena, Frank; Wong, Peter; Starostine, Andrei; Canete, Myra M; Vlasblom, James; Wu, Samuel; Orsi, Chris; Collins, Sean R; Chandran, Shamanta; Haw, Robin; Rilstone, Jennifer J; Gandi, Kiran; Thompson, Natalie J; Musso, Gabe; St Onge, Peter; Ghanny, Shaun; Lam, Mandy H Y; Butland, Gareth; Altaf-Ul, Amin M; Kanaya, Shigehiko; Shilatifard, Ali; O'Shea, Erin; Weissman, Jonathan S; Ingles, C James; Hughes, Timothy R; Parkinson, John; Gerstein, Mark; Wodak, Shoshana J; Emili, Andrew; Greenblatt, Jack F

    2006-03-30

    Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.

  12. Global subcellular characterization of protein degradation using quantitative proteomics.

    PubMed

    Larance, Mark; Ahmad, Yasmeen; Kirkwood, Kathryn J; Ly, Tony; Lamond, Angus I

    2013-03-01

    Protein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyze the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting data set analyzed more than 74,000 peptides, corresponding to ~5000 proteins, from nuclear, cytosolic, membrane, and cytoskeletal compartments. These data identified rapidly degraded proteasome targets, such as PRR11 and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterized as rapidly degraded through analysis of whole cell lysates. Bioinformatic analysis of the entire data set is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution.

  13. Obesity, cancer, and acetyl-CoA metabolism

    PubMed Central

    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 regulating inflammatory processes in obesity-linked cancer. PMID:23878588

  14. Erasers of Histone Acetylation: The Histone Deacetylase Enzymes

    PubMed Central

    Seto, Edward; Yoshida, Minoru

    2014-01-01

    Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl functional groups from the lysine residues of both histone and nonhistone proteins. In humans, there are 18 HDAC enzymes that use either zinc- or NAD+-dependent mechanisms to deacetylate acetyl lysine substrates. Although removal of histone acetyl epigenetic modification by HDACs regulates chromatin structure and transcription, deacetylation of nonhistones controls diverse cellular processes. HDAC inhibitors are already known potential anticancer agents and show promise for the treatment of many diseases. PMID:24691964

  15. Involvement of phosphotransacetylase, acetate kinase, and acetyl phosphate synthesis in control of the phosphate regulon in Escherichia coli.

    PubMed

    Wanner, B L; Wilmes-Riesenberg, M R

    1992-04-01

    Two controls of the phosphate (PHO) regulon require sensor proteins that are protein kinases that phosphorylate the regulator, PhoB, which in turn activates transcription only when phosphorylated. Pi control requires the Pi sensor PhoR; the other control is Pi independent and requires the sensor CreC (formerly called PhoM). Here we describe an additional control of the PHO regulon which is Pi independent and requires neither PhoR nor CreC. This control is regulated by a two-step pathway in carbon metabolism in which acetyl coenzyme A, Pi, and ADP are converted into acetate, coenzyme A, and ATP via the enzymes phosphotransacetylase (Pta) and acetate kinase (AckA). It responds to the synthesis of acetyl phosphate, an intermediate in the Pta-AckA pathway. Since the synthesis of acetyl phosphate via this pathway leads to the incorporation of Pi into ATP, the primary phosphoryl donor in metabolism, we propose that a regulatory coupling(s) may exist between the PHO regulon, which encodes genes for Pi uptake, and genes for enzymes in central metabolism for incorporation of Pi into ATP. Regulatory interactions of this sort may be important in global control. Further, it provides a functional basis for the concept of cross-regulation in the PHO regulon. This is also the first evidence that acetyl phosphate may have a role as an effector of gene regulation.

  16. Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite-specific functions during Plasmodium falciparum intraerythrocytic development

    PubMed Central

    Miao, Jun; Lawrence, Matthew; Jeffers, Victoria; Zhao, Fangqing; Parker, Daniel; Ge, Ying; Sullivan, William J.; Cui, Liwang

    2013-01-01

    Summary Lysine acetylation has emerged as a major posttranslational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine-acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion, and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely-related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium-specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites. PMID:23796209

  17. Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite-specific functions during Plasmodium falciparum intraerythrocytic development.

    PubMed

    Miao, Jun; Lawrence, Matthew; Jeffers, Victoria; Zhao, Fangqing; Parker, Daniel; Ge, Ying; Sullivan, William J; Cui, Liwang

    2013-08-01

    Lysine acetylation has emerged as a major post-translational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine-acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium-specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites. © 2013 John Wiley & Sons Ltd.

  18. Global landscape of HIV-human protein complexes.

    PubMed

    Jäger, Stefanie; Cimermancic, Peter; Gulbahce, Natali; Johnson, Jeffrey R; McGovern, Kathryn E; Clarke, Starlynn C; Shales, Michael; Mercenne, Gaelle; Pache, Lars; Li, Kathy; Hernandez, Hilda; Jang, Gwendolyn M; Roth, Shoshannah L; Akiva, Eyal; Marlett, John; Stephens, Melanie; D'Orso, Iván; Fernandes, Jason; Fahey, Marie; Mahon, Cathal; O'Donoghue, Anthony J; Todorovic, Aleksandar; Morris, John H; Maltby, David A; Alber, Tom; Cagney, Gerard; Bushman, Frederic D; Young, John A; Chanda, Sumit K; Sundquist, Wesley I; Kortemme, Tanja; Hernandez, Ryan D; Craik, Charles S; Burlingame, Alma; Sali, Andrej; Frankel, Alan D; Krogan, Nevan J

    2011-12-21

    Human immunodeficiency virus (HIV) has a small genome and therefore relies heavily on the host cellular machinery to replicate. Identifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how HIV rewires the host's cellular machinery during the course of infection. Here we report the use of affinity tagging and purification mass spectrometry to determine systematically the physical interactions of all 18 HIV-1 proteins and polyproteins with host proteins in two different human cell lines (HEK293 and Jurkat). Using a quantitative scoring system that we call MiST, we identified with high confidence 497 HIV-human protein-protein interactions involving 435 individual human proteins, with ∼40% of the interactions being identified in both cell types. We found that the host proteins hijacked by HIV, especially those found interacting in both cell types, are highly conserved across primates. We uncovered a number of host complexes targeted by viral proteins, including the finding that HIV protease cleaves eIF3d, a subunit of eukaryotic translation initiation factor 3. This host protein is one of eleven identified in this analysis that act to inhibit HIV replication. This data set facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of HIV infection.

  19. Metabolism and acetylation contribute to leucine-mediated inhibition of cardiac glucose uptake.

    PubMed

    Renguet, Edith; Ginion, Audrey; Gélinas, Roselle; Bultot, Laurent; Auquier, Julien; Robillard Frayne, Isabelle; Daneault, Caroline; Vanoverschelde, Jean-Louis; Des Rosiers, Christine; Hue, Louis; Horman, Sandrine; Beauloye, Christophe; Bertrand, Luc

    2017-08-01

    High plasma leucine levels strongly correlate with type 2 diabetes. Studies of muscle cells have suggested that leucine alters the insulin response for glucose transport by activating an insulin-negative feedback loop driven by the mammalian target of rapamycin/p70 ribosomal S6 kinase (mTOR/p70S6K) pathway. Here, we examined the molecular mechanism involved in leucine's action on cardiac glucose uptake. Leucine was indeed able to curb glucose uptake after insulin stimulation in both cultured cardiomyocytes and perfused hearts. Although leucine activated mTOR/p70S6K, the mTOR inhibitor rapamycin did not prevent leucine's inhibitory action on glucose uptake, ruling out the contribution of the insulin-negative feedback loop. α-Ketoisocaproate, the first metabolite of leucine catabolism, mimicked leucine's effect on glucose uptake. Incubation of cardiomyocytes with [(13)C]leucine ascertained its metabolism to ketone bodies (KBs), which had a similar negative impact on insulin-stimulated glucose transport. Both leucine and KBs reduced glucose uptake by affecting translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Finally, we found that leucine elevated the global protein acetylation level. Pharmacological inhibition of lysine acetyltransferases counteracted this increase in protein acetylation and prevented leucine's inhibitory action on both glucose uptake and GLUT4 translocation. Taken together, these results indicate that leucine metabolism into KBs contributes to inhibition of cardiac glucose uptake by hampering the translocation of GLUT4-containing vesicles via acetylation. They offer new insights into the establishment of insulin resistance in the heart.NEW & NOTEWORTHY Catabolism of the branched-chain amino acid leucine into ketone bodies efficiently inhibits cardiac glucose uptake through decreased translocation of glucose transporter 4 to the plasma membrane. Leucine increases protein acetylation. Pharmacological inhibition of acetylation

  20. Characterization of nucleolin K88 acetylation defines a new pool of nucleolin colocalizing with pre-mRNA splicing factors.

    PubMed

    Das, Sadhan; Cong, Rong; Shandilya, Jayasha; Senapati, Parijat; Moindrot, Benoit; Monier, Karine; Delage, Hélène; Mongelard, Fabien; Kumar, Sanjeev; Kundu, Tapas K; Bouvet, Philippe

    2013-03-01

    Nucleolin is a multifunctional protein that carries several post-translational modifications. We characterized nucleolin acetylation and developed antibodies specific to nucleolin K88 acetylation. Using this antibody we show that nucleolin is acetylated in vivo and is not localized in the nucleoli, but instead is distributed throughout the nucleoplasm. Immunofluorescence studies indicate that acetylated nucleolin is co-localized with the splicing factor SC35 and partially with Y12. Acetylated nucleolin is expressed in all tested proliferating cell types. Our findings show that acetylation defines a new pool of nucleolin which support a role for nucleolin in the regulation of mRNA maturation and transcription by RNA polymerase II.

  1. Acetylation of Tau Inhibits Its Degradation and Contributes to Tauopathy

    PubMed Central

    Min, Sang-Won; Cho, Seo-Hyun; Zhou, Yungui; Schroeder, Sebastian; Haroutunian, Vahram; Seeley, William W.; Huang, Eric J.; Shen, Yong; Masliah, Eliezer; Mukherjee, Chandrani; Meyers, David; Cole, Philip A.; Ott, Melanie; Gan, Li

    2011-01-01

    Summary Neurodegenerative tauopathies characterized by hyperphosphorylated tau include frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and Alzheimer's disease (AD). Reducing tau levels improves cognitive function in mouse models of AD and FTDP-17, but the mechanisms regulating the turnover of pathogenic tau are unknown. We found that tau is acetylated and that tau acetylation prevents degradation of phosphorylated tau (p-tau). Using two antibodies specific for acetylated tau, we showed that tau acetylation is elevated in patients at early and moderate Braak stages of tauopathy. Histone acetyltransferase p300 was involved in tau acetylation and the class III protein deacetylase SIRT1 in deacetylation. Deleting SIRT1 enhanced levels of acetylated-tau and pathogenic forms of p-tau in vivo, likely by blocking proteasome-mediated degradation. Inhibiting p300 with a small molecule promoted tau deacetylation and eliminated p-tau associated with tauopathy. Modulating tau acetylation could be a new therapeutic strategy to reduce tau-mediated neurodegeneration. PMID:20869593

  2. Histone Acetylation Inhibitors Promote Axon Growth in Adult DRG neurons

    PubMed Central

    Lin, Shen; Nazif, Kutaiba; Smith, Alexander; Baas, Peter W; Smith, George M

    2015-01-01

    Intrinsic mechanisms that guide damaged axons to regenerate following spinal cord injury remain poorly understood. Manipulation of posttranslational modifications of key proteins in mature neurons could re-invigorate growth machinery after injury. One such modification is acetylation, a reversible process controlled by two enzyme families acting in opposition, the Histone Deacetylases (HDACs) and the Histone Acetyl Transferases (HATs). While acetylated histones in the nucleus is associated with upregulation of growth promoting genes, de-acetylated tubulin in the axoplasm is associated with more labile microtubules, conducive to axon growth. In this study we investigated the effects of HAT inhibitors and HDAC inhibitors on cultured adult dorsal root ganglia (DRG) neurons. We found that inhibition of HATs, using Anacardic Acid or CPTH2, improved axon outgrowth, while inhibition of HDACs using TSA or Tubacin, inhibited axon growth. Furthermore, Anacardic Acid increased the number of axons able to cross an inhibitory chondroitin sulfate proteoglycan (CSPG) border. Histone acetylation, but not tubulin acetylation levels, was affected by HAT inhibitors, whereas tubulin acetylation levels were increased in the presence of HDAC inhibitor Tubacin. Although microtubule stabilizing drug taxol did not have an effect on the lengths of DRG axons, nocodazole decreased axon lengths. While the mechanistic basis will require future studies, our data show that inhibitors of HAT can augment axon growth in adult DRG neurons, with the potential of aiding axon growth over inhibitory substrates produced by the glial scar. PMID:25702820

  3. Chaperone-mediated acetylation of histones by Rtt109 identified by quantitative proteomics

    PubMed Central

    Abshiru, Nebiyu; Ippersiel, Kevin; Tang, Yong; Yuan, Hua; Marmorstein, Ronen; Verreault, Alain; Thibault, Pierre

    2014-01-01

    Rtt109 is a fungal-specific histone acetyltransferase (HAT) that associates with either Vps75 or Asf1 to acetylate histone H3. Recent biochemical and structural studies suggest that site-specific acetylation of H3 by Rtt109 is dictated by the binding chaperone where Rtt109-Asf1 acetylates K56, while Rtt109-Vps75 acetylates K9 and K27. To gain further insights into the roles of Vps75 and Asf1 in directing site-specific acetylation of H3, we used quantitative proteomics to profile the global and site-specific changes in H3 and H4 during in vitro acetylation assays with Rtt109 and its chaperones. Our analyses showed that Rtt109-Vps75 preferentially acetylates H3 K9 and K23, the former residue being the major acetylation site. At high enzyme to substrate ratio, Rtt109 also acetylated K14, K18, K27 and to a lower extent K56 of histone H3. Importantly, this study revealed that in contrast to Rtt109-Vps75, Rtt109-Asf1 displayed a far greater site-specificity, with K56 being the primary site of acetylation. For the first time, we also report the acetylation of histone H4 K12 by Rtt109-Vps75, whereas Rtt109-Asf1 showed no detectable activity toward H4. PMID:23036725

  4. Annotating N termini for the human proteome project: N termini and Nα-acetylation status differentiate stable cleaved protein species from degradation remnants in the human erythrocyte proteome.

    PubMed

    Lange, Philipp F; Huesgen, Pitter F; Nguyen, Karen; Overall, Christopher M

    2014-04-04

    A goal of the Chromosome-centric Human Proteome Project is to identify all human protein species. With 3844 proteins annotated as "missing", this is challenging. Moreover, proteolytic processing generates new protein species with characteristic neo-N termini that are frequently accompanied by altered half-lives, function, interactions, and location. Enucleated and largely void of internal membranes and organelles, erythrocytes are simple yet proteomically challenging cells due to the high hemoglobin content and wide dynamic range of protein concentrations that impedes protein identification. Using the N-terminomics procedure TAILS, we identified 1369 human erythrocyte natural and neo-N-termini and 1234 proteins. Multiple semitryptic N-terminal peptides exhibited improved mass spectrometric identification properties versus the intact tryptic peptide enabling identification of 281 novel erythrocyte proteins and six missing proteins identified for the first time in the human proteome. With an improved bioinformatics workflow, we developed a new classification system and the Terminus Cluster Score. Thereby we described a new stabilizing N-end rule for processed protein termini, which discriminates novel protein species from degradation remnants, and identified protein domain hot spots susceptible to cleavage. Strikingly, 68% of the N-termini were within genome-encoded protein sequences, revealing alternative translation initiation sites, pervasive endoproteolytic processing, and stabilization of protein fragments in vivo. The mass spectrometry proteomics data have been deposited to ProteomeXchange with the data set identifier .

  5. Olig1 Acetylation and Nuclear Export Mediate Oligodendrocyte Development

    PubMed Central

    Dai, Jinxiang; Bercury, Kathryn K.; Jin, Weilin

    2015-01-01

    The oligodendrocyte transcription factor Olig1 is critical for both oligodendrocyte development and remyelination in mice. Nuclear to cytoplasmic translocation of Olig1 protein occurs during brain development and in multiple sclerosis, but the detailed molecular mechanism of this translocation remains elusive. Here, we report that Olig1 acetylation and deacetylation drive its active translocation between the nucleus and the cytoplasm in both mouse and rat oligodendrocytes. We identified three functional nuclear export sequences (NES) localized in the basic helix-loop-helix domain and one specific acetylation site at Lys 150 (human Olig1) in NES1. Olig1 acetylation and deacetylation are regulated by the acetyltransferase CREB-binding protein and the histone deacetylases HDAC1, HDAC3, and HDAC10. Acetylation of Olig1 decreased its chromatin association, increased its interaction with inhibitor of DNA binding 2 and facilitated its retention in the cytoplasm of mature oligodendrocytes. These studies establish that acetylation of Olig1 regulates its chromatin dissociation and subsequent translocation to the cytoplasm and is required for its function in oligodendrocyte maturation. SIGNIFICANCE STATEMENT The nuclear to cytoplasmic translocation of Olig1 protein has been observed during mouse and human brain development and in multiple sclerosis in several studies, but the detailed molecular mechanism of this translocation remains elusive. Here, we provide insight into the mechanism by which acetylation of Olig1 regulates its unique nuclear-cytoplasmic shuttling during oligodendrocyte development and how the acetylation status of Olig1 modulates its distinct function in the nucleus versus the cytoplasm. The current study provides a unique example of a lineage-specific transcription factor that is actively translocated from the nucleus to the cytoplasm as the cell differentiates. Importantly, we demonstrate that this process is tightly controlled by acetylation at a single

  6. Bayesian Proteoform Modeling Improves Protein Quantification of Global Proteomic Measurements

    SciTech Connect

    Webb-Robertson, Bobbie-Jo M.; Matzke, Melissa M.; Datta, Susmita; Payne, Samuel H.; Kang, Jiyun; Bramer, Lisa M.; Nicora, Carrie D.; Shukla, Anil K.; Metz, Thomas O.; Rodland, Karin D.; Smith, Richard D.; Tardiff, Mark F.; McDermott, Jason E.; Pounds, Joel G.; Waters, Katrina M.

    2014-12-01

    As the capability of mass spectrometry-based proteomics has matured, tens of thousands of peptides can be measured simultaneously, which has the benefit of offering a systems view of protein expression. However, a major challenge is that with an increase in throughput, protein quantification estimation from the native measured peptides has become a computational task. A limitation to existing computationally-driven protein quantification methods is that most ignore protein variation, such as alternate splicing of the RNA transcript and post-translational modifications or other possible proteoforms, which will affect a significant fraction of the proteome. The consequence of this assumption is that statistical inference at the protein level, and consequently downstream analyses, such as network and pathway modeling, have only limited power for biomarker discovery. Here, we describe a Bayesian model (BP-Quant) that uses statistically derived peptides signatures to identify peptides that are outside the dominant pattern, or the existence of multiple over-expressed patterns to improve relative protein abundance estimates. It is a research-driven approach that utilizes the objectives of the experiment, defined in the context of a standard statistical hypothesis, to identify a set of peptides exhibiting similar statistical behavior relating to a protein. This approach infers that changes in relative protein abundance can be used as a surrogate for changes in function, without necessarily taking into account the effect of differential post-translational modifications, processing, or splicing in altering protein function. We verify the approach using a dilution study from mouse plasma samples and demonstrate that BP-Quant achieves similar accuracy as the current state-of-the-art methods at proteoform identification with significantly better specificity. BP-Quant is available as a MatLab ® and R packages at https://github.com/PNNL-Comp-Mass-Spec/BP-Quant.

  7. [Effect of acetylation and oxidation on some properties of breadfruit (Artocarpus altilis) seed starch].

    PubMed

    Rincón, Alicia Mariela; Bou Rached, Lizet; Aragoza, Luis E; Padilla, Fanny

    2007-09-01

    Starch extracted from seeds of Artocarpus altilis (Breadfruit) was chemically modified by acetylation and oxidation, and its functional properties were evaluated and compared with these of native starch. Analysis of the chemical composition showed that moisture content was higher for modified starches. Ash, protein, crude fiber and amylose contents were reduced by the modifications, but did not alter the native starch granules' irregularity, oval shape and smooth surface. Acetylation produced changes in water absorption, swelling power and soluble solids, these values were higher for acetylated starch, while values for native and oxidized starches were similar. Both modifications reduced pasting temperature; oxidation reduced maximum peak viscosity but it was increased by acetylation. Hot paste viscosity was reduced by both modifications, whereas cold paste viscosity was lower in the oxidized starch and higher in the acetylated starch. Breakdown was increased by acetylation and reduced with oxidation. Setback value was reduced after acetylation, indicating it could minimize retrogradation of the starch.

  8. Male-specific lethal complex in Drosophila counteracts histone acetylation and does not mediate dosage compensation

    PubMed Central

    Sun, Lin; Fernandez, Harvey R.; Donohue, Ryan C.; Li, Jilong; Cheng, Jianlin; Birchler, James A.

    2013-01-01

    Dosage compensation is achieved in male Drosophila by a twofold up-regulation of the single X chromosome to reach the level of the two X chromosomes in females. A popular hypothesis to explain this phenomenon is that the male-specific lethal (MSL) complex, which is present at high levels on the male X, mediates this modulation of gene expression. One member of the complex, MOF, a histone acetyltransferase, acetylates lysine 16 of histone H4 and another, MSL2, which is only expressed in males, triggers its assembly. Here, we find that when a GAL4-MOF fusion protein is targeted to an upstream-activating sequence linked to a miniwhite reporter, up-regulation occurs in females but down-regulation in males, even though in the latter the whole MSL complex is recruited to the reporter genes and produces an increased histone acetylation. The expression of a GAL4-MSL2 fusion protein does not cause dosage compensation of X and autosomal reporters in females, although its expression causes the organization of the MSL complex on the reporter genes, leading to increased histone acetylation. RNAseq analysis of global endogenous gene expression in females with ectopic expression of MSL2 to coat the X chromosomes shows no evidence of increased expression compared with normal females. These data from multiple approaches indicate that the MSL complex does not mediate dosage compensation directly, but rather its activity overrides the high level of histone acetylation and counteracts the potential overexpression of X-linked genes to achieve the proper twofold up-regulation in males. PMID:23382189

  9. Global conformations of proteins as predicted from the modeling of their CZE mobility data.

    PubMed

    Deiber, Julio A; Piaggio, María V; Peirotti, Marta B

    2011-10-01

    Estimations of protein global conformations in well-specified physicochemical microenvironments are obtained through global structural parameters defined from polypeptide-scale analyses. For this purpose protein electrophoretic mobility data must be interpreted through a physicochemical CZE model to obtain estimates of protein equivalent hydrodynamic radius, effective and total charge numbers, hydration, actual ionizing pK and pH-near molecule. The electrical permittivity of protein domain is also required. In this framework, the solvent drag on proteins is obtained via the characteristic friction power coefficient associated with the number of amino acid residues defining the global chain conformation in solution. Also, the packing dimension related to the spatial distribution of amino acid residues within the protein domain is evaluated and discussed. These scaling coefficients together with the effective and total charge number fractions of proteins provide relevant interpretations of protein global conformations mainly from collapsed globule to hybrid chain regimes. Also, protein transport properties may be estimated within this framework. In this regard, the central role played by the friction power coefficient in the evaluation of these properties is highlighted.

  10. Proteome-wide analysis of lysine acetylation in the plant pathogen Botrytis cinerea

    PubMed Central

    Lv, Binna; Yang, Qianqian; Li, Delong; Liang, Wenxing; Song, Limin

    2016-01-01

    Lysine acetylation is a dynamic and reversible post-translational modification that plays an important role in diverse cellular processes. Botrytis cinerea is the most thoroughly studied necrotrophic species due to its broad host range and huge economic impact. However, to date, little is known about the functions of lysine acetylation in this plant pathogen. In this study, we determined the lysine acetylome of B. cinerea through the combination of affinity enrichment and high-resolution LC-MS/MS analysis. Overall, 1582 lysine acetylation sites in 954 proteins were identified. Bioinformatics analysis shows that the acetylated proteins are involved in diverse biological functions and show multiple cellular localizations. Several particular amino acids preferred near acetylation sites, including KacY, KacH, Kac***R, KacF, FKac and Kac***K, were identified in this organism. Protein interaction network analysis demonstrates that a variety of interactions are modulated by protein acetylation. Interestingly, 6 proteins involved in virulence of B. cinerea, including 3 key components of the high-osmolarity glycerol pathway, were found to be acetylated, suggesting that lysine acetylation plays regulatory roles in pathogenesis. These data provides the first comprehensive view of the acetylome of B. cinerea and serves as a rich resource for functional analysis of lysine acetylation in this plant pathogen. PMID:27381557

  11. Aspirin-mediated acetylation induces structural alteration and aggregation of bovine pancreatic insulin.

    PubMed

    Yousefi, Reza; Taheri, Behnaz; Alavi, Parnian; Shahsavani, Mohammad Bagher; Asadi, Zahra; Ghahramani, Maryam; Niazi, Ali; Alavianmehr, Mohammad Mehdi; Moosavi-Movahedi, Ali Akbar

    2016-01-01

    The simple aggregation of insulin under various chemical and physical stresses is still an important challenge for both pharmaceutical production and clinical formulation. In the storage form, this protein is subjected to various chemical modifications which alter its physicochemical and aggregation properties. Aspirin (acetylsalicylic acid) which is the most widely used medicine worldwide has been indicated to acetylate a large number of proteins both in vitro and in vivo. In this study, as insulin treated with aspirin at 37°C, a significant level of acetylation was observed by flourescamine and o-phthalaldehyde assay. Also, different spectroscopic techniques, gel electrophoresis, and microscopic assessment were applied to compare the structural variation and aggregation/fibrillation propensity among acetylated and non-acetylated insulin samples. The results of spectroscopic assessments elucidate that acetylation induces insulin unfolding which is accompanied with the exposure of protein hydrophobic patches, a transition from alpha-helix to beta-sheet and increased propensity of the protein for aggregation. The kinetic studies propose that acetylation increases aggregation rate of insulin under both thermal and chemical stresses. Also, gel electrophoresis and dynamic light scattering experiments suggest that acetylation induces insulin oligomerization. Additionally, the results of Thioflavin T fluorescence study, Congo red absorption assessment, and microscopic analysis suggest that acetylation with aspirin enhances the process of insulin fibrillation. Overall, the increased susceptibility of acetylated insulin for aggregation may reflect the fact that this type of modification has significant structural destabilizing effect which finally makes the protein more vulnerable for pathogenic aggregation/fibrillation.

  12. The Cation-Responsive Protein NhaR of Escherichia coli Activates pgaABCD Transcription, Required for Production of the Biofilm Adhesin Poly-β-1,6-N-Acetyl-d-Glucosamine▿

    PubMed Central

    Goller, Carlos; Wang, Xin; Itoh, Yoshikane; Romeo, Tony

    2006-01-01

    The pgaABCD operon of Escherichia coli is required for production of the biofilm adhesin poly-β-1,6-N-acetyl-d-glucosamine (PGA). We establish here that NhaR, a DNA-binding protein of the LysR family of transcriptional regulators, activates transcription of this operon. Disruption of the nhaR gene decreased biofilm formation without affecting planktonic growth. PGA production was undetectable in an nhaR mutant strain. Expression of a pgaA′-′lacZ translational fusion was induced by NaCl and alkaline pH, but not by CaCl2 or sucrose, in an nhaR-dependent fashion. Primer extension and quantitative real-time reverse transcription-PCR analyses further revealed that NhaR affects the steady-state level of pga mRNA. A purified recombinant NhaR protein bound specifically and with high affinity within the pgaABCD promoter region; one apparent binding site overlaps the −35 element, and a second site lies immediately upstream of the first. This protein was necessary and sufficient for activation of in vitro transcription from the pgaA promoter. These results define a novel mechanism for regulation of biofilm formation in response to environmental conditions and suggest an expanded role for NhaR in promoting bacterial survival. PMID:16997959

  13. Acetylation of C/EBPα inhibits its granulopoietic function

    PubMed Central

    Bararia, Deepak; Kwok, Hui Si; Welner, Robert S.; Numata, Akihiko; Sárosi, Menyhárt B.; Yang, Henry; Wee, Sheena; Tschuri, Sebastian; Ray, Debleena; Weigert, Oliver; Levantini, Elena; Ebralidze, Alexander K.; Gunaratne, Jayantha; Tenen, Daniel G.

    2016-01-01

    CCAAT/enhancer-binding protein alpha (C/EBPα) is an essential transcription factor for myeloid lineage commitment. Here we demonstrate that acetylation of C/EBPα at lysine residues K298 and K302, mediated at least in part by general control non-derepressible 5 (GCN5), impairs C/EBPα DNA-binding ability and modulates C/EBPα transcriptional activity. Acetylated C/EBPα is enriched in human myeloid leukaemia cell lines and acute myeloid leukaemia (AML) samples, and downregulated upon granulocyte-colony stimulating factor (G-CSF)- mediated granulocytic differentiation of 32Dcl3 cells. C/EBPα mutants that mimic acetylation failed to induce granulocytic differentiation in C/EBPα-dependent assays, in both cell lines and in primary hematopoietic cells. Our data uncover GCN5 as a negative regulator of C/EBPα and demonstrate the importance of C/EBPα acetylation in myeloid differentiation. PMID:27005833

  14. Recognition Imaging of Acetylated Chromatin Using a DNA Aptamer

    PubMed Central

    Lin, Liyun; Fu, Qiang; Williams, Berea A.R.; Azzaz, Abdelhamid M.; Shogren-Knaak, Michael A.; Chaput, John C.; Lindsay, Stuart

    2009-01-01

    Histone acetylation plays an important role in the regulation of gene expression. A DNA aptamer generated by in vitro selection to be highly specific for histone H4 protein acetylated at lysine 16 was used as a recognition element for atomic force microscopy-based recognition imaging of synthetic nucleosomal arrays with precisely controlled acetylation. The aptamer proved to be reasonably specific at recognizing acetylated histones, with recognition efficiencies of 60% on-target and 12% off-target. Though this selectivity is much poorer than the >2000:1 equilibrium specificity of the aptamer, it is a large improvement on the performance of a ChIP-quality antibody, which is not selective at all in this application, and it should permit high-fidelity recognition with repeated imaging. The ability to image the precise location of posttranslational modifications may permit nanometer-scale investigation of their effect on chromatin structure. PMID:19751687

  15. Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling*

    PubMed Central

    Larance, Mark; Kirkwood, Kathryn J.; Tinti, Michele; Brenes Murillo, Alejandro; Ferguson, Michael A. J.; Lamond, Angus I.

    2016-01-01

    We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754). PMID:27114452

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

    PubMed Central

    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

  17. Global multiple protein-protein interaction network alignment by combining pairwise network alignments

    PubMed Central

    2015-01-01

    Background A wealth of protein interaction data has become available in recent years, creating an urgent need for powerful analysis techniques. In this context, the problem of finding biologically meaningful correspondences between different protein-protein interaction networks (PPIN) is of particular interest. The PPIN of a species can be compared with that of other species through the process of PPIN alignment. Such an alignment can provide insight into basic problems like species evolution and network component function determination, as well as translational problems such as target identification and elucidation of mechanisms of disease spread. Furthermore, multiple PPINs can be aligned simultaneously, expanding the analytical implications of the result. While there are several pairwise network alignment algorithms, few methods are capable of multiple network alignment. Results We propose SMAL, a MNA algorithm based on the philosophy of scaffold-based alignment. SMAL is capable of converting results from any global pairwise alignment algorithms into a MNA in linear time. Using this method, we have built multiple network alignments based on combining pairwise alignments from a number of publicly available (pairwise) network aligners. We tested SMAL using PPINs of eight species derived from the IntAct repository and employed a number of measures to evaluate performance. Additionally, as part of our experimental investigations, we compared the effectiveness of SMAL while aligning up to eight input PPINs, and examined the effect of scaffold network choice on the alignments. Conclusions A key advantage of SMAL lies in its ability to create MNAs through the use of pairwise network aligners for which native MNA implementations do not exist. Experiments indicate that the performance of SMAL was comparable to that of the native MNA implementation of established methods such as IsoRankN and SMETANA. However, in terms of computational time, SMAL was significantly faster

  18. Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 4: intercellular bridges, mitochondria, nuclear envelope, apoptosis, ubiquitination, membrane/voltage-gated channels, methylation/acetylation, and transcription factors.

    PubMed

    Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

    2010-04-01

    As germ cells divide and differentiate from spermatogonia to spermatozoa, they share a number of structural and functional features that are common to all generations of germ cells and these features are discussed herein. Germ cells are linked to one another by large intercellular bridges which serve to move molecules and even large organelles from the cytoplasm of one cell to another. Mitochondria take on different shapes and features and topographical arrangements to accommodate their specific needs during spermatogenesis. The nuclear envelope and pore complex also undergo extensive modifications concomitant with the development of germ cell generations. Apoptosis is an event that is normally triggered by germ cells and involves many proteins. It occurs to limit the germ cell pool and acts as a quality control mechanism. The ubiquitin pathway comprises enzymes that ubiquitinate as well as deubiquitinate target proteins and this pathway is present and functional in germ cells. Germ cells express many proteins involved in water balance and pH control as well as voltage-gated ion channel movement. In the nucleus, proteins undergo epigenetic modifications which include methylation, acetylation, and phosphorylation, with each of these modifications signaling changes in chromatin structure. Germ cells contain specialized transcription complexes that coordinate the differentiation program of spermatogenesis, and there are many male germ cell-specific differences in the components of this machinery. All of the above features of germ cells will be discussed along with the specific proteins/genes and abnormalities to fertility related to each topic. Copyright 2009 Wiley-Liss, Inc.

  19. SDU: A Semidefinite Programming-Based Underestimation Method for Stochastic Global Optimization in Protein Docking

    PubMed Central

    Paschalidis, Ioannis Ch.; Shen, Yang; Vakili, Pirooz; Vajda, Sandor

    2007-01-01

    This paper introduces a new stochastic global optimization method targeting protein-protein docking problems, an important class of problems in computational structural biology. The method is based on finding general convex quadratic underestimators to the binding energy function that is funnel-like. Finding the optimum underestimator requires solving a semidefinite programming problem, hence the name semidefinite programming-based underestimation (SDU). The underestimator is used to bias sampling in the search region. It is established that under appropriate conditions SDU locates the global energy minimum with probability approaching one as the sample size grows. A detailed comparison of SDU with a related method of convex global underestimator (CGU), and computational results for protein-protein docking problems are provided. PMID:19759849

  20. SDU: A Semidefinite Programming-Based Underestimation Method for Stochastic Global Optimization in Protein Docking.

    PubMed

    Paschalidis, Ioannis Ch; Shen, Yang; Vakili, Pirooz; Vajda, Sandor

    2007-04-01

    This paper introduces a new stochastic global optimization method targeting protein-protein docking problems, an important class of problems in computational structural biology. The method is based on finding general convex quadratic underestimators to the binding energy function that is funnel-like. Finding the optimum underestimator requires solving a semidefinite programming problem, hence the name semidefinite programming-based underestimation (SDU). The underestimator is used to bias sampling in the search region. It is established that under appropriate conditions SDU locates the global energy minimum with probability approaching one as the sample size grows. A detailed comparison of SDU with a related method of convex global underestimator (CGU), and computational results for protein-protein docking problems are provided.

  1. Acetylation of αA-crystallin in the human lens: Effects on structure and chaperone function

    PubMed Central

    Nagaraj, Ram H.; Nahomi, Rooban B.; Shanthakumar, Shilpa; Linetsky, Mikhail; Padmanabha, Smitha; Pasupuleti, Nagarekha; Wang, Benlian; Santhoshkumar, Puttur; Panda, Alok Kumar; Biswas, Ashis

    2011-01-01

    α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens.To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a Nε-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it could affect the chaperone function of αA-crystallin. PMID:22120592

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

    PubMed

    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.

  3. The Effect of Acetyl Salicylic Acid Induced Nitric Oxide Synthesis in the Normalization of Hypertension through the Stimulation of Renal Cortexin Synthesis and by the Inhibition of Dermcidin Isoform 2, A Hypertensive Protein Production.

    PubMed

    Ghosh, Rajeshwary; Bank, Sarbashri; Maji, Uttam K; Bhattacharya, Rabindra; Guha, Santanu; Khan, Nighat N; Sinha, A Kumar

    2014-09-01

    Currently, there is no specific medication for essential hypertension (EH), a major form of the condition, in man. As acetyl salicylic acid (aspirin) is reported to stimulate the synthesis of renal (r)-cortexin, an anti-essential hypertensive protein, and, as aspirin is reported to inhibit dermcidin isoform 2 (dermcidin), a causative protein for EH, the role of aspirin in the control of EH in man was studied. Oral administration of 150 mg aspirin/70 kg body weight in subjects with EH was found to reduce both the elevated systolic and diastolic blood pressures to normal levels within 3 h due to the normalization of dermcidin level in these subjects. The plasma cortexin level at day 0, 1, 30 and 90 were 0.5 pmol/ml, 155.5 pmol/ml, 160.2 pmol/ml, 190.5 pmol/ml respectively with increased NO synthesis (r=+0.994). In vitro studies demonstrated that the incubation of the goat kidney cortex cells with aspirin stimulated (r)-cortexin synthesis due to NO synthesis. It could be suggested that the use of aspirin might control EH in man.

  4. The Effect of Acetyl Salicylic Acid Induced Nitric Oxide Synthesis in the Normalization of Hypertension through the Stimulation of Renal Cortexin Synthesis and by the Inhibition of Dermcidin Isoform 2, A Hypertensive Protein Production

    PubMed Central

    Ghosh, Rajeshwary; Bank, Sarbashri; Maji, Uttam K.; Bhattacharya, Rabindra; Guha, Santanu; Khan, Nighat N.; Sinha, A. Kumar

    2014-01-01

    Currently, there is no specific medication for essential hypertension (EH), a major form of the condition, in man. As acetyl salicylic acid (aspirin) is reported to stimulate the synthesis of renal (r)-cortexin, an anti-essential hypertensive protein, and, as aspirin is reported to inhibit dermcidin isoform 2 (dermcidin), a causative protein for EH, the role of aspirin in the control of EH in man was studied. Oral administration of 150 mg aspirin/70 kg body weight in subjects with EH was found to reduce both the elevated systolic and diastolic blood pressures to normal levels within 3 h due to the normalization of dermcidin level in these subjects. The plasma cortexin level at day 0, 1, 30 and 90 were 0.5 pmol/ml, 155.5 pmol/ml, 160.2 pmol/ml, 190.5 pmol/ml respectively with increased NO synthesis (r=+0.994). In vitro studies demonstrated that the incubation of the goat kidney cortex cells with aspirin stimulated (r)-cortexin synthesis due to NO synthesis. It could be suggested that the use of aspirin might control EH in man. PMID:25324696

  5. In the absence of cellular poly (A) binding protein, the glycolytic enzyme GAPDH translocated to the cell nucleus and activated the GAPDH mediated apoptotic pathway by enhancing acetylation and serine 46 phosphorylation of p53

    SciTech Connect

    Thangima Zannat, Mst.; Bhattacharjee, Rumpa B.; Bag, Jnanankur

    2011-06-03

    Highlights: {yields} PABP knock down and cell apoptosis. {yields} Nuclear translocation of GAPDH in PABP depleted cells. {yields} Role of p53 in apoptosis of PABP depleted cells. {yields} Bax translocation and cytochrome C release and caspase 3 activation following PABP depletion. {yields} Association of p53 with Bcl2 and Bax. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) interacts with 3' poly (A) tract of eukaryotic mRNA and is important for both translation and stability of mRNA. Previously, we have shown that depletion of PABP by siRNA prevents protein synthesis and consequently leads to cell death through apoptosis. In the present investigation, we studied the mechanism of cell apoptosis. We show that in the absence of PABP, the glycolytic enzyme GAPDH translocated to the cell nucleus and activated the GAPDH mediated apoptotic pathway by enhancing acetylation and serine 46 phosphorylation of p53. As a result, p53 translocated to the mitochondria to initiate Bax mediated apoptosis.

  6. Smad Acetylation: A New Level of Regulation in TGF-Beta Signaling

    DTIC Science & Technology

    2007-07-01

    AD_________________ Award Number: W81XWH-04-1-0357 TITLE: Smad Acetylation : A New Level of...TYPE Annual Summary 3. DATES COVERED (From - To) 1 JUL 2004 - 30 JUN 2007 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Smad Acetylation : A New...proposal suggests a series of experiments designed to study the acetylation of Smad proteins. We have determined that Smad2 can be efficiently

  7. ASEB: a web server for KAT-specific acetylation site prediction.

    PubMed

    Wang, Likun; Du, Yipeng; Lu, Ming; Li, Tingting

    2012-07-01

    Protein lysine acetylation plays an important role in the normal functioning of cells, including gene expression regulation, protein stability and metabolism regulation. Although large amounts of lysine acetylation sites have been identified via large-scale mass spectrometry or traditional experimental methods, the lysine (K)-acetyl-transferase (KAT) responsible for the acetylation of a given protein or lysine site remains largely unknown due to the experimental limitations of KAT substrate identification. Hence, the in silico prediction of KAT-specific acetylation sites may provide direction for further experiments. In our previous study, we developed the acetylation set enrichment based (ASEB) computer program to predict which KAT-families are responsible for the acetylation of a given protein or lysine site. In this article, we provide KAT-specific acetylation site prediction as a web service. This web server not only provides the online tool and R package for the method in our previous study, but several useful services are also included, such as the integration of protein-protein interaction information to enhance prediction accuracy. This web server can be freely accessed at http://cmbi.bjmu.edu.cn/huac.

  8. Trends in global warming and evolution of matrix protein 2 family from influenza A virus.

    PubMed

    Yan, Shao-Min; Wu, Guang

    2009-12-01

    The global warming is an important factor affecting the biological evolution, and the influenza is an important disease that threatens humans with possible epidemics or pandemics. In this study, we attempted to analyze the trends in global warming and evolution of matrix protein 2 family from influenza A virus, because this protein is a target of anti-flu drug, and its mutation would have significant effect on the resistance to anti-flu drugs. The evolution of matrix protein 2 of influenza A virus from 1959 to 2008 was defined using the unpredictable portion of amino-acid pair predictability. Then the trend in this evolution was compared with the trend in the global temperature, the temperature in north and south hemispheres, and the temperature in influenza A virus sampling site, and species carrying influenza A virus. The results showed the similar trends in global warming and in evolution of M2 proteins although we could not correlate them at this stage of study. The study suggested the potential impact of global warming on the evolution of proteins from influenza A virus.

  9. Global analysis of the glycoproteome in Saccharomyces cerevisiae reveals new roles for protein glycosylation in eukaryotes

    PubMed Central

    Kung, Li A; Tao, Sheng-Ce; Qian, Jiang; Smith, Michael G; Snyder, Michael; Zhu, Heng

    2009-01-01

    To further understand the roles of protein glycosylation in eukaryotes, we globally identified glycan-containing proteins in yeast. A fluorescent lectin binding assay was developed and used to screen protein microarrays containing over 5000 proteins purified from yeast. A total of 534 yeast proteins were identified that bound either Concanavalin A (ConA) or Wheat-Germ Agglutinin (WGA); 406 of them were novel. Among the novel glycoproteins, 45 were validated by mobility shift upon treatment with EndoH and PNGase F, thereby extending the number of validated yeast glycoproteins to 350. In addition to many components of the secretory pathway, we identified other types of proteins, such as transcription factors and mitochondrial proteins. To further explore the role of glycosylation in mitochondrial function, the localization of four mitochondrial proteins was examined in the presence and absence of tunicamycin, an inhibitor of N-linked protein glycosylation. For two proteins, localization to the mitochondria is diminished upon tunicamycin treatment, indicating that protein glycosylation is important for protein function. Overall, our studies greatly extend our understanding of protein glycosylation in eukaryotes through the cataloguing of glycoproteins, and describe a novel role for protein glycosylation in mitochondrial protein function and localization. PMID:19756047

  10. Native state interconversion of a metamorphic protein requires global unfolding

    PubMed Central

    Tyler, Robert C.; Murray, Nathan J.; Peterson, Francis C.; Volkman, Brian F.

    2011-01-01

    Lymphotactin (Ltn) is a unique chemokine that under physiological solution conditions displays large-scale structural heterogeneity, defining a new category of ‘metamorphic proteins’. Previous Ltn studies have indicated that each form is required for proper function, but the mechanism of interconversion remains unknown. Here we have investigated the temperature dependence of kinetic rates associated with interconversion and unfolding by stopped-flow fluorescence to determine transition-state free energies. Comparisons of derived thermodynamic parameters revealed striking similarities between interconversion and protein unfolding. We conclude that Ltn native state rearrangement proceeds by way of a large-scale unfolding process rather than a unique intermediate structure. PMID:21776971

  11. Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs.

    PubMed

    Smits, Arne H; Lindeboom, Rik G H; Perino, Matteo; van Heeringen, Simon J; Veenstra, Gert Jan C; Vermeulen, Michiel

    2014-09-01

    While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs.

  12. Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs

    PubMed Central

    Smits, Arne H.; Lindeboom, Rik G.H.; Perino, Matteo; van Heeringen, Simon J.; Veenstra, Gert Jan C.; Vermeulen, Michiel

    2014-01-01

    While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs. PMID:25056316

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

    PubMed Central

    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

  14. Sirtuin-dependent reversible lysine acetylation of glutamine synthetases reveals an autofeedback loop in nitrogen metabolism

    PubMed Central

    You, Di; Yin, Bin-Cheng; Li, Zhi-Hai; Zhou, Ying; Yu, Wen-Bang; Zuo, Peng; Ye, Bang-Ce

    2016-01-01

    In cells of all domains of life, reversible lysine acetylation modulates the function of proteins involved in central cellular processes such as metabolism. In this study, we demonstrate that the nitrogen regulator GlnR of the actinomycete Saccharopolyspora erythraea directly regulates transcription of the acuA gene (SACE_5148), which encodes a Gcn5-type lysine acetyltransferase. We found that AcuA acetylates two glutamine synthetases (GlnA1 and GlnA4) and that this lysine acetylation inactivated GlnA4 (GSII) but had no significant effect on GlnA1 (GSI-β) activity under the conditions tested. Instead, acetylation of GlnA1 led to a gain-of-function that modulated its interaction with the GlnR regulator and enhanced GlnR–DNA binding. It was observed that this regulatory function of acetylated GSI-β enzymes is highly conserved across actinomycetes. In turn, GlnR controls the catalytic and regulatory activities (intracellular acetylation levels) of glutamine synthetases at the transcriptional and posttranslational levels, indicating an autofeedback loop that regulates nitrogen metabolism in response to environmental change. Thus, this GlnR-mediated acetylation pathway provides a signaling cascade that acts from nutrient sensing to acetylation of proteins to feedback regulation. This work presents significant new insights at the molecular level into the mechanisms underlying the regulation of protein acetylation and nitrogen metabolism in actinomycetes. PMID:27247389

  15. Protein Structure and Evolution: Are They Constrained Globally by a Principle Derived from Information Theory?

    PubMed Central

    Hatton, Leslie; Warr, Gregory

    2015-01-01

    That the physicochemical properties of amino acids constrain the structure, function and evolution of proteins is not in doubt. However, principles derived from information theory may also set bounds on the structure (and thus also the evolution) of proteins. Here we analyze the global properties of the full set of proteins in release 13-11 of the SwissProt database, showing by experimental test of predictions from information theory that their collective structure exhibits properties that are consistent with their being guided by a conservation principle. This principle (Conservation of Information) defines the global properties of systems composed of discrete components each of which is in turn assembled from discrete smaller pieces. In the system of proteins, each protein is a component, and each protein is assembled from amino acids. Central to this principle is the inter-relationship of the unique amino acid count and total length of a protein and its implications for both average protein length and occurrence of proteins with specific unique amino acid counts. The unique amino acid count is simply the number of distinct amino acids (including those that are post-translationally modified) that occur in a protein, and is independent of the number of times that the particular amino acid occurs in the sequence. Conservation of Information does not operate at the local level (it is independent of the physicochemical properties of the amino acids) where the influences of natural selection are manifest in the variety of protein structure and function that is well understood. Rather, this analysis implies that Conservation of Information would define the global bounds within which the whole system of proteins is constrained; thus it appears to be acting to constrain evolution at a level different from natural selection, a conclusion that appears counter-intuitive but is supported by the studies described herein. PMID:25970335

  16. Protein structure and evolution: are they constrained globally by a principle derived from information theory?

    PubMed

    Hatton, Leslie; Warr, Gregory

    2015-01-01

    That the physicochemical properties of amino acids constrain the structure, function and evolution of proteins is not in doubt. However, principles derived from information theory may also set bounds on the structure (and thus also the evolution) of proteins. Here we analyze the global properties of the full set of proteins in release 13-11 of the SwissProt database, showing by experimental test of predictions from information theory that their collective structure exhibits properties that are consistent with their being guided by a conservation principle. This principle (Conservation of Information) defines the global properties of systems composed of discrete components each of which is in turn assembled from discrete smaller pieces. In the system of proteins, each protein is a component, and each protein is assembled from amino acids. Central to this principle is the inter-relationship of the unique amino acid count and total length of a protein and its implications for both average protein length and occurrence of proteins with specific unique amino acid counts. The unique amino acid count is simply the number of distinct amino acids (including those that are post-translationally modified) that occur in a protein, and is independent of the number of times that the particular amino acid occurs in the sequence. Conservation of Information does not operate at the local level (it is independent of the physicochemical properties of the amino acids) where the influences of natural selection are manifest in the variety of protein structure and function that is well understood. Rather, this analysis implies that Conservation of Information would define the global bounds within which the whole system of proteins is constrained; thus it appears to be acting to constrain evolution at a level different from natural selection, a conclusion that appears counter-intuitive but is supported by the studies described herein.

  17. Are Proteins Just Coiled Cords? Local and Global Analysis of Contact Maps Reveals the Backbone-Dependent Nature of Proteins.

    PubMed

    Santoni, Daniele; Paci, Paola; Di Paola, Luisa; Giuliani, Alessandro

    2016-01-01

    In this work, we present an extensive analysis of protein contact network topology applied to a wide data set. We extended the concept of degree distribution to graphlets, describing local connectivity patterns. We compared results to those derived from artificial networks of the same size (number of nodes), reproducing the average degree of each protein network. The artificial networks resemble the coiling of immaterial cords and we tried to understand if they could catch the protein structure topology upon the sole constraint of backbone (cord). We found a surprisingly similar pattern for local topological descriptors (graphlets distribution) while real proteins and cords differ at large extent in the global topological invariant average shortest path that presumably catches the systemic nature of protein and the non negligible encumbrance of backbone (residues steric hindrance). We demonstrated average shortest path to link polymer length and physical size of the molecule, and its minimization plays the role of `target function` of folding process.

  18. Acetylation mimic of lysine 280 exacerbates human Tau neurotoxicity in vivo

    PubMed Central

    Gorsky, Marianna Karina; Burnouf, Sylvie; Dols, Jacqueline; Mandelkow, Eckhard; Partridge, Linda

    2016-01-01

    Dysfunction and accumulation of the microtubule-associated human Tau (hTau) protein into intraneuronal aggregates is observed in many neurodegenerative disorders including Alzheimer’s disease (AD). Reversible lysine acetylation has recently emerged as a post-translational modification that may play an important role in the modulation of hTau pathology. Acetylated hTau species have been observed within hTau aggregates in human AD brains and multi-acetylation of hTau in vitro regulates its propensity to aggregate. However, whether lysine acetylation at position 280 (K280) modulates hTau-induced toxicity in vivo is unknown. We generated new Drosophila transgenic models of hTau pathology to evaluate the contribution of K280 acetylation to hTau toxicity, by analysing the respective toxicity of pseudo-acetylated (K280Q) and pseudo-de-acetylated (K280R) mutant forms of hTau. We observed that mis-expression of pseudo-acetylated K280Q-hTau in the adult fly nervous system potently exacerbated fly locomotion defects and photoreceptor neurodegeneration. In addition, modulation of K280 influenced total hTau levels and phosphorylation without changing hTau solubility. Altogether, our results indicate that pseudo-acetylation of the single K280 residue is sufficient to exacerbate hTau neurotoxicity in vivo, suggesting that acetylated K280-hTau species contribute to the pathological events leading to neurodegeneration in AD. PMID:26940749

  19. Standardised extract of Bacopa monniera (CDRI-08) improves contextual fear memory by differentially regulating the activity of histone acetylation and protein phosphatases (PP1α, PP2A) in hippocampus.

    PubMed

    Preethi, Jayakumar; Singh, Hemant K; Venkataraman, Jois Shreyas; Rajan, Koilmani Emmanuvel

    2014-05-01

    Contextual fear conditioning is a paradigm for investigating cellular mechanisms involved in hippocampus-dependent memory. Earlier, we showed that standardised extract of Bacopa monniera (CDRI-08) improves hippocampus-dependent learning in postnatal rats by elevating the level of serotonin (5-hydroxytryptamine, 5-HT), activate 5-HT3A receptors, and cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein. In this study, we have further examined the molecular mechanism of CDRI-08 in hippocampus-dependent memory and compared to the histone deacetylase (HDACs) inhibitor sodium butyrate (NaB). To assess the hippocampus-dependent memory, wistar rat pups were subjected to contextual fear conditioning (CFC) following daily (postnatal days 15-29) administration of vehicle solution (0.5 % gum acacia + 0.9 % saline)/CDRI-08 (80 mg/kg, p.o.)/NaB (1.2 g/kg in PBS, i.p.). CDRI-08/NaB treated group showed enhanced freezing behavior compared to control group when re-exposed to the same context. Administration of CDRI-08/NaB resulted in activation of extracellular signal-regulated kinase ERK/CREB signaling cascade and up-regulation of p300, Ac-H3 and Ac-H4 levels, and down-regulation of HDACs (1, 2) and protein phosphatases (PP1α, PP2A) in hippocampus following CFC. This would subsequently result in an increased brain-derived neurotrophic factor (Bdnf) (exon IV) mRNA in hippocampus. Altogether, our results indicate that CDRI-08 enhances hippocampus-dependent contextual memory by differentially regulating histone acetylation and protein phosphatases in hippocampus.

  20. An acetylation rheostat for the control of muscle energy homeostasis

    PubMed Central

    Menzies, Keir; Auwerx, Johan

    2013-01-01

    In recent years the role of acetylation has gained ground as an essential modulator of intermediary metabolism in skeletal muscle. Imbalance in energy homeostasis or chronic cellular stress, due to diet, aging or disease, translate into alterations in the acetylation levels of key proteins which governs bioenergetics, cellular substrate use and/or changes in mitochondrial content and function. For example, cellular stress induced by exercise or caloric restriction can alter the coordinated activity of acetyltransferases and deacetylases to increase mitochondrial biogenesis and function in order to adapt to low energetic levels. The natural duality of these enzymes, as metabolic sensors and effector proteins, have helped biologists understand how the body can integrate seemingly distinct signaling pathways to control mitochondrial biogenesis, insulin sensitivity, glucose transport, reactive oxygen species handling, angiogenesis and muscle satellite cell proliferation/differentiation. Our review will summarize the recent developments related to acetylation dependent responses following metabolic stress in skeletal muscle. PMID:23999889

  1. Assays for Acetylation and Other Acylations of Lysine Residues.

    PubMed

    Pelletier, Nadine; Grégoire, Serge; Yang, Xiang-Jiao

    2017-02-02

    Lysine acetylation refers to addition of an acetyl moiety to the epsilon-amino group of a lysine residue and is important for regulating protein functions in various organisms from bacteria to humans. This is a reversible and precisely controlled covalent modification that either serves as an on/off switch or participates in a codified manner with other post-translational modifications to regulate different cellular and developmental processes in normal and pathological states. This unit describes methods for in vitro and in vivo determination of lysine acetylation. Such methods can be easily extended for analysis of other acylations (such as propionylation, butyrylation, crotonylation, and succinylation) that are also present in histones and many other proteins. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  2. An acetylation rheostat for the control of muscle energy homeostasis.

    PubMed

    Menzies, Keir; Auwerx, Johan

    2013-12-01

    In recent years, the role of acetylation has gained ground as an essential modulator of intermediary metabolism in skeletal muscle. Imbalance in energy homeostasis or chronic cellular stress, due to diet, aging, or disease, translate into alterations in the acetylation levels of key proteins which govern bioenergetics, cellular substrate use, and/or changes in mitochondrial content and function. For example, cellular stress induced by exercise or caloric restriction can alter the coordinated activity of acetyltransferases and deacetylases to increase mitochondrial biogenesis and function in order to adapt to low energetic levels. The natural duality of these enzymes, as metabolic sensors and effector proteins, has helped biologists to understand how the body can integrate seemingly distinct signaling pathways to control mitochondrial biogenesis, insulin sensitivity, glucose transport, reactive oxygen species handling, angiogenesis, and muscle satellite cell proliferation/differentiation. Our review will summarize the recent developments related to acetylation-dependent responses following metabolic stress in skeletal muscle.

  3. Local-global alignment for finding 3D similarities in protein structures

    DOEpatents

    Zemla, Adam T [Brentwood, CA

    2011-09-20

    A method of finding 3D similarities in protein structures of a first molecule and a second molecule. The method comprises providing preselected information regarding the first molecule and the second molecule. Comparing the first molecule and the second molecule using Longest Continuous Segments (LCS) analysis. Comparing the first molecule and the second molecule using Global Distance Test (GDT) analysis. Comparing the first molecule and the second molecule using Local Global Alignment Scoring function (LGA_S) analysis. Verifying constructed alignment and repeating the steps to find the regions of 3D similarities in protein structures.

  4. Alanine and proline content modulate global sensitivity to discrete perturbations in disordered proteins.

    PubMed

    Perez, Romel B; Tischer, Alexander; Auton, Matthew; Whitten, Steven T

    2014-12-01

    Molecular transduction of biological signals is understood primarily in terms of the cooperative structural transitions of protein macromolecules, providing a mechanism through which discrete local structure perturbations affect global macromolecular properties. The recognition that proteins lacking tertiary stability, commonly referred to as intrinsically disordered proteins (IDPs), mediate key signaling pathways suggests that protein structures without cooperative intramolecular interactions may also have the ability to couple local and global structure changes. Presented here are results from experiments that measured and tested the ability of disordered proteins to couple local changes in structure to global changes in structure. Using the intrinsically disordered N-terminal region of the p53 protein as an experimental model, a set of proline (PRO) and alanine (ALA) to glycine (GLY) substitution variants were designed to modulate backbone conformational propensities without introducing non-native intramolecular interactions. The hydrodynamic radius (R(h)) was used to monitor changes in global structure. Circular dichroism spectroscopy showed that the GLY substitutions decreased polyproline II (PP(II)) propensities relative to the wild type, as expected, and fluorescence methods indicated that substitution-induced changes in R(h) were not associated with folding. The experiments showed that changes in local PP(II) structure cause changes in R(h) that are variable and that depend on the intrinsic chain propensities of PRO and ALA residues, demonstrating a mechanism for coupling local and global structure changes. Molecular simulations that model our results were used to extend the analysis to other proteins and illustrate the generality of the observed PRO and alanine effects on the structures of IDPs.

  5. Nε-lysine acetylation determines dissociation from GAP junctions and lateralization of connexin 43 in normal and dystrophic heart

    PubMed Central

    Colussi, Claudia; Rosati, Jessica; Straino, Stefania; Spallotta, Francesco; Berni, Roberta; Stilli, Donatella; Rossi, Stefano; Musso, Ezio; Macchi, Emilio; Mai, Antonello; Sbardella, Gianluca; Castellano, Sabrina; Chimenti, Cristina; Frustaci, Andrea; Nebbioso, Angela; Altucci, Lucia; Capogrossi, Maurizio C.; Gaetano, Carlo

    2011-01-01

    Wanting to explore the epigenetic basis of Duchenne cardiomyopathy, we found that global histone acetylase activity was abnormally elevated and the acetylase P300/CBP-associated factor (PCAF) coimmunoprecipitated with connexin 43 (Cx43), which was Nε-lysine acetylated and lateralized in mdx heart. This observation was paralleled by Cx43 dissociation from N-cadherin and zonula occludens 1, whereas pp60-c-Src association was unaltered. In vivo treatment of mdx with the pan-histone acetylase inhibitor anacardic acid significantly reduced Cx43 Nε-lysine acetylation and restored its association to GAP junctions (GJs) at intercalated discs. Noteworthy, in normal as well as mdx mice, the class IIa histone deacetylases 4 and 5 constitutively colocalized with Cx43 either at GJs or in the lateralized compartments. The class I histone deacetylase 3 was also part of the complex. Treatment of normal controls with the histone deacetylase pan-inhibitor suberoylanilide hydroxamic acid (MC1568) or the class IIa-selective inhibitor 3-{4-[3-(3-fluorophenyl)-3-oxo-1-propen-1-yl]-1-methyl-1H-pyrrol-2-yl}-N-hydroxy-2-propenamide (MC1568) determined Cx43 hyperacetylation, dissociation from GJs, and distribution along the long axis of ventricular cardiomyocytes. Consistently, the histone acetylase activator pentadecylidenemalonate 1b (SPV106) hyperacetylated cardiac proteins, including Cx43, which assumed a lateralized position that partly reproduced the dystrophic phenotype. In the presence of suberoylanilide hydroxamic acid, cell to cell permeability was significantly diminished, which is in agreement with a Cx43 close conformation in the consequence of hyperacetylation. Additional experiments, performed with Cx43 acetylation mutants, revealed, for the acetylated form of the molecule, a significant reduction in plasma membrane localization and a tendency to nuclear accumulation. These results suggest that Cx43 Nε-lysine acetylation may have physiopathological consequences for cell to

  6. Fatal Intoxication with Acetyl Fentanyl.

    PubMed

    Cunningham, Susan M; Haikal, Nabila A; Kraner, James C

    2016-01-01

    Among the new psychoactive substances encountered in forensic investigations is the opioid, acetyl fentanyl. The death of a 28-year-old man from recreational use of this compound is reported. The decedent was found in the bathroom of his residence with a tourniquet secured around his arm and a syringe nearby. Postmortem examination findings included marked pulmonary and cerebral edema and needle track marks. Toxicological analysis revealed acetyl fentanyl in subclavian blood, liver, vitreous fluid, and urine at concentrations of 235 ng/mL, 2400 ng/g, 131 ng/mL, and 234 ng/mL, respectively. Acetyl fentanyl was also detected in the accompanying syringe. Death was attributed to recreational acetyl fentanyl abuse, likely through intravenous administration. The blood acetyl fentanyl concentration is considerably higher than typically found in fatal fentanyl intoxications. Analysis of this case underscores the need for consideration of a wide range of compounds with potential opioid-agonist activity when investigating apparent recreational drug-related deaths. © 2015 American Academy of Forensic Sciences.

  7. Acetylome analysis reveals the involvement of lysine acetylation in diverse biological processes in Phytophthora sojae

    PubMed Central

    Li, Delong; Lv, Binna; Tan, Lingling; Yang, Qianqian; Liang, Wenxing

    2016-01-01

    Lysine acetylation is a dynamic and highly conserved post-translational modification that plays an important regulatory role in almost every aspects of cell metabolism in both eukaryotes and prokaryotes. Phytophthora sojae is one of the most important plant pathogens due to its huge economic impact. However, to date, little is known about the functions of lysine acetylation in this Phytopthora. Here, we conducted a lysine acetylome in P. sojae. Overall, 2197 lysine acetylation sites in 1150 proteins were identified. The modified proteins are involved in diverse biological processes and are localized to multiple cellular compartments. Importantly, 7 proteins involved in the pathogenicity or the secretion pathway of P. sojae were found to be acetylated. These data provide the first comprehensive view of the acetylome of P. sojae and serve as an important resource for functional analysis of lysine acetylation in plant pathogens. PMID:27412925

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

    PubMed

    Choudhary, Chunaram; Weinert, Brian T; Nishida, Yuya; Verdin, Eric; Mann, Matthias

    2014-08-01

    Lysine acetylation is a conserved protein post-translational modification that links acetyl-coenzyme A metabolism and cellular signalling. Recent advances in the identification and quantification of lysine acetylation by mass spectrometry have increased our understanding of lysine acetylation, 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 and crotonylation. The intricate link between lysine acylation and cellular metabolism has been clarified by the occurrence of several such metabolite-sensitive acylations and their selective removal by sirtuin deacylases. These emerging findings point to new functions for different lysine acylations and deacylating enzymes and also highlight the mechanisms by which acetylation regulates various cellular processes.

  9. Acetylome analysis reveals the involvement of lysine acetylation in diverse biological processes in Phytophthora sojae.

    PubMed

    Li, Delong; Lv, Binna; Tan, Lingling; Yang, Qianqian; Liang, Wenxing

    2016-07-14

    Lysine acetylation is a dynamic and highly conserved post-translational modification that plays an important regulatory role in almost every aspects of cell metabolism in both eukaryotes and prokaryotes. Phytophthora sojae is one of the most important plant pathogens due to its huge economic impact. However, to date, little is known about the functions of lysine acetylation in this Phytopthora. Here, we conducted a lysine acetylome in P. sojae. Overall, 2197 lysine acetylation sites in 1150 proteins were identified. The modified proteins are involved in diverse biological processes and are localized to multiple cellular compartments. Importantly, 7 proteins involved in the pathogenicity or the secretion pathway of P. sojae were found to be acetylated. These data provide the first comprehensive view of the acetylome of P. sojae and serve as an important resource for functional analysis of lysine acetylation in plant pathogens.

  10. Lysine Acetylation of CREBH Regulates Fasting-Induced Hepatic Lipid Metabolism.

    PubMed

    Kim, Hyunbae; Mendez, Roberto; Chen, Xuequn; Fang, Deyu; Zhang, Kezhong

    2015-12-01

    Cyclic AMP-responsive element-binding protein 3-like 3, hepatocyte specific (CREBH), is a hepatic transcription factor that functions as a key regulator of energy homeostasis. Here, we defined a regulatory CREBH posttranslational modification process, namely, lysine-specific acetylation, and its functional involvement in fasting-induced hepatic lipid metabolism. Fasting induces CREBH acetylation in mouse livers in a time-dependent manner, and this event is critical for CREBH transcriptional activity in regulating hepatic lipid homeostasis. The histone acetyltransferase PCAF-mediated acetylation and the deacetylase sirtuin-1-mediated deacetylation coexist to maintain CREBH acetylation states under fasting conditions. Site-directed mutagenesis and functional analyses revealed that the lysine (K) residue at position 294 (K294) within the bZIP domain of the CREBH protein is the site where fasting-induced acetylation/deacetylation occurs. Introduction of the acetylation-deficient (K294R) or acetylation-mimicking (K294Q) mutation inhibited or enhanced CREBH transcriptional activity, respectively. Importantly, CREBH acetylation at lysine 294 was required for the interaction and synergy between CREBH and peroxisome proliferator-activated receptor α (PPARα) in activating their target genes upon fasting or glucagon stimulation. Introduction of the CREBH lysine 294 mutation in the liver leads to hepatic steatosis and hyperlipidemia in animals under prolonged fasting. In summary, our study reveals a molecular mechanism by which fasting or glucagon stimulation modulates lipid homeostasis through acetylation of CREBH. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. Lysine Acetylation of CREBH Regulates Fasting-Induced Hepatic Lipid Metabolism

    PubMed Central

    Kim, Hyunbae; Mendez, Roberto; Chen, Xuequn; Fang, Deyu

    2015-01-01

    Cyclic AMP-responsive element-binding protein 3-like 3, hepatocyte specific (CREBH), is a hepatic transcription factor that functions as a key regulator of energy homeostasis. Here, we defined a regulatory CREBH posttranslational modification process, namely, lysine-specific acetylation, and its functional involvement in fasting-induced hepatic lipid metabolism. Fasting induces CREBH acetylation in mouse livers in a time-dependent manner, and this event is critical for CREBH transcriptional activity in regulating hepatic lipid homeostasis. The histone acetyltransferase PCAF-mediated acetylation and the deacetylase sirtuin-1-mediated deacetylation coexist to maintain CREBH acetylation states under fasting conditions. Site-directed mutagenesis and functional analyses revealed that the lysine (K) residue at position 294 (K294) within the bZIP domain of the CREBH protein is the site where fasting-induced acetylation/deacetylation occurs. Introduction of the acetylation-deficient (K294R) or acetylation-mimicking (K294Q) mutation inhibited or enhanced CREBH transcriptional activity, respectively. Importantly, CREBH acetylation at lysine 294 was required for the interaction and synergy between CREBH and peroxisome proliferator-activated receptor α (PPARα) in activating their target genes upon fasting or glucagon stimulation. Introduction of the CREBH lysine 294 mutation in the liver leads to hepatic steatosis and hyperlipidemia in animals under prolonged fasting. In summary, our study reveals a molecular mechanism by which fasting or glucagon stimulation modulates lipid homeostasis through acetylation of CREBH. PMID:26438600

  12. Observed surface lysine acetylation of human carbonic anhydrase II expressed in Escherichia coli

    PubMed Central

    Mahon, Brian P; Lomelino, Carrie L; Salguero, Antonieta L; Driscoll, Jenna M; Pinard, Melissa A; McKenna, Robert

    2015-01-01

    Acetylation of surface lysine residues of proteins has been observed in Escherichia coli (E. coli), an organism that has been extensively utilized for recombinant protein expression. This post-translational modification is shown to be important in various processes such as metabolism, stress-response, transcription, and translation. As such, utilization of E. coli expression systems for protein production may yield non-native acetylation events of surface lysine residues. Here we present the crystal structures of wild-type and a variant of human carbonic anhydrase II (hCA II) that have been expressed in E. coli and exhibit surface lysine acetylation and we speculate on the effect this has on the conformational stability of each enzyme. Both structures were determined to 1.6 Å resolution and show clear electron density for lysine acetylation. The lysine acetylation does not distort the structure and the surface lysine acetylation events most likely do not interfere with the biological interpretation. However, there is a reduction in conformational stability in the hCA II variant compared to wild type (∼4°C decrease). This may be due to other lysine acetylation events that have occurred but are not visible in the crystal structure due to intrinsic disorder. Therefore, surface lysine acetylation events may affect overall protein stability and crystallization, and should be considered when using E. coli expression systems. PMID:26266677

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

    PubMed Central

    Wagner, Gregory R.; Hirschey, Matthew D.

    2014-01-01

    Cellular proteins are decorated with a wide range of acetyl and other acyl modifications. Many studies have demonstrated regulation of site-specific acetylation by acetyltransferases and deacetylases. Acylation is emerging as a new type of lysine modification, but less is known about its overall regulatory role. Furthermore, the mechanisms of lysine acylation, its overlap with protein acetylation, and how it influences cellular function are major unanswered questions in the field. In this review, we discuss the known roles of acetyltransferases and deacetylases, and the sirtuins as a conserved family of NAD+-dependent protein deacylases that are important for response to cellular stress and homeostasis. We also consider the evidence for an emerging idea of non-enzymatic protein acylation. Finally, we put forward the hypothesis that protein acylation is a form of protein “carbon stress”, that the deacylases evolved to remove as a part of a global protein quality control network. PMID:24725594

  14. Temporal Regulation of the Bacillus subtilis Acetylome and Evidence for a Role of MreB Acetylation in Cell Wall Growth

    PubMed Central

    Carabetta, Valerie J.; Greco, Todd M.; Tanner, Andrew W.

    2016-01-01

    ABSTRACT Nε-Lysine acetylation has been recognized as a ubiquitous regulatory posttranslational modification that influences a variety of important biological processes in eukaryotic cells. Recently, it has been realized that acetylation is also prevalent in bacteria. Bacteria contain hundreds of acetylated proteins, with functions affecting diverse cellular pathways. Still, little is known about the regulation or biological relevance of nearly all of these modifications. Here we characterize the cellular growth-associated regulation of the Bacillus subtilis acetylome. Using acetylation enrichment and quantitative mass spectrometry, we investigate the logarithmic and stationary growth phases, identifying over 2,300 unique acetylation sites on proteins that function in essential cellular pathways. We determine an acetylation motif, EK(ac)(D/Y/E), which resembles the eukaryotic mitochondrial acetylation signature, and a distinct stationary-phase-enriched motif. By comparing the changes in acetylation with protein abundances, we discover a subset of critical acetylation events that are temporally regulated during cell growth. We functionally characterize the stationary-phase-enriched acetylation on the essential shape-determining protein MreB. Using bioinformatics, mutational analysis, and fluorescence microscopy, we define a potential role for the temporal acetylation of MreB in restricting cell wall growth and cell diameter. IMPORTANCE The past decade highlighted Nε-lysine acetylation as a prevalent posttranslational modification in bacteria. However, knowledge regarding the physiological importance and temporal regulation of acetylation has remained limited. To uncover potential regulatory roles for acetylation, we analyzed how acetylation patterns and abundances change between growth phases in B. subtilis. To demonstrate that the identification of cell growth-dependent modifications can point to critical regulatory acetylation events, we further characterized

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

    SciTech Connect

    Saare, Mario; Rebane, Ana; Rajashekar, Balaji; Vilo, Jaak; Peterson, Paert

    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

  16. Regulation of global protein translation and protein degradation in aerobic dormancy.

    PubMed

    Ramnanan, Christopher J; Allan, Marcus E; Groom, Amy G; Storey, Kenneth B

    2009-03-01

    We hypothesized that protein turnover would be substantially suppressed during estivation in the land snail, Otala lactea, as part of a wholesale move to conserve ATP in the hypometabolic state, and that decreased rates of protein synthesis and degradation would be mediated by altering the phosphorylation state of key proteins. Rates of protein translation, measured in vitro, decreased by approximately 80% in extracts of foot muscle and hepatopancreas after 2 days of estivation, and this reduction was associated with strong increases in the phosphorylation of ribosomal factors, eIF2 alpha and eEF2, as well as decreased phosphorylation of 4E-BP1. Reductions in levels of markers of ribosomal biogenesis and a tissue-specific reduction in the phosphorylation state of eIF4E and eIF4GI were also evident after 14 days of estivation. Activity of the 20S proteasome decreased by 60-80% after 2 days of estivation and this decrease was mediated by protein kinase G in vitro, whereas protein phosphatase 2A activated the proteasome. Levels of protein carbonyls did not change in snail tissues during estivation whereas the expression heat shock proteins increased, suggesting that protein resistance to damage is enhanced in estivation. In conclusion, protein synthesis and degradation rates were coordinately suppressed during estivation in O. lactea and this is associated with the phosphorylation of ribosomal initiation and elongation factors and the 20S proteasome.

  17. Protein-energy malnutrition alters hippocampal plasticity-associated protein expression following global ischemia in the gerbil.

    PubMed

    Prosser-Loose, Erin J; Verge, Valerie M K; Cayabyab, Francisco S; Paterson, Phyllis G

    2010-11-01

    Previously it has been demonstrated that protein-energy malnutrition (PEM) impairs habituation in the open field test following global ischemia. The present study examined the hypothesis that PEM exerts some of its deleterious effects on functional outcome by altering the post-ischemic expression of the plasticity-associated genes brain-derived neurotrophic factor (BDNF), its receptor tropomyosin-related kinase B (trkB), and growth-associated protein-43 (GAP-43). Male, Mongolian gerbils (11-12 wk) were randomized to either control diet (12.5% protein) or PEM (2% protein) for 4 wk, and then underwent 5 min bilateral common carotid artery occlusion or sham surgery. Tympanic temperature was maintained at 36.5 ± 0.5°C during surgery. Brains collected at 1, 3 and 7 d post-surgery were processed by in-situ hybridization or immunofluorescence. BDNF and trkB mRNA expression was increased in hippocampal CA1 neurons after ischemia at all time points and was not significantly influenced by diet. However, increased trkB protein expression after ischemia was exacerbated by PEM at 7 d in the CA1 region. Post-ischemic GAP-43 protein increased at 3 and 7 d in the CA1 region, and PEM intensified this response and extended it to the CA3 and hilar regions. PEM exerted these effects without exacerbating CA1 neuron loss caused by global ischemia. The findings suggest that PEM increases the stress response and/or hyper-excitability in the hippocampus after global ischemia. Nutritional care appears to have robust effects on plasticity mechanisms important to recovery after brain ischemia.

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

    PubMed

    Weinert, Brian T; Wagner, Sebastian A; Horn, Heiko; Henriksen, Peter; Liu, Wenshe R; Olsen, Jesper V; Jensen, Lars J; Choudhary, Chunaram

    2011-07-26

    Posttranslational modification of proteins by acetylation and phosphorylation regulates most cellular processes in living organisms. Surprisingly, the evolutionary conservation of phosphorylated serine and threonine residues is only marginally higher than that of unmodified serines and threonines. With high-resolution mass spectrometry, we identified 1981 lysine acetylation sites in the proteome of Drosophila melanogaster. We used data sets of experimentally identified acetylation and phosphorylation sites in Drosophila and humans to analyze the evolutionary conservation of these modification sites between flies and humans. Site-level conservation analysis revealed that acetylation sites are highly conserved, significantly more so than phosphorylation sites. Furthermore, comparison of lysine conservation in Drosophila and humans with that in nematodes and zebrafish revealed that acetylated lysines 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 that acetylation of ubiquitin-conjugating E2 enzymes was evolutionarily conserved, and mutation of a conserved acetylation site impaired the function of the human E2 enzyme UBE2D3. This systems-level analysis of comparative posttranslational modification showed that acetylation is an anciently conserved modification and suggests that phosphorylation sites may have evolved faster than acetylation sites.

  19. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in any...

  20. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in any...

  1. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in any...

  2. Methylene blue decreases mitochondrial lysine acetylation in the diabetic heart.

    PubMed

    Berthiaume, Jessica M; Hsiung, Chia-Heng; Austin, Alison B; McBrayer, Sean P; Depuydt, Mikayla M; Chandler, Margaret P; Miyagi, Masaru; Rosca, Mariana G

    2017-08-01

    Diabetic cardiomyopathy is preceded by mitochondrial alterations, and progresses to heart failure. We studied whether treatment with methylene blue (MB), a compound that was reported to serve as an alternate electron carrier within the mitochondrial electron transport chain (ETC), improves mitochondrial metabolism and cardiac function in type 1 diabetes. MB was administered at 10 mg/kg/day to control and diabetic rats. Both echocardiography and hemodynamic studies were performed to assess cardiac function. Mitochondrial studies comprised the measurement of oxidative phosphorylation and specific activities of fatty acid oxidation enzymes. Proteomic studies were employed to compare the level of lysine acetylation on cardiac mitochondrial proteins between the experimental groups. We found that MB facilitates NADH oxidation, increases NAD(+), and the activity of deacetylase Sirtuin 3, and reduces protein lysine acetylation in diabetic cardiac mitochondria. We identified that lysine acetylation on 83 sites in 34 proteins is lower in the MB-treated diabetic group compared to the same sites in the untreated diabetic group. These changes occur across critical mitochondrial metabolic pathways including fatty acid transport and oxidation, amino acid metabolism, tricarboxylic acid cycle, ETC, transport, and regulatory proteins. While the MB treatment has no effect on the activities of acyl-CoA dehydrogenases, it decreases 3-hydroxyacyl-CoA dehydrogenase activity and long-chain fatty acid oxidation, and improves cardiac function. Providing an alternative route for mitochondrial electron transport is a novel therapeutic approach to decrease lysine acetylation, alleviate cardiac metabolic inflexibility, and improve cardiac function in diabetes.

  3. Global stability of protein folding from an empirical free energy function.

    PubMed

    Ruiz-Blanco, Yasser B; Marrero-Ponce, Yovani; Paz, Waldo; García, Yamila; Salgado, Jesús

    2013-03-21

    The principles governing protein folding stand as one of the biggest challenges of Biophysics. Modeling the global stability of proteins and predicting their tertiary structure are hard tasks, due in part to the variety and large number of forces involved and the difficulties to describe them with sufficient accuracy. We have developed a fast, physics-based empirical potential, intended to be used in global structure prediction methods. This model considers four main contributions: Two entropic factors, the hydrophobic effect and configurational entropy, and two terms resulting from a decomposition of close-packing interactions, namely the balance of the dispersive interactions of folded and unfolded states and electrostatic interactions between residues. The parameters of the model were fixed from a protein data set whose unfolding free energy has been measured at the "standard" experimental conditions proposed by Maxwell et al. (2005) and a large data set of 1151 monomeric proteins obtained from the PDB. A blind test with proteins taken from ProTherm database, at similar experimental conditions, was carried out. We found a good correlation with the test data set, proving the effectiveness of our model for predicting protein folding free energies in considered standard conditions. Such a prediction compares favorably against estimations made with FoldX's function and the force field GROMOS96. This model constitutes a valuable tool for the fast evaluation of protein structure stability in 3D structure prediction methods.

  4. N-ACETYL GROUPS IN VITELLENIN,

    DTIC Science & Technology

    The presence of acetyl groups in vitellenin was confirmed by hydrazinolysis according to the DNP method of Phillips. After hydrazinolysis of 10-30...hydrazinolysis at room temperature for 1 hour, vitellenin contains N- acetyl , but no Oacetyl, groups. (Author)

  5. Evolution of a Histone H4-K16 Acetyl-Specific DNA Aptamer

    PubMed Central

    Williams, Berea A. R.; Lin, Liyun; Lindsay, Stuart M.; Chaput, John C.

    2009-01-01

    We report the in vitro selection of DNA aptamers that bind to histone H4 proteins acetylated at lysine 16. The best aptamer identified in this selection binds to the target protein with a Kd of 21 nM, and discriminates against both the non-acetylated protein and histone H4 proteins acetylated at lysine 8. Comparative binding assays performed with a chip-quality antibody reveal that this aptamer binds to the acetylated histone target with similar affinity to a commercial antibody, but shows significantly greater specificity (15-fold versus 2,400-fold) for the target molecule. This result demonstrates that aptamers that are both modification and location specific can be generated to bind specific protein post-translational modifications. PMID:19385619

  6. Protein ranking: From local to global structure in the protein similarity network

    PubMed Central

    Weston, Jason; Elisseeff, Andre; Zhou, Dengyong; Leslie, Christina S.; Noble, William Stafford

    2004-01-01

    Biologists regularly search databases of DNA or protein sequences for evolutionary or functional relationships to a given query sequence. We describe a ranking algorithm that exploits the entire network structure of similarity relationships among proteins in a sequence database by performing a diffusion operation on a precomputed, weighted network. The resulting ranking algorithm, evaluated by using a human-curated database of protein structures, is efficient and provides significantly better rankings than a local network search algorithm such as psi-blast. PMID:15087500

  7. Conserved Lysine Acetylation within the Microtubule-Binding Domain Regulates MAP2/Tau Family Members

    PubMed Central

    Hwang, Andrew W.; Trzeciakiewicz, Hanna; Friedmann, Dave; Yuan, Chao-Xing; Marmorstein, Ronen; Lee, Virginia M. Y.; Cohen, Todd J.

    2016-01-01

    Lysine acetylation has emerged as a dominant post-translational modification (PTM) regulating tau proteins in Alzheimer’s disease (AD) and related tauopathies. Mass spectrometry studies indicate that tau acetylation sites cluster within the microtubule-binding region (MTBR), a region that is highly conserved among tau, MAP2, and MAP4 family members, implying that acetylation could represent a conserved regulatory mechanism for MAPs beyond tau. Here, we combined mass spectrometry, biochemical assays, and cell-based approaches to demonstrate that the tau family members MAP2 and MAP4 are also subject to reversible acetylation. We identify a cluster of lysines in the MAP2 and MAP4 MTBR that undergo CBP-catalyzed acetylation, many of which are conserved in tau. Similar to tau, MAP2 acetylation can occur in a cysteine-dependent auto-regulatory manner in the presence of acetyl-CoA. Furthermore, tubulin reduced MAP2 acetylation, suggesting tubulin binding dictates MAP acetylation status. Taken together, these results uncover a striking conservation of MAP2/Tau family post-translational modifications that could expand our understanding of the dynamic mechanisms regulating microtubules. PMID:28002468

  8. Conserved Lysine Acetylation within the Microtubule-Binding Domain Regulates MAP2/Tau Family Members.

    PubMed

    Hwang, Andrew W; Trzeciakiewicz, Hanna; Friedmann, Dave; Yuan, Chao-Xing; Marmorstein, Ronen; Lee, Virginia M Y; Cohen, Todd J

    2016-01-01

    Lysine acetylation has emerged as a dominant post-translational modification (PTM) regulating tau proteins in Alzheimer's disease (AD) and related tauopathies. Mass spectrometry studies indicate that tau acetylation sites cluster within the microtubule-binding region (MTBR), a region that is highly conserved among tau, MAP2, and MAP4 family members, implying that acetylation could represent a conserved regulatory mechanism for MAPs beyond tau. Here, we combined mass spectrometry, biochemical assays, and cell-based approaches to demonstrate that the tau family members MAP2 and MAP4 are also subject to reversible acetylation. We identify a cluster of lysines in the MAP2 and MAP4 MTBR that undergo CBP-catalyzed acetylation, many of which are conserved in tau. Similar to tau, MAP2 acetylation can occur in a cysteine-dependent auto-regulatory manner in the presence of acetyl-CoA. Furthermore, tubulin reduced MAP2 acetylation, suggesting tubulin binding dictates MAP acetylation status. Taken together, these results uncover a striking conservation of MAP2/Tau family post-translational modifications that could expand our understanding of the dynamic mechanisms regulating microtubules.

  9. MQAPRank: improved global protein model quality assessment by learning-to-rank.

    PubMed

    Jing, Xiaoyang; Dong, Qiwen

    2017-05-25

    Protein structure prediction has achieved a lot of progress during the last few decades and a greater number of models for a certain sequence can be predicted. Consequently, assessing the qualities of predicted protein models in perspective is one of the key components of successful protein structure prediction. Over the past years, a number of methods have been developed to address this issue, which could be roughly divided into three categories: single methods, quasi-single methods and clustering (or consensus) methods. Although these methods achieve much success at different levels, accurate protein model quality assessment is still an open problem. Here, we present the MQAPRank, a global protein model quality assessment program based on learning-to-rank. The MQAPRank first sorts the decoy models by using single method based on learning-to-rank algorithm to indicate their relative qualities for the target protein. And then it takes the first five models as references to predict the qualities of other models by using average GDT_TS scores between reference models and other models. Benchmarked on CASP11 and 3DRobot datasets, the MQAPRank achieved better performances than other leading protein model quality assessment methods. Recently, the MQAPRank participated in the CASP12 under the group name FDUBio and achieved the state-of-the-art performances. The MQAPRank provides a convenient and powerful tool for protein model quality assessment with the state-of-the-art performances, it is useful for protein structure prediction and model quality assessment usages.

  10. The RNA-binding protein Gemin5 binds directly to the ribosome and regulates global translation

    PubMed Central

    Francisco-Velilla, Rosario; Fernandez-Chamorro, Javier; Ramajo, Jorge; Martinez-Salas, Encarnación

    2016-01-01

    RNA-binding proteins (RBPs) play crucial roles in all organisms. The protein Gemin5 harbors two functional domains. The N-terminal domain binds to snRNAs targeting them for snRNPs assembly, while the C-terminal domain binds to IRES elements through a non-canonical RNA-binding site. Here we report a comprehensive view of the Gemin5 interactome; most partners copurified with the N-terminal domain via RNA bridges. Notably, Gemin5 sediments with the subcellular ribosome fraction, and His-Gemin5 binds to ribosome particles via its N-terminal domain. The interaction with the ribosome was lost in F381A and Y474A Gemin5 mutants, but not in W14A and Y15A. Moreover, the ribosomal proteins L3 and L4 bind directly with Gemin5, and conversely, Gemin5 mutants impairing the binding to the ribosome are defective in the interaction with L3 and L4. The overall polysome profile was affected by Gemin5 depletion or overexpression, concomitant to an increase or a decrease, respectively, of global protein synthesis. Gemin5, and G5-Nter as well, were detected on the polysome fractions. These results reveal the ribosome-binding capacity of the N-ter moiety, enabling Gemin5 to control global protein synthesis. Our study uncovers a crosstalk between this protein and the ribosome, and provides support for the view that Gemin5 may control translation elongation. PMID:27507887

  11. PPARα Activation Induces Nε-Lys-Acetylation of Rat Liver Peroxisomal Multifunctional Enzyme Type 1

    PubMed Central

    Contreras, Miguel A.; Alzate, Oscar; Singh, Avtar K.

    2013-01-01

    Peroxisomes are ubiquitous subcellular organelles that participate in metabolic and disease processes, with few of its proteins undergoing posttranslational modifications. As the role of lysine-acetylation has expanded into the cellular intermediary metabolism, we used a combination of differential centrifugation, organelle isolation by linear density gradient centrifugation, western blot analysis, and peptide fingerprinting and amino acid sequencing by mass spectrometry to investigate protein acetylation in control and ciprofibrate-treated rat liver peroxisomes. Organelle protein samples isolated by density gradient centrifugation from PPARα-agonist treated rat liver screened with an anti-Nε-acetyl lysine antibody revealed a single protein band of 75 kDa. Immunoprecipitation with this antibody resulted in the precipitation of a protein from the protein pool of ciprofibrate-induced peroxisomes, but not from the protein pool of non-induced peroxisomes. Peptide mass fingerprinting analysis identified the protein as the peroxisomal multifunctional enzyme type 1. In addition, mass spectrometry-based amino acid sequencing resulted in the identification of unique peptides containing 4 acetylated-Lys residues (K155, K173, K190, and K583). This is the first report that demonstrates posttranslational acetylation of a peroxisomal enzyme in PPARα-dependent proliferation of peroxisomes in rat liver. PMID:24092543

  12. The Sorcerer II Global Ocean Sampling expedition: expanding the universe of protein families.

    PubMed

    Yooseph, Shibu; Sutton, Granger; Rusch, Douglas B; Halpern, Aaron L; Williamson, Shannon J; Remington, Karin; Eisen, Jonathan A; Heidelberg, Karla B; Manning, Gerard; Li, Weizhong; Jaroszewski, Lukasz; Cieplak, Piotr; Miller, Christopher S; Li, Huiying; Mashiyama, Susan T; Joachimiak, Marcin P; van Belle, Christopher; Chandonia, John-Marc; Soergel, David A; Zhai, Yufeng; Natarajan, Kannan; Lee, Shaun; Raphael, Benjamin J; Bafna, Vineet; Friedman, Robert; Brenner, Steven E; Godzik, Adam; Eisenberg, David; Dixon, Jack E; Taylor, Susan S; Strausberg, Robert L; Frazier, Marvin; Venter, J Craig

    2007-03-01

    Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

  13. On simplified global nonlinear function for fitness landscape: a case study of inverse protein folding.

    PubMed

    Xu, Yun; Hu, Changyu; Dai, Yang; Liang, Jie

    2014-01-01

    The construction of fitness landscape has broad implication in understanding molecular evolution, cellular epigenetic state, and protein structures. We studied the problem of constructing fitness landscape of inverse protein folding or protein design, with the aim to generate amino acid sequences that would fold into an a priori determined structural fold which would enable engineering novel or enhanced biochemistry. For this task, an effective fitness function should allow identification of correct sequences that would fold into the desired structure. In this study, we showed that nonlinear fitness function for protein design can be constructed using a rectangular kernel with a basis set of proteins and decoys chosen a priori. The full landscape for a large number of protein folds can be captured using only 480 native proteins and 3,200 non-protein decoys via a finite Newton method. A blind test of a simplified version of fitness function for sequence design was carried out to discriminate simultaneously 428 native sequences not homologous to any training proteins from 11 million challenging protein-like decoys. This simplified function correctly classified 408 native sequences (20 misclassifications, 95% correct rate), which outperforms several other statistical linear scoring function and optimized linear function. Our results further suggested that for the task of global sequence design of 428 selected proteins, the search space of protein shape and sequence can be effectively parametrized with just about 3,680 carefully chosen basis set of proteins and decoys, and we showed in addition that the overall landscape is not overly sensitive to the specific choice of this set. Our results can be generalized to construct other types of fitness landscape.

  14. On Simplified Global Nonlinear Function for Fitness Landscape: A Case Study of Inverse Protein Folding

    PubMed Central

    Xu, Yun; Hu, Changyu; Dai, Yang; Liang, Jie

    2014-01-01

    The construction of fitness landscape has broad implication in understanding molecular evolution, cellular epigenetic state, and protein structures. We studied the problem of constructing fitness landscape of inverse protein folding or protein design, with the aim to generate amino acid sequences that would fold into an a priori determined structural fold which would enable engineering novel or enhanced biochemistry. For this task, an effective fitness function should allow identification of correct sequences that would fold into the desired structure. In this study, we showed that nonlinear fitness function for protein design can be constructed using a rectangular kernel with a basis set of proteins and decoys chosen a priori. The full landscape for a large number of protein folds can be captured using only 480 native proteins and 3,200 non-protein decoys via a finite Newton method. A blind test of a simplified version of fitness function for sequence design was carried out to discriminate simultaneously 428 native sequences not homologous to any training proteins from 11 million challenging protein-like decoys. This simplified function correctly classified 408 native sequences (20 misclassifications, 95% correct rate), which outperforms several other statistical linear scoring function and optimized linear function. Our results further suggested that for the task of global sequence design of 428 selected proteins, the search space of protein shape and sequence can be effectively parametrized with just about 3,680 carefully chosen basis set of proteins and decoys, and we showed in addition that the overall landscape is not overly sensitive to the specific choice of this set. Our results can be generalized to construct other types of fitness landscape. PMID:25110986

  15. The Sorcerer II Global Ocean Sampling Expedition: Expanding theUniverse of Protein Families

    SciTech Connect

    Yooseph, Shibu; Sutton, Granger; Rusch, Douglas B.; Halpern,Aaron L.; Williamson, Shannon J.; Remington, Karin; Eisen, Jonathan A.; Heidelberg, Karla B.; Manning, Gerard; Li, Weizhong; Jaroszewski, Lukasz; Cieplak, Piotr; Miller, Christopher S.; Li, Huiying; Mashiyama, Susan T.; Joachimiak, Marcin P.; van Belle, Christopher; Chandonia, John-Marc; Soergel, David A.; Zhai, Yufeng; Natarajan, Kannan; Lee, Shaun; Raphael,Benjamin J.; Bafna, Vineet; Friedman, Robert; Brenner, Steven E.; Godzik,Adam; Eisenberg, David; Dixon, Jack E.; Taylor, Susan S.; Strausberg,Robert L.; Frazier, Marvin; Venter, J.Craig

    2006-03-23

    Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

  16. The Sorcerer II Global Ocean Sampling Expedition: Expanding the Universe of Protein Families

    PubMed Central

    Yooseph, Shibu; Sutton, Granger; Rusch, Douglas B; Halpern, Aaron L; Williamson, Shannon J; Remington, Karin; Eisen, Jonathan A; Heidelberg, Karla B; Manning, Gerard; Li, Weizhong; Jaroszewski, Lukasz; Cieplak, Piotr; Miller, Christopher S; Li, Huiying; Mashiyama, Susan T; Joachimiak, Marcin P; van Belle, Christopher; Chandonia, John-Marc; Soergel, David A; Zhai, Yufeng; Natarajan, Kannan; Lee, Shaun; Raphael, Benjamin J; Bafna, Vineet; Friedman, Robert; Brenner, Steven E; Godzik, Adam; Eisenberg, David; Dixon, Jack E; Taylor, Susan S; Strausberg, Robert L; Frazier, Marvin; Venter, J. Craig

    2007-01-01

    Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature. PMID:17355171

  17. Estimation of benchmark dose as the threshold levels of urinary cadmium, based on excretion of total protein, {beta} {sub 2}-microglobulin, and N-acetyl-{beta}-D-glucosaminidase in cadmium nonpolluted regions in Japan

    SciTech Connect

    Kobayashi, Etsuko . E-mail: ekoba@faculty.chiba-u.jp; Suwazono, Yasushi; Uetani, Mirei; Inaba, Takeya; Oishi, Mitsuhiro; Kido, Teruhiko; Nishijo, Muneko; Nakagawa, Hideaki; Nogawa, Koji

    2006-07-15

    Previously, we investigated the association between urinary cadmium (Cd) concentration and indicators of renal dysfunction, including total protein, {beta} {sub 2}-microglobulin ({beta} {sub 2}-MG), and N-acetyl-{beta}-D-glucosaminidase (NAG). In 2778 inhabitants {>=}50 years of age (1114 men, 1664 women) in three different Cd nonpolluted areas in Japan, we showed that a dose-response relationship existed between renal effects and Cd exposure in the general environment without any known Cd pollution. However, we could not estimate the threshold levels of urinary Cd at that time. In the present study, we estimated the threshold levels of urinary Cd as the benchmark dose low (BMDL) using the benchmark dose (BMD) approach. Urinary Cd excretion was divided into 10 categories, and an abnormality rate was calculated for each. Cut-off values for urinary substances were defined as corresponding to the 84% and 95% upper limit values of the target population who have not smoked. Then we calculated the BMD and BMDL using a log-logistic model. The values of BMD and BMDL for all urinary substances could be calculated. The BMDL for the 84% cut-off value of {beta} {sub 2}-MG, setting an abnormal value at 5%, was 2.4 {mu}g/g creatinine (cr) in men and 3.3 {mu}g/g cr in women. In conclusion, the present study demonstrated that the threshold level of urinary Cd could be estimated in people living in the general environment without any known Cd-pollution in Japan, and the value was inferred to be almost the same as that in Belgium, Sweden, and China.

  18. SNaPP: Simplified Nanoproteomics Platform for Reproducible Global Proteomic Analysis of Nanogram Protein Quantities

    SciTech Connect

    Huang, Eric L.; Piehowski, Paul D.; Orton, Daniel J.; Moore, Ronald J.; Qian, Wei-Jun; Casey, Cameron P.; Sun, Xiaofei; Dey, Sudhansu K.; Burnum-Johnson, Kristin E.; Smith, Richard D.

    2016-03-01

    Global proteomic analyses are now widely applied across biological research to study changes in an organism(s) proteome correlated with a perturbation, phenotype and/or time series of interest.[1-3] Further, it has been broadly established that efficient and reproducible sample preparation workflows are crucial to successful quantitative proteome comparisons, especially when applying label free methods.[4-8] However, clinical samples are often severely limited in quantity and can preclude the application of more robust bulk sample processing workflows due to e.g. contamination, carry-over, or sample losses.[9] This has limited the effective application of global proteomics for many sample types of great interest, e.g. LCM dissected tissues, FACS sorted cells, circulating tumor cells (CTC), and early embryos. In a typical proteomics experiment, bulk homogenization is applied to generate sufficient protein for processing (> 10 µg protein), and can blend the proteomes of many different cell types and disparate tissue regions. The resulting “average” proteome, can effectively render unobservable proteome changes of interest, and preclude important applications. Global proteomic analyses of complex protein samples in nanogram quantities require a fastidious approach to achieve in-depth protein coverage and quantitative reproducibility.

  19. An improved hybrid global optimization method for protein tertiary structure prediction

    PubMed Central

    McAllister, Scott R.

    2009-01-01

    First principles approaches to the protein structure prediction problem must search through an enormous conformational space to identify low-energy, near-native structures. In this paper, we describe the formulation of the tertiary structure prediction problem as a nonlinear constrained minimization problem, where the goal is to minimize the energy of a protein conformation subject to constraints on torsion angles and interatomic distances. The core of the proposed algorithm is a hybrid global optimization method that combines the benefits of the αBB deterministic global optimization approach with conformational space annealing. These global optimization techniques employ a local minimization strategy that combines torsion angle dynamics and rotamer optimization to identify and improve the selection of initial conformations and then applies a sequential quadratic programming approach to further minimize the energy of the protein conformations subject to constraints. The proposed algorithm demonstrates the ability to identify both lower energy protein structures, as well as larger ensembles of low-energy conformations. PMID:20357906

  20. AMPK Represses TOP mRNA Translation But Not Global Protein Synthesis in Liver

    PubMed Central

    Reiter, Ali K.; Bolster, Douglas R.; Crozier, Stephen J.; Kimball, Scot R.; Jefferson, Leonard S.

    2008-01-01

    The AMP-activated protein kinase (AMPK) represses signaling through the mammalian target of rapamycin complex 1 (mTORC1). In muscle, repression of mTORC1 leads to a reduction in global protein synthesis. In contrast, repression of mTORC1 in the liver has no immediate effect on global protein synthesis. In the present study, signaling through mTORC1 and translation of specific mRNAs such as those bearing a 5’-terminal oligopyrimidine (TOP) tract and were examined in rat liver following activation of AMPK after treadmill running. Activation of AMPK repressed translation of the TOP mRNAs encoding rpS6, rpS8, and eEF1α. In contrast, neither global protein synthesis nor translation of mRNAs encoding GAPDH or β-actin was changed. Basal phosphorylation of the mTORC1 target 4E-BP1, but not S6K1 or rpS6, was reduced following activation of AMPK. Thus, in liver, AMPK activation repressed translation of TOP mRNAs through a mechanism distinct from downregulated phosphorylation of S6K1 or rpS6. PMID:18638456

  1. Evaluation of Affinity-Tagged Protein Expression Strategies using Local and Global Isotope Ratio Measurements

    SciTech Connect

    Hervey, IV, William Judson; Khalsa-Moyers, Gurusahai K; Lankford, Patricia K; Owens, Elizabeth T; McKeown, Catherine K; Lu, Tse-Yuan S; Foote, Linda J; Morrell-Falvey, Jennifer L; McDonald, W Hayes; Pelletier, Dale A; Hurst, Gregory {Greg} B

    2009-01-01

    Protein enrichments of engineered, affinity-tagged (or bait ) fusion proteins with interaction partners are often laden with background, non-specific proteins, due to interactions that occur in vitro as an artifact of the technique. Furthermore, the in vivo expression of the bait protein may itself affect physiology or metabolism. In this study, intrinsic affinity purification challenges were investigated in a model protein complex, DNA-dependent RNA polymerase (RNAP), encompassing chromosome- and plasmid-encoding strategies for bait proteins in two different microbial species: Escherichia coli and Rhodopseudomonas palustris. Isotope ratio measurements of bait protein expression strains relative to native, wild-type strains were performed by liquid chromatography tandem mass spectrometry (LC-MS-MS) to assess bait protein expression strategies in each species. Authentic interacting proteins of RNAP were successfully discerned from artifactual co-isolating proteins by the isotopic differentiation of interactions as random or targeted (I-DIRT) method (A. J. Tackett et al. J. Proteome Res. 2005, 4 (5), 1752-1756). To investigate broader effects of bait protein production in the bacteria, we compared proteomes from strains harboring a plasmid that encodes an affinity-tagged subunit (RpoA) of the RNAP complex with the corresponding wild-type strains using stable isotope metabolic labeling. The ratio of RpoA abundance in plasmid strains versus wild type was 0.8 for R. palustris and 1.7 for E. coli. While most other proteins showed no appreciable difference, proteins significantly increased in abundance in plasmid-encoded bait-expressing strains of both species included the plasmid encoded antibiotic resistance protein, GenR and proteins involved in amino acid biosynthesis. Together, these local, complex-specific and more global, whole proteome isotopic abundance ratio measurements provided a tool for evaluating both in vivo and in vitro effects of plasmid

  2. From local structure to a global framework: recognition of protein folds

    PubMed Central

    Joseph, Agnel Praveen; de Brevern, Alexandre G.

    2014-01-01

    Protein folding has been a major area of research for many years. Nonetheless, the mechanisms leading to the formation of an active biological fold are still not fully apprehended. The huge amount of available sequence and structural information provides hints to identify the putative fold for a given sequence. Indeed, protein structures prefer a limited number of local backbone conformations, some being characterized by preferences for certain amino acids. These preferences largely depend on the local structural environment. The prediction of local backbone conformations has become an important factor to correctly identifying the global protein fold. Here, we review the developments in the field of local structure prediction and especially their implication in protein fold recognition. PMID:24740960

  3. Kinetic and Thermodynamic Analysis of Acetyl-CoA Activation of Staphylococcus aureus Pyruvate Carboxylase.

    PubMed

    Westerhold, Lauren E; Bridges, Lance C; Shaikh, Saame Raza; Zeczycki, Tonya N

    2017-07-11

    Allosteric regulation of pyruvate carboxylase (PC) activity is pivotal to maintaining metabolic homeostasis. In contrast, dysregulated PC activity contributes to the pathogenesis of numerous diseases, rendering PC a possible target for allosteric therapeutic development. Recent research efforts have focused on demarcating the role of acetyl-CoA, one of the most potent activators of PC, in coordinating catalytic events within the multifunctional enzyme. Herein, we report a kinetic and thermodynamic analysis of acetyl-CoA activation of the Staphylococcus aureus PC (SaPC)-catalyzed carboxylation of pyruvate to identify novel means by which acetyl-CoA synchronizes catalytic events within the PC tetramer. Kinetic and linked-function analysis, or thermodynamic linkage analysis, indicates that the substrates of the biotin carboxylase and carboxyl transferase domain are energetically coupled in the presence of acetyl-CoA. In contrast, both kinetic and energetic coupling between the two domains is lost in the absence of acetyl-CoA, suggesting a functional role for acetyl-CoA in facilitating the long-range transmission of substrate-induced conformational changes within the PC tetramer. Interestingly, thermodynamic activation parameters for the SaPC-catalyzed carboxylation of pyruvate are largely independent of acetyl-CoA. Our results also reveal the possibility that global conformational changes give rise to observed species-specific thermodynamic activation parameters. Taken together, our kinetic and thermodynamic results provide a possible allosteric mechanism by which acetyl-CoA coordinates catalysis within the PC tetramer.

  4. A semisynthetic Atg3 reveals that acetylation promotes Atg3 membrane binding and Atg8 lipidation

    NASA Astrophysics Data System (ADS)

    Li, Yi-Tong; Yi, Cong; Chen, Chen-Chen; Lan, Huan; Pan, Man; Zhang, Shao-Jin; Huang, Yi-Chao; Guan, Chao-Jian; Li, Yi-Ming; Yu, Li; Liu, Lei

    2017-03-01

    Acetylation of Atg3 regulates the lipidation of the protein Atg8 in autophagy. The molecular mechanism behind this important biochemical event remains to be elucidated. We describe the first semi-synthesis of homogeneous K19/K48-diacetylated Atg3 through sequential hydrazide-based native chemical ligation. In vitro reconstitution experiments with the semi-synthetic proteins confirm that Atg3 acetylation can promote the lipidation of Atg8. We find that acetylation of Atg3 enhances its binding to phosphatidylethanolamine-containing liposomes and to endoplasmic reticulum, through which it promotes the lipidation process.

  5. Local and global structural drivers for the photoactivation of the orange carotenoid protein

    SciTech Connect

    Gupta, Sayan; Guttman, Miklos; Leverenz, Ryan L.; Zhumadilova, Kulyash; Pawlowski, Emily G.; Petzold, Christopher J.; Lee, Kelly K.; Ralston, Corie Y.; Kerfeld, Cheryl A.

    2015-09-18

    Here, photoprotective mechanisms are of fundamental importance for the survival of photosynthetic organisms. In cyanobacteria, the orange carotenoid protein (OCP), when activated by intense blue light, binds to the light-harvesting antenna and triggers the dissipation of excess captured light energy. Using a combination of small angle X-ray scattering (SAXS), X-ray hydroxyl radical footprinting, circular dichroism, and H/D exchange mass spectrometry, we identified both the local and global structural changes in the OCP upon photoactivation. SAXS and H/D exchange data showed that global tertiary structural changes, including complete domain dissociation, occur upon photoactivation, but with alteration of secondary structure confined to only the N terminus of the OCP. Microsecond radiolytic labeling identified rearrangement of the H-bonding network associated with conserved residues and structural water molecules. Collectively, these data provide experimental evidence for an ensemble of local and global structural changes, upon activation of the OCP, that are essential for photoprotection.

  6. Local and global structural drivers for the photoactivation of the orange carotenoid protein

    PubMed Central

    Gupta, Sayan; Guttman, Miklos; Leverenz, Ryan L.; Zhumadilova, Kulyash; Pawlowski, Emily G.; Petzold, Christopher J.; Lee, Kelly K.; Ralston, Corie Y.; Kerfeld, Cheryl A.

    2015-01-01

    Photoprotective mechanisms are of fundamental importance for the survival of photosynthetic organisms. In cyanobacteria, the orange carotenoid protein (OCP), when activated by intense blue light, binds to the light-harvesting antenna and triggers the dissipation of excess captured light energy. Using a combination of small angle X-ray scattering (SAXS), X-ray hydroxyl radical footprinting, circular dichroism, and H/D exchange mass spectrometry, we identified both the local and global structural changes in the OCP upon photoactivation. SAXS and H/D exchange data showed that global tertiary structural changes, including complete domain dissociation, occur upon photoactivation, but with alteration of secondary structure confined to only the N terminus of the OCP. Microsecond radiolytic labeling identified rearrangement of the H-bonding network associated with conserved residues and structural water molecules. Collectively, these data provide experimental evidence for an ensemble of local and global structural changes, upon activation of the OCP, that are essential for photoprotection. PMID:26385969

  7. Prediction of Protein Structure by Ab Initio Global Optimization of Potential Energy

    NASA Astrophysics Data System (ADS)

    Scheraga, Harold; Liwo, Adam; Pillardy, Jarek; Czaplewski, Czarek; Lee, Jooyoung; Ripoll, Daniel; Kazmierkiewicz, Rajmund; Oldziej, Stanislaw; Arnautova, Yelena; Wedemeyer, William; Saunders, Jeff

    2002-03-01

    A hierarchical ab initio method is used to predict the three-dimensional structures of globular proteins. The protein is described initially as a virtual-bond chain, with its side chains represented as ellipsoids. After searching the conformational space with this united-residue (UNRES) model, with many-body interactions, the UNRES model is converted to an all-atom model, and the global optimization of the potential energy is continued with the ECEPP/3 force field and the SRFOPT hydration free-energy. Results of the application of this methodology in the CASP3 and CASP4 exercises, and more-recent methodological improvements, will be presented.

  8. Effects of histone deacetylase inhibitors on amygdaloid histone acetylation and neuropeptide Y expression: a role in anxiety-like and alcohol-drinking behaviours.

    PubMed

    Sakharkar, Amul J; Zhang, Huaibo; Tang, Lei; Baxstrom, Kathryn; Shi, Guangbin; Moonat, Sachin; Pandey, Subhash C

    2014-08-01

    Recent studies have demonstrated the involvement of epigenetic mechanisms in psychiatric disorders, including alcoholism. Here, we investigated the effects of histone deacetylase (HDAC) inhibitor, trichostatin A (TSA) on amygdaloid HDAC-induced histone deacetylation and neuropeptide Y (NPY) expression and on anxiety-like and alcohol-drinking behaviours in alcohol-preferring (P) and -non-preferring (NP) rats. It was found that P rats displayed higher anxiety-like and alcohol-drinking behaviours, higher amygdaloid nuclear, but not cytosolic, HDAC activity, which was associated with increased HDAC2 protein levels and deficits in histone acetylation and NPY expression in the central (CeA) and medial nucleus of amygdala (MeA), as compared to NP rats. TSA treatment attenuated the anxiety-like and alcohol-drinking behaviours, with concomitant reductions in amygdaloid nuclear, but not cytosolic HDAC activity, and HDAC2, but not HDAC4, protein levels in the CeA and MeA of P rats, without effect in NP rats. TSA treatment also increased global histone acetylation (H3-K9 and H4-K8) and NPY expression in the CeA and MeA of P, but not in NP rats. Histone H3 acetylation within the NPY promoter was also innately lower in the amygdala of P rats compared with NP rats; which was normalized by TSA treatment. Voluntary ethanol intake in P, but not NP rats, produced anxiolytic effects and decreased the HDAC2 levels and increased histone acetylation in the CeA and MeA. These results suggest that higher HDAC2 expression-related deficits in histone acetylation may be involved in lower NPY expression in the amygdala of P rats, and operative in controlling anxiety-like and alcohol-drinking behaviours.

  9. The E. coli sirtuin CobB shows no preference for enzymatic and nonenzymatic lysine acetylation substrate sites

    PubMed Central

    AbouElfetouh, Alaa; Kuhn, Misty L; Hu, Linda I; Scholle, Michael D; Sorensen, Dylan J; Sahu, Alexandria K; Becher, Dörte; Antelmann, Haike; Mrksich, Milan; Anderson, Wayne F; Gibson, Bradford W; Schilling, Birgit; Wolfe, Alan J

    2015-01-01

    Nε-lysine acetylation is an abundant posttranslational modification of thousands of proteins involved in diverse cellular processes. In the model bacterium Escherichia coli, the ε-amino group of a lysine residue can be acetylated either catalytically by acetyl-coenzyme A (acCoA) and lysine acetyltransferases, or nonenzymatically by acetyl phosphate (acP). It is well known that catalytic acCoA-dependent Nε-lysine acetylation can be reversed by deacetylases. Here, we provide genetic, mass spectrometric, structural and immunological evidence that CobB, a deacetylase of the sirtuin family of NAD+-dependent deacetylases, can reverse acetylation regardless of acetyl donor or acetylation mechanism. We analyzed 69 lysines on 51 proteins that we had previously detected as robustly, reproducibly, and significantly more acetylated in a cobB mutant than in its wild-type parent. Functional and pathway enrichment analyses supported the hypothesis that CobB regulates protein function in diverse and often essential cellular processes, most notably translation. Combined mass spectrometry, bioinformatics, and protein structural data provided evidence that the accessibility and three-dimensional microenvironment of the target acetyllysine help determine CobB specificity. Finally, we provide evidence that CobB is the predominate deacetylase in E. coli. PMID:25417765

  10. The E. coli sirtuin CobB shows no preference for enzymatic and nonenzymatic lysine acetylation substrate sites.

    PubMed

    AbouElfetouh, Alaa; Kuhn, Misty L; Hu, Linda I; Scholle, Michael D; Sorensen, Dylan J; Sahu, Alexandria K; Becher, Dörte; Antelmann, Haike; Mrksich, Milan; Anderson, Wayne F; Gibson, Bradford W; Schilling, Birgit; Wolfe, Alan J

    2015-02-01

    N(ε) -lysine acetylation is an abundant posttranslational modification of thousands of proteins involved in diverse cellular processes. In the model bacterium Escherichia coli, the ε-amino group of a lysine residue can be acetylated either catalytically by acetyl-coenzyme A (acCoA) and lysine acetyltransferases, or nonenzymatically by acetyl phosphate (acP). It is well known that catalytic acCoA-dependent N(ε) -lysine acetylation can be reversed by deacetylases. Here, we provide genetic, mass spectrometric, structural and immunological evidence that CobB, a deacetylase of the sirtuin family of NAD(+) -dependent deacetylases, can reverse acetylation regardless of acetyl donor or acetylation mechanism. We analyzed 69 lysines on 51 proteins that we had previously detected as robustly, reproducibly, and significantly more acetylated in a cobB mutant than in its wild-type parent. Functional and pathway enrichment analyses supported the hypothesis that CobB regulates protein function in diverse and often essential cellular processes, most notably translation. Combined mass spectrometry, bioinformatics, and protein structural data provided evidence that the accessibility and three-dimensional microenvironment of the target acetyllysine help determine CobB specificity. Finally, we provide evidence that CobB is the predominate deacetylase in E. coli.

  11. Picosecond phase grating spectroscopy of hemoglobin and myoglobin: energetics and dynamics of global protein motion.

    PubMed

    Richard, L; Genberg, L; Deak, J; Chiu, H L; Miller, R J

    1992-11-10

    Phase grating spectroscopy has been used to follow the optically triggered tertiary structural changes of carboxymyoglobin (MbCO) and carboxyhemoglobin (HbCO). Probe wavelength and temperature dependencies have shown that the grating signal arises from nonthermal density changes induced by the protein structural changes. The material displaced through the protein structural changes leads to the excitation of coherent acoustic modes of the surrounding water. The coupling of the structural changes to the fluid hydrodynamics demonstrates that a global change in the protein structure is occurring in less than 30 ps. The global relaxation is on the same time scale as the local changes in structure in the vicinity of the heme pocket. The observed dynamics for global relaxation and correspondence between the local and global structural changes provides evidence for the involvement of collective modes in the propagation of the initial tertiary conformational changes. The energetics can also be derived from the acoustic signal. For MbCO, the photodissociation process is endothermic by 21 +/- 2 kcal/mol, which corresponds closely to the expected Fe-CO bond enthalpy. In contrast, HbCO dissipates approximately 10 kcal/mol more energy relative to myoglobin during its initial tertiary structural relaxation. The difference in energetics indicates that significantly more energy is stored in the hemoglobin structure and is believed to be related to the quaternary structure of hemoglobin not present in the monomeric form of myoglobin. These findings provide new insight into the biomechanics of conformational changes in proteins and lend support to theoretical models invoking stored strain energy as the driving force for large amplitude correlated motions.

  12. Acetylation of lysine 92 improves the chaperone and anti-apoptotic activities of human αB-crystallin.

    PubMed

    Nahomi, Rooban B; Huang, Rong; Nandi, Sandip K; Wang, Benlian; Padmanabha, Smitha; Santhoshkumar, Puttur; Filipek, Slawomir; Biswas, Ashis; Nagaraj, Ram H

    2013-11-12

    αB-Crystallin is a chaperone and an anti-apoptotic protein that is strongly expressed in many tissues, including the lens, retina, heart, and kidney. In the human lens, several lysine residues in αB-crystallin are acetylated. We have previously shown that such acetylation is predominant at lysine 92 (K92) and lysine 166 (K166). We have investigated the effect of lysine acetylation on the structure and functions of αB-crystallin by the specific introduction of an N(ε)-acetyllysine (AcK) mimic at K92. The introduction of AcK slightly altered the secondary and tertiary structures of the protein. The introduction of AcK also resulted in an increase in the molar mass and hydrodynamic radius of the protein, and the protein became structurally more open and more stable than the native protein. The acetyl protein acquired higher surface hydrophobicity and exhibited 25-55% higher chaperone activity than the native protein. The acetyl protein had more client protein binding per subunit of the protein and higher binding affinity relative to that of the native protein. The acetyl protein was at least 20% more effective in inhibiting chemically induced apoptosis than the native protein. Molecular modeling suggests that acetylation of K92 makes the "α-crystallin domain" more hydrophobic. Together, our results reveal that the acetylation of a single lysine residue in αB-crystallin makes the protein structurally more stable and improves its chaperone and anti-apoptotic activities. Our findings suggest that lysine acetylation of αB-crystallin is an important chemical modification for enhancing αB-crystallin's protective functions in the eye.

  13. Global optimum protein threading with gapped alignment and empirical pair score functions.

    PubMed

    Lathrop, R H; Smith, T F

    1996-02-02

    We describe a branch-and-bound search algorithm for finding the exact global optimum gapped sequence-structure alignment ("threading") between a protein sequence and a protein core or structural model, using an arbitrary amino acid pair score function (e.g. contact potentials, knowledge-based potentials, potentials of mean force, etc.). The search method imposes minimal conditions on how structural environments are defined or the form of the score function, and allows arbitrary sequence-specific functions for scoring loops and active site residues. Consequently the search method can be used with many different score functions and threading methodologies; this paper illustrates five from the literature. On a desktop workstation running LISP, we have found the global optimum protein sequence-structure alignment in NP-hard search spaces as large as 9.6 x 10(31), at rates ranging as high as 6.8 x 10(28) equivalent threadings per second (most of which are pruned before they ever are examined explicitly). Continuing the procedure past the global optimum enumerates successive candidate threadings in monotonically increasing score order. We give efficient algorithms for search space size, uniform random sampling, segment placement probabilities, mean, standard deviation and partition function. The method should prove useful for structure prediction, as well as for critical evaluation of new pair score functions.

  14. Stimulation of V(D)J recombination by histone acetylation.

    PubMed

    McBlane, F; Boyes, J

    2000-04-20

    V(D)J recombination assembles functional immunoglobulin and T cell receptor genes from individual gene segments [1]. A common recombination mechanism, initiated by the proteins RAG1 and RAG2 at conserved recombination signal sequences (RSSs), operates at all rearranging loci [2] [3]. It has been proposed that the key regulator of the reaction is 'accessibility' of the RSS within chromatin [4]. Recently, the packaging of RSSs into nucleosomes was shown to inhibit initiation of V(D)J recombination [5] [6]. Nevertheless, the tight tissue specificity of regulation cannot be explained by nucleosome-mediated repression alone because a significant fraction of RSSs would be predicted to lie in linker regions between nucleosomes. Therefore, some aspect of the regulation of the recombination reaction must rely on the disruption of higher-order chromatin structure. Here, we report that histone acetylation directly stimulates the recombination reaction in vivo in the correct cell- and stage-specific manner. Neither expression of RAG genes nor activity of RAG proteins was increased by acetylation. Furthermore, histone acetylation failed to overcome nucleosome-mediated repression of RSS recognition and cleavage in vitro. Our data suggest a role for histone acetylation in stimulating recombination in vivo through disruption of higher-order chromatin structures.

  15. Using Local States To Drive the Sampling of Global Conformations in Proteins

    PubMed Central

    2016-01-01

    Conformational changes associated with protein function often occur beyond the time scale currently accessible to unbiased molecular dynamics (MD) simulations, so that different approaches have been developed to accelerate their sampling. Here we investigate how the knowledge of backbone conformations preferentially adopted by protein fragments, as contained in precalculated libraries known as structural alphabets (SA), can be used to explore the landscape of protein conformations in MD simulations. We find that (a) enhancing the sampling of native local states in both metadynamics and steered MD simulations allows the recovery of global folded states in small proteins; (b) folded states can still be recovered when the amount of information on the native local states is reduced by using a low-resolution version of the SA, where states are clustered into macrostates; and (c) sequences of SA states derived from collections of structural motifs can be used to sample alternative conformations of preselected protein regions. The present findings have potential impact on several applications, ranging from protein model refinement to protein folding and design. PMID:26808351

  16. Acetylation of RNA polymerase II regulates growth-factor-induced gene transcription in mammalian cells.

    PubMed

    Schröder, Sebastian; Herker, Eva; Itzen, Friederike; He, Daniel; Thomas, Sean; Gilchrist, Daniel A; Kaehlcke, Katrin; Cho, Sungyoo; Pollard, Katherine S; Capra, John A; Schnölzer, Martina; Cole, Philip A; Geyer, Matthias; Bruneau, Benoit G; Adelman, Karen; Ott, Melanie

    2013-11-07

    Lysine acetylation regulates transcription by targeting histones and nonhistone proteins. Here we report that the central regulator of transcription, RNA polymerase II, is subject to acetylation in mammalian cells. Acetylation occurs at eight lysines within the C-terminal domain (CTD) of the largest polymerase subunit and is mediated by p300/KAT3B. CTD acetylation is specifically enriched downstream of the transcription start sites of polymerase-occupied genes genome-wide, indicating a role in early stages of transcription initiation or elongation. Mutation of lysines or p300 inhibitor treatment causes the loss of epidermal growth-factor-induced expression of c-Fos and Egr2, immediate-early genes with promoter-proximally paused polymerases, but does not affect expression or polymerase occupancy at housekeeping genes. Our studies identify acetylation as a new modification of the mammalian RNA polymerase II required for the induction of growth factor response genes.

  17. Mutations of Arabidopsis TBL32 and TBL33 affect xylan acetylation and secondary wall deposition

    DOE PAGES

    Yuan, Youxi; Teng, Quincy; Zhong, Ruiqin; ...

    2016-01-08

    Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be monoand di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reductionmore » in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-Omonoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. Furthermore, these results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls.« less

  18. Mutations of Arabidopsis TBL32 and TBL33 Affect Xylan Acetylation and Secondary Wall Deposition

    PubMed Central

    Yuan, Youxi; Teng, Quincy; Zhong, Ruiqin; Haghighat, Marziyeh; Richardson, Elizabeth A.; Ye, Zheng-Hua

    2016-01-01

    Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be mono- and di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reduction in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-O-monoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. These results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls. PMID:26745802

  19. Polycomb inhibits histone acetylation by CBP by binding directly to its catalytic domain

    PubMed Central

    Tie, Feng; Banerjee, Rakhee; Fu, Chen; Stratton, Carl A.; Fang, Ming; Harte, Peter J.

    2016-01-01

    Drosophila Polycomb (PC), a subunit of Polycomb repressive complex 1 (PRC1), is well known for its role in maintaining repression of the homeotic genes and many others and for its binding to trimethylated histone H3 on Lys 27 (H3K27me3) via its chromodomain. Here, we identify a novel activity of PC: inhibition of the histone acetylation activity of CREB-binding protein (CBP). We show that PC and its mammalian CBX orthologs interact directly with the histone acetyltransferase (HAT) domain of CBP, binding to the previously identified autoregulatory loop, whose autoacetylation greatly enhances HAT activity. We identify a conserved PC motif adjacent to the chromodomain required for CBP binding and show that PC binding inhibits acetylation of histone H3. CBP autoacetylation impairs PC binding in vitro, and PC is preferentially associated with unacetylated CBP in vivo. PC knockdown elevates the acetylated H3K27 (H3K27ac) level globally and at promoter regions of some genes that are bound by both PC and CBP. Conversely, PC overexpression decreases the H3K27ac level in vivo and also suppresses CBP-dependent Polycomb phenotypes caused by overexpression of Trithorax, an antagonist of Polycomb silencing. We find that PC is physically associated with the initiating form of RNA polymerase II (Pol II) and that many promoters co-occupied by PC and CBP are associated with paused Pol II, suggesting that PC may play a role in Pol II pausing. These results suggest that PC/PRC1 inhibition of CBP HAT activity plays a role in regulating transcription of both repressed and active PC-regulated genes. PMID:26802126

  20. Acetylated histone H3 increases nucleosome dissociation

    NASA Astrophysics Data System (ADS)

    Simon, Marek; Manohar, Mridula; Ottesen, Jennifer; Poirier, Michael

    2009-03-01

    Chromatin's basic unit structure is the nucleosome, i.e. genomic DNA wrapped around a particular class of proteins -- histones -- which due to their physical hindrance, block vital biological processes, such as DNA repair, DNA replication, and RNA transcription. Histone post-translational modifications, which are known to exist in vivo, are hypothesized to regulate these biological processes by directly altering DNA-histone interactions and thus nucleosome structure and stability. Using magnetic tweezers technique we studied the acetylation of histone H3 in the dyad region, i.e. at K115 and K122, on reconstituted arrays of nucleosomes under constant external force. Based on the measured increase in the probability of dissociation of modified nucleosomes, we infer that this double modification could facilitate histone chaperone mediated nucleosome disassembly in vivo.

  1. Human 14-3-3 Paralogs Differences Uncovered by Cross-Talk of Phosphorylation and Lysine Acetylation

    PubMed Central

    Uhart, Marina; Bustos, Diego M.

    2013-01-01

    The 14-3-3 protein family interacts with more than 700 different proteins in mammals, in part as a result of its specific phospho-serine/phospho-threonine binding activity. Upon binding to 14-3-3, the stability, subcellular localization and/or catalytic activity of the ligands are modified. Seven paralogs are strictly conserved in mammalian species. Although initially thought as redundant, the number of studies showing specialization is growing. We created a protein-protein interaction network for 14-3-3, kinases and their substrates signaling in human cells. We included information of phosphorylation, acetylation and other PTM sites, obtaining a complete representation of the 14-3-3 binding partners and their modifications. Using a computational system approach we found that networks of each 14-3-3 isoform are statistically different. It was remarkable to find that Tyr was the most phosphorylatable amino acid in domains of 14-3-3 epsilon partners. This, together with the over-representation of SH3 and Tyr_Kinase domains, suggest that epsilon could be involved in growth factors receptors signaling pathways particularly. We also found that within zeta’s network, the number of acetylated partners (and the number of modify lysines) is significantly higher compared with each of the other isoforms. Our results imply previously unreported hidden differences of the 14-3-3 isoforms interaction networks. The phosphoproteome and lysine acetylome within each network revealed post-transcriptional regulation intertwining phosphorylation and lysine acetylation. A global understanding of these networks will contribute to predict what could occur when regulatory circuits become dysfunctional or are modified in response to external stimuli. PMID:23418452

  2. Characterization of Semisynthetic and Naturally Nα-Acetylated α-Synuclein in Vitro and in Intact Cells

    PubMed Central

    Fauvet, Bruno; Fares, Mohamed-Bilal; Samuel, Filsy; Dikiy, Igor; Tandon, Anurag; Eliezer, David; Lashuel, Hilal A.

    2012-01-01

    N-terminal acetylation is a very common post-translational modification, although its role in regulating protein physical properties and function remains poorly understood. α-Synuclein (α-syn), a protein that has been linked to the pathogenesis of Parkinson disease, is constitutively Nα-acetylated in vivo. Nevertheless, most of the biochemical and biophysical studies on the structure, aggregation, and function of α-syn in vitro utilize recombinant α-syn from Escherichia coli, which is not N-terminally acetylated. To elucidate the effect of Nα-acetylation on the biophysical and biological properties of α-syn, we produced Nα-acetylated α-syn first using a semisynthetic methodology based on expressed protein ligation (Berrade, L., and Camarero, J. A. (2009) Cell. Mol. Life Sci. 66, 3909–3922) and then a recombinant expression strategy, to compare its properties to unacetylated α-syn. We demonstrate that both WT and Nα-acetylated α-syn share a similar secondary structure and oligomeric state using both purified protein preparations and in-cell NMR on E. coli overexpressing Nα-acetylated α-syn. The two proteins have very close aggregation propensities as shown by thioflavin T binding and sedimentation assays. Furthermore, both Nα-acetylated and WT α-syn exhibited similar ability to bind synaptosomal membranes in vitro and in HeLa cells, where both internalized proteins exhibited prominent cytosolic subcellular distribution. We then determined the effect of attenuating Nα-acetylation in living cells, first by using a nonacetylable mutant and then by silencing the enzyme responsible for α-syn Nα-acetylation. Both approaches revealed similar subcellular distribution and membrane binding for both the nonacetylable mutant and WT α-syn, suggesting that N-terminal acetylation does not significantly affect its structure in vitro and in intact cells. PMID:22718772

  3. Large-scale co-evolution analysis of protein structural interlogues using the global protein structural interactome map (PSIMAP).

    PubMed

    Kim, Wan K; Bolser, Dan M; Park, Jong H

    2004-05-01

    Interacting pairs of proteins should co-evolve to maintain functional and structural complementarity. Consequently, such a pair of protein families shows similarity between their phylogenetic trees. Although the tendency of co-evolution has been known for various ligand-receptor pairs, it has not been studied systematically in the widest possible scope. We investigated the degree of co-evolution for more than 900 family pairs in a global protein structural interactome map (PSIMAP--a map of all the structural domain-domain interactions in the PDB). There was significant correlation in 45% of the total SCOPs Family level pairs, rising to 78% in 454 reliable family interactions. Expectedly, the intra-molecular interactions between protein families showed stronger co-evolution than inter-molecular interactions. However, both types of interaction have a fundamentally similar pattern of co-evolution except for cases where different interfaces are involved. These results validate the use of co-evolution analysis with predictive methods such as PSIMAP to improve the accuracy of prediction based on "homologous interaction". The tendency of co-evolution enabled a nearly 5-fold enrichment in the identification of true interactions among the potential interlogues in PSIMAP. The estimated sensitivity was 79.2%, and the specificity was 78.6%. The results of co-evolution analysis are available online at http://www.biointeraction.org

  4. Neural crest development and craniofacial morphogenesis is coordinated by nitric oxide and histone acetylation</