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Sample records for acetylation methylation phosphorylation

  1. Interfacing protein lysine acetylation and protein phosphorylation

    PubMed Central

    Tran, Hue T.; Uhrig, R. Glen; Nimick, Mhairi; Moorhead, Greg B.

    2012-01-01

    Recognition that different protein covalent modifications can operate in concert to regulate a single protein has forced us to re-think the relationship between amino acid side chain modifications and protein function. Results presented by Tran et al. 2012 demonstrate the association of a protein phosphatase (PP2A) with a histone/lysine deacetylase (HDA14) on plant microtubules along with a histone/lysine acetyltransferase (ELP3). This finding reveals a regulatory interface between two prevalent covalent protein modifications, protein phosphorylation and acetylation, emphasizing the integrated complexity of post-translational protein regulation found in nature. PMID:22827947

  2. 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

  3. Rapid test for acetyl-methyl-carbinol formation by Enterobacteriaceae.

    PubMed Central

    Qadri, S M; Nichols, C W; Qadri, S G; Villarreal, A

    1978-01-01

    A modified Voges-Proskauer test is described which distinguishes within 4 to 8 hours between organisms that can produce acetyl-methyl-carbinol (acetoin) from glucose fermentation and those that cannot. PMID:363745

  4. Rapid test for acetyl-methyl-carbinol formation by Enterobacteriaceae.

    PubMed

    Qadri, S M; Nichols, C W; Qadri, S G; Villarreal, A

    1978-10-01

    A modified Voges-Proskauer test is described which distinguishes within 4 to 8 hours between organisms that can produce acetyl-methyl-carbinol (acetoin) from glucose fermentation and those that cannot. PMID:363745

  5. Methods to detect NF-κB Acetylation and Methylation

    PubMed Central

    Chen, JinJing; Chen, Lin-Feng

    2015-01-01

    Summary Post-translational modifications of NF-κB, including acetylation and methylation, have emerged as an important regulatory mechanism for determining the duration and strength of NF-κB nuclear activity as well as its transcriptional output. Within the seven NF-κB family proteins, the RelA subunit of NF-κB is the most studied for its regulation by lysine acetylation and methylation. Acetylation or methylation at different lysine residues modulates distinct functions of NF-κB, including DNA binding and transcription activity, protein stability, and its interaction with NF-κB modulators. Here, we describe the experimental methods to monitor the in vitro and in vivo acetylated or methylated forms of NF-κB. These methods include radiolabeling the acetyl- or methyl- groups and immunoblotting with pan or site-specific acetyl- or methyl-lysine antibodies. Radiolabeling is useful in the initial validation of the modifications. Immunoblotting with antibodies provides a rapid and powerful approach to detect and analyze the functions of these modifications in vitro and in vivo. PMID:25736763

  6. Modulation of the cytoplasmic functions of mammalian post-transcriptional regulatory proteins by methylation and acetylation: a key layer of regulation waiting to be uncovered?

    PubMed

    Blee, Tajekesa K P; Gray, Nicola K; Brook, Matthew

    2015-12-01

    Post-transcriptional control of gene expression is critical for normal cellular function and viability and many of the proteins that mediate post-transcriptional control are themselves subject to regulation by post-translational modification (PTM), e.g. phosphorylation. However, proteome-wide studies are revealing new complexities in the PTM status of mammalian proteins, in particular large numbers of novel methylated and acetylated residues are being identified. Here we review studied examples of methylation/acetylation-dependent regulation of post-transcriptional regulatory protein (PTRP) function and present collated PTM data that points to the huge potential for regulation of mRNA fate by these PTMs. PMID:26614674

  7. Acetylation of insulin receptor substrate-1 is permissive for tyrosine phosphorylation

    PubMed Central

    Kaiser, Christina; James, Stephen R

    2004-01-01

    Background Insulin receptor substrate (IRS) proteins are key moderators of insulin action. Their specific regulation determines downstream protein-protein interactions and confers specificity on growth factor signalling. Regulatory mechanisms that have been identified include phosphorylation of IRS proteins on tyrosine and serine residues and ubiquitination of lysine residues. This study investigated other potential molecular mechanisms of IRS-1 regulation. Results Using the sos recruitment yeast two-hybrid system we found that IRS-1 and histone deacetylase 2 (HDAC2) interact in the cytoplasmic compartment of yeast cells. The interaction mapped to the C-terminus of IRS-1 and was confirmed through co-immunoprecipitation in vitro of recombinant IRS-1 and HDAC2. HDAC2 bound to IRS-1 in mammalian cells treated with phorbol ester or after prolonged treatment with insulin/IGF-1 and also in the livers of ob/ob mice but not PTP1B knockout mice. Thus, the association occurs under conditions of compromised insulin signalling. We found that IRS-1 is an acetylated protein, of which the acetylation is increased by treatment of cells with Trichostatin A (TSA), an inhibitor of HDAC activity. TSA-induced increases in acetylation of IRS-1 were concomitant with increases in tyrosine phosphorylation in response to insulin. These effects were confirmed using RNA interference against HDAC2, indicating that HDAC2 specifically prevents phosphorylation of IRS-1 by the insulin receptor. Conclusions Our results show that IRS-1 is an acetylated protein, a post-translational modification that has not been previously described. Acetylation of IRS-1 is permissive for tyrosine phosphorylation and facilitates insulin-stimulated signal transduction. Specific inhibition of HDAC2 may increase insulin sensitivity in otherwise insulin resistant conditions. PMID:15522123

  8. Effects of methyl acetyl phosphate, a covalent antisickling agent, on the density profiles of sickle erythrocytes.

    PubMed

    Ueno, H; Yatco, E; Benjamin, L J; Manning, J M

    1992-07-01

    Methyl acetyl phosphate specifically acetylates valine-1, lysine-82, and lysine-144 in the 2,3-diphosphoglycerate binding cleft of hemoglobin S, thereby inhibiting its gelation (greater than 32 gm/dl) at pH 7.4. To extend these findings, the effect of methyl acetyl phosphate on the density of sickle cells has been evaluated by phthalate ester gradient centrifugation and by Larex-Percoll density centrifugation. After treatment with methyl acetyl phosphate (40% modification of the intracellular hemoglobin S), oxygenated sickle erythrocytes had a lowered density profile, as measured in a phthalate ester gradient. Thus 83% of untreated oxygenated sickle cells had densities greater than 1.098 gm/ml, whereas after treatment with methyl acetyl phosphate, 52% of the cells were in this density range. Under anaerobic conditions, methyl acetyl phosphate was even more effective in lowering cell density. For example, 50% of untreated deoxygenated cells had densities greater than 1.098 gm/ml, but none of the cells treated with methyl acetyl phosphate were this dense. For studies with Larex-Percoll density gradients, sickle erythrocytes were first separated into two fractions (densities greater than and less than 1.1 gm/ml) by Percoll-Hypaque centrifugation. The amount of oxygenated sickle cells exhibiting densities greater than 1.074 gm/ml decreased by about 32% on treatment with methyl acetyl phosphate. For deoxygenated sickle cells, treatment with methyl acetyl phosphate resulted in an average decrease of approximately 24% in the number of cells with densities greater than 1.074 gm/ml.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1613320

  9. Cross-talk between phosphorylation and lysine acetylation in a genome-reduced bacterium

    PubMed Central

    van Noort, Vera; Seebacher, Jan; Bader, Samuel; Mohammed, Shabaz; Vonkova, Ivana; Betts, Matthew J; Kühner, Sebastian; Kumar, Runjun; Maier, Tobias; O'Flaherty, Martina; Rybin, Vladimir; Schmeisky, Arne; Yus, Eva; Stülke, Jörg; Serrano, Luis; Russell, Robert B; Heck, Albert JR; Bork, Peer; Gavin, Anne-Claude

    2012-01-01

    Protein post-translational modifications (PTMs) represent important regulatory states that when combined have been hypothesized to act as molecular codes and to generate a functional diversity beyond genome and transcriptome. We systematically investigate the interplay of protein phosphorylation with other post-transcriptional regulatory mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae. Systematic perturbations by deletion of its only two protein kinases and its unique protein phosphatase identified not only the protein-specific effect on the phosphorylation network, but also a modulation of proteome abundance and lysine acetylation patterns, mostly in the absence of transcriptional changes. Reciprocally, deletion of the two putative N-acetyltransferases affects protein phosphorylation, confirming cross-talk between the two PTMs. The measured M. pneumoniae phosphoproteome and lysine acetylome revealed that both PTMs are very common, that (as in Eukaryotes) they often co-occur within the same protein and that they are frequently observed at interaction interfaces and in multifunctional proteins. The results imply previously unreported hidden layers of post-transcriptional regulation intertwining phosphorylation with lysine acetylation and other mechanisms that define the functional state of a cell. PMID:22373819

  10. SIRT1 phosphorylation by AMP-activated protein kinase regulates p53 acetylation

    PubMed Central

    Lau, Alan W; Liu, Pengda; Inuzuka, Hiroyuki; Gao, Daming

    2014-01-01

    The deacetylase SIRT1 regulates multiple biological processes including cellular metabolism and aging. Importantly, SIRT1 can also inactivate the p53 tumor suppressor via deacetylation, suggesting a role in oncogenesis. Recently, SIRT1 was shown to be released from its endogenous inhibitor DBC1 by a process requiring AMPK and the phosphorylation of SIRT1 by yet undefined kinase(s). Here we provide further evidence that AMPK directly phosphorylates SIRT1 on T344, releasing it from DBC1. Furthermore, a phospho-mimetic SIRT1 (T334E) showed decreased binding to DBC1, supporting the importance of this phosphorylation in AMPK-mediated regulation of SIRT1 activity. In addition, inhibition of AMPK by Compound C led to increased p53 acetylation, suggesting a role for the AMPK/SIRT1 pathway in regulating p53 signaling. Together, our results support a hypothesis that AMPK negatively regulates p53 acetylation via phosphorylation of SIRT1 on T344. Furthermore, our findings also define the AMPK/SIRT1 axis as a possible targetable pathway to regulate p53 function. PMID:24959379

  11. N-acetylation and phosphorylation of Sec complex subunits in the ER membrane

    PubMed Central

    2012-01-01

    Background Covalent modifications of proteins provide a mechanism to control protein function. Here, we have investigated modifications of the heptameric Sec complex which is responsible for post-translational protein import into the endoplasmic reticulum (ER). It consists of the Sec61 complex (Sec61p, Sbh1p, Sss1p) which on its own mediates cotranslational protein import into the ER and the Sec63 complex (Sec63p, Sec62p, Sec71p, Sec72p). Little is known about the biogenesis and regulation of individual Sec complex subunits. Results We show that Sbh1p when it is part of the Sec61 complex is phosphorylated on T5 which is flanked by proline residues. The phosphorylation site is conserved in mammalian Sec61ß, but only partially in birds, and not in other vertebrates or unicellular eukaryotes, suggesting convergent evolution. Mutation of T5 to A did not affect the ability of mutant Sbh1p to complement the growth defect in a Δsbh1Δsbh2 strain, and did not result in a hypophosphorylated protein which shows that alternate sites can be used by the T5 kinase. A survey of yeast phosphoproteome data shows that Sbh1p can be phosphorylated on multiple sites which are organized in two patches, one at the N-terminus of its cytosolic domain, the other proximal to the transmembrane domain. Surprisingly, although N-acetylation has been shown to interfere with ER targeting, we found that both Sbh1p and Sec62p are cotranslationally N-acetylated by NatA, and N-acetyl-proteome data indicate that Sec61p is modified by the same enzyme. Mutation of the N-acetylation site, however, did not affect Sec62p function in posttranslational protein import into the ER. Disabling NatA resulted in growth retardation, but not in co- or posttranslational translocation defects or instability of Sec62p or Sbh1p. Conclusions We conclude that N-acetylation of transmembrane and tail-anchored proteins does not interfere with their ER-targeting, and that Sbh1p phosphorylation on T5, which is not present in

  12. The Intracellular Concentration of Acetyl Phosphate in Escherichia coli Is Sufficient for Direct Phosphorylation of Two-Component Response Regulators▿

    PubMed Central

    Klein, Adam H.; Shulla, Ana; Reimann, Sylvia A.; Keating, David H.; Wolfe, Alan J.

    2007-01-01

    Acetyl phosphate, the intermediate of the AckA-Pta pathway, acts as a global signal in Escherichia coli. Although acetyl phosphate clearly signals through two-component response regulators, it remains unclear whether acetyl phosphate acts as a direct phospho donor or functions through an indirect mechanism. We used two-dimensional thin-layer chromatography to measure the relative concentrations of acetyl phosphate, acetyl coenzyme A, ATP, and GTP over the course of the entire growth curve. We estimated that the intracellular concentration of acetyl phosphate in wild-type cells reaches at least 3 mM, a concentration sufficient to activate two-component response regulators via direct phosphoryl transfer. PMID:17545286

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

    NASA Astrophysics Data System (ADS)

    Zhang, Kangling

    2008-01-01

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

  14. Kinetic studies on the acid hydrolysis of the methyl ketoside of unsubstituted and O-acetylated N-acetylneuraminic acid

    PubMed Central

    Neuberger, A.; Ratcliffe, Wendy A.

    1973-01-01

    The hydrolysis of the model compound 2-O-methyl-4,7,8,9-tetra-O-acetyl-N-acetyl-α-d-neuraminic acid and neuraminidase (Vibrio cholerae) closely resembled that of the O-acetylated sialic acid residues of rabbit Tamm–Horsfall glycoprotein. This confirmed that O-acetylation was responsible for the unusually slow rate of acid hydrolysis of O-acetylated sialic acid residues observed in rabbit Tamm–Horsfall glycoprotein and their resistance to hydrolysis by neuraminidase. The first-order rate constant of hydrolysis of 2-methyl-N-acetyl-α-d-neuraminic acid by 0.05m-H2SO4 was 56-fold greater than that of 2-O-methyl-4,7,8,9-tetra-O-acetyl-N-acetyl -α-d-neuraminic acid. Kinetic studies have shown that in the pH range 1.00–3.30, the observed rate of hydrolysis of 2-methyl-N-acetyl-α-d-neuraminic acid can be attributed to acid-catalysed hydrolysis of the negatively charged CO2− form of the methyl ketoside. PMID:4748825

  15. Dynamics of DNA methylation and Histone H4 acetylation during floral bud differentiation in azalea

    PubMed Central

    2010-01-01

    Background The ability to control the timing of flowering is a key strategy for planning production in ornamental species such as azalea, however it requires a thorough understanding of floral transition. Floral transition is achieved through a complex genetic network and regulated by multiple environmental and endogenous cues. Dynamic changes between chromatin states facilitating or inhibiting DNA transcription regulate the expression of floral induction pathways in response to environmental and developmental signals. DNA methylation and histone modifications are involved in controlling the functional state of chromatin and gene expression. Results The results of this work indicate that epigenetic mechanisms such as DNA methylation and histone H4 acetylation have opposite and particular dynamics during the transition from vegetative to reproductive development in the apical shoots of azalea. Global levels of DNA methylation and histone H4 acetylation as well as immunodetection of 5-mdC and acetylated H4, in addition to a morphological study have permitted the delimitation of four basic phases in the development of the azalea bud and allowed the identification of a stage of epigenetic reprogramming which showed a sharp decrease of whole DNA methylation similar to that is defined in other developmental processes in plants and in mammals. Conclusion The epigenetic control and reorganization of chromatin seem to be decisive for coordinating floral development in azalea. DNA methylation and H4 deacetylation act simultaneously and co-ordinately, restructuring the chromatin and regulating the gene expression during soot apical meristem development and floral differentiation. PMID:20067625

  16. Acetyl Methyl Torsion in N-Ethylacetamide: A Challenge for Microwave Spectroscopy and Quantum Chemistry.

    PubMed

    Kannengießer, Raphaela; Lach, Marcel J; Stahl, Wolfgang; Nguyen, Ha Vinh Lam

    2015-06-22

    The gas-phase structures and parameters describing acetyl methyl torsion of N-ethylacetamide are determined with high accuracy, using a combination of molecular beam Fourier-transform microwave spectroscopy and quantum chemical calculations. Conformational studies at the MP2 level of theory yield four minima on the energy surface. The most energetically favorable conformer, which possesses C1 symmetry, is assigned. Due to the torsional barrier of 73.4782(1) cm(-1) of the acetyl methyl group, fine splitting up to 4.9 GHz is found in the spectrum. The conformational structure is not only confirmed by the rotational constants, but also by the orientation of the internal rotor. The (14) N quadrupole hyperfine splittings are analyzed and the deduced coupling constants are compared with the calculated values. PMID:25916631

  17. Structural Basis for Phosphorylation and Lysine Acetylation Cross-talk in a Kinase Motif Associated with Myocardial Ischemia and Cardioprotection*

    PubMed Central

    Parker, Benjamin L.; Shepherd, Nicholas E.; Trefely, Sophie; Hoffman, Nolan J.; White, Melanie Y.; Engholm-Keller, Kasper; Hambly, Brett D.; Larsen, Martin R.; James, David E.; Cordwell, Stuart J.

    2014-01-01

    Myocardial ischemia and cardioprotection by ischemic pre-conditioning induce signal networks aimed at survival or cell death if the ischemic period is prolonged. These pathways are mediated by protein post-translational modifications that are hypothesized to cross-talk with and regulate each other. Phosphopeptides and lysine-acetylated peptides were quantified in isolated rat hearts subjected to ischemia or ischemic pre-conditioning, with and without splitomicin inhibition of lysine deacetylation. We show lysine acetylation (acetyl-Lys)-dependent activation of AMP-activated protein kinase, AKT, and PKA kinases during ischemia. Phosphorylation and acetyl-Lys sites mapped onto tertiary structures were proximal in >50% of proteins investigated, yet they were mutually exclusive in 50 ischemic pre-conditioning- and/or ischemia-associated peptides containing the KXXS basophilic protein kinase consensus motif. Modifications in this motif were modeled in the C terminus of muscle-type creatine kinase. Acetyl-Lys increased proximal dephosphorylation by 10-fold. Structural analysis of modified muscle-type creatine kinase peptide variants by two-dimensional NMR revealed stabilization via a lysine-phosphate salt bridge, which was disrupted by acetyl-Lys resulting in backbone flexibility and increased phosphatase accessibility. PMID:25008320

  18. Rapid changes in chick liver acetyl-CoA carboxylase indicative of phosphorylation control.

    PubMed

    Clarke, S D

    1983-10-31

    Liver fatty acid synthesis was suppressed 75,95 and 90% within 1, 2 and 4 hrs respectively of depriving chicks of food. Accompanying this rapid drop in lipogenesis was a marked reduction in acetyl-CoA carboxylase activity, i.e., 40 and 75% decrease after 2 and 4 hrs of fasting. Adding 10 mM citrate to the crude liver supernatant, or incubating the supernatant at 37 degrees, 30 min increased activity of the briefly fasted birds, but neither method restored carboxylase activity to fed level. Heat and citrate activation were additive and together resulted in an activity comparable to the fed condition. The heat-dependent activation was accelerated by exogenous phosphoprotein phosphatase, and completely blocked by 100 mM NaF. Thus, enhancement of carboxylase activity from liver of briefly fasted chicks appears to be a dephosphorylation process. This is the first report indicating acute changes in chick carboxylase activity may involve a phosphorylation-dephosphorylation mechanism. PMID:6140006

  19. 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. PMID:27185186

  20. Histone H3 Acetylation and H3 K4 Methylation Define Distinct Chromatin Regions Permissive for Transgene Expression

    PubMed Central

    Yan, Chunhong; Boyd, Douglas D.

    2006-01-01

    Histone modifications are associated with distinct transcription states and serve as heritable epigenetic markers for chromatin structure and function. While H3 K9 methylation defines condensed heterochromatin that is able to silence a nearby gene, how gene silencing within euchromatin regions is achieved remains elusive. We report here that histone H3 K4 methylation or K9/K14 acetylation defines distinct chromatin regions permissive or nonpermissive for transgene expression. A permissive chromatin region is enriched in H3 K4 methylation and H3 acetylation, while a nonpermissive region is poor in or depleted of these two histone modifications. The histone modification states of the permissive chromatin can spread to transgenic promoters. However, de novo histone H3 acetylation and H3 K4 methylation at a transgenic promoter in a nonpermissive chromatin region are stochastic, leading to variegated transgene expression. Moreover, nonpermissive chromatin progressively silences a transgene, an event that is accompanied by the reduction of H3 K4 methylation and H3 acetylation levels at the transgenic promoter. These repressive effects of nonpermissive chromatin cannot be completely countered by strong transcription activators, indicating the dominance of the chromatin effects. We therefore propose a model in which histone H3 acetylation and H3 K4 methylation localized to discrete sites in the mammalian genome mark distinct chromatin functions that dictate transgene expression or silencing. PMID:16914722

  1. Phosphorylation of Methyl-α-d-Glucopyranoside in Polymyxin B-Treated Salmonella typhimurium

    PubMed Central

    Teuber, Michael

    1969-01-01

    Phosphoenolpyruvate-dependent phosphorylation of methyl-α-d-glucopyranoside in Salmonella typhimurium is increased by the membrane active polypeptide antibiotic polymyxin B whereas active transport ability is abolished. PMID:4311870

  2. Aurora B and Cdk1 mediate Wapl activation and release of acetylated cohesin from chromosomes by phosphorylating Sororin

    PubMed Central

    Nishiyama, Tomoko; Sykora, Martina M.; Huis in ’t Veld, Pim J.; Mechtler, Karl; Peters, Jan-Michael

    2013-01-01

    Sister chromatid cohesion depends on Sororin, a protein that stabilizes acetylated cohesin complexes on DNA by antagonizing the cohesin release factor Wings-apart like protein (Wapl). Cohesion is essential for chromosome biorientation but has to be dissolved to enable sister chromatid separation. To achieve this, the majority of cohesin is removed from chromosome arms in prophase and prometaphase in a manner that depends on Wapl and phosphorylation of cohesin’s subunit stromal antigen 2 (SA2), whereas centromeric cohesin is cleaved in metaphase by the protease separase. Here we show that the mitotic kinases Aurora B and Cyclin-dependent kinase 1 (Cdk1) destabilize interactions between Sororin and the cohesin subunit precocious dissociation of sisters protein 5 (Pds5) by phosphorylating Sororin, leading to release of acetylated cohesin from chromosome arms and loss of cohesion. At centromeres, the cohesin protector shugoshin (Sgo1)-protein phosphatase 2A (PP2A) antagonizes Aurora B and Cdk1 partly by dephosphorylating Sororin and thus maintains cohesion until metaphase. We propose that the stepwise loss of cohesion between chromosome arms and centromeres is caused by local regulation of Wapl activity, which is controlled by the phosphorylation state of Sororin. PMID:23901111

  3. Interplay between lysine methylation and Cdk phosphorylation in growth control by the retinoblastoma protein

    PubMed Central

    Carr, Simon M; Munro, Shonagh; Kessler, Benedikt; Oppermann, Udo; La Thangue, Nicholas B

    2011-01-01

    As a critical target for cyclin-dependent kinases (Cdks), the retinoblastoma tumour suppressor protein (pRb) controls early cell cycle progression. We report here a new type of regulation that influences Cdk recognition and phosphorylation of substrate proteins, mediated through the targeted methylation of a critical lysine residue in the Cdk substrate recognition site. In pRb, lysine (K) 810 represents the essential and conserved basic residue (SPXK) required for cyclin/Cdk recognition and phosphorylation. Methylation of K810 by the methyltransferase Set7/9 impedes binding of Cdk and thereby prevents subsequent phosphorylation of the associated serine (S) residue, retaining pRb in the hypophosphorylated growth-suppressing state. Methylation of K810 is under DNA damage control, and methylated K810 impacts on phosphorylation at sites throughout the pRb protein. Set7/9 is required for efficient cell cycle arrest, and significantly, a mutant derivative of pRb that cannot be methylated at K810 exhibits compromised cell cycle arrest. Thus, the regulation of phosphorylation by Cdks reflects the combined interplay with methylation events, and more generally the targeted methylation of a lysine residue within a Cdk-consensus site in pRb represents an important point of control in cell cycle progression. PMID:21119616

  4. Histone deacetylase inhibitors valproic acid and depsipeptide sensitize retinoblastoma cells to radiotherapy by increasing H2AX phosphorylation and p53 acetylation-phosphorylation.

    PubMed

    Kawano, Takeshi; Akiyama, Masaharu; Agawa-Ohta, Miyuki; Mikami-Terao, Yoko; Iwase, Satsuki; Yanagisawa, Takaaki; Ida, Hiroyuki; Agata, Naoki; Yamada, Hisashi

    2010-10-01

    Although p53 is intact in most cases of retinoblastoma, it is largely inactivated by the ubiqutin-proteasome system through interaction with murine double minute 2 (MDM2) and murine double minute X (MDMX). The present study showed that the histone deacetylase (HDAC) inhibitors valproic acid (VPA) and depsipeptide (FK228) synergistically enhanced ionizing radiation (IR)-induced apoptosis, associated with activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase in Y79 and WER1-Rb1 human retinoblastoma cells. Both VPA and FK228 enhanced IR-induced phosphorylation of histone H2AX on Ser139 preceding apoptosis. Exposure of cells to IR in the presence of VPA or FK228 induced the accumulation of p53 acetylated at Lys382 and phosphorylated at Ser46 through the reduction of binding affinity with MDM2 and MDMX. These results suggest that acetylation of p53 by HDAC inhibitors is a promising new therapeutic target in refractory retinoblastoma. PMID:20811699

  5. New polygalacturonases from Trichoderma reesei: characterization and their specificities to partially methylated and acetylated pectins.

    PubMed

    Mohamed, Saleh A; Christensen, Tove M I E; Mikkelsen, Jorn Dalgaard

    2003-03-14

    Two extracellular isoenzymes of polygalacturonases PG1 and PG2 were isolated from 3-day-old culture filtrates of Trichoderma reesei. The two enzymes were purified to homogeneity by ion-exchange, gel filtration and hydrophobic interaction chromatographies. PG1 and PG2 exhibit similar molecular weights from gel filtration and SDS-PAGE. Their properties, including optimal pH and temperature, thermal stability and Km were compared. Characterization of substrate specificity showed that the two enzymes had higher affinity toward PGA (B0100) derived from sugar beet pectin (SBP) than PGA from lime pectin. A series of SBPs with different distribution patterns of methyl and acetyl groups, produced by treatment with either plant pectin methylesterase (P-series) or fungal pectin methylesterase (F-series) or base catalysis (B-series), was used as substrates for PG1 and PG2. Substrates with a low degree of esterification were preferred substrates. The activities of PG1 and PG2 were strongly correlated to the degree of methylation and very little effect from acetylation. The products generated by digestion of selected lime and SBPs were analysed using matrix assisted laser desorption ionisation time of flight (MALDI TOF) MS. A mode of action revealed a random cleavage pattern for PG1 and PG2, confirming that these enzymes are endopolygalacturonases. PMID:12668107

  6. Reduction and Methyl Transfer Kinetics of the Alpha Subunit from Acetyl-Coenzyme A Synthase

    SciTech Connect

    Xiangshi Tan; Christopher Sewell; Qingwu Yang; Paul A. Lindahl

    2003-01-15

    OAK-B135 Stopped-flow was used to evaluate the methylation and reduction kinetics of the isolated alpha subunit of acetyl-Coenzyme A synthase from Moorella thermoacetica. This catalytically active subunit contains a novel Ni-X-Fe4S4 cluster and a putative unidentified n =2 redox site called D. The D-site must be reduced for a methyl group to transfer from a corrinoid-iron-sulfur protein, a key step in the catalytic synthesis of acetyl-CoA. The Fe4S4 component of this cluster is also redox active, raising the possibility that it is the D-site or a portion thereof. Results presented demonstrate that the D-site reduces far faster than the Fe4S4 component, effectively eliminating this possibility. Rather, this component may alter catalytically important properties of the Ni center. The D-site is reduced through a pathway that probably does not involve the Fe4S4 component of this active-site cluster.

  7. Regulation of DNA methylation patterns by CK2-mediated phosphorylation of Dnmt3a.

    PubMed

    Deplus, Rachel; Blanchon, Loïc; Rajavelu, Arumugam; Boukaba, Abdelhalim; Defrance, Matthieu; Luciani, Judith; Rothé, Françoise; Dedeurwaerder, Sarah; Denis, Hélène; Brinkman, Arie B; Simmer, Femke; Müller, Fabian; Bertin, Benjamin; Berdasco, Maria; Putmans, Pascale; Calonne, Emilie; Litchfield, David W; de Launoit, Yvan; Jurkowski, Tomasz P; Stunnenberg, Hendrik G; Bock, Christoph; Sotiriou, Christos; Fraga, Mario F; Esteller, Manel; Jeltsch, Albert; Fuks, François

    2014-08-01

    DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns. PMID:25066127

  8. Histone H3 lysine 4 acetylation and methylation dynamics define breast cancer subtypes

    PubMed Central

    Messier, Terri L.; Gordon, Jonathan A. R.; Boyd, Joseph R.; Tye, Coralee E.; Browne, Gillian; Stein, Janet L.; Lian, Jane B.; Stein, Gary S.

    2016-01-01

    The onset and progression of breast cancer are linked to genetic and epigenetic changes that alter the normal programming of cells. Epigenetic modifications of DNA and histones contribute to chromatin structure that result in the activation or repression of gene expression. Several epigenetic pathways have been shown to be highly deregulated in cancer cells. Targeting specific histone modifications represents a viable strategy to prevent oncogenic transformation, tumor growth or metastasis. Methylation of histone H3 lysine 4 has been extensively studied and shown to mark genes for expression; however this residue can also be acetylated and the specific function of this alteration is less well known. To define the relative roles of histone H3 methylation (H3K4me3) and acetylation (H3K4ac) in breast cancer, we determined genomic regions enriched for both marks in normal-like (MCF10A), transformed (MCF7) and metastatic (MDA-MB-231) cells using a genome-wide ChIP-Seq approach. Our data revealed a genome-wide gain of H3K4ac associated with both early and late breast cancer cell phenotypes, while gain of H3K4me3 was predominantly associated with late stage cancer cells. Enrichment of H3K4ac was over-represented at promoters of genes associated with cancer-related phenotypic traits, such as estrogen response and epithelial-to-mesenchymal transition pathways. Our findings highlight an important role for H3K4ac in predicting epigenetic changes associated with early stages of transformation. In addition, our data provide a valuable resource for understanding epigenetic signatures that correlate with known breast cancer-associated oncogenic pathways. PMID:26783963

  9. Histone H3 lysine 4 acetylation and methylation dynamics define breast cancer subtypes.

    PubMed

    Messier, Terri L; Gordon, Jonathan A R; Boyd, Joseph R; Tye, Coralee E; Browne, Gillian; Stein, Janet L; Lian, Jane B; Stein, Gary S

    2016-02-01

    The onset and progression of breast cancer are linked to genetic and epigenetic changes that alter the normal programming of cells. Epigenetic modifications of DNA and histones contribute to chromatin structure that result in the activation or repression of gene expression. Several epigenetic pathways have been shown to be highly deregulated in cancer cells. Targeting specific histone modifications represents a viable strategy to prevent oncogenic transformation, tumor growth or metastasis. Methylation of histone H3 lysine 4 has been extensively studied and shown to mark genes for expression; however this residue can also be acetylated and the specific function of this alteration is less well known. To define the relative roles of histone H3 methylation (H3K4me3) and acetylation (H3K4ac) in breast cancer, we determined genomic regions enriched for both marks in normal-like (MCF10A), transformed (MCF7) and metastatic (MDA-MB-231) cells using a genome-wide ChIP-Seq approach. Our data revealed a genome-wide gain of H3K4ac associated with both early and late breast cancer cell phenotypes, while gain of H3K4me3 was predominantly associated with late stage cancer cells. Enrichment of H3K4ac was over-represented at promoters of genes associated with cancer-related phenotypic traits, such as estrogen response and epithelial-to-mesenchymal transition pathways. Our findings highlight an important role for H3K4ac in predicting epigenetic changes associated with early stages of transformation. In addition, our data provide a valuable resource for understanding epigenetic signatures that correlate with known breast cancer-associated oncogenic pathways. PMID:26783963

  10. Computational Study of Environmental Effects on Torsional Free Energy Surface of N-Acetyl-N'-methyl-L-alanylamide Dipeptide

    ERIC Educational Resources Information Center

    Carlotto, Silvia; Zerbetto, Mirco

    2014-01-01

    We propose an articulated computational experiment in which both quantum mechanics (QM) and molecular mechanics (MM) methods are employed to investigate environment effects on the free energy surface for the backbone dihedral angles rotation of the small dipeptide N-Acetyl-N'-methyl-L-alanylamide. This computation exercise is appropriate for an…

  11. Receptor interactions through phosphorylation and methylation pathways in bacterial chemotaxis.

    PubMed Central

    Sanders, D A; Koshland, D E

    1988-01-01

    The effects of messages initiated by one receptor on the covalent modification of a second receptor were studied by use of a technique for rapidly separating the receptors. Methylation of the bacterial-chemotactic serine receptor increases as a result of aspartate binding to the aspartate receptor. The aspartate-induced methylation on the serine receptor is absent in a strain that lacks cheA and cheW genes and is not the result of physical interaction, such as the formation of heterodimers between the aspartate and serine receptors, or of alterations in the affinity of the serine receptor for the methyltransferase and the methylesterase. Serine-induced methylation of the serine receptor did not require cheA and cheW. A model is presented in which the receptor methylation level depends on the combination of (i) a ligand-induced conformational change on the receptor substrate of the methylation enzymes and (ii) an indirect cytoplasmic signal that operates through the methylesterase. PMID:2847160

  12. DNA methylation and histone acetylation regulate the expression of MGMT and chemosensitivity to temozolomide in malignant melanoma cell lines.

    PubMed

    Chen, Ya-Ping; Hou, Xiao-Yang; Yang, Chun-Sheng; Jiang, Xiao-Xiao; Yang, Ming; Xu, Xi-Feng; Feng, Shou-Xin; Liu, Yan-Qun; Jiang, Guan

    2016-08-01

    Malignant melanoma is an aggressive, highly lethal dermatological malignancy. Chemoresistance and rapid metastasis limit the curative effect of multimodal therapies like surgery or chemotherapy. The suicide enzyme O6-methylguanine-DNA methyltransferase (MGMT) removes adducts from the O6-position of guanine to repair DNA damage. High MGMT expression is associated with resistance to therapy in melanoma. However, it is unknown if MGMT is regulated by DNA methylation or histone acetylation in melanoma. We examined the effects of the DNA methylation inhibitor 5-Aza-2'-deoxycytidine and histone deacetylase inhibitor Trichostatin A alone or in combination on MGMT expression and promoter methylation and histone acetylation in A375, MV3, and M14 melanoma cells. This study demonstrates that MGMT expression, CpG island methylation, and histone acetylation vary between melanoma cell lines. Combined treatment with 5-Aza-2'-deoxycytidine and Trichostatin A led to reexpression of MGMT, indicating that DNA methylation and histone deacetylation are associated with silencing of MGMT in melanoma. This study provides information on the role of epigenetic modifications in malignant melanoma that may enable the development of new strategies for treating malignant melanoma. PMID:26943799

  13. Mechanism of glucagon inhibition of liver acetyl-CoA carboxylase. Interrelationship of the effects of phosphorylation, polymer-protomer transition, and citrate on enzyme activity.

    PubMed

    Swenson, T L; Porter, J W

    1985-03-25

    The short-term regulation of rat liver acetyl-CoA carboxylase by glucagon has been studied in hepatocytes from rats that had been fasted and refed a fat-free diet. Glucagon inhibition of the activity of this enzyme can be accounted for by a direct correlation between phosphorylation, polymer-protomer ratio, and activity. Glucagon rapidly inactivates acetyl-CoA carboxylase with an accompanying 4-fold increase in the phosphorylation of the enzyme and 3-fold increase in the protomer-polymer ratio of enzyme protein. Citrate, an allosteric activator of acetyl-CoA carboxylase required for enzyme activity, has no effect on these phenomena, indicating a mechanism that is independent of citrate concentration within the cell. The observation of these effects of glucagon on acetyl-CoA carboxylase activity is absolutely dependent upon the minimization of proteolytic degradation of the enzyme after cell lysis. Therefore, for the first time, an interrelationship has been demonstrated between phosphorylation, protomer-polymer ratio, and citrate for the inactivation of acetyl-CoA carboxylase by glucagon. PMID:2857722

  14. 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

  15. Neuroprotection in rabbit retina with N-acetyl-aspartylglutamate and 2-phosphonyl-methyl pentanedioic acid

    NASA Astrophysics Data System (ADS)

    Hacker, Henry D.; Yourick, Debra L.; Koenig, Michael K.; Slusher, Barbara S.; Meyerhoff, James L.

    1999-06-01

    Retinal tissue is subject to ischemia from diabetic retinopathy and other conditions that affect the retinal vasculature such as lupus erythematosus and temporal arteritis. There is evidence in animal models of reversible ischemia that a therapeutic window exists during early recovery when agents that reduce glutamate activity at its receptor sites can rescue neurons from injury. To model ischemia, we used sodium cyanide (NaCN), to inhibit oxidative metabolism, and 2-deoxyglucose (2-DG) to inhibit glycolysis. Dissociated rabbit retina cells were studied to evaluate the potential neuroprotective effects of N-acetyl-aspartyl-glutamate (MAAG), which competes with glutamate as a low-potency agonist at the NMDA receptor complex. N-acetylated α-linked acidic dipeptidase (NAALADase; the NAAG-hydrolyzing enzyme) is responsible for the hydrolysis of NAAG into glutamate, a neurotransmitter and potent excitotoxin, and N-acetylaspartate. 2-Phosphonyl-methyl pentanedioic acid (PMPA) and β-linked NAAG (β-NAAG), inhibitors of NAALADase, were also tested, since inhibition of NAALADase could reduce synaptic glutamate and increase the concentration of NAAG. We found that metabolic inhibition with NaCN/2-DG for 1 hour caused 50% toxicity as assessed with the MTT assay. Co-treatment with NAAG resulted in dose-dependent protection of up to 55% (p<0.005). When the non-hydrolyzable, NAALADase inhibitor β-NAAG was employed dose-dependent protection of up to 37% was observed (p<0.001). PMPA also showed 48% protection (p<.05-.001) against these insults. These data suggest that NAAG may antagonize the effect of glutamate at the NMDA receptor complex in retina. Inhibition of NAALADase by PMPA and β-NAAG may increase the activity of endogenous NAAG.

  16. Unique epigenetic influence of H2AX phosphorylation and H3K56 acetylation on normal stem cell radioresponses

    PubMed Central

    Jacobs, Keith M.; Misri, Sandeep; Meyer, Barbara; Raj, Suyash; Zobel, Cheri L.; Sleckman, Barry P.; Hallahan, Dennis E.; Sharma, Girdhar G.

    2016-01-01

    Normal tissue injury resulting from cancer radiotherapy is often associated with diminished regenerative capacity. We examined the relative radiosensitivity of normal stem cell populations compared with non–stem cells within several radiosensitive tissue niches and culture models. We found that these stem cells are highly radiosensitive, in contrast to their isogenic differentiated progeny. Of interest, they also exhibited a uniquely attenuated DNA damage response (DDR) and muted DNA repair. Whereas stem cells exhibit reduced ATM activation and ionizing radiation–induced foci, they display apoptotic pannuclear H2AX-S139 phosphorylation (γH2AX), indicating unique radioresponses. We also observed persistent phosphorylation of H2AX-Y142 along the DNA breaks in stem cells, which promotes apoptosis while inhibiting DDR signaling. In addition, down-regulation of constitutively elevated histone-3 lysine-56 acetylation (H3K56ac) in stem cells significantly decreased their radiosensitivity, restored DDR function, and increased survival, signifying its role as a key contributor to stem cell radiosensitivity. These results establish that unique epigenetic landscapes affect cellular heterogeneity in radiosensitivity and demonstrate the nonubiquitous nature of radiation responses. We thus elucidate novel epigenetic rheostats that promote ionizing radiation hypersensitivity in various normal stem cell populations, identifying potential molecular targets for pharmacological radioprotection of stem cells and hopefully improving the efficacy of future cancer treatment. PMID:26941327

  17. Leptin Effect on Acetylation and Phosphorylation of Pgc1α in Muscle Cells Associated With Ampk and Akt Activation in High-Glucose Medium.

    PubMed

    García-Carrizo, Francisco; Nozhenko, Yuriy; Palou, Andreu; Rodríguez, Ana M

    2016-03-01

    Leptin is crucial in energy metabolism, including muscle regulation. Peroxisome proliferator activated receptor gamma co-activator 1α (PGC1α) orchestrates energy metabolism and is tightly controlled by post-translational covalent modifications such as phosphorylation and acetylation. We aimed to further the knowledge of PGC1α control by leptin (at physiological levels) in muscle cells by time-sequentially analysing the activation of AMP activated protein kinase (AMPK), P38 mitogen-activated protein kinase (P38 MAPK) and Akt (Protein kinase B)--all known to phosphorylate PGC1α and to be involved in the regulation of its acetylation status--in C2C12 myotubes placed in a high-glucose serum-free medium. We also studied the protein levels of PGC1α, Sirtuin 1, adiponectin, COX IV, mitofusin 2 (Mfn2), and pyruvate dehydrogenase kinase 4 (PDK4). Our main findings suggest an important role of leptin regulating AMPK and Akt phosphorylation, Mfn2 induction and PGC1α acetylation status, with the novelty that the latter in transitorily increased in response to leptin, an effect dependent, at least in part, on AMPK regulation. These post-translational reversible changes in PGC1α in response to leptin, especially the increase in acetylation status, may be related to the physiological role of the hormone in modulating muscle cell response to the physiological/nutritional status. PMID:26218179

  18. MODE OF ACTION OF ACETYLXYLAN ESTERASE FROM STREPTOMYCES LIVIDANS: A STUDY WITH DEOXY AND DEOXY-FLUORO ANALOGUES OF ACETYLATED METHYL BETA-D-XYLOPYRANOSIDE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The action of Streptomyces lividans acetylxylan esterase on methyl 2,4-di-O-acetyl- and 3,4-di-O-acetyl beta-D-xylopyranoside was compared with its action on the 2- and 3-deoxy and 2- and 3-deoxy-fluoro-analogues of the two diacetates in order to elucidate the role of the free hydroxyl group in the ...

  19. Phosphorylation and arginine methylation mark histone H2A prior to deposition during Xenopus laevis development

    PubMed Central

    2014-01-01

    Background Stored, soluble histones in eggs are essential for early development, in particular during the maternally controlled early cell cycles in the absence of transcription. Histone post-translational modifications (PTMs) direct and regulate chromatin-templated transactions, so understanding the nature and function of pre-deposition maternal histones is essential to deciphering mechanisms of regulation of development, chromatin assembly, and transcription. Little is known about histone H2A pre-deposition modifications nor known about the transitions that occur upon the onset of zygotic control of the cell cycle and transcription at the mid-blastula transition (MBT). Results We isolated histones from staged Xenopus laevis oocytes, eggs, embryos, and assembled pronuclei to identify changes in histone H2A modifications prior to deposition and in chromatin. Soluble and chromatin-bound histones from eggs and embryos demonstrated distinct patterns of maternal and zygotic H2A PTMs, with significant pre-deposition quantities of S1ph and R3me1, and R3me2s. We observed the first functional distinction between H2A and H4 S1 phosphorylation, as we showed that H2A and H2A.X-F (also known as H2A.X.3) serine 1 (S1) is phosphorylated concomitant with germinal vesicle breakdown (GVBD) while H4 serine 1 phosphorylation occurs post-MBT. In egg extract H2A/H4 S1 phosphorylation is independent of the cell cycle, chromatin assembly, and DNA replication. H2AS1ph is highly enriched on blastula chromatin during repression of zygotic gene expression while H4S1ph is correlated with the beginning of maternal gene expression and the lengthening of the cell cycle, consistent with distinct biological roles for H2A and H4 S1 phosphorylation. We isolated soluble H2A and H2A.X-F from the egg and chromatin-bound in pronuclei and analyzed them by mass spectrometry analysis to quantitatively determine abundances of S1ph and R3 methylation. We show that H2A and H4 S1ph, R3me1 and R3me2s are

  20. PSD-93 MEDIATES TYROSINE-PHOSPHORYLATION OF THE N-METHYL-D-ASPARTATE RECEPTORS

    PubMed Central

    Sato, Yuko; Tao, Yuan-Xiang; Su, Qingning; Johns, Roger A

    2009-01-01

    Src family protein kinases (SFKs)-mediated tyrosine-phosphorylation regulates N-methyl-D-aspartate (NMDA) receptor synaptic function. Some members of the membrane-associated guanylate kinase (MAGUK) family of proteins bind to both SFKs and NMDA receptors, but it is unclear whether the MAGUK family of proteins is required for SFKs-mediated tyrosine-phosphorylation of the NMDA receptors. Here, we showed by co-immunoprecipitation that PSD-93, a member of the MAGUK family of proteins, interacts with the NMDA receptor subunits NR2A and NR2B as well as with Fyn, a member of the SFKs, in mouse cerebral cortex. Using a biochemical fractionation approach to isolate subcellular compartments revealed that the expression of Fyn, but not of other members of the SFKs (Lyn, Src, and Yes), was significantly decreased in synaptosomal membrane fractions derived from the cerebral cortex of PSD-93 knockout mice. Interestingly, we found that PSD-93 disruption causes reduction of tyrosine-phosphorylated NR2A and NR2B in the same fraction. Moreover, PSD-93 deletion markedly blocked the SFKs-mediated increase in tyrosine-phosphorylated NR2A and NR2B through the protein kinase C pathway after induction with 4β-PMA in cultured cortical neurons. Our findings indicate that PSD-93 appears to mediate tyrosine-phosphorylation of the NMDA receptors and synaptic localization of Fyn. PMID:18423999

  1. 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

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

    PubMed

    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

  3. Phosphorylation and Methylation of Proteasomal Proteins of the Haloarcheon Haloferax volcanii

    DOE PAGESBeta

    Humbard, Matthew A.; Reuter, Christopher J.; Zuobi-Hasona, Kheir; Zhou, Guangyin; Maupin-Furlow, Julie A.

    2010-01-01

    Promore » teasomes are composed of 20S core particles (CPs) of α - and β -type subunits that associate with regulatory particle AAA ATPases such as the proteasome-activating nucleotidase (PAN) complexes of archaea. In this study, the roles and additional sites of post-translational modification of proteasomes were investigated using the archaeon Haloferax volcanii as a model. Indicative of phosphorylation, phosphatase-sensitive isoforms of α 1 and α 2 were detected by 2-DE immunoblot. To map these and other potential sites of post-translational modification, proteasomes were purified and analyzed by tandem mass spectrometry (MS/MS). Using this approach, several phosphosites were mapped including α 1 Thr147, α 2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped to α 1 , thus, revealing a new type of proteasomal modification.bing the biological role of α 1 and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation for α 1 variants including Thr147Ala, Thr158Ala and Ser58Ala. An H. volcanii Rio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer to α 1 . The α 1 variants in Thr and Ser residues that displayed dominant negative phenotypes were significantly reduced in their ability to accept phosphoryl groups from Rio1p, thus, providing an important link between cell physiology and proteasomal phosphorylation.« less

  4. Methyl tetra-O-acetyl-α-D-glucopyranuronate: crystal structure and influence on the crystallisation of the β anomer.

    PubMed

    Hayes, John A; Eccles, Kevin S; Lawrence, Simon E; Moynihan, Humphrey A

    2016-04-29

    Methyl tetra-O-acetyl-β-D-glucopyranuronate (1) and methyl tetra-O-acetyl-α-D-glucopyranuronate (3) were isolated as crystalline solids and their crystal structures were obtained. That of the β anomer (1) was the same as that reported by Root et al., while anomer (3) was found to crystallise in the orthorhombic space group P212121 with two independent molecules in the asymmetric unit. No other crystal forms were found for either compound upon recrystallisation from a range of solvents. The α anomer (3) was found to be an impurity in initially precipitated batches of β-anomer (1) in quantities <3%; however, it was possible to remove the α impurity either by recrystallisation or by efficient washing, i.e. the α anomer is not incorporated inside the β anomer crystals. The β anomer (1) was found to grow as prisms or needles elongated in the a crystallographic direction in the absence of the α impurity, while the presence of the α anomer (3) enhanced this elongation. PMID:27031190

  5. Liver histone H3 methylation and acetylation may associate with type 2 diabetes development.

    PubMed

    Tu, Peipei; Li, Xiaodan; Ma, Baicheng; Duan, Huikun; Zhang, Yaofang; Wu, Ri; Ni, Zaizhong; Jiang, Pingzhe; Wang, Haisong; Li, Miao; Zhu, Jianhong; Li, Minggang

    2015-03-01

    Type 2 diabetes (T2D) is a complicated systemic disease, and the exact pathogenetic molecular mechanism is unclear. Distinct histone modifications regulate gene expression in certain diseases, but little is known about histone epigenetics in diabetes. In the current study, C57BL/6 J mice were used to build T2D model, then treated with exendin-4 (10 μg/kg). Histone H3K9 and H3K23 acetylation, H3K4 monomethylation and H3K9 dimethylation were explored by Western blotting of liver histone extracts. Real-time polymerase chain reaction (PCR) was used to examine expression levels of diabetes-related genes, while chromatin immunoprecipitation (ChIP) was applied to analyze H3 and H3K9 acetylation, H3K4 monomethylation, and H3K9 dimethylation in the promoter of facilitated glucose transporter member 2 (Glut2) gene. The results showed that liver's total H3K4 monomethylation and H3K9 dimethylation was increased in diabetic mice, which was abrogated with the treatment of exendin-4. In contrast, H3K9 and H3K23 acetylation were reduced in diabetic mice, while exendin-4 only alleviated the reduction of H3K9 acetylation. Our data indicated that the progression of type 2 diabetes mellitus (T2D) is associated with global liver histone H3K9 and H3K23 acetylation, H3K4 monomethylation, and H3K9 dimethylation. Exploiting exact histone modify enzyme inhibitors, which may represent a novel strategy to prevent T2D. PMID:25666660

  6. The DNA methylation inhibitor 5-azacytidine decreases melanin synthesis by inhibiting CREB phosphorylation.

    PubMed

    Shin, Jun Seob; Jeong, Hyo-Soon; Kim, Myo-Kyoung; Yun, Hye-Young; Baek, Kwang Jin; Kwon, Nyoun Soo; Kim, Dong-Seok

    2015-10-01

    Here we examined the effects of a DNA methylation inhibitor, 5-azacytidine, on melanogenesis in Mel-Ab cells. We found that 5-azacytidine decreased the melanin content and tyrosinase activity in these cells in a dose-dependent manner; importantly, 5-azacytidine was not cytotoxic at the concentrations used in these experiments. On the other hand, 5-azacytidine did not affect tyrosinase activity in a cell-free system, indicating that 5-azacytidine is not a direct tyrosinase inhibitor. Instead, 5-azacytidine decreased the protein levels of microphthalmia-associated transcription factor (MITF) and tyrosinase. Thus, we investigated the effects of 5-azacytidine on signal transduction pathways related to melanogenesis. However, 5-azacytidine did not have any effect on either Akt or glycogen synthase kinase 3β (GSK3β) phosphorylation. The phosphorylation of cAMP response element-binding protein (CREB) is well known to regulate MITF expression, thereby also regulating tyrosinase expression. We found that 5-azacytidine decreased the phosphorylation of CREB. Therefore, we propose that 5-azacytidine may decrease melanin synthesis by downregulating MITF and tyrosinase via CREB inactivation. PMID:26601420

  7. Pharmacokinetics and N-acetylation metabolism of S-methyl-l-cysteine and trans-S-1-propenyl-l-cysteine in rats and dogs.

    PubMed

    Amano, Hirotaka; Kazamori, Daichi; Itoh, Kenji

    2016-11-01

    1. Pharmacokinetics and N-acetylation metabolism of S-methyl-L-cysteine (SMC) and trans-S-1-propenyl-L-cysteine (S1PC) were examined in rats and dogs. SMC and S1PC (2-5 mg/kg) were well absorbed in both species with high bioavailability (88-100%). 2. SMC and S1PC were excreted only to a small extent in the urine of rats and dogs. The small renal clearance values (<0.03 l/h/kg) indicated the extensive renal reabsorption of SMC and S1PC, which potentially contributed to their long elimination half-lives (>5 h) in dogs. 3. S1PC, but not SMC, underwent N-acetylation extensively in vivo, which can be explained by the relative activities of N-acetylation of S1PC/SMC and deacetylation of their N-acetylated forms, N-acetyl-S1PC/N-acetyl-SMC, in the liver and kidney in vitro. The activities for S1PC N-acetylation were similar to or higher than those for N-acetyl-S1PC deacetylation in liver S9 fractions of rat and dog, whereas liver and kidney S9 fractions of rat and dog had little activity for SMC N-acetylation or considerably higher activities for N-acetyl-SMC deacetylation. 4. Our study demonstrated that the pharmacokinetics of SMC and S1PC in rats and dogs was characterized by high bioavailability and extensive renal reabsorption; however, the extent of undergoing the N-acetylation metabolism was extremely different between SMC and S1PC. PMID:26887651

  8. Bivalent Regions of Cytosine Methylation and H3K27 Acetylation Suggest an Active Role for DNA Methylation at Enhancers.

    PubMed

    Charlet, Jessica; Duymich, Christopher E; Lay, Fides D; Mundbjerg, Kamilla; Dalsgaard Sørensen, Karina; Liang, Gangning; Jones, Peter A

    2016-05-01

    The role of cytosine methylation in the structure and function of enhancers is not well understood. In this study, we investigate the role of DNA methylation at enhancers by comparing the epigenomes of the HCT116 cell line and its highly demethylated derivative, DKO1. Unlike promoters, a portion of regular and super- or stretch enhancers show active H3K27ac marks co-existing with extensive DNA methylation, demonstrating the unexpected presence of bivalent chromatin in both cultured and uncultured cells. Furthermore, our findings also show that bivalent regions have fewer nucleosome-depleted regions and transcription factor-binding sites than monovalent regions. Reduction of DNA methylation genetically or pharmacologically leads to a decrease of the H3K27ac mark. Thus, DNA methylation plays an unexpected dual role at enhancer regions, being anti-correlated focally at transcription factor-binding sites but positively correlated globally with the active H3K27ac mark to ensure structural enhancer integrity. PMID:27153539

  9. Accumulation of galactosylsphingosine (psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse

    SciTech Connect

    Yoshimura, T.; Kobayashi, T.; Shinnoh, N.; Goto, I. )

    1990-10-01

    In attempts to elucidate mechanisms of demyelination in the twitcher mouse (Twi), phosphorylation and methylation of myelin basic protein (MBP) were examined in the brainstem and spinal cord of this species. Phosphorylation of MBP in isolated myelin by an endogenous kinase and an exogenous (32P)ATP was not impaired and protein kinase C activity in the brain cytosol was not reduced. When the methylation of an arginine residue of MBP was examined in slices of the brainstem and spinal cord, using (3H)methionine as a donor of the methyl groups, no difference was found between Twi and the controls. Radioactivity of the (3H) methionine residue of MBP of Twi was also similar to that of the controls. Thus, accumulation of psychosine in Twi does not interfere with the activity of endogenous kinase, methylation of MBP, and the synthesis and transport of MBP into myelin membrane.

  10. Trichostatin A and 5-azacytidine both cause an increase in global histone H4 acetylation and a decrease in global DNA and H3K9 methylation during mitosis in maize

    PubMed Central

    2010-01-01

    Background Modifications of DNA and histones in various combinations are correlated with many cellular processes. In this study, we investigated the possible relationship between histone H4 tetraacetylation, DNA methylation and histone H3 dimethylation at lysine 9 during mitosis in maize root meristems. Results Treatment with trichostatin A, which inhibits histone deacetylases, resulted in increased histone H4 acetylation accompanied by the decondensation of interphase chromatin and a decrease in both global H3K9 dimethylation and DNA methylation during mitosis in maize root tip cells. These observations suggest that histone acetylation may affect DNA and histone methylation during mitosis. Treatment with 5-azacytidine, a cytosine analog that reduces DNA methylation, caused chromatin decondensation and mediated an increase in H4 acetylation, in addition to reduced DNA methylation and H3K9 dimethylation during interphase and mitosis. These results suggest that decreased DNA methylation causes a reduction in H3K9 dimethylation and an increase in H4 acetylation. Conclusions The interchangeable effects of 5-azacytidine and trichostatin A on H4 acetylation, DNA methylation and H3K9 dimethylation indicate a mutually reinforcing action between histone acetylation, DNA methylation and histone methylation with respect to chromatin modification. Treatment with trichostatin A and 5-azacytidine treatment caused a decrease in the mitotic index, suggesting that H4 deacetylation and DNA and H3K9 methylation may contain the necessary information for triggering mitosis in maize root tips. PMID:20718950

  11. Breaking the HAC Barrier: Histone H3K9 acetyl/methyl balance regulates CENP-A assembly

    PubMed Central

    Ohzeki, Jun-ichirou; Bergmann, Jan H; Kouprina, Natalay; Noskov, Vladimir N; Nakano, Megumi; Kimura, Hiroshi; Earnshaw, William C; Larionov, Vladimir; Masumoto, Hiroshi

    2012-01-01

    The kinetochore is responsible for accurate chromosome segregation. However, the mechanism by which kinetochores assemble and are maintained remains unclear. Here we report that de novo CENP-A assembly and kinetochore formation on human centromeric alphoid DNA arrays is regulated by a histone H3K9 acetyl/methyl balance. Tethering of histone acetyltransferases (HATs) to alphoid DNA arrays breaks a cell type-specific barrier for de novo stable CENP-A assembly and induces assembly of other kinetochore proteins at the ectopic alphoid site. Similar results are obtained following tethering of CENP-A deposition factors hMis18α or HJURP. HAT tethering bypasses the need for hMis18α, but HJURP is still required for de novo kinetochore assembly. In contrast, H3K9 methylation following tethering of H3K9 tri-methylase (Suv39h1) to the array prevents de novo CENP-A assembly and kinetochore formation. CENP-A arrays assembled de novo by this mechanism can form human artificial chromosomes (HACs) that are propagated indefinitely in human cells. PMID:22473132

  12. Cross-talk between Arg methylation and Ser phosphorylation modulates apoptosis signal–regulating kinase 1 activation in endothelial cells

    PubMed Central

    Chen, Ming; Qu, Xiaosheng; Zhang, Zhiqing; Wu, Huayu; Qin, Xia; Li, Fuji; Liu, Zhenfang; Tian, Liyuan; Miao, Jianhua; Shu, Wei

    2016-01-01

    We describe a novel functional interaction between ASK1 and PRMT5. We show that PRMT5 interacts with and methylates ASK1 at arginine residue 89 and thereby negatively regulates its activity by promoting the interaction between ASK1 and Akt and thus phosphorylating ASK1 at serine residue 83. Furthermore, the association between ASK1 and Akt is enhanced by VEGF stimulation, and PRMT5 is required for this association. Moreover, PRMT5-mediated ASK1 methylation impaired the H2O2-induced activity of ASK1, and this inhibitory effect of PRMT5 was abolished by replacement of arginine 89 with Trp or depletion of PRMT5 expression by RNA interference. Together the results demonstrate cross-talk between arginine methylation and serine phosphorylation in ASK1. PMID:26912789

  13. Alternative synthesis of 3-acetyl, 3-epoxy, and 3-formyl chlorins from a 3-vinyl chlorin, methyl pyropheophorbide-a, via iodination.

    PubMed

    Oba, Toru; Masuya, Takuto; Yasuda, Satoru; Ito, Satoshi

    2015-08-01

    We developed novel methods to convert the C3-vinyl group of a chlorophyll derivative, methyl pyropheophorbide-a, into an acetyl group, an epoxy group, and a formyl group via iodination with I2 and phenyliodine(III) bis(trifluoroacetate). Reaction of the iodinated intermediate with ethylene glycol and subsequent treatment with base led to formation of the C3-acetyl chlorin. Reaction of the iodinated intermediate with ethylenediamine afforded the C3-oxiranyl chlorin. The C3-formyl chlorin was readily derived from the epoxide without hazardous reagents such as OsO4. These reactions were facile and useful alternatives to the previous methods. PMID:26048784

  14. Specificity of sites within eight-membered ring zeolite channels for carbonylation of methyls to acetyls.

    PubMed

    Bhan, Aditya; Allian, Ayman D; Sunley, Glenn J; Law, David J; Iglesia, Enrique

    2007-04-25

    The acid-catalyzed formation of carbon-carbon bonds from C1 precursors via CO insertion into chemisorbed methyl groups occurs selectively within eight-membered ring (8-MR) zeolite channels. This elementary step controls catalytic carbonylation rates of dimethyl ether (DME) to methyl acetate. The number of O-H groups within 8-MR channels was measured by rigorous deconvolution of the infrared bands for O-H groups in cation-exchanged and acid forms of mordenite (M,H-MOR) and ferrierite (H-FER) after adsorption of basic probe molecules of varying size. DME carbonylation rates are proportional to the number of O-H groups within 8-MR channels. Na+ cations selectively replaced protons within 8-MR channels and led to a disproportionate decrease in carbonylation turnover rates (per total H+). These conclusions are consistent with the low or undetectable rates of carbonylation on zeolites without 8-MR channels (H-BEA, H-FAU, H-MFI). Such specificity of methyl reactivity upon confinement within small channels appears to be unprecedented in catalysis by microporous solids, which typically select reactions by size exclusion of bulkier transition states. PMID:17397162

  15. 2-Acetyl-amino-1,3,4,6-tetra-O-(tri-methyl-silyl)-2-de-oxy-α-d-gluco-pyran-ose.

    PubMed

    Cheng, Zhao-Dong; Cui, Yan-Li; Mao, Jian-Wei

    2013-06-01

    The title compound, C20H47NO6Si4, was synthesized by per-O-tri-methyl-silylation of N-acetyl-d-glucosa-mine using chloro-tri-methyl-silane in the presence of hexa-methyl-disiloxane. The tri-methyl-silyl group and acetamido group are located on the same side of the pyran ring, showing an α-configuration glycoside. One of the tri-methyl-silyl groups is disordered over two orientations, with site-occupancy factors of 0.625 (9) and 0.375 (9). In the crystal, N-H⋯O hydrogen bonds link the mol-ecules into supra-molecular chains along the a-axis direction. PMID:23795087

  16. Cancer-preventive peptide lunasin from Solanum nigrum L. inhibits acetylation of core histones H3 and H4 and phosphorylation of retinoblastoma protein (Rb).

    PubMed

    Jeong, Jin Boo; Jeong, Hyung Jin; Park, Jae Ho; Lee, Sun Hee; Lee, Jeong Rak; Lee, Hee Kyeong; Chung, Gyu Young; Choi, Jeong Doo; de Lumen, Ben O

    2007-12-26

    Lunasin, a unique 43 amino acid, 4.8 kDa cancer-chemopreventive peptide initially reported in soybean and now found in barley and wheat, has been shown to be cancer-chemopreventive in mammalian cells and in a skin cancer mouse model against oncogenes and chemical carcinogens. To identify bioactive components in traditional herbal medicines and in search for new sources of lunasin, we report here the properties of lunasin from Solanum nigrum L. (SNL), a plant indigenous to northeast Asia. Lunasin was screened in the crude extracts of five varieties of the medicinal plants of Solanaceae origin and seven other major herbal plants. An in vitro digestion stability assay for measuring bioavailability was carried out on SNL crude protein and autoclaved SNL using pepsin and pancreatin. A nonradioactive histone acetyltransferase (HAT) assay and HAT activity colorimetric assay were used to measure the inhibition of core histone acetylation. The inhibitory effect of lunasin on the phosphorylation of retinoblastoma protein (Rb) was determined by immunoblotting against phospho-Rb. Lunasin isolated from autoclaved SNL inhibited core histone H3 and H4 acetylation, the activities of the HATs, and the phosphorylation of the Rb protein. Lunasin in the crude protein and in the autoclaved crude protein was very stable to pepsin and pancreatin in vitro digestion, while the synthetic pure lunasin was digested at 2 min after the reaction. We conclude that lunasin is a bioactive and bioavailable component in SNL and that consumption of SNL may play an important role in cancer prevention. PMID:18038993

  17. DNA Methylation and Histone Acetylation Patterns in Cultured Bovine Adipose Tissue-Derived Stem Cells (BADSCs)

    PubMed Central

    Abouhamzeh, Beheshteh; Salehi, Mohammad; Hosseini, Ahmad; Masteri-Farahani, Ali Reza; Fadai, Fatemeh; Heidari, Mohammad Hasan; Nourozian, Mohsen; Soleimani, Masoud; Khorashadizadeh, Mohsen; Mossahebi-Mohammadi, Majid; Mansouri, Ardalan

    2015-01-01

    Objective Many studies have focused on the epigenetic characteristics of donor cells to improve somatic cell nuclear transfer (SCNT). We hypothesized that the epigenetic status and chromatin structure of undifferentiated bovine adipose tissue-derived stem cells (BADSCs) would not remain constant during different passages. The objective of this study was to determine the mRNA expression patterns of DNA methyltransferases (DNMT1, DNMT3a, DNMT3b) and histone deacetyltransferses (HDAC1, HDAC2, HDAC3) in BADSCs. In addition, we compared the measured levels of octamer binding protein-4 expression (OCT4) and acetylation of H3K9 (H3K9ac) in BADSCs cultures and different passages in vitro. Materials and Methods In this experimental study, subcutaneous fat was obtained from adult cows immediately post-mortem. Relative level of DNMTs and HDACs was examined using quantitative real time polymerase chain reaction (q-PCR), and the level of OCT4 and H3K9ac was analyzed by flow cytometry at passages 3 (P3), 5 (P5) and 7 (P7). Results The OCT4 protein level was similar at P3 and P5 but a significant decrease in its level was seen at P7. The highest and lowest levels of H3K9ac were observed at P5 and P7, respectively. At P5, the expression of HDACs and DNMTs was significantly decreased. In contrast, a remarkable increase in the expression of DNMTs was observed at P7. Conclusion Our data demonstrated that the epigenetic status of BADSCs was variable during culture. The P5 cells showed the highest level of stemness and multipotency and the lowest level of chromatin compaction. Therefore, we suggest that P5 cells may be more efficient for SCNT compared with other passages. PMID:25685737

  18. Identification of active site lysyl residues of phenylalanine dehydrogenase by chemical modification with methyl acetyl phosphate combined with site-directed mutagenesis.

    PubMed

    Kataoka, K; Tanizawa, K; Fukui, T; Ueno, H; Yoshimura, T; Esaki, N; Soda, K

    1994-12-01

    A monoanionic acetylation reagent, methyl acetyl phosphate, was used to acetylate lysyl residues of the recombinant thermostable phenylalanine dehydrogenase from Thermoactinomyces intermedius. The enzyme was irreversibly inactivated with the reagent in a time- and dose-dependent manner. Simultaneous addition of substrate and coenzyme markedly protected the enzyme from inactivation. Acetylated lysyl residues presumably occurring at the active site were determined by differential modification; the enzyme was first modified with a cold reagent in the presence of both substrate and coenzyme and, after removal of the added substances by gel filtration, was then labeled with a radioactive reagent. At least 7 lysyl residues per enzyme subunit were radiolabeled by this method. To further specify the lysyl residue(s) whose modification results in inactivation of the enzyme, 5 lysyl residues highly conserved in various amino acid dehydrogenase sequences were replaced with Ala by site-directed mutagenesis. Although all of the single mutant enzymes were inactivated with the reagent as effectively as the wild-type enzyme, a double mutant enzyme in which both Lys-69 and Lys-81 were replaced with Ala was found to be inactivated very slowly. These results suggest that the reagent can acetylate both of these lysyl residues and inactivate the enzyme. Kinetic analyses of the single Lys-69 and Lys-81 mutant enzymes revealed that they are involved in substrate binding and catalysis, respectively, like the corresponding residues in the homologous leucine dehydrogenase. PMID:7706231

  19. RelA Ser276 Phosphorylation-Coupled Lys310 Acetylation Controls Transcriptional Elongation of Inflammatory Cytokines in Respiratory Syncytial Virus Infection▿

    PubMed Central

    Brasier, Allan R.; Tian, B.; Jamaluddin, M.; Kalita, Mridul K.; Garofalo, Roberto P.; Lu, Muping

    2011-01-01

    Respiratory syncytial virus (RSV) is a negative-sense single-stranded RNA virus responsible for lower respiratory tract infections (LRTIs) in humans. In experimental models of RSV LRTI, the actions of the nuclear factor κB (NF-κB) transcription factor mediate inflammation and pathology. We have shown that RSV replication induces a mitogen-and-stress-related kinase 1 (MSK-1) pathway that activates NF-κB RelA transcriptional activity by a process involving serine phosphorylation at serine (Ser) residue 276. In this study, we examined the mechanism by which phospho-Ser276 RelA mediates expression of the NF-κB-dependent gene network. RelA-deficient mouse embryonic fibroblasts (MEFs) complemented with the RelA Ser276Ala mutant are deficient in CXCL2/Groβ, KC, and interleukin-6 (IL-6) expression, but NFKBIA/IκBα is preserved. We show that RSV-induced RelA Ser276 phosphorylation is required for acetylation at Lys310, an event required for transcriptional activity and stable association of RelA with the activated positive transcriptional elongation factor (PTEF-b) complex proteins, bromodomain 4 (Brd4), and cyclin-dependent kinase 9 (CDK9). In contrast to gene loading pattern of PTEF-b proteins produced by tumor necrosis factor (TNF) stimulation, RSV induces their initial clearance followed by partial reaccumulation coincident with RelA recruitment. The RSV-induced binding patterns of the CDK9 substrate, phospho-Ser2 RNA polymerase (Pol) II, follows a similar pattern of clearance and downstream gene reaccumulation. The functional role of CDK9 was examined using CDK9 small interfering RNA (siRNA) and CDK inhibitors, where RSV-induced NF-κB-dependent gene expression was significantly inhibited. Finally, although RSV induces a transition from short transcripts to fully spliced mRNA in wild-type RelA (RelA WT)-expressing cells, this transition is not seen in cells expressing RelA Ser276Ala. We conclude that RelA Ser276 phosphorylation mediates RelA acetylation, Brd4

  20. RelA Ser276 phosphorylation-coupled Lys310 acetylation controls transcriptional elongation of inflammatory cytokines in respiratory syncytial virus infection.

    PubMed

    Brasier, Allan R; Tian, B; Jamaluddin, M; Kalita, Mridul K; Garofalo, Roberto P; Lu, Muping

    2011-11-01

    Respiratory syncytial virus (RSV) is a negative-sense single-stranded RNA virus responsible for lower respiratory tract infections (LRTIs) in humans. In experimental models of RSV LRTI, the actions of the nuclear factor κB (NF-κB) transcription factor mediate inflammation and pathology. We have shown that RSV replication induces a mitogen-and-stress-related kinase 1 (MSK-1) pathway that activates NF-κB RelA transcriptional activity by a process involving serine phosphorylation at serine (Ser) residue 276. In this study, we examined the mechanism by which phospho-Ser276 RelA mediates expression of the NF-κB-dependent gene network. RelA-deficient mouse embryonic fibroblasts (MEFs) complemented with the RelA Ser276Ala mutant are deficient in CXCL2/Groβ, KC, and interleukin-6 (IL-6) expression, but NFKBIA/IκBα is preserved. We show that RSV-induced RelA Ser276 phosphorylation is required for acetylation at Lys310, an event required for transcriptional activity and stable association of RelA with the activated positive transcriptional elongation factor (PTEF-b) complex proteins, bromodomain 4 (Brd4), and cyclin-dependent kinase 9 (CDK9). In contrast to gene loading pattern of PTEF-b proteins produced by tumor necrosis factor (TNF) stimulation, RSV induces their initial clearance followed by partial reaccumulation coincident with RelA recruitment. The RSV-induced binding patterns of the CDK9 substrate, phospho-Ser2 RNA polymerase (Pol) II, follows a similar pattern of clearance and downstream gene reaccumulation. The functional role of CDK9 was examined using CDK9 small interfering RNA (siRNA) and CDK inhibitors, where RSV-induced NF-κB-dependent gene expression was significantly inhibited. Finally, although RSV induces a transition from short transcripts to fully spliced mRNA in wild-type RelA (RelA WT)-expressing cells, this transition is not seen in cells expressing RelA Ser276Ala. We conclude that RelA Ser276 phosphorylation mediates RelA acetylation, Brd4

  1. Chromatin remodeling in plant cell culture: patterns of DNA methylation and histone H3 and H4 acetylation vary during growth of asynchronous potato cell suspensions.

    PubMed

    Law, R David; Suttle, Jeffrey C

    2005-06-01

    Changes in DNA cytosine methylation and core histone multi-acetylation were determined in cell suspension cultures of potato (Solanum tuberosum L. cv. Russet Burbank) during 15 days of in vitro culture. Cell subculture induced a transient 33% decrease in genome-wide 5-methylcytosine (5mC) content and a transient threefold increase in transcription rates that were most evident at 6 and 9 days after subculture, respectively. In contrast to the global reduction in 5mC content, subculture resulted in a transient twofold increase in 5mC levels within 5'-CCGG-3' sequences and no detectable change in 5'-CG-3' methylation. Multi-acetylation of histones H3.1, H3.2 and H4 rose 2-, 1.5- and 3-fold by 9, 9 and 12 days after subculture, respectively. All observed epigenetic changes were reset during aging of cell cultures. Inclusion of the histone deacetylase inhibitor trichostatin A (TSA) and/or the cytosine methylation inhibitor 5-azacytidine (5AC) in culture sequentially decreased genome-wide 5mC levels by approximately 25% at day 9, then decreased 5'-mCmCGG-3' by 30-50% and increased H3 and H4 multi-acetylation by 30-60% at day 15, compared to controls. Treatment with 5AC or TSA alone or in combination had no effect on RNA synthesis at day 9. At day 15, 5AC treatment remained ineffective, while de novo RNA synthesis was approximately twofold higher in cells grown in both inhibitors or in TSA alone. Collectively, these results demonstrate that in potato suspension cultures, rapid, reversible changes in 5mC levels precede regulatory post-translational acetylation of core histones, and suggest that interactions between these epigenetic processes appear to be necessary to power transcription and growth induction in potato cells. PMID:15922608

  2. A novel one-pot and one-step microwave-assisted cyclization-methylation reaction of amino alcohols and acetylated derivatives with dimethyl carbonate and TBAC.

    PubMed

    Ochoa-Terán, Adrián; Guerrero, Leticia; Rivero, Ignacio A

    2014-01-01

    A simple and efficient microwave-assisted methodology for the synthesis of 4-substituted-3-methyl-1,3-oxazolidin-2-ones from amino alcohols catalyzed by a ionic liquid was developed. This novel one-pot and one-step cyclization-methylation reaction represents an easier and faster method than any other reported protocols that can be used to obtain the desired products in good yields and high purity. Applying microwave irradiation at 130°C in the presence of TBAC, dimethyl carbonate acts simultaneously as carbonylating and methylating agent and surprisingly promotes an in situ basic trans esterification when a N-acetylated amino alcohol is used as starting material. Furthermore, dimethyl carbonate worked better than diethyl carbonate in performing this reaction. PMID:25692177

  3. A Novel One-Pot and One-Step Microwave-Assisted Cyclization-Methylation Reaction of Amino Alcohols and Acetylated Derivatives with Dimethyl Carbonate and TBAC

    PubMed Central

    Ochoa-Terán, Adrián; Guerrero, Leticia; Rivero, Ignacio A.

    2014-01-01

    A simple and efficient microwave-assisted methodology for the synthesis of 4-substituted-3-methyl-1,3-oxazolidin-2-ones from amino alcohols catalyzed by a ionic liquid was developed. This novel one-pot and one-step cyclization-methylation reaction represents an easier and faster method than any other reported protocols that can be used to obtain the desired products in good yields and high purity. Applying microwave irradiation at 130°C in the presence of TBAC, dimethyl carbonate acts simultaneously as carbonylating and methylating agent and surprisingly promotes an in situ basic trans esterification when a N-acetylated amino alcohol is used as starting material. Furthermore, dimethyl carbonate worked better than diethyl carbonate in performing this reaction. PMID:25692177

  4. PGC-1α Serine 570 Phosphorylation and GCN5-mediated Acetylation by Angiotensin II Drive Catalase Down-regulation and Vascular Hypertrophy*

    PubMed Central

    Xiong, Shiqin; Salazar, Gloria; San Martin, Alejandra; Ahmad, Mushtaq; Patrushev, Nikolay; Hilenski, Lula; Nazarewicz, Rafal Robert; Ma, Minhui; Ushio-Fukai, Masuko; Alexander, R. Wayne

    2010-01-01

    Angiotensin II (Ang II) is a pleuripotential hormone that is important in the pathophysiology of multiple conditions including aging, cardiovascular and renal diseases, and insulin resistance. Reactive oxygen species (ROS) are important mediators of Ang II-induced signaling generally and have a well defined role in vascular hypertrophy, which is inhibited by overexpression of catalase, inferring a specific role of H2O2. The molecular mechanisms are understood incompletely. The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a key regulator of energy metabolism and ROS-scavenging enzymes including catalase. We show that Ang II stimulates Akt-dependent PGC-1α serine 570 phosphorylation, which is required for the binding of the histone acetyltransferase GCN5 (general control nonderepressible 5) to PGC-1α and for its lysine acetylation. These sequential post-translational modifications suppress PGC-1α activity and prevent its binding to the catalase promoter through the forkhead box O1 transcription factor, thus decreasing catalase expression. We demonstrate that overexpression of the phosphorylation-defective mutant PGC-1α (S570A) prevents Ang II-induced increases in H2O2 levels and hypertrophy ([3H]leucine incorporation). Knockdown of PGC-1α by small interfering RNA promotes basal and Ang II-stimulated ROS and hypertrophy, which is reversed by polyethylene glycol-conjugated catalase. Thus, endogenous PGC-1α is a negative regulator of vascular hypertrophy by up-regulating catalase expression and thus reducing ROS levels. We provide novel mechanistic insights by which Ang II may mediate its ROS-dependent pathophysiologic effects on multiple cardiometabolic diseases. PMID:19940161

  5. Structure of NDP-forming Acetyl-CoA synthetase ACD1 reveals a large rearrangement for phosphoryl transfer.

    PubMed

    Weiße, Renato H-J; Faust, Annette; Schmidt, Marcel; Schönheit, Peter; Scheidig, Axel J

    2016-02-01

    The NDP-forming acyl-CoA synthetases (ACDs) catalyze the conversion of various CoA thioesters to the corresponding acids, conserving their chemical energy in form of ATP. The ACDs are the major energy-conserving enzymes in sugar and peptide fermentation of hyperthermophilic archaea. They are considered to be primordial enzymes of ATP synthesis in the early evolution of life. We present the first crystal structures, to our knowledge, of an ACD from the hyperthermophilic archaeon Candidatus Korachaeum cryptofilum. These structures reveal a unique arrangement of the ACD subunits alpha and beta within an α2β2-heterotetrameric complex. This arrangement significantly differs from other members of the superfamily. To transmit an activated phosphoryl moiety from the Ac-CoA binding site (within the alpha subunit) to the NDP-binding site (within the beta subunit), a distance of 51 Å has to be bridged. This transmission requires a larger rearrangement within the protein complex involving a 21-aa-long phosphohistidine-containing segment of the alpha subunit. Spatial restraints of the interaction of this segment with the beta subunit explain the necessity for a second highly conserved His residue within the beta subunit. The data support the proposed four-step reaction mechanism of ACDs, coupling acyl-CoA thioesters with ATP synthesis. Furthermore, the determined crystal structure of the complex with bound Ac-CoA allows first insight, to our knowledge, into the determinants for acyl-CoA substrate specificity. The composition and size of loops protruding into the binding pocket of acyl-CoA are determined by the individual arrangement of the characteristic subdomains. PMID:26787904

  6. Methyl-CpG-binding protein 2 is phosphorylated by homeodomain-interacting protein kinase 2 and contributes to apoptosis.

    PubMed

    Bracaglia, Giorgia; Conca, Barbara; Bergo, Anna; Rusconi, Laura; Zhou, Zhaolan; Greenberg, Michael E; Landsberger, Nicoletta; Soddu, Silvia; Kilstrup-Nielsen, Charlotte

    2009-12-01

    Mutations in the methyl-CpG-binding protein 2 (MeCP2) are associated with Rett syndrome and other neurological disorders. MeCP2 represses transcription mainly by recruiting various co-repressor complexes. Recently, MeCP2 phosphorylation at Ser 80, Ser 229 and Ser 421 was shown to occur in the brain and modulate MeCP2 silencing activities. However, the kinases directly responsible for this are largely unknown. Here, we identify the homeodomain-interacting protein kinase 2 (HIPK2) as a kinase that binds MeCP2 and phosphorylates it at Ser 80 in vitro and in vivo. HIPK2 modulates cell proliferation and apoptosis, and the neurological defects of Hipk2-null mice indicate its role in proper brain functions. We show that MeCP2 cooperates with HIPK2 in induction of apoptosis and that Ser 80 phosphorylation is required together with the DNA binding of MeCP2. These data are, to our knowledge, the first that describe a kinase associating with MeCP2, causing its specific phosphorylation in vivo and, furthermore, they reinforce the role of MeCP2 in regulating cell growth. PMID:19820693

  7. Methyl-CpG-binding protein 2 is phosphorylated by homeodomain-interacting protein kinase 2 and contributes to apoptosis

    PubMed Central

    Bracaglia, Giorgia; Conca, Barbara; Bergo, Anna; Rusconi, Laura; Zhou, Zhaolan; Greenberg, Michael E; Landsberger, Nicoletta; Soddu, Silvia; Kilstrup-Nielsen, Charlotte

    2009-01-01

    Mutations in the methyl-CpG-binding protein 2 (MeCP2) are associated with Rett syndrome and other neurological disorders. MeCP2 represses transcription mainly by recruiting various co-repressor complexes. Recently, MeCP2 phosphorylation at Ser 80, Ser 229 and Ser 421 was shown to occur in the brain and modulate MeCP2 silencing activities. However, the kinases directly responsible for this are largely unknown. Here, we identify the homeodomain-interacting protein kinase 2 (HIPK2) as a kinase that binds MeCP2 and phosphorylates it at Ser 80 in vitro and in vivo. HIPK2 modulates cell proliferation and apoptosis, and the neurological defects of Hipk2-null mice indicate its role in proper brain functions. We show that MeCP2 cooperates with HIPK2 in induction of apoptosis and that Ser 80 phosphorylation is required together with the DNA binding of MeCP2. These data are, to our knowledge, the first that describe a kinase associating with MeCP2, causing its specific phosphorylation in vivo and, furthermore, they reinforce the role of MeCP2 in regulating cell growth. PMID:19820693

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

    PubMed Central

    Garay, Pablo G.; Martin, Osvaldo A.; Scheraga, Harold 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 13C chemical shifts of different nuclei to detect methylation, acetylation and glycosylation of protein residues by monitoring the deviation of the 13C chemical shifts from the expected (mean) experimental value of the non-modified residue. As a proof-of-concept, we used 13C 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 13Cε 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 13C-nucleus, with which to determine other modifications commonly seen, such as methylation of arginine and glycosylation of serine, asparagine and threonine, shows encouraging results. PMID:27547559

  9. 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. PMID:27547559

  10. Adenine Nucleotide Translocase Is Acetylated in Vivo in Human Muscle: Modeling Predicts a Decreased ADP Affinity and Altered Control of Oxidative Phosphorylation

    PubMed Central

    2015-01-01

    Proteomics techniques have revealed that lysine acetylation is abundant in mitochondrial proteins. This study was undertaken (1) to determine the relationship between mitochondrial protein acetylation and insulin sensitivity in human skeletal muscle, identifying key acetylated proteins, and (2) to use molecular modeling techniques to understand the functional consequences of acetylation of adenine nucleotide translocase 1 (ANT1), which we found to be abundantly acetylated. Eight lean and eight obese nondiabetic subjects had euglycemic clamps and muscle biopsies for isolation of mitochondrial proteins and proteomics analysis. A number of acetylated mitochondrial proteins were identified in muscle biopsies. Overall, acetylation of mitochondrial proteins was correlated with insulin action (r = 0.60; P < 0.05). Of the acetylated proteins, ANT1, which catalyzes ADP–ATP exchange across the inner mitochondrial membrane, was acetylated at lysines 10, 23, and 92. The extent of acetylation of lysine 23 decreased following exercise, depending on insulin sensitivity. Molecular dynamics modeling and ensemble docking simulations predicted the ADP binding site of ANT1 to be a pocket of positively charged residues, including lysine 23. Calculated ADP–ANT1 binding affinities were physiologically relevant and predicted substantial reductions in affinity upon acetylation of lysine 23. Insertion of these derived binding affinities as parameters into a complete mathematical description of ANT1 kinetics predicted marked reductions in adenine nucleotide flux resulting from acetylation of lysine 23. Therefore, acetylation of ANT1 could have dramatic physiological effects on ADP–ATP exchange. Dysregulation of acetylation of mitochondrial proteins such as ANT1 therefore could be related to changes in mitochondrial function that are associated with insulin resistance. PMID:24884163

  11. Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility.

    PubMed

    Dudiki, Tejasvi; Kadunganattil, Suraj; Ferrara, John K; Kline, Douglas W; Vijayaraghavan, Srinivasan

    2015-01-01

    Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function. PMID:26569399

  12. Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility

    PubMed Central

    Dudiki, Tejasvi; Kadunganattil, Suraj; Ferrara, John K.; Kline, Douglas W.; Vijayaraghavan, Srinivasan

    2015-01-01

    Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function. PMID:26569399

  13. Expression and methylation of microsomal triglyceride transfer protein and acetyl-CoA carboxylase are associated with fatty liver syndrome in chicken.

    PubMed

    Liu, Zhen; Li, Qinghe; Liu, Ranran; Zhao, Guiping; Zhang, Yonghong; Zheng, Maiqing; Cui, Huanxian; Li, Peng; Cui, Xiaoyan; Liu, Jie; Wen, Jie

    2016-06-01

    The typical characteristic of fatty liver syndrome (FLS) is an increased hepatic triacylglycerol content, and a sudden decline in egg production often occurs. FLS may develop into fatty liver hemorrhagic syndrome (FLHS), characterized by sudden death from hepatic rupture and hemorrhage. DNA methylation is associated with transcriptional silencing, leading to the etiology and pathogenesis of some animal diseases. The roles of DNA methylation in the genesis of FLS, however, are largely unknown. The lipogenic methyl-deficient diet (MDD) caused FLS similar to human nonalcoholic steatohepatitis (NASH). After 16 Jingxing-Huang (JXH) hens were fed MDD for 10 wk, eight exhibited FLS (designated as FLS-susceptible birds); the remainder, without FLS, served as controls (NFLS). Physiological and biochemical variables, gene expression levels, and DNA methylation were determined in the liver. The development of FLS in JXH hens was accompanied by abnormal lipid accumulation. Relative expression of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and microsomal triglyceride transfer protein (MTTP) were significantly up-regulated in the FLS group in comparison with the NFLS group. The transcript abundance of sterol regulatory element binding protein 1 (SREBP-1c), stearoyl-CoA desaturase (SCD), liver X receptor alpha (LXRα), peroxisome proliferator-activated receptor alpha (PPARα), and peroxisome proliferator-activated receptor gamma (PPARγ) did not differ between the two groups. Interestingly, MTTP and ACC mRNA abundance were negatively correlated with the level of promoter methylation. The extent of DNA methylation of the cytosine-guanine (CpG) sites in the SREBP-1c, FAS, PPARα, and LXRα promoter regions was also analyzed by direct sequencing but none differed between FLS and NFLS birds. Taken together, these results specify link DNA methylation to the pathogenesis of FLS in chickens. PMID:27083546

  14. Both H4K20 mono-methylation and H3K56 acetylation mark transcription-dependent histone turnover in fission yeast.

    PubMed

    Yang, Hanna; Kwon, Chang Seob; Choi, Yoonjung; Lee, Daeyoup

    2016-08-01

    Nucleosome dynamics facilitated by histone turnover is required for transcription as well as DNA replication and repair. Histone turnover is often associated with various histone modifications such as H3K56 acetylation (H3K56Ac), H3K36 methylation (H3K36me), and H4K20 methylation (H4K20me). In order to correlate histone modifications and transcription-dependent histone turnover, we performed genome wide analyses for euchromatic regions in G2/M-arrested fission yeast. The results show that transcription-dependent histone turnover at 5' promoter and 3' termination regions is directly correlated with the occurrence of H3K56Ac and H4K20 mono-methylation (H4K20me1) in actively transcribed genes. Furthermore, the increase of H3K56Ac and H4K20me1 and antisense RNA production was observed in the absence of the histone H3K36 methyltransferase Set2 and histone deacetylase complex (HDAC) that are involved in the suppression of histone turnover within the coding regions. These results together indicate that H4K20me1 as well as H3K56Ac are bona fide marks for transcription-dependent histone turnover in fission yeast. PMID:27268234

  15. Identifying dominant conformations of N-acetyl-L-cysteine methyl ester and N-acetyl-L-cysteine in water: VCD signatures of the amide I and the Cdbnd O stretching bands

    NASA Astrophysics Data System (ADS)

    Poopari, Mohammad Reza; Dezhahang, Zahra; Xu, Yunjie

    2015-02-01

    Infrared (IR) and vibrational circular dichroism (VCD) spectra of N-Acetyl-L-Cysteine Methyl Ester (NALCME) and N-Acetyl-L-Cysteine (NALC) in D2O under different pHs were measured. We focus on the VCD signatures of the amide I and the Cdbnd O stretching spectral signatures of the neutral NALCME and NALC species and the related ones of the deprotonated NALC species in the region of 1800-1500 cm-1. A sign inversion is observed for the amide I VCD band going from the neutral NALCME and NALC to the deprotonated NALC species. Density functional theory (DFT) calculations were carried out to search for the possible conformations of these three species and to simulate their IR and VCD spectra at the B3LYP/aug-cc-pVTZ level in the gas phase and with the polarization continuum model of water solvent. The most stable conformations found for neutral NALCME and NALC exhibit drastically difference VCD patterns, whereas those of deprotonated NALC show similar patterns. We establish an empirical structural-spectral relationship where the aforementioned VCD signatures can be used as spectral markers to identify dominant conformations of these two amino acid derivatives under different pHs. It is recognized that the dominant conformers identified using the VCD spectral markers differ from those based on the relative DFT energies for neutral NALCME and NALC. The influence of solvent on both the conformational geometries and their relative stabilities is discussed. The aforementioned discrepancy can be attributed to the explicit solute-solvent hydrogen-bonding interactions which are not accounted for in the calculations. The empirical structural-spectral relationship identified can potentially be applied to large, related amino acids and polypeptides in water.

  16. Determination of endotoxin by the measurement of the acetylated methyl glycoside derivative of Kdo with gas-liquid chromatography-mass spectrometry.

    PubMed

    Rybka, J; Gamian, A

    2006-02-01

    A gas-liquid chromatographic-mass spectrometric (GLC-MS) method was applied to the detection of 3-deoxy-d-manno-2-octulosonic acid (Kdo), a constituent of bacterial lipopolysaccharide (LPS, endotoxin). Samples containing LPS were dried, methanolyzed with 2 M HCl in methanol at 60 degrees C for 1 h and acetylated with acetic anhydride and pyridine (1:1, v/v) solution at 100 degrees C for 30 min, then the products were analyzed by GLC-MS or GLC-MSMS. Four acetylated methylglycoside methyl ester derivatives of Kdo are formed in these conditions, namely one with pyranose ring (Kdo1), two derivatives in the furanose form (Kdo2 and 3) and one derivative of anhydro Kdo (Kdo4), as results from their mass fragmentation patterns. Synthetic Kdo produced mainly Kdo4 derivative, whereas Kdo1 of pyranose ring shape was the predominating derivative formed from LPS. The ion fragment of m/z 375 was selected for the specific detection of this Kdo1 derivative, which might be applied for the endotoxin determination. That approach was used for the analysis of preparations of bacteria, bacteriophages and samples of animal sera. In order to ensure the removal of phosphate substitutions from Kdo, methanolyzed samples can be treated with alkaline phosphatase (2.6 U, pH 9.2, 37 degrees C, 15 min), what was elaborated on Vibrio LPS preparation. PMID:15932775

  17. The human protein kinase HIPK2 phosphorylates and downregulates the methyl-binding transcription factor ZBTB4.

    PubMed

    Yamada, D; Pérez-Torrado, R; Filion, G; Caly, M; Jammart, B; Devignot, V; Sasai, N; Ravassard, P; Mallet, J; Sastre-Garau, X; Schmitz, M L; Defossez, P-A

    2009-07-01

    HIPK2 is a eukaryotic Serine-Threonine kinase that controls cellular proliferation and survival in response to exogenous signals. Here, we show that the human transcription factor ZBTB4 is a new target of HIPK2. The two proteins interact in vitro, colocalize and associate in vivo, and HIPK2 phosphorylates several conserved residues of ZBTB4. Overexpressing HIPK2 causes the degradation of ZBTB4, whereas overexpressing a kinase-deficient mutant of HIPK2 has no effect. The chemical activation of HIPK2 also decreases the amount of ZBTB4 in cells. Conversely, the inhibition of HIPK2 by drugs or by RNA interference causes a large increase in ZBTB4 levels. This negative regulation of ZBTB4 by HIPK2 occurs under normal conditions of cell growth. In addition, the degradation is increased by DNA damage. These findings have two consequences. First, we have recently shown that ZBTB4 inhibits the transcription of p21. Therefore, the activation of p21 by HIPK2 is two-pronged: stimulation of the activator p53, and simultaneous repression of the inhibitor ZBTB4. Second, ZBTB4 is also known to bind methylated DNA and repress methylated sequences. Consequently, our findings raise the possibility that HIPK2 might influence the epigenetic regulation of gene expression at loci that remain to be identified. PMID:19448668

  18. 3-Acetyl-1-(3-methyl­phen­yl)-5-phenyl-1H-pyrazole-4-carbonitrile

    PubMed Central

    Abdel-Aziz, Hatem A.; Al-Obaid, Abdul-Rahman M.; Ghabbour, Hazem A.; Hemamalini, Madhukar; Fun, Hoong-Kun

    2012-01-01

    In the title compound, C19H15N3O, the central pyrazole ring makes dihedral angles of 35.52 (12) and 62.21 (11)° with the attached phenyl and methyl-substituted phenyl rings, respectively. The corresponding angle between the phenyl and methyl-substituted phenyl rings is 62.90 (11)°. In the crystal, mol­ecules are connected by weak C—H⋯O hydrogen bonds, forming supra­molecular chains propagating along the a-axis direction. PMID:22347089

  19. Extended string-like binding of the phosphorylated HP1α N-terminal tail to the lysine 9-methylated histone H3 tail

    PubMed Central

    Shimojo, Hideaki; Kawaguchi, Ayumi; Oda, Takashi; Hashiguchi, Nobuto; Omori, Satoshi; Moritsugu, Kei; Kidera, Akinori; Hiragami-Hamada, Kyoko; Nakayama, Jun-ichi; Sato, Mamoru; Nishimura, Yoshifumi

    2016-01-01

    The chromodomain of HP1α binds directly to lysine 9-methylated histone H3 (H3K9me). This interaction is enhanced by phosphorylation of serine residues in the N-terminal tail of HP1α by unknown mechanism. Here we show that phosphorylation modulates flexibility of HP1α’s N-terminal tail, which strengthens the interaction with H3. NMR analysis of HP1α’s chromodomain with N-terminal tail reveals that phosphorylation does not change the overall tertiary structure, but apparently reduces the tail dynamics. Small angle X-ray scattering confirms that phosphorylation contributes to extending HP1α’s N-terminal tail. Systematic analysis using deletion mutants and replica exchange molecular dynamics simulations indicate that the phosphorylated serines and following acidic segment behave like an extended string and dynamically bind to H3 basic residues; without phosphorylation, the most N-terminal basic segment of HP1α inhibits interaction of the acidic segment with H3. Thus, the dynamic string-like behavior of HP1α’s N-terminal tail underlies the enhancement in H3 binding due to phosphorylation. PMID:26934956

  20. Simvastatin enhances NMDA receptor GluN2B expression and phosphorylation of GluN2B and GluN2A through increased histone acetylation and Src signaling in hippocampal CA1 neurons.

    PubMed

    Chen, Tingting; Zhang, Baofeng; Li, Guoxi; Chen, Lei; Chen, Ling

    2016-08-01

    Simvastatin (SV) can improve cognitive deficits in Alzheimer's disease patients and mice. Herein, we report that the administration of SV (20 mg/kg) for 5 days in mice (SV-mice) or the treatment of slices with SV (10 μM) for 4 h (SV-slices) could increase the density of NMDA-evoked inward currents (INMDA) in hippocampal CA1 pyramidal cells, which were blocked by farnesol (FOH) that converts farnesyl pyrophosphate (FPP), but not geranylgeraniol (GGOH) that increases geranylgeranylpyrophosphate (GGPP). Sensitivity of INMDA to ifenprodil in SV-mice or SV-slices was significantly increased. The levels of hippocampal GluN2B and GluN2A or Src phosphorylation in SV-mice or SV-slices were higher than controls, which were sensitive to FOH. The Src inhibitor PP2 could inhibit the SV-enhanced phosphorylation of GluN2B and GluN2A and SV-augmented INMDA, but PI3K inhibitor LY294002 did not. The levels of GluN2B mRNA and protein were elevated in SV-mice, which was abolished by FOH, but not by GGOH or PP2. Furthermore, the histone H3K9 and H3K27 acetylation of GluN2B promoter was increased in SV-mice, which was suppressed by FOH rather than GGOH or PP2. In control mice and slices, the reduction of FPP by farnesyl transferase inhibitor could increase the levels of GluN2B expression, the histone H3K9 and H3K27 acetylation and enhance the phosphorylation of GluN2B, GluN2A and Src. The findings indicate that the administration of SV can enhance GluN2B expression and GluN2B and GluN2A phosphorylation leading to augmentation of NMDAR activity through reducing FPP to increase histone acetylation of GluN2B and Src signaling. PMID:27016018

  1. Acetylation of RNA Processing Proteins and Cell Cycle Proteins in Mitosis

    PubMed Central

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

    2010-01-01

    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 posttranslational 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 anti-acetyl-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. PMID:20812760

  2. [DNA methylation in obesity].

    PubMed

    Pokrywka, Małgorzata; Kieć-Wilk, Beata; Polus, Anna; Wybrańska, Iwona

    2014-01-01

    The number of overweight and obese people is increasing at an alarming rate, especially in the developed and developing countries. Obesity is a major risk factor for diabetes, cardiovascular disease, and cancer, and in consequence for premature death. The development of obesity results from the interplay of both genetic and environmental factors, which include sedentary life style and abnormal eating habits. In the past few years a number of events accompanying obesity, affecting expression of genes which are not directly connected with the DNA base sequence (e.g. epigenetic changes), have been described. Epigenetic processes include DNA methylation, histone modifications such as acetylation, methylation, phosphorylation, ubiquitination, and sumoylation, as well as non-coding micro-RNA (miRNA) synthesis. In this review, the known changes in the profile of DNA methylation as a factor affecting obesity and its complications are described. PMID:25531701

  3. 2-Acetyl­amino-1,3,4,6-tetra-O-(tri­methyl­silyl)-2-de­oxy-α-d-gluco­pyran­ose

    PubMed Central

    Cheng, Zhao-Dong; Cui, Yan-Li; Mao, Jian-Wei

    2013-01-01

    The title compound, C20H47NO6Si4, was synthesized by per-O-tri­methyl­silylation of N-acetyl-d-glucosa­mine using chloro­tri­methyl­silane in the presence of hexa­methyl­disiloxane. The tri­methyl­silyl group and acetamido group are located on the same side of the pyran ring, showing an α-configuration glycoside. One of the tri­methyl­silyl groups is disordered over two orientations, with site-occupancy factors of 0.625 (9) and 0.375 (9). In the crystal, N—H⋯O hydrogen bonds link the mol­ecules into supra­molecular chains along the a-axis direction. PMID:23795087

  4. Metal Complexes of New Bioactive Pyrazolone Phenylhydrazones; Crystal Structure of 4-Acetyl-3-methyl-1-phenyl-2-pyrazoline-5-one phenylhydrazone Ampp-Ph

    PubMed Central

    Idemudia, Omoruyi G.; Sadimenko, Alexander P.; Hosten, Eric C.

    2016-01-01

    The condensation reaction of phenylhydrazine and dinitrophenylhydrazine with 4-acetyl and 4-benzoyl pyrazolone precipitated air-stable acetyldinitrophenylhydrazone Ampp-Dh, benzoylphenylhydrazone Bmpp-Ph and benzoyldinitrophenylhydrazone Bmpp-Dh in their keto imine form; a study inspired by the burning interest for the development of new bioactive materials with novel properties that may become alternative therapeutic agents. Elemental analysis, FTIR, 1H, and 13C NMR, and mass spectroscopy have been used to justify their proposed chemical structures, which were in agreement with the single crystal structure of Bmpp-Dh earlier reported according to X-ray crystallography. The single crystal structure of 4-acetyl-3-methyl-1-phenyl--pyrazoline-5-one phenylhydrazone Ampp-Ph, which crystallizes in a triclinic crystal system with a P-1 (No. 2) space group is presented. Octahedral Mn(II), Ni(II), Co(II), and Cu(II) complexes of these respective ligands with two molecules each of the bidentate Schiff base, coordinating to the metal ion through the azomethine nitrogen C=N and the keto oxygen C=O, which were afforded by the reaction of aqueous solutions of the corresponding metal salts with the ligands are also reported. Their identity and proposed structures were according to elemental analysis, FTIR spectroscopy, UV-VIS spectrophotometry (electronic spectra) and Bohr magnetic moments, as well as thermogravimetric analysis (TGA) results. A look at the antibacterial and antioxidant activities of synthesized compounds using the methods of the disc diffusion against some selected bacterial isolates and 1,1-diphenyl-2-picryl-hydrazil (DPPH) respectively, showed biological activities in relation to employed standard medicinal drugs. PMID:27213342

  5. Metal Complexes of New Bioactive Pyrazolone Phenylhydrazones; Crystal Structure of 4-Acetyl-3-methyl-1-phenyl-2-pyrazoline-5-one phenylhydrazone Ampp-Ph.

    PubMed

    Idemudia, Omoruyi G; Sadimenko, Alexander P; Hosten, Eric C

    2016-01-01

    The condensation reaction of phenylhydrazine and dinitrophenylhydrazine with 4-acetyl and 4-benzoyl pyrazolone precipitated air-stable acetyldinitrophenylhydrazone Ampp-Dh, benzoylphenylhydrazone Bmpp-Ph and benzoyldinitrophenylhydrazone Bmpp-Dh in their keto imine form; a study inspired by the burning interest for the development of new bioactive materials with novel properties that may become alternative therapeutic agents. Elemental analysis, FTIR, ¹H, and (13)C NMR, and mass spectroscopy have been used to justify their proposed chemical structures, which were in agreement with the single crystal structure of Bmpp-Dh earlier reported according to X-ray crystallography. The single crystal structure of 4-acetyl-3-methyl-1-phenyl--pyrazoline-5-one phenylhydrazone Ampp-Ph, which crystallizes in a triclinic crystal system with a P-1 (No. 2) space group is presented. Octahedral Mn(II), Ni(II), Co(II), and Cu(II) complexes of these respective ligands with two molecules each of the bidentate Schiff base, coordinating to the metal ion through the azomethine nitrogen C=N and the keto oxygen C=O, which were afforded by the reaction of aqueous solutions of the corresponding metal salts with the ligands are also reported. Their identity and proposed structures were according to elemental analysis, FTIR spectroscopy, UV-VIS spectrophotometry (electronic spectra) and Bohr magnetic moments, as well as thermogravimetric analysis (TGA) results. A look at the antibacterial and antioxidant activities of synthesized compounds using the methods of the disc diffusion against some selected bacterial isolates and 1,1-diphenyl-2-picryl-hydrazil (DPPH) respectively, showed biological activities in relation to employed standard medicinal drugs. PMID:27213342

  6. Regulation of platelet activating factor synthesis: modulation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase by phosphorylation and dephosphorylation in rat spleen microsomes

    SciTech Connect

    Lenihan, D.J.; Lee, T.C.

    1984-05-16

    1-Alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase plays an important regulatory role in the biosynthesis of platelet activating factor, a potent bioactive mediator. The authors tested the hypothesis that the activity of acetyltransferase may be modulated by enzymatic phosphorylation and dephosphorylation. The results showed that acetyltransferase activity in rat spleens was 2- to 3-fold higher in microsomes isolated in the presence of F/sup -/ than in those isolated in the presence of Cl/sup -/. The microsomal acetyltransferase could be activated by preincubation of microsomes, isolated in the presence of Cl/sup -/, with ATP, Mg/sup 2 +/, and the soluble fraction from rat spleen. Addition of phosphatidylserine, diacylglycerols, plus Ca/sup 2 +/ further enhanced the activity. The increase in the activity of acetyltranferase was abolished by treatment of the activated microsomes with alkaline phosphatase. Conversely, the activity of acetyltransferase can be reactivated in the alkaline phosphatase-treated microsomes with incubation conditions that favor phosphorylation. Therefore, the findings suggest that acetyltransferase activity is regulated by reversible activation/inactivation through phosphorylation/dephosphorylation.

  7. Growth, optical, luminescence, thermal and mechanical behavior of an organic single crystal: 3-Acetyl-2-methyl-4-phenylquinolin-1-ium chloride.

    PubMed

    Nirosha, M; Kalainathan, S; Sarveswari, S; Vijayakumar, V

    2014-04-01

    A single crystal of 3-acetyl-2-methyl-4-phenylquinolin-1-ium chloride has grown by slow evaporation solution growth technique using ethanol as solvent. The structural, thermal, optical and mechanical property has studied for the grown crystal. Single crystal XRD revealed that the crystal belongs to monoclinic system with space group P21/c. The presences of Functional groups in the crystallized material have confirmed using the FTIR vibrational spectrum. The optical absorbance spectrum recorded from 190 to 1100nm shows the cut-off wavelength occurs at 371nm. The material shows its transparency in the entire region of the visible spectrum. The photoluminescence spectrum shows the ultraviolet and blue emission in the crystal. Thermogravimetric and differential thermal analysis reveal the thermal stability of the grown crystal. Etching study shows the grown mechanism and surface features of the crystal. Vickers microhardness studies have carried out on the (01-1) plane to understand the mechanical properties of the grown crystal. The hardness of the title compound increases on increasing the load. The Meyer's index number (n), and the stiffness constants for different loads has calculated and reported. PMID:24389003

  8. A novel phosphorylation site of N-methyl-D-aspartate receptor GluN2B at S1284 is regulated by Cdk5 in neuronal ischemia.

    PubMed

    Lu, Wen; Ai, Heng; Peng, Lin; Wang, Jie-jie; Zhang, Bin; Liu, Xiao; Luo, Jian-hong

    2015-09-01

    N-methyl-D-aspartate receptors (NMDARs) are a key player in synaptic and several neurological diseases, such as stroke. Phosphorylation of NMDAR subunits at their cytoplasmic carboxyl termini has been considered to be an important mechanism to regulate the receptor function. Cyclin-dependent kinase 5 (Cdk5) has been demonstrated to be responsible for regulating phosphorylation and function of NMDARs. Besides, it is also suggested that Cdk5 is involved in ischemic insult. In the present study, we showed that GluN2B subunit serine 1284 at its cytoplasmic carboxyl termini was regulated by Cdk5 in neuronal ischemia. Interestingly, both oxygen glucose deprivation (OGD) in cultured hippocampal neurons and transient global ischemia in mice induce dramatic changes in the phosphorylated level of GluN2B at S1284. However, no significant changes in the phosphorylation of this site are found neither in chemical LTP stimulation in cultured hippocampal neurons nor fear conditioning in adult mice. Taken together, our study identified NMDAR GluN2B S1284 as a novel phosphorylation site regulated by Cdk5 with implication in neuronal ischemia. PMID:26093036

  9. Mechanisms of HIV-tat-Induced Phosphorylation of N-Methyl-d-Aspartate Receptor Subunit 2A in Human Primary Neurons

    PubMed Central

    King, Jessie E.; Eugenin, Eliseo A.; Hazleton, Joy E.; Morgello, Susan; Berman, Joan W.

    2010-01-01

    HIV infection of the central nervous system results in neurological dysfunction in a large number of individuals. NeuroAIDS is characterized by neuronal injury and loss, yet there is no evidence of HIV-infected neurons. Neuronal damage and dropout must therefore be due to indirect effects of HIV infection of other central nervous system cells through elaboration of inflammatory factors and neurotoxic viral proteins, including the viral transactivator, tat. We previously demonstrated that HIV-tat-induced apoptosis in human primary neurons is dependent on N-methyl-d-aspartate receptor (NMDAR) activity. NMDAR activity is regulated by various mechanisms including NMDAR phosphorylation, which may lead to neuronal dysfunction and apoptosis in pathological conditions. We now demonstrate that tat treatment of human neurons results in tyrosine (Y) phosphorylation of the NMDAR subunit 2A (NR2A) in a src kinase–dependent manner. In vitro kinase assays and in vivo data indicated that NR2A Y1184, Y1325, and Y1425 are phosphorylated. Tat treatment of neuronal cultures enhanced phosphorylation of NR2A Y1325, indicating that this site is tat sensitive. Human brain tissue sections from HIV-infected individuals with encephalitis showed an increased phosphorylation of NR2A Y1325 in neurons as compared with uninfected and HIV-infected individuals without encephalitis. These findings suggest new avenues of treatment for HIV-associated cognitive impairment. PMID:20448061

  10. Acetyl-l-carnitine (ALCAR) prevents hypobaric hypoxia-induced spatial memory impairment through extracellular related kinase-mediated nuclear factor erythroid 2-related factor 2 phosphorylation.

    PubMed

    Barhwal, K; Hota, S K; Jain, V; Prasad, D; Singh, S B; Ilavazhagan, G

    2009-06-30

    Exposure to hypobaric hypoxia, a condition involving decreased availability of oxygen is known to be associated with oxidative stress, neurodegeneration and memory impairment. The multifactorial response of the brain and the complex signaling pathways involved therewith limits the therapeutic efficacy of several antioxidants in ameliorating hypobaric hypoxia-induced memory impairment. The present study was therefore aimed at investigating the potential of acetyl-l-carnitine (ALCAR), a known antioxidant that has been reported to augment neurotrophin-mediated survival mechanisms, in ameliorating hypoxia-induced neurodegeneration and memory impairment. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor involved in the cellular defense mechanism against oxidative stress related to brain injury and neurological disorders. The study was designed to understand the mechanisms involving Nrf2 stabilization following exposure to hypobaric hypoxia. The results displayed reference memory impairment in Sprague-Dawley rats exposed to hypobaric hypoxia (7620 m) for 14 consecutive days which however improved on administration of ALCAR during hypoxic exposure. The study also revealed Nrf2 regulated augmented antioxidant response on administration of ALCAR which was through a novel tyrosine kinase A (TrkA) receptor-mediated mechanism. A decrease in free radical generation, lipid peroxidation and protein oxidation was also observed along with a concomitant increase in thioredoxin and reduced glutathione levels on administration of ALCAR during exposure to hypobaric hypoxia. The present study therefore reveals the therapeutic potential of ALCAR under conditions of hypobaric hypoxia and elucidates a novel mechanism of action of the drug. PMID:19318118

  11. 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.

  12. Phosphorylation of N-methyl-D-aspartic acid receptor-associated neuronal nitric oxide synthase depends on estrogens and modulates hypothalamic nitric oxide production during the ovarian cycle

    PubMed Central

    Parkash, Jyoti; D'Anglemont De Tassigny, Xavier; Bellefontaine, Nicole; Campagne, Celine; Mazure, Danièle; Buée-Scherrer, Valérie; Prevot, Vincent

    2010-01-01

    Within the preoptic region, nitric oxide (NO) production varies during the ovarian cycle and has the ability to impact hypothalamic reproductive function. One mechanism for the regulation of NO release mediated by estrogens during the estrous cycle includes physical association of the calcium-activated neuronal NO synthase (nNOS) enzyme with the glutamate N-methyl-D-aspartate (NMDA) receptor channels via the postsynaptic density 95 (PSD 95) scaffolding protein. Here, we demonstrate that endogenous variations in estrogens levels during the estrous cycle also coincide with corresponding changes in the state of nNOS Ser1412 phosphorylation, the level of association of this isoform with the NMDA receptor/PSD-95 complex at the plasma membrane and the activity of NOS. Neuronal NOS Ser1412 phosphorylation is maximal on the afternoon of proestrus, when both the levels of estrogens and the physical association of nNOS with NMDA receptors are highest. Estradiol mimicked these effects in ovariectomized (OVX) rats. In addition, the catalytic activity of NOS in membrane protein extracts from the preoptic region, i.e., independent of any functional protein-protein interactions or cell-cell signaling, was significantly increased in estradioltreated OVX rats compared to OVX rats. Finally, λ phosphatase-mediated nNOS dephosphorylation dramatically impaired NOS activity in preoptic region protein extracts, thus demonstrating the important role of phosphorylation in the regulation of NO production in the preoptic region. Taken together, these results yield new insights into the regulation of neuron-derived NO production by gonadal steroids within the preoptic region and raise the possibility that changes in nNOS phosphorylation during fluctuating physiological conditions may be involved in the hypothalamic control of key neuroendocrine functions, such as reproduction. PMID:20371700

  13. 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

  14. Prebiotic Fiber Increases Hepatic Acetyl CoA Carboxylase Phosphorylation and Suppresses Glucose-Dependent Insulinotropic Polypeptide Secretion More Effectively When Used with Metformin in Obese Rats1,2

    PubMed Central

    Pyra, Kim A.; Saha, Dolan C.; Reimer, Raylene A.

    2013-01-01

    Independently, metformin (MET) and the prebiotic, oligofructose (OFS), have been shown to increase glucagon-like peptide (GLP-1) secretion. Our objective was to determine whether using OFS as an adjunct with MET augments GLP-1 secretion in obese rats. Male, diet-induced obese Sprague Dawley rats were randomized to: 1) high-fat/-sucrose diet [HFHS; control (C); 20% fat, 50% sucrose wt:wt]; 2) HFHS+10% OFS (OFS); 3) HFHS + MET [300 mg/kg/d (MET)]; 4) HFHS+10% OFS+MET (OFS +MET). Body composition, glycemia, satiety hormones, and mechanisms related to dipeptidyl peptidase 4 (DPP4) activity in plasma, hepatic AMP-activated protein kinase (AMPK; Western blots), and gut microbiota (qPCR) were examined. Direct effects of MET and SCFA were examined in human enteroendocrine cells. The interaction between OFS and MET affected fat mass, hepatic TG, secretion of glucose-dependent insulinotropic polypeptide (GIP) and leptin, and AMPKα2 mRNA and phosphorylated acetyl CoA carboxylase (pACC) levels (P < 0.05). Combined, OFS and MET reduced GIP secretion to a greater extent than either treatment alone (P < 0.05). The hepatic pACC level was increased by OFS+MET by at least 50% above all other treatments, which did not differ from each other (P < 0.05). OFS decreased plasma DPP4 activity (P < 0.001). Cecal Bifidobacteria (P < 0.001) were markedly increased and C. leptum decreased (P < 0.001) with OFS consumption. In human enteroendocrine cells, the interaction between MET and SCFA affected GLP-1 secretion (P < 0.04) but was not associated with higher GLP-1 than the highest individual doses. In conclusion, the combined actions of OFS and MET were associated with important interaction effects that have the potential to improve metabolic outcomes associated with obesity. PMID:22223580

  15. Altered protein phosphatase 2A methylation and Tau phosphorylation in the young and aged brain of methylenetetrahydrofolate reductase (MTHFR) deficient mice

    PubMed Central

    Sontag, Jean-Marie; Wasek, Brandi; Taleski, Goce; Smith, Josephine; Arning, Erland; Sontag, Estelle; Bottiglieri, Teodoro

    2014-01-01

    Common functional polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, a key enzyme in folate and homocysteine metabolism, influence risk for a variety of complex disorders, including developmental, vascular, and neurological diseases. MTHFR deficiency is associated with elevation of homocysteine levels and alterations in the methylation cycle. Here, using young and aged Mthfr knockout mouse models, we show that mild MTHFR deficiency can lead to brain-region specific impairment of the methylation of Ser/Thr protein phosphatase 2A (PP2A). Relative to wild-type controls, decreased expression levels of PP2A and leucine carboxyl methyltransferase (LCMT1) were primarily observed in the hippocampus and cerebellum, and to a lesser extent in the cortex of young null Mthfr−/− and aged heterozygous Mthfr+/− mice. A marked down regulation of LCMT1 correlated with the loss of PP2A/Bα holoenzymes. Dietary folate deficiency significantly decreased LCMT1, methylated PP2A and PP2A/Bα levels in all brain regions examined from aged Mthfr+/+ mice, and further exacerbated the regional effects of MTHFR deficiency in aged Mthfr+/− mice. In turn, the down regulation of PP2A/Bα was associated with enhanced phosphorylation of Tau, a neuropathological hallmark of Alzheimer’s disease (AD). Our findings identify hypomethylation of PP2A enzymes, which are major CNS phosphatases, as a novel mechanism by which MTHFR deficiency and Mthfr gene-diet interactions could lead to disruption of neuronal homeostasis, and increase the risk for a variety of neuropsychiatric disorders, including age-related diseases like sporadic AD. PMID:25202269

  16. Altered protein phosphatase 2A methylation and Tau phosphorylation in the young and aged brain of methylenetetrahydrofolate reductase (MTHFR) deficient mice.

    PubMed

    Sontag, Jean-Marie; Wasek, Brandi; Taleski, Goce; Smith, Josephine; Arning, Erland; Sontag, Estelle; Bottiglieri, Teodoro

    2014-01-01

    Common functional polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, a key enzyme in folate and homocysteine metabolism, influence risk for a variety of complex disorders, including developmental, vascular, and neurological diseases. MTHFR deficiency is associated with elevation of homocysteine levels and alterations in the methylation cycle. Here, using young and aged Mthfr knockout mouse models, we show that mild MTHFR deficiency can lead to brain-region specific impairment of the methylation of Ser/Thr protein phosphatase 2A (PP2A). Relative to wild-type controls, decreased expression levels of PP2A and leucine carboxyl methyltransferase (LCMT1) were primarily observed in the hippocampus and cerebellum, and to a lesser extent in the cortex of young null Mthfr (-/-) and aged heterozygous Mthfr (+/-) mice. A marked down regulation of LCMT1 correlated with the loss of PP2A/Bα holoenzymes. Dietary folate deficiency significantly decreased LCMT1, methylated PP2A and PP2A/Bα levels in all brain regions examined from aged Mthfr (+/+) mice, and further exacerbated the regional effects of MTHFR deficiency in aged Mthfr (+/-) mice. In turn, the down regulation of PP2A/Bα was associated with enhanced phosphorylation of Tau, a neuropathological hallmark of Alzheimer's disease (AD). Our findings identify hypomethylation of PP2A enzymes, which are major CNS phosphatases, as a novel mechanism by which MTHFR deficiency and Mthfr gene-diet interactions could lead to disruption of neuronal homeostasis, and increase the risk for a variety of neuropsychiatric disorders, including age-related diseases like sporadic AD. PMID:25202269

  17. Multifaceted Histone H3 Methylation and Phosphorylation Readout by the Plant Homeodomain Finger of Human Nuclear Antigen Sp100C.

    PubMed

    Zhang, Xiaojie; Zhao, Dan; Xiong, Xiaozhe; He, Zhimin; Li, Haitao

    2016-06-10

    The decoding of histone post-translational modifications by chromatin-binding modules ("readers") constitutes one major mechanism of epigenetic regulation. Nuclear antigen Sp100 (SPECKLED, 100 kDa), a constitutive component of the promyelocytic leukemia nuclear bodies, plays key roles in intrinsic immunity and transcriptional repression. Sp100C, a splicing isoform specifically up-regulated upon interferon stimulation, harbors a unique tandem plant homeodomain (PHD) finger and bromodomain at its C terminus. Combining structural, quantitative binding, and cellular co-localization studies, we characterized Sp100C PHD finger as an unmethylated histone H3 Lys(4) (H3K4me0) reader that tolerates histone H3 Thr(3) phosphorylation (H3T3ph), histone H3 Lys(9) trimethylation (H3K9me3), and histone H3 Ser(10) phosphorylation (H3S10ph), hallmarks associated with the mitotic chromosome. In contrast, whereas H3K4me0 reader activity is conserved in Sp140, an Sp100C paralog, the multivalent tolerance of H3T3ph, H3K9me3, and H3S10ph was lost for Sp140. The complex structure determined at 2.1 Å revealed a highly coordinated lysine ϵ-amine recognition sphere formed by an extended N-terminal motif for H3K4me0 readout. Interestingly, reader pocket rigidification by disulfide bond formation enhanced H3K4me0 binding by Sp100C. An additional complex structure solved at 2.7 Å revealed that H3T3ph is recognized by the arginine residue, Arg(713), that is unique to the PHD finger of Sp100C. Consistent with a restrictive cellular role of Sp100C, these results establish a direct chromatin targeting function of Sp100C that may regulate transcriptional gene silencing and promyelocytic leukemia nuclear body-mediated intrinsic immunity in response to interferon stimulation. PMID:27129259

  18. Effect of Histone Acetylation on N-Methyl-D-Aspartate 2B Receptor Subunits and Interleukin-1 Receptors in Association with Nociception-Related Somatosensory Cortex Dysfunction in a Mouse Model of Sepsis.

    PubMed

    Imamura, Yukio; Yoshikawa, Nao; Murkami, Yuki; Mitani, Satoko; Matsumoto, Naoya; Matsumoto, Hisatake; Yamada, Tomoki; Yamakawa, Kazuma; Nakagawa, Junichiro; Ogura, Hiroshi; Shimazu, Takeshi; Jin, Takashi

    2016-06-01

    Whole-body inflammation (i.e., sepsis) often results in brain-related sensory dysfunction. We previously reported that interleukin (IL)-1 resulted in synaptic dysfunction of septic encephalopathy, but the underlying molecular mechanisms remain unknown, as do effective treatments. Using mice, we examined immunohistochemistry, co-immunoprecipitation, enzyme-linked immunosorbent assay, and behavior analyses, and investigated the role of the N-methyl-D-aspartate 2B subunit (NR2B) of NMDA receptor, IL-1 receptor, and histone acetylation in the pathophysiology underlying sensory dysfunction induced by lipopolysaccharide (LPS). Mice groups of sham-operated, LPS, LPS with an NR2B antagonist, or LPS with resveratrol (a histone acetylation activator) were analyzed. We found that LPS increased NR2B and interleukin-1 receptor (IL-1R) immunoreactivity. The expression of Iba1, a marker for microglia and/or macrophages, increased more significantly in the brain than in the spinal cord, implicating NR2B and IL-1R in brain inflammation. Immunoprecipitation with NR2B and IL-1R revealed related antibodies. Blood levels of IL-1β (i.e., the IL-1R ligand) increased, though not significantly, suggesting that inflammation peaked at 20 h. Behavioral assessments of central (CNS) and peripheral sensory (PNS) function indicated that LPS delayed CNS but not PNS escape latency. Finally, NR2B antagonist or resveratrol in the lateral ventricle antagonized the effects of LPS in the brain and improved animal survival. In summary, histone acetylation may control expression of NR2B and IL-1R, alleviating inflammation-induced sensory neuronal dysfunction caused by LPS. PMID:26682951

  19. The double PHD finger domain of MOZ/MYST3 induces α-helical structure of the histone H3 tail to facilitate acetylation and methylation sampling and modification.

    PubMed

    Dreveny, Ingrid; Deeves, Sian E; Fulton, Joel; Yue, Baigong; Messmer, Marie; Bhattacharya, Amit; Collins, Hilary M; Heery, David M

    2014-01-01

    Histone tail modifications control many nuclear processes by dictating the dynamic exchange of regulatory proteins on chromatin. Here we report novel insights into histone H3 tail structure in complex with the double PHD finger (DPF) of the lysine acetyltransferase MOZ/MYST3/KAT6A. In addition to sampling H3 and H4 modification status, we show that the DPF cooperates with the MYST domain to promote H3K9 and H3K14 acetylation, although not if H3K4 is trimethylated. Four crystal structures of an extended DPF alone and in complex with unmodified or acetylated forms of the H3 tail reveal the molecular basis of crosstalk between H3K4me3 and H3K14ac. We show for the first time that MOZ DPF induces α-helical conformation of H3K4-T11, revealing a unique mode of H3 recognition. The helical structure facilitates sampling of H3K4 methylation status, and proffers H3K9 and other residues for modification. Additionally, we show that a conserved double glycine hinge flanking the H3 tail helix is required for a conformational change enabling docking of H3K14ac with the DPF. In summary, our data provide the first observations of extensive helical structure in a histone tail, revealing the inherent ability of the H3 tail to adopt alternate conformations in complex with chromatin regulators. PMID:24150941

  20. The upregulation of NR2A-containing N-methyl-D-aspartate receptor function by tyrosine phosphorylation of postsynaptic density 95 via facilitating Src/proline-rich tyrosine kinase 2 activation.

    PubMed

    Zhao, Chao; Du, Cai-Ping; Peng, Yan; Xu, Zhen; Sun, Chang-Cheng; Liu, Yong; Hou, Xiao-Yu

    2015-04-01

    The activation of postsynaptic N-methyl-D-aspartate (NMDA) receptors is required for long-term potentiation (LTP) of synaptic transmission. Postsynaptic density 95 (PSD-95) serves as a scaffold protein that tethers NMDA receptor subunits, kinases, and signal molecules. Our previous study proves that PSD-95 is a substrate of Src/Fyn and identifies Y523 on PSD-95 as a principal phosphorylation site. In this paper, we try to define an involvement and molecular consequences of PSD-95 phosphorylation by Src in NMDA receptor regulation. We found that either NMDA or chemical LTP induction leads to rapid phosphorylation of PSD-95 by Src in cultured cortical neurons. The phosphorylation of Y523 on PSD-95 potentiates NR2A-containing NMDA receptor current amplitude, implying an important role of Src-mediated PSD-95 phosphorylation in NMDA receptor activation. Comparing to wild-type PSD-95, overexpression of nonphosphorylatable mutant PSD-95Y523F attenuated the NMDA-stimulated NR2A tyrosine phosphorylation that enhances electrophysiological responses of NMDA receptor channels, while did not affect the membrane localization of NR2A subunits. PSD-95Y523D, a phosphomimetic mutant of PSD-95, induced NR2A tyrosine phosphorylation even if there was no NMDA treatment. In addition, the deficiency of Y523 phosphorylation on PSD-95 impaired the facilitatory effect of PSD-95 on the activation of Src and proline-rich tyrosine kinase 2 (Pyk2) and decreased the binding of Pyk2 with PSD-95. These results indicate that PSD-95 phosphorylation by Src facilitates the integration of Pyk2 to PSD-95 signal complex, the activation of Pyk2/Src, as well as the subsequent tyrosine phosphorylation of NR2A, which ultimately results in the upregulation of NMDA receptor function and synaptic transmission. PMID:24981431

  1. Histone H3 phosphorylation – A versatile chromatin modification for different occasions

    PubMed Central

    Sawicka, Anna; Seiser, Christian

    2012-01-01

    Post-translation modifications of histones modulate the accessibility and transcriptional competence of specific chromatin regions within the eukaryotic genome. Phosphorylation of histone H3 is unique in the sense that it associates on one hand with open chromatin during gene activation and marks on the other hand highly condensed chromatin during mitosis. Phosphorylation of serine residues at histone H3 is a highly dynamic process that creates together with acetylation and methylation marks at neighboring lysine residues specific combinatorial patterns that are read by specific detector proteins. In this review we describe the importance of different histone H3 phosphorylation marks for chromatin condensation during mitosis. In addition, we review the signals that trigger histone H3 phosphorylation and the factors that control this reversible modification during interphase and mediate the biological readout of the signal. Finally, we discuss different models describing the role of histone H3 phosphorylation in the activation of transcription of poised genes or by transient derepression of epigenetically silenced genes. We propose that histone H3 phosphorylation in the context with lysine methylation might temporarily relieve the silencing of specific genes without affecting the epigenetic memory. PMID:22564826

  2. The metal complexes of heterocyclic β-diketones and their derivatives—V. The synthesis, structure and i.r. spectral studies of metal(II) complexes of 1-phenyl-3-methyl-4-acetyl-pyrazolone-5 (HPMAP)

    NASA Astrophysics Data System (ADS)

    Okafor, Emmanuel Chukwuemeka

    The divalent metal complexes of Be, Mn, Co, Ni, Cu, Zn, Pb, Hg and Ba with 1-phenyl-3-methyl-4-acetyl-pyrazolone-5 (HPMAP), have been synthesized for the first time. It is shown that even though the ligand, HPMAP, has four potential coordination sites, it behaves like a bidentate enol forming neutral metal chelates through the carbonyl and enolic hydroxyl groups. The complexes were characterized by means of elemental analyses, conductivity measurements, i.r. proton NMR spectroscopy. Proton magnetic resonance spectral data for some of the chelates have been recorded and discussed. The i.r. spectra of HPMAP and its chelates have been measured between 4000 and 200 cm -1 and assignments are proposed for the observed frequencies. Replacement of a methyl group in the acetylacetonate by the pyrazolone moiety fused to the chelate ring has been found to strengthen the C …O and MO bonds and weaken the C …C bond of the chelate ring. The magnitudes of MO stretching frequencies for the transition metals follow very closely the Irving-Williams stability order Cu > Ni > Zn > Co > Mn.

  3. Methyl 9-Oxo-(10E,12E)-octadecadienoate Isolated from Fomes fomentarius Attenuates Lipopolysaccharide-Induced Inflammatory Response by Blocking Phosphorylation of STAT3 in Murine Macrophages

    PubMed Central

    Choe, Ji-Hyun; Yi, Young-Joo; Lee, Myeong-Seok; Seo, Dong-Won

    2015-01-01

    Fomes fomentarius is a fungus of the Polyporaceae family and is used in traditional oriental therapies. Although the anti-inflammatory activities of this species have been previously reported, the identity of the bioactive compounds responsible for this activity remains unknown. Here, we investigated whether methyl 9-oxo-(10E,12E)-octadecadienoate (FF-8) purified from F. fomentarius exerts anti-inflammatory activity in murine macrophages stimulated with lipopolysaccharide (LPS). FF-8 suppressed secretion of nitric oxide (NO) and prostaglandin E2 through downregulation of inducible NO synthase and cyclooxygenase-2 expression induced by LPS. In addition, pretreatment of cells with FF-8 led to a reduction in levels of secreted inflammatory cytokines such as tumor necrosis factor-α and interleukin-6 in macrophages stimulated with LPS. Conversely, FF-8 did not affect nuclear factor κB, p38, c-Jun NH2-terminal kinase, and extracellular signal-regulated kinase pathways. Instead, FF-8 specifically interfered with signal transducer and activator of transcription 3 (STAT3) phosphorylation induced by LPS. Collectively, this study demonstrated that FF-8 purified from F. fomentarius suppresses inflammatory responses in macrophages stimulated with LPS by inhibiting STAT3 activation. Further studies will be required to elucidate the anti-inflammatory effect of FF-8 in vivo. PMID:26539049

  4. Imaging Histone Methylations in Living Animals.

    PubMed

    Sekar, Thillai V; Paulmurugan, Ramasamy

    2016-01-01

    Histone modifications (methylation, acetylation, phosphorylation, sumoylation, etc.,) are at the heart of cellular regulatory mechanisms, which control expression of genes in an orderly fashion and control the entire cellular regulatory networks. Histone lysine methylation has been identified as one of the several posttranslational histone modifications that plays crucial role in regulating gene expressions in facultative heterochromatic DNA regions while maintaining structural integrity in constitutive heterochromatic DNA regions. Since histone methylation is dysregulated in various cellular diseases, it has been considered a potential therapeutic target for drug development. Currently there is no simple method available to screen and preclinically evaluate drugs modulating this cellular process, we recently developed two different methods by adopting reporter gene technology to screen drugs and to preclinically evaluate them in living animals. Method detects and quantitatively monitors the level of histone methylations in intact cells, is of a prerequisite to screen small molecules that modulate histone lysine methylation. Here, we describe two independent optical imaging sensors developed to image histone methylations in cells and in living animals. Since we used standard PCR-based cloning strategies to construct different plasmid vectors shown in this chapter, we are not providing any details regarding the construction methods, instead, we focus on detailing various methods used for measuring histone methylation-assisted luciferase quantitation in cells and imaging in living animals. PMID:27424907

  5. IFNγ Induces DNA Methylation-Silenced GPR109A Expression via pSTAT1/p300 and H3K18 Acetylation in Colon Cancer.

    PubMed

    Bardhan, Kankana; Paschall, Amy V; Yang, Dafeng; Chen, May R; Simon, Priscilla S; Bhutia, Yangzom D; Martin, Pamela M; Thangaraju, Muthusamy; Browning, Darren D; Ganapathy, Vadivel; Heaton, Christopher M; Gu, Keni; Lee, Jeffrey R; Liu, Kebin

    2015-07-01

    Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress proinflammatory diseases. GPR109A is the receptor for short-chain fatty acids. The functions of GPR109A have been the subject of extensive studies; however, the molecular mechanisms underlying GPR109A expression is largely unknown. We show that GPR109A is highly expressed in normal human colon tissues, but is silenced in human colon carcinoma cells. The GPR109A promoter DNA is methylated in human colon carcinoma. Strikingly, we observed that IFNγ, a cytokine secreted by activated T cells, activates GPR109A transcription without altering its promoter DNA methylation. Colon carcinoma grows significantly faster in IFNγ-deficient mice than in wild-type mice in an orthotopic colon cancer mouse model. A positive correlation was observed between GPR109A protein level and tumor-infiltrating T cells in human colon carcinoma specimens, and IFNγ expression level is higher in human colon carcinoma tissues than in normal colon tissues. We further demonstrated that IFNγ rapidly activates pSTAT1 that binds to the promoter of p300 to activate its transcription. p300 then binds to the GPR109A promoter to induce H3K18 hyperacetylation, resulting in chromatin remodeling in the methylated GPR109A promoter. The IFNγ-activated pSTAT1 then directly binds to the methylated but hyperacetylated GPR109 promoter to activate its transcription. Overall, our data indicate that GPR109A acts as a tumor suppressor in colon cancer, and the host immune system might use IFNγ to counteract DNA methylation-mediated GPR109A silencing as a mechanism to suppress tumor development. PMID:25735954

  6. Phosphorylation of Tyrosine 1070 at the GluN2B Subunit Is Regulated by Synaptic Activity and Critical for Surface Expression of N-Methyl-D-aspartate (NMDA) Receptors.

    PubMed

    Lu, Wen; Fang, Weiqing; Li, Jian; Zhang, Bin; Yang, Qian; Yan, Xunyi; Peng, Lin; Ai, Heng; Wang, Jie-jie; Liu, Xiao; Luo, Jianhong; Yang, Wei

    2015-09-18

    The number and subunit composition of synaptic N-methyl-d-aspartate receptors (NMDARs) play critical roles in synaptic plasticity, learning, and memory and are implicated in neurological disorders. Tyrosine phosphorylation provides a powerful means of regulating NMDAR function, but the underling mechanism remains elusive. In this study we identified a tyrosine site on the GluN2B subunit, Tyr-1070, which was phosphorylated by a proto-oncogene tyrosine-protein (Fyn) kinase and critical for the surface expression of GluN2B-containing NMDARs. The phosphorylation of GluN2B at Tyr-1070 was required for binding of Fyn kinase to GluN2B, which up-regulated the phosphorylation of GluN2B at Tyr-1472. Moreover, our results revealed that the phosphorylation change of GluN2B at Tyr-1070 accompanied the Tyr-1472 phosphorylation and Fyn associated with GluN2B in synaptic plasticity induced by both chemical and contextual fear learning. Taken together, our findings provide a new mechanism for regulating the surface expression of NMDARs with implications for synaptic plasticity. PMID:26229100

  7. Methylation of RNA polymerase II non-consensus Lysine residues marks early transcription in mammalian cells

    PubMed Central

    Dias, João D; Rito, Tiago; Torlai Triglia, Elena; Kukalev, Alexander; Ferrai, Carmelo; Chotalia, Mita; Brookes, Emily; Kimura, Hiroshi; Pombo, Ana

    2015-01-01

    Dynamic post-translational modification of RNA polymerase II (RNAPII) coordinates the co-transcriptional recruitment of enzymatic complexes that regulate chromatin states and processing of nascent RNA. Extensive phosphorylation of serine residues at the largest RNAPII subunit occurs at its structurally-disordered C-terminal domain (CTD), which is composed of multiple heptapeptide repeats with consensus sequence Y1-S2-P3-T4-S5-P6-S7. Serine-5 and Serine-7 phosphorylation mark transcription initiation, whereas Serine-2 phosphorylation coincides with productive elongation. In vertebrates, the CTD has eight non-canonical substitutions of Serine-7 into Lysine-7, which can be acetylated (K7ac). Here, we describe mono- and di-methylation of CTD Lysine-7 residues (K7me1 and K7me2). K7me1 and K7me2 are observed during the earliest transcription stages and precede or accompany Serine-5 and Serine-7 phosphorylation. In contrast, K7ac is associated with RNAPII elongation, Serine-2 phosphorylation and mRNA expression. We identify an unexpected balance between RNAPII K7 methylation and acetylation at gene promoters, which fine-tunes gene expression levels. DOI: http://dx.doi.org/10.7554/eLife.11215.001 PMID:26687004

  8. Synthesis of Novel 3-Aryl-N-Methyl-1,2,5,6-Tetrahydropyridine Derivatives by Suzuki coupling: As Acetyl Cholinesterase Inhibitors

    PubMed Central

    Prasad, S.B. Benaka; Kumar, Y.C. Sunil; Kumar, C.S. Ananda; Sadashiva, C.T; Vinaya, K; Rangappa, K.S

    2007-01-01

    Alzheimer’s disease (AD) is a neurodegenerative disorder affecting the central nervous system, which is also associated with progressive loss of memory and cognition. The development of numerous structural classes of compounds with different pharmacological profile could be an evolving, promising therapeutic approach for the treatment of AD. Thus, providing a symptomatic treatment for this disease are cholinomimetics with the pharmacological profile of Acetylcholinesterase (AChE) inhibitors. In view of this, we have synthesized novel 3-aryl-N-methyl-1,2,5,6-tetrahydropyridine derivatives 5a-k by Suzuki coupling and screened the efficacy of these derivatives for their AChE inhibitor activity. PMID:19662135

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Determination of Acetylation of the Gli Transcription Factors.

    PubMed

    Coni, Sonia; Di Magno, Laura; Canettieri, Gianluca

    2015-01-01

    The Gli transcription factors (Gli1, Gli2, and Gli3) are the final effectors of the Hedgehog (Hh) signaling and play a key role in development and cancer. The activity of the Gli proteins is finely regulated by covalent modifications, such as phosphorylation, ubiquitination, and acetylation. Both Gli1 and Gli2 are acetylated at a conserved lysine, and this modification causes the inhibition of their transcriptional activity. Thus, the acetylation status of these proteins represents a useful marker to monitor Hh activation in pathophysiological conditions. Herein we describe the techniques utilized to detect in vitro and intracellular acetylation of the Gli transcription factors. PMID:26179046

  11. Acetylation and deacetylation of Cdc25A constitutes a novel mechanism for modulating Cdc25A functions with implications for cancer

    PubMed Central

    Lozada, Enerlyn M.; Andrysik, Zdenek; Yin, Moying; Redilla, Nicholas; Rice, Kathryn; Stambrook, Peter J.

    2016-01-01

    The dual specificity phosphatase Cdc25A is a key regulator of the cell cycle that promotes cell cycle progression by dephosphorylating and activating cyclin-dependent kinases. In response to genotoxicants, Cdc25A undergoes posttranslational modifications which contribute to its proteasome-mediated degradation and consequent cell cycle checkpoint arrest. The most thoroughly studied Cdc25A modification is phosphorylation. We now provide the first evidence that Cdc25A can be acetylated and that it directly interacts with the ARD1 acetyltransferase which acetylates Cdc25A both biochemically and in cultured cells. When acetylated, Cdc25A has an extended half-life. We have also identified the class IV histone deacetylase, HDAC11, as a Cdc25A deacetylase. We further show that DNA damage, such as exposure to methyl methanesulfonate (MMS), etoposide or arsenic, increases Cdc25A acetylation. Importantly, this acetylation modulates Cdc25A phosphatase activity and its function as a cell cycle regulator, and may reflect a cellular response to DNA damage. Since Cdc25A, ARD1, and HDAC11 are frequently dysregulated in multiple types of cancer, our findings may provide insight into a novel mechanism in carcinogenesis. PMID:26967250

  12. Histone deacetylase inhibitors block IFNγ-induced STAT1 phosphorylation.

    PubMed

    Ginter, Torsten; Bier, Carolin; Knauer, Shirley K; Sughra, Kalsoom; Hildebrand, Dagmar; Münz, Tobias; Liebe, Theresa; Heller, Regine; Henke, Andreas; Stauber, Roland H; Reichardt, Werner; Schmid, Johannes A; Kubatzky, Katharina F; Heinzel, Thorsten; Krämer, Oliver H

    2012-07-01

    Signal transducer and activator of transcription 1 (STAT1) is important for innate and adaptive immunity. Histone deacetylase inhibitors (HDACi) antagonize unbalanced immune functions causing chronic inflammation and cancer. Phosphorylation and acetylation regulate STAT1 and different IFNs induce phosphorylated STAT1 homo-/heterodimers, e.g. IFNα activates several STATs whereas IFNγ only induces phosphorylated STAT1 homodimers. In transformed cells HDACi trigger STAT1 acetylation linked to dephosphorylation by the phosphatase TCP45. It is unclear whether acetylation differentially affects STAT1 activated by IFNα or IFNγ, and if cellular responses to both cytokines depend on a phosphatase-dependent inactivation of acetylated STAT1. Here, we report that HDACi counteract IFN-induced phosphorylation of a critical tyrosine residue in the STAT1 C-terminus in primary cells and hematopoietic cells. STAT1 mutants mimicking a functionally inactive DNA binding domain (DBD) reveal that the number of acetylation-mimicking sites in STAT1 determines whether STAT1 is recruited to response elements after stimulation with IFNγ. Furthermore, we show that IFNα-induced STAT1 heterodimers carrying STAT1 molecules mimicking acetylation bind cognate DNA and provide innate anti-viral immunity. IFNγ-induced acetylated STAT1 homodimers are though inactive, suggesting that heterodimerization and complex formation can rescue STAT1 lacking a functional DBD. Apparently, the type of cytokine determines how acetylation affects the nuclear entry and DNA binding of STAT1. Our data contribute to a better understanding of STAT1 regulation by acetylation. PMID:22425562

  13. {gamma}-aminobutyric acid{sub A} (GABA{sub A}) receptor regulates ERK1/2 phosphorylation in rat hippocampus in high doses of Methyl Tert-Butyl Ether (MTBE)-induced impairment of spatial memory

    SciTech Connect

    Zheng Gang; Zhang Wenbin; Zhang Yun; Chen Yaoming; Liu Mingchao; Yao Ting; Yang Yanxia; Zhao Fang; Li Jingxia; Huang Chuanshu; Luo Wenjing Chen Jingyuan

    2009-04-15

    Experimental and occupational exposure to Methyl Tert-Butyl Ether (MTBE) has been reported to induce neurotoxicological and neurobehavioral effects, such as headache, nausea, dizziness, and disorientation, etc. However, the molecular mechanisms involved in MTBE-induced neurotoxicity are still not well understood. In the present study, we investigated the effects of MTBE on spatial memory and the expression and function of GABA{sub A} receptor in the hippocampus. Our results demonstrated that intraventricular injection of MTBE impaired the performance of the rats in a Morris water maze task, and significantly increased the expression of GABA{sub A} receptor {alpha}1 subunit in the hippocampus. The phosphorylation of ERK1/2 decreased after the MTBE injection. Furthermore, the decreased ability of learning and the reduction of phosphorylated ERK1/2 level of the MTBE-treated rats was partly reversed by bicuculline injected 30 min before the training. These results suggested that MTBE exposure could result in impaired spatial memory. GABA{sub A} receptor may play an important role in the MTBE-induced impairment of learning and memory by regulating the phosphorylation of ERK in the hippocampus.

  14. Structural analysis of three methyl N-phosphorylated 5,6-dihydroxy-2-azabicyclo[2.2.1]heptane-3-carboxylates

    NASA Astrophysics Data System (ADS)

    Sousa, Carlos A. D.; Vale, M. Luísa C.; Rodríguez-Borges, José E.; Garcia-Mera, Xerardo

    2012-01-01

    Both exo and endo isomers of (±)-methyl N-diphenylphosphoryl-2-azabicyclo[2.2.1]hept-5-ene-3-carboxylate and (±)-methyl N-diphenylphosphoryloxy-2-azabicyclo[2.2.1]hept-5-ene-3-carboxylate were dihydroxylated with OsO 4. The unexpected formation of (±)-methyl 5,6-dihydroxy -N-diphenylphosphoryl-2-azabicyclo[2.2.1]heptane-3- endo-carboxylate from (±)-methyl N-diphenylphosphoryloxy-2-azabicyclo[2.2.1]hept-5-ene-3- endo-carboxylate is discussed based on NMR analyses and experimental observations. The two N-diphenylphosphoryl dihydroxybicycles are analyzed in terms of their crystalline structure by X-ray crystallography.

  15. Homeodomain-interacting protein kinase-2 (HIPK2) phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77 and modulates its DNA binding affinity.

    PubMed

    Zhang, Qingchun; Wang, Yinsheng

    2007-12-01

    The chromosomal high-mobility group A (HMGA) proteins, composed of HMGA1a, HMGA1b and HMGA2, play important roles in the regulation of numerous processes in eukaryotic cells, such as transcriptional regulation, DNA repair, RNA processing, and chromatin remodeling. The biological activities of HMGA1 proteins are highly regulated by their post-translational modifications (PTMs), including acetylation, methylation, and phosphorylation. Recently, it was found that the homeodomain-interacting protein kinase-2 (HIPK2), a newly identified serine/threonine kinase, co-immunoprecipitated with, and phosphorylated, HMGA1 proteins. However, the sites and the biological significance of the phosphorylation have not been elucidated. Here, we found that HIPK2 phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77, and HMGA1b at Thr-41 and Thr-66. In addition, we demonstrated that cdc2, which is known to phosphorylate HMGA1 proteins, could induce the phosphorylation of HMGA1 proteins at the same Ser/Thr sites. The two kinases, however, exhibited different site preferences for the phosphorylation: The preference for HIPK2 phosphorylation followed the order of Thr-77 > Thr-52 > Ser-35, whereas the order for cdc2 phosphorylation was Thr-52 > Thr-77 > Ser-35. Moreover, we found that the HIPK2-phosphorylated HMGA1a reduced the binding affinity of HMGA1a to human germ line promoter, and the drop in binding affinity induced by HIPK2 phosphorylation was lower than that introduced by cdc2 phosphorylation, which is consistent with the notion that the second AT-hook in HMGA1a is more important for DNA binding than the third AT-hook. PMID:17960875

  16. Histone H4 Lysine 20 (H4K20) Methylation, Expanding the Signaling Potential of the Proteome One Methyl Moiety at a Time.

    PubMed

    van Nuland, Rick; Gozani, Or

    2016-03-01

    Covalent post-translational modifications (PTMs) of proteins can regulate the structural and functional state of a protein in the absence of primary changes in the underlying sequence. Common PTMs include phosphorylation, acetylation, and methylation. Histone proteins are critical regulators of the genome and are subject to a highly abundant and diverse array of PTMs. To highlight the functional complexity added to the proteome by lysine methylation signaling, here we will focus on lysine methylation of histone proteins, an important modification in the regulation of chromatin and epigenetic processes. We review the signaling pathways and functions associated with a single residue, H4K20, as a model chromatin and clinically important mark that regulates biological processes ranging from the DNA damage response and DNA replication to gene expression and silencing. PMID:26598646

  17. Histone phosphorylation

    PubMed Central

    Rossetto, Dorine; Avvakumov, Nikita; Côté, Jacques

    2012-01-01

    Histone posttranslational modifications are key components of diverse processes that modulate chromatin structure. These marks function as signals during various chromatin-based events, and act as platforms for recruitment, assembly or retention of chromatin-associated factors. The best-known function of histone phosphorylation takes place during cellular response to DNA damage, when phosphorylated histone H2A(X) demarcates large chromatin domains around the site of DNA breakage. However, multiple studies have also shown that histone phosphorylation plays crucial roles in chromatin remodeling linked to other nuclear processes. In this review, we summarize the current knowledge of histone phosphorylation and describe the many kinases and phosphatases that regulate it. We discuss the key roles played by this histone mark in DNA repair, transcription and chromatin compaction during cell division and apoptosis. Additionally, we describe the intricate crosstalk that occurs between phosphorylation and other histone modifications and allows for sophisticated control over the chromatin remodeling processes. PMID:22948226

  18. Dysregulation of AKT Pathway by SMYD2-Mediated Lysine Methylation on PTEN1,2

    PubMed Central

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

    2015-01-01

    Phosphatase and tensin homologue (PTEN), one of the well-characterized tumor suppressor proteins, counteracts the phosphatidylinositol 3-kinase-AKT pathway through its unique lipid phosphatase activity. The functions of PTEN are regulated by a variety of posttranslational modifications such as acetylation, oxidation, ubiquitylation, phosphorylation, and SUMOylation. However, methylation of PTEN has not been reported so far. In this study, we demonstrated that the oncogenic protein lysine methyltransferase SET and MYND domain containing 2 (SMYD2) methylates PTEN at lysine 313 in vitro and in vivo. Knockdown of SMYD2 suppressed the cell growth of breast cancer cells and attenuated phosphorylation levels of AKT, indicating that SMYD2-mediated methylation negatively regulates PTEN tumor suppressor activity and results in activation of the phosphatidylinositol 3-kinase-AKT pathway. Furthermore, PTEN protein with lysine 313 substitution diminished phosphorylation of PTEN at serine 380, which is known to inactivate tumor suppressor functions of PTEN. Taken together, our findings unveil a novel mechanism of PTEN dysregulation regulated by lysine methylation in human cancer. PMID:25925379

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

    PubMed

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

    2015-04-01

    Phosphatase and tensin homologue (PTEN), one of the well-characterized tumor suppressor proteins, counteracts the phosphatidylinositol 3-kinase-AKT pathway through its unique lipid phosphatase activity. The functions of PTEN are regulated by a variety of posttranslational modifications such as acetylation, oxidation, ubiquitylation, phosphorylation, and SUMOylation. However, methylation of PTEN has not been reported so far. In this study, we demonstrated that the oncogenic protein lysine methyltransferase SET and MYND domain containing 2 (SMYD2) methylates PTEN at lysine 313 in vitro and in vivo. Knockdown of SMYD2 suppressed the cell growth of breast cancer cells and attenuated phosphorylation levels of AKT, indicating that SMYD2-mediated methylation negatively regulates PTEN tumor suppressor activity and results in activation of the phosphatidylinositol 3-kinase-AKT pathway. Furthermore, PTEN protein with lysine 313 substitution diminished phosphorylation of PTEN at serine 380, which is known to inactivate tumor suppressor functions of PTEN. Taken together, our findings unveil a novel mechanism of PTEN dysregulation regulated by lysine methylation in human cancer. PMID:25925379

  20. Ghrelin receptor activity amplifies hippocampal N-methyl-d-aspartate receptor-mediated postsynaptic currents and increases phosphorylation of the GluN1 subunit at Ser896 and Ser897.

    PubMed

    Muniz, Brandon G; Isokawa, Masako

    2015-12-01

    Although ghrelin and its cognate receptor growth hormone secretagogue receptor (GHSR1a) are highly localized in the hypothalamic nuclei for the regulation of metabolic states and feeding, GHSR1a is also highly localized in the hippocampus, suggesting its involvement in extra-hypothalamic functions. Indeed, exogenous application of ghrelin has been reported to improve hippocampal learning and memory. However, the underlying mechanism of ghrelin regulation of hippocampal functions is poorly understood. Here, we report ghrelin-promoted phosphorylation of GluN1 and amplified N-methyl-d-aspartate receptor (NMDAR)-mediated excitatory postsynaptic currents in the CA1 pyramidal cells of the hippocampus in slice preparations. The ghrelin-induced responses were sensitive to a GHSR1a antagonist and inverse agonist, and were absent in GHSR1a homozygous knock-out mice. These results indicated that activation of GHSR1a was critical in the ghrelin-induced enhancement of the NMDAR function. Interestingly, heterozygous mouse hippocampi were also insensitive to ghrelin treatment, suggesting that a slight reduction in the availability of GHSR1a may be sufficient to negate the effect of ghrelin on GluN1 phosphorylation and NMDAR channel activities. In addition, NMDAR-mediated spike currents, which are of dendritic origin, were blocked by the GHSR1a antagonist, suggesting the presence of GHSR1a on the pyramidal cell dendrites in physical proximity to NMDAR. Together with our findings on the localization of GHSR1a in the CA1 region of the hippocampus, which was shown by fluorescent ghrelin binding, immunoreactivity, and enhanced green fluorescent protein reporter gene expression, we conclude that the activation of GHSR1a favours rapid modulation of the NMDAR-mediated glutamatergic synaptic transmission by phosphorylating GluN1 in the hippocampus. PMID:26490687

  1. Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation.

    PubMed

    Hunter, Gerald O; Fox, Melanie J; Smith-Kinnaman, Whitney R; Gogol, Madelaine; Fleharty, Brian; Mosley, Amber L

    2016-09-01

    In eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y1S2P3T4S5P6S7)n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1 Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes. PMID:27247267

  2. SIAH-mediated ubiquitination and degradation of acetyl-transferases regulate the p53 response and protein acetylation.

    PubMed

    Grishina, Inna; Debus, Katherina; García-Limones, Carmen; Schneider, Constanze; Shresta, Amit; García, Carlos; Calzado, Marco A; Schmitz, M Lienhard

    2012-12-01

    Posttranslational modification of proteins by lysine acetylation regulates many biological processes ranging from signal transduction to chromatin compaction. Here we identify the acetyl-transferases CBP/p300, Tip60 and PCAF as new substrates for the ubiquitin E3 ligases SIAH1 and SIAH2. While CBP/p300 can undergo ubiquitin/proteasome-dependent degradation by SIAH1 and SIAH2, the two other acetyl-transferases are exclusively degraded by SIAH2. Accordingly, SIAH-deficient cells show enhanced protein acetylation, thus revealing SIAH proteins as indirect regulators of the cellular acetylation status. Functional experiments show that Tip60/PCAF-mediated acetylation of the tumor suppressor p53 is antagonized by the p53 target gene SIAH2 which mediates ubiquitin/proteasome-mediated degradation of both acetyl-transferases and consequently diminishes p53 acetylation and transcriptional activity. The p53 kinase HIPK2 mediates hierarchical phosphorylation of SIAH2 at 5 sites, which further boosts its activity as a ubiquitin E3 ligase for several substrates and therefore dampens the late p53 response. PMID:23044042

  3. Regulation of AU-Rich Element RNA Binding Proteins by Phosphorylation and the Prolyl Isomerase Pin1

    PubMed Central

    Shen, Zhong-Jian; Malter, James S.

    2015-01-01

    The accumulation of 3' untranslated region (3'-UTR), AU-rich element (ARE) containing mRNAs, are predominantly controlled at the post-transcriptional level. Regulation appears to rely on a variable and dynamic interaction between mRNA target and ARE-specific binding proteins (AUBPs). The AUBP-ARE mRNA recognition is directed by multiple intracellular signals that are predominantly targeted at the AUBPs. These include (but are unlikely limited to) methylation, acetylation, phosphorylation, ubiquitination and isomerization. These regulatory events ultimately affect ARE mRNA location, abundance, translation and stability. In this review, we describe recent advances in our understanding of phosphorylation and its impact on conformation of the AUBPs, interaction with ARE mRNAs and highlight the role of Pin1 mediated prolyl cis-trans isomerization in these biological process. PMID:25874604

  4. Unexpected ring-closure products derived from 3-(2-allylanilino)-3-phenylacrylate esters: crystal and molecular structures of 3-acetyl-8-allyl-6-methyl-2-phenylquinolin-4-yl acetate and (2RS)-2,8-dimethyl-4-phenyl-1,2-dihydro-6H-pyrrolo[3,2,1-ij]quinolin-6-one.

    PubMed

    Luque, Adriana L; Sanabria, Carlos M; Palma, Alirio; Cobo, Justo; Glidewell, Christopher

    2016-08-01

    The reactions of two 3-(2-allylanilino)-3-phenylacrylate esters with acetic anhydride and with strong acids has revealed a richly diverse reactivity providing a number of unexpected products. Thus, acetylation of ethyl 3-(2-allylanilino)-3-phenylacrylate, (Ia), or ethyl 3-(2-allyl-4-methylanilino)-3-phenylacrylate, (Ib), with acetic anhydride yields not only the expected acetylated esters, (II), as the major products but also the unexpected polysubstituted quinolines 3-acetyl-8-allyl-2-phenylquinolin-4-yl acetate, (IIIa), and 3-acetyl-8-allyl-6-methyl-2-phenylquinolin-4-yl acetate, (IIIb), as minor products. Subsequent reaction of the major product ethyl 2-[(2-allyl-4-methylanilino)(phenyl)methylidene]-3-oxobutanoate, (IIb), with concentrated sulfuric acid did not provide the expected 3-acetylquinoline derivative, but instead two unexpected products, namely ethyl 4-ethyl-2-phenyl-1,4-dihydroquinoline-3-carboxylate, (IV), and ethyl 3-acetyl-4-ethyl-2-phenyl-3,4-dihydroquinoline-3-carboxylate, (V), in yields of 39 and 22%, respectively. The reaction of (Ib) with Eaton's reagent gave both the quinoline (Z)-6-methyl-2-phenyl-8-(prop-1-en-1-yl)quinolin-4(1H)-one, (VI), and the unexpected tricyclic product (2RS)-2,8-dimethyl-4-phenyl-1,2-dihydro-6H-pyrrolo[3,2,1-ij]quinolin-6-one, (VII), in yields of 71 and 12%, respectively. The products (II)-(VII) have all been fully characterized spectroscopically and the crystal structures of two of the unexpected products, i.e. (IIIb) (C23H21NO3) and (VII) (C19H17NO), are reported here. The formation of compounds (IV), (V) and (VII) all require an isomerization of the initial allyl substituent, with migration of the C=C double bond from the terminal site to the internal site. In (IIIb), the two acetyl substituents are oriented such that the intramolecular distance between the two carbonyl O atoms is only 3.243 (2) Å, and in (VII), the five-membered ring adopts a twisted half-chair conformation. The molecules of compound (IIIb

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

    PubMed

    Chen, Yongcan; Zhu, Wei-Guo

    2016-07-01

    DNA damage response (DDR) signaling network is initiated to protect cells from various exogenous and endogenous damage resources. Timely and accurate regulation of DDR proteins is required for distinct DNA damage repair pathways. Post-translational modifications of histone and non-histone proteins play a vital role in the DDR factor foci formation and signaling pathway. Phosphorylation, ubiquitylation, SUMOylation, neddylation, poly(ADP-ribosyl)ation, acetylation, and methylation are all involved in the spatial-temporal regulation of DDR, among which phosphorylation and ubiquitylation are well studied. Studies in the past decade also revealed extensive roles of lysine methylation in response to DNA damage. Lysine methylation is finely regulated by plenty of lysine methyltransferases, lysine demethylases, and can be recognized by proteins with chromodomain, plant homeodomain, Tudor domain, malignant brain tumor domain, or proline-tryptophan-tryptophan-proline domain. In this review, we outline the dynamics and regulation of histone lysine methylation at canonical (H3K4, H3K9, H3K27, H3K36, H3K79, and H4K20) and non-canonical sites after DNA damage, and discuss their context-specific functions in DDR protein recruitment or extraction, chromatin environment establishment, and transcriptional regulation. We also present the emerging advances of lysine methylation in non-histone proteins during DDR. PMID:27217472

  6. Structure and function of histone methylation-binding proteins in plants.

    PubMed

    Liu, Yanli; Min, Jinrong

    2016-06-15

    Post-translational modifications of histones play important roles in modulating many essential biological processes in both animals and plants. These covalent modifications, including methylation, acetylation, phosphorylation, ubiquitination, SUMOylation and so on, are laid out and erased by histone-modifying enzymes and read out by effector proteins. Recent studies have revealed that a number of developmental processes in plants are under the control of histone post-translational modifications, such as floral transition, seed germination, organogenesis and morphogenesis. Therefore, it is critical to identify those protein domains, which could specifically recognize these post-translational modifications to modulate chromatin structure and regulate gene expression. In the present review, we discuss the recent progress in understanding the structure and function of the histone methylation readers in plants, by focusing on Arabidopsis thaliana proteins. PMID:27288029

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

    PubMed

    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 signalling 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

  8. 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

  9. 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

  10. 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

  11. Acetylation Targets the M2 Isoform of Pyruvate Kinase for Degradation through Chaperone-Mediated Autophagy and Promotes Tumor Growth

    PubMed Central

    Lv, Lei; Li, Dong; Zhao, Di; Lin, Ruiting; Chu, Yajing; Zhang, Heng; Zha, Zhengyu; Liu, Ying; Li, Zi; Xu, Yanping; Wang, Gang; Huang, Yiran; Xiong, Yue; Guan, Kun-Liang; Lei, Qun-Ying

    2016-01-01

    SUMMARY Most tumor cells take up more glucose than normal cells but metabolize glucose via glycolysis even in the presence of normal levels of oxygen, a phenomenon known as the Warburg effect. Tumor cells commonly express the embryonic M2 isoform of pyruvate kinase (PKM2) that may contribute to the metabolism shift from oxidative phosphorylation to aerobic glycolysis and tumorigenesis. Here we show that PKM2 is acetylated on lysine 305 and that this acetylation is stimulated by high glucose concentration. PKM2 K305 acetylation decreases PKM2 enzyme activity and promotes its lysosomal-dependent degradation via chaperone-mediated autophagy (CMA). Acetylation increases PKM2 interaction with HSC70, a chaperone for CMA, and association with lysosomes. Ectopic expression of an acetylation mimetic K305Q mutant accumulates glycolytic intermediates and promotes cell proliferation and tumor growth. These results reveal an acetylation regulation of pyruvate kinase and the link between lysine acetylation and CMA. PMID:21700219

  12. Protein Acetylation and Acetyl Coenzyme A Metabolism in Budding Yeast

    PubMed Central

    Galdieri, Luciano; Zhang, Tiantian; Rogerson, Daniella; Lleshi, Rron

    2014-01-01

    Cells sense and appropriately respond to the physical conditions and availability of nutrients in their environment. This sensing of the environment and consequent cellular responses are orchestrated by a multitude of signaling pathways and typically involve changes in transcription and metabolism. Recent discoveries suggest that the signaling and transcription machineries are regulated by signals which are derived from metabolism and reflect the metabolic state of the cell. Acetyl coenzyme A (CoA) is a key metabolite that links metabolism with signaling, chromatin structure, and transcription. Acetyl-CoA is produced by glycolysis as well as other catabolic pathways and used as a substrate for the citric acid cycle and as a precursor in synthesis of fatty acids and steroids and in other anabolic pathways. This central position in metabolism endows acetyl-CoA with an important regulatory role. Acetyl-CoA serves as a substrate for lysine acetyltransferases (KATs), which catalyze the transfer of acetyl groups to the epsilon-amino groups of lysines in histones and many other proteins. Fluctuations in the concentration of acetyl-CoA, reflecting the metabolic state of the cell, are translated into dynamic protein acetylations that regulate a variety of cell functions, including transcription, replication, DNA repair, cell cycle progression, and aging. This review highlights the synthesis and homeostasis of acetyl-CoA and the regulation of transcriptional and signaling machineries in yeast by acetylation. PMID:25326522

  13. p53 Ser15 phosphorylation and histone modifications contribute to IR-induced miR-34a transcription in mammary epithelial cells

    PubMed Central

    Wang, Bo; Li, Dongping

    2013-01-01

    Previous studies have demonstrated that miR-34a is a direct transcriptional target of tumor suppressor p53 and plays a crucial role in p53-mediated biological processes, such as cell cycle arrest, apoptosis and senescence. However, the role of p53 phosphorylation at Ser15 and histone modifications in ionizing radiation (IR)-induced miR-34a transcription in human mammary epithelial cells remains unknown. The present study showed that IR triggers miR-34a induction in rat mammary gland tissue and human mammary epithelial cells in a dose- and time-dependent fashion. Gene copy number and CpG methylation exhibit no effect on IR-inducible miR-34a expression, while the levels of phosphorylated p53 at Ser15 are markedly elevated in human mammary epithelial cells 96 h post-IR, which correlates with IR-inducible miR-34a transcription and the p38 MAPK pathway. Conversely, suppression of p38 MAPK with SB239063 inhibits IR-induced p53 phosphorylation at Ser15 and miR-34a expression in a dose-dependent manner. Our study found that wild-type p53 is enriched at miR-34a promoter, and luciferase activity of miR-34a promoter reporter is attenuated by either mutant p53 (Ser15Ala) or mutant miR-34a promoter. Furthermore, IR also triggers phosphorylation, tri-methylation and acetylation of histone H3 and acetylation of histone H4, which correlates with IR-inducible miR-34a transcription, while SAHA potentiates IR-inducible miR-34a expression. Moreover, acetyl-histone H3 is significantly enriched at miR-34a promoter in IR-exposed HMEC cells. Yet, we show that there is no correlation between IR-inducible miR-34a expression and IR-induced rapid and transient G2/M arrest. In sum, our novel data for the first time demonstrate that IR-induced p53 Ser15 phosphorylation via p38 MAPK is essential for its functional regulation of IR-inducible miR-34a transcription in human mammary epithelial cells, and that histone modifications may also play a key role in IR-inducible miR-34a expression. PMID

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

    SciTech Connect

    Lee, Juhyung; Yun, Nuri; Kim, Chiho; Song, Min-Young; Park, Kang-Sik; Oh, Young J.

    2014-04-25

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

  15. A SUMO-acetyl switch in PXR biology.

    PubMed

    Cui, Wenqi; Sun, Mengxi; Zhang, Shupei; Shen, Xunan; Galeva, Nadezhda; Williams, Todd D; Staudinger, Jeff L

    2016-09-01

    Post-translational modification (PTM) of nuclear receptor superfamily members regulates various aspects of their biology to include sub-cellular localization, the repertoire of protein-binding partners, as well as their stability and mode of degradation. The nuclear receptor pregnane X receptor (PXR, NR1I2) is a master-regulator of the drug-inducible gene expression in liver and intestine. The PXR-mediated gene activation program is primarily recognized to increase drug metabolism, drug transport, and drug efflux pathways in these tissues. The activation of PXR also has important implications in significant human diseases including inflammatory bowel disease and cancer. Our recent investigations reveal that PXR is modified by multiple PTMs to include phosphorylation, SUMOylation, and ubiquitination. Using both primary cultures of hepatocytes and cell-based assays, we show here that PXR is modified through acetylation on lysine residues. Further, we show that increased acetylation of PXR stimulates its increased SUMO-modification to support active transcriptional suppression. Pharmacologic inhibition of lysine de-acetylation using trichostatin A (TSA) alters the sub-cellular localization of PXR in cultured hepatocytes, and also has a profound impact upon PXR transactivation capacity. Both the acetylation and SUMOylation status of the PXR protein is affected by its ability to associate with the lysine de-acetylating enzyme histone de-acetylase (HDAC)3 in a complex with silencing mediator of retinoic acid and thyroid hormone receptor (SMRT). Taken together, our data support a model in which a SUMO-acetyl 'switch' occurs such that acetylation of PXR likely stimulates SUMO-modification of PXR to promote the active repression of PXR-target gene expression. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie. PMID:26883953

  16. The world of protein acetylation.

    PubMed

    Drazic, Adrian; Myklebust, Line M; Ree, Rasmus; Arnesen, Thomas

    2016-10-01

    Acetylation is one of the major post-translational protein modifications in the cell, with manifold effects on the protein level as well as on the metabolome level. The acetyl group, donated by the metabolite acetyl-coenzyme A, can be co- or post-translationally attached to either the α-amino group of the N-terminus of proteins or to the ε-amino group of lysine residues. These reactions are catalyzed by various N-terminal and lysine acetyltransferases. In case of lysine acetylation, the reaction is enzymatically reversible via tightly regulated and metabolism-dependent mechanisms. The interplay between acetylation and deacetylation is crucial for many important cellular processes. In recent years, our understanding of protein acetylation has increased significantly by global proteomics analyses and in depth functional studies. This review gives a general overview of protein acetylation and the respective acetyltransferases, and focuses on the regulation of metabolic processes and physiological consequences that come along with protein acetylation. PMID:27296530

  17. Regulation of gap junction channels and hemichannels by phosphorylation and redox changes: a revision.

    PubMed

    Pogoda, Kristin; Kameritsch, Petra; Retamal, Mauricio A; Vega, José L

    2016-01-01

    Post-translational modifications of connexins play an important role in the regulation of gap junction and hemichannel permeability. The prerequisite for the formation of functional gap junction channels is the assembly of connexin proteins into hemichannels and their insertion into the membrane. Hemichannels can affect cellular processes by enabling the passage of signaling molecules between the intracellular and extracellular space. For the intercellular communication hemichannels from one cell have to dock to its counterparts on the opposing membrane of an adjacent cell to allow the transmission of signals via gap junctions from one cell to the other. The controlled opening of hemichannels and gating properties of complete gap junctions can be regulated via post-translational modifications of connexins. Not only channel gating, but also connexin trafficking and assembly into hemichannels can be affected by post-translational changes. Recent investigations have shown that connexins can be modified by phosphorylation/dephosphorylation, redox-related changes including effects of nitric oxide (NO), hydrogen sulfide (H2S) or carbon monoxide (CO), acetylation, methylation or ubiquitination. Most of the connexin isoforms are known to be phosphorylated, e.g. Cx43, one of the most studied connexin at all, has 21 reported phosphorylation sites. In this review, we provide an overview about the current knowledge and relevant research of responsible kinases, connexin phosphorylation sites and reported effects on gap junction and hemichannel regulation. Regarding the effects of oxidants we discuss the role of NO in different cell types and tissues and recent studies about modifications of connexins by CO and H2S. PMID:27229925

  18. Remarkable beta-selectivity in the synthesis of beta-1-C-arylglucosides: stereoselective reduction of acetyl-protected methyl 1-C-arylglucosides without acetoxy-group participation.

    PubMed

    Deshpande, Prashant P; Ellsworth, Bruce A; Buono, Frederic G; Pullockaran, Annie; Singh, Janak; Kissick, Thomas P; Huang, Ming-H; Lobinger, Hildegard; Denzel, Theodor; Mueller, Richard H

    2007-12-01

    An efficient and practical process to generate beta-C-arylglucoside derivatives was achieved. The process described involves Lewis acid mediated ionic reduction of a peracetylated 1-C-aryl methyl glucoside derived from the addition of an aryl-Li to selectively protected delta-D-gluconolactone. The reduction of the 2-acetoxy-1-C-oxacarbenium ion intermediates proceeds with a high degree of selectivity to give beta-C-arylglucosides without 2-acetoxy group participation. Furthermore, during the reduction process we also identified an unprecedented critical role of water. By changing from the usual benzyl ether protecting groups because of cost and chemical compatibility concerns, the new process is made additionally efficient and highly selective. PMID:17997568

  19. Scope and Limitations of 3-Iodo-Kdo Fluoride-Based Glycosylation Chemistry using N-Acetyl Glucosamine Acceptors.

    PubMed

    Pokorny, Barbara; Kosma, Paul

    2015-12-01

    The ketosidic linkage of 3-deoxy-d-manno-octulosonic acid (Kdo) to lipid A constitutes a general structural feature of the bacterial lipopolysaccharide core. Glycosylation reactions of Kdo donors, however, are challenging due to the absence of a directing group at C-3 and elimination reactions resulting in low yields and anomeric selectivities of the glycosides. While 3-iodo-Kdo fluoride donors showed excellent glycosyl donor properties for the assembly of Kdo oligomers, glycosylation of N-acetyl-glucosamine derivatives was not straightforward. Specifically, oxazoline formation of a β-anomeric methyl glycoside, as well as iodonium ion transfer to an allylic aglycon was found. In addition, dehalogenation of the directing group by hydrogen atom transfer proved to be incompatible with free hydroxyl groups next to benzyl groups. In contrast, glycosylation of a suitably protected methyl 2-acetamido-2-deoxy-α-d-glucopyranoside derivative and subsequent deiodination proceeded in excellent yields and α-specificity, and allowed for subsequent 4-O-phosphorylation. This way, the disaccharides α-Kdo-(2→6)-α-GlcNAcOMe and α-Kdo-(2→6)-α-GlcNAcOMe-4-phosphate were obtained in good overall yields. PMID:27308198

  20. AMPK/Snf1 signaling regulates histone acetylation: Impact on gene expression and epigenetic functions.

    PubMed

    Salminen, Antero; Kauppinen, Anu; Kaarniranta, Kai

    2016-08-01

    AMP-activated protein kinase (AMPK) and its yeast homolog, Snf1, are critical regulators in the maintenance of energy metabolic balance not only stimulating energy production but also inhibiting energy-consuming processes. The AMPK/Snf1 signaling controls energy metabolism by specific phosphorylation of many metabolic enzymes and transcription factors, enhancing or suppressing their functions. The AMPK/Snf1 complexes can be translocated from cytoplasm into nuclei where they are involved in the regulation of transcription. Recent studies have indicated that AMPK/Snf1 activation can control histone acetylation through different mechanisms affecting not only gene transcription but also many other epigenetic functions. For instance, AMPK/Snf1 enzymes can phosphorylate the histone H3S10 (yeast) and H2BS36 (mammalian) sites which activate specific histone acetyltransferases (HAT), consequently enhancing histone acetylation. Moreover, nuclear AMPK can phosphorylate type 2A histone deacetylases (HDAC), e.g. HDAC4 and HDAC5, triggering their export from nuclei thus promoting histone acetylation reactions. AMPK activation can also increase the level of acetyl CoA, e.g. by inhibiting fatty acid and cholesterol syntheses. Acetyl CoA is a substrate for HATs, thus increasing their capacity for histone acetylation. On the other hand, AMPK can stimulate the activity of nicotinamide phosphoribosyltransferase (NAMPT) which increases the level of NAD(+). NAD(+) is a substrate for nuclear sirtuins, especially for SIRT1 and SIRT6, which deacetylate histones and transcription factors, e.g. those regulating ribosome synthesis and circadian clocks. Histone acetylation is an important epigenetic modification which subsequently can affect chromatin remodeling, e.g. via bromodomain proteins. We will review the signaling mechanisms of AMPK/Snf1 in the control of histone acetylation and subsequently clarify their role in the epigenetic regulation of ribosome synthesis and circadian clocks

  1. Nuclear c-Abl-mediated tyrosine phosphorylation induces chromatin structural changes through histone modifications that include H4K16 hypoacetylation

    SciTech Connect

    Aoyama, Kazumasa; Fukumoto, Yasunori; Ishibashi, Kenichi; Kubota, Sho; Morinaga, Takao; Horiike, Yasuyoshi; Yuki, Ryuzaburo; Takahashi, Akinori; Nakayama, Yuji; Yamaguchi, Naoto

    2011-12-10

    c-Abl tyrosine kinase, which is ubiquitously expressed, has three nuclear localization signals and one nuclear export signal and can shuttle between the nucleus and the cytoplasm. c-Abl plays important roles in cell proliferation, adhesion, migration, and apoptosis. Recently, we developed a pixel imaging method for quantitating the level of chromatin structural changes and showed that nuclear Src-family tyrosine kinases are involved in chromatin structural changes upon growth factor stimulation. Using this method, we show here that nuclear c-Abl induces chromatin structural changes in a manner dependent on the tyrosine kinase activity. Expression of nuclear-targeted c-Abl drastically increases the levels of chromatin structural changes, compared with that of c-Abl. Intriguingly, nuclear-targeted c-Abl induces heterochromatic profiles of histone methylation and acetylation, including hypoacetylation of histone H4 acetylated on lysine 16 (H4K16Ac). The level of heterochromatic histone modifications correlates with that of chromatin structural changes. Adriamycin-induced DNA damage stimulates translocation of c-Abl into the nucleus and induces chromatin structural changes together with H4K16 hypoacetylation. Treatment with trichostatin A, a histone deacetylase inhibitor, blocks chromatin structural changes but not nuclear tyrosine phosphorylation by c-Abl. These results suggest that nuclear c-Abl plays an important role in chromatin dynamics through nuclear tyrosine phosphorylation-induced heterochromatic histone modifications.

  2. Mechanism and Regulation of Acetylated Histone Binding by the Tandem PHD Finger of DPF3b

    PubMed Central

    Zeng, Lei; Zhang, Qiang; Li, SiDe; Plotnikov, Alexander N.; Walsh, Martin J.; Zhou, Ming-Ming

    2010-01-01

    Histone lysine acetylation and methylation are important during gene transcription in a chromatin context1,2. Our knowledge about the types of protein modules that can interact with acetyl-lysine has so far been limited to bromodomains1. Recently, a tandem PHD (plant homeodomain) finger3 (PHD12) of human DPF3b, which functions in association with the BAF chromatin remodelling complex to initiate transcription in the heart and muscle development, was reported to bind histones H3 and H4 in an acetylation sensitive manner4, making it a first alternative to bromodomains for acetyl-lysine binding5. Here, we report the structural mechanism of acetylated histone binding by the double PHD fingers of DPF3b. Our three-dimensional solution structures and biochemical analysis of DPF3b illuminate the molecular basis of the integrated tandem PHD finger, which acts as one functional unit in the sequence-specific recognition of lysine 14-acetylated histone H3 (H3K14ac). Whereas the interaction with H3 is promoted by acetylation at lysine 14, it is inhibited by methylation at lysine 4, and these opposing influences are important during transcriptional activation of DPF3b target genes Pitx2 and Jmjd1c. Binding of this tandem protein module to chromatin can thus be regulated by different histone modifications during the initiation of gene transcription. PMID:20613843

  3. Middle-Down and Chemical Proteomic Approaches to Reveal Histone H4 Modification Dynamics in Cell Cycle: Label-Free Semi-Quantification of Histone Tail Peptide Modifications Including Phosphorylation and Highly Sensitive Capture of Histone PTM Binding Proteins Using Photo-Reactive Crosslinkers

    PubMed Central

    Yamamoto, Kazuki; Chikaoka, Yoko; Hayashi, Gosuke; Sakamoto, Ryosuke; Yamamoto, Ryuji; Sugiyama, Akira; Kodama, Tatsuhiko; Okamoto, Akimitsu; Kawamura, Takeshi

    2015-01-01

    Mass spectrometric proteomics is an effective approach for identifying and quantifying histone post-translational modifications (PTMs) and their binding proteins, especially in the cases of methylation and acetylation. However, another vital PTM, phosphorylation, tends to be poorly quantified because it is easily lost and inefficiently ionized. In addition, PTM binding proteins for phosphorylation are sometimes resistant to identification because of their variable binding affinities. Here, we present our efforts to improve the sensitivity of detection of histone H4 tail peptide phosphorylated at serine 1 (H4S1ph) and our successful identification of an H4S1ph binder candidate by means of a chemical proteomics approach. Our nanoLC-MS/MS system permitted semi-quantitative label-free analysis of histone H4 PTM dynamics of cell cycle-synchronized HeLa S3 cells, including phosphorylation, methylation, and acetylation. We show that H4S1ph abundance on nascent histone H4 unmethylated at lysine 20 (H4K20me0) peaks from late S-phase to M-phase. We also attempted to characterize effects of phosphorylation at H4S1 on protein–protein interactions. Specially synthesized photoaffinity bait peptides specifically captured 14-3-3 proteins as novel H4S1ph binding partners, whose interaction was otherwise undetectable by conventional peptide pull-down experiments. This is the first report that analyzes dynamics of PTM pattern on the whole histone H4 tail during cell cycle and enables the identification of PTM binders with low affinities using high-resolution mass spectrometry and photo-affinity bait peptides. PMID:26819910

  4. Is lys-Ne-acetylation the next big thing in post-translational modifications?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lys-N'-acetylation (KAC) has recently ascended from a posttranslational modification (PTM) of limited distribution to one approaching the abundance of O-phosphorylation. Thousands of KAC-proteins have been identified in archaea, bacteria, and eukarya, and the KAC system of acetyltransferases, deace...

  5. The dynamic organization of fungal acetyl-CoA carboxylase

    PubMed Central

    Hunkeler, Moritz; Stuttfeld, Edward; Hagmann, Anna; Imseng, Stefan; Maier, Timm

    2016-01-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control. PMID:27073141

  6. The dynamic organization of fungal acetyl-CoA carboxylase.

    PubMed

    Hunkeler, Moritz; Stuttfeld, Edward; Hagmann, Anna; Imseng, Stefan; Maier, Timm

    2016-01-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control. PMID:27073141

  7. The dynamic organization of fungal acetyl-CoA carboxylase

    NASA Astrophysics Data System (ADS)

    Hunkeler, Moritz; Stuttfeld, Edward; Hagmann, Anna; Imseng, Stefan; Maier, Timm

    2016-04-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.

  8. Lysine Acetylation: Elucidating the Components of an Emerging Global Signaling Pathway in Trypanosomes

    PubMed Central

    Alonso, Victoria Lucia; Serra, Esteban Carlos

    2012-01-01

    In the past ten years the number of acetylated proteins reported in literature grew exponentially. Several authors have proposed that acetylation might be a key component in most eukaryotic signaling pathways, as important as phosphorylation. The enzymes involved in this process are starting to emerge; acetyltransferases and deacetylases are found inside and outside the nuclear compartment and have different regulatory functions. In trypanosomatids several of these enzymes have been described and are postulated to be novel antiparasitic targets for the rational design of drugs. In this paper we overview the most important known acetylated proteins and the advances made in the identification of new acetylated proteins using high-resolution mass spectrometry. Also, we summarize what is known so far about the acetyltransferases and deacetylases in eukaryotes, focusing on trypanosomes and their potential use as chemotherapeutic targets. PMID:23093844

  9. Acetyl transfer in arylamine metabolism

    PubMed Central

    Booth, J.

    1966-01-01

    1. N-Hydroxyacetamidoaryl compounds (hydroxamic acids) are metabolites of arylamides, and an enzyme that transfers the acetyl group from these derivatives to arylamines has been found in rat tissues. The reaction products were identified by thin-layer chromatography and a spectrophotometric method, with 4-amino-azobenzene as acetyl acceptor, was used to measure enzyme activity. 2. The acetyltransferase was in the soluble fraction of rat liver, required a thiol for maximum activity and had a pH optimum between 6·0 and 7·5. 3. The soluble fractions of various rat tissues showed decreasing activity in the following order: liver, adrenal, kidney, lung, spleen, testis, heart; brain was inactive. 4. With the exception of aniline and aniline derivatives all the arylamines tested were effective as acetyl acceptors but aromatic compounds with side-chain amino groups were inactive. 5. The N-hydroxyacetamido derivatives of 2-naphthylamine, 4-amino-biphenyl and 2-aminofluorene were active acetyl donors but N-hydroxyacetanilide showed only slight activity. Acetyl-CoA was not a donor. 6. Some properties of the enzyme are compared with those of other acetyltransferases. PMID:5969287

  10. 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. PMID:26389815

  11. Druggability of methyl-lysine binding sites.

    PubMed

    Santiago, C; Nguyen, K; Schapira, M

    2011-12-01

    Structural modules that specifically recognize--or read--methylated or acetylated lysine residues on histone peptides are important components of chromatin-mediated signaling and epigenetic regulation of gene expression. Deregulation of epigenetic mechanisms is associated with disease conditions, and antagonists of acetyl-lysine binding bromodomains are efficacious in animal models of cancer and inflammation, but little is known regarding the druggability of methyl-lysine binding modules. We conducted a systematic structural analysis of readers of methyl marks and derived a predictive druggability landscape of methyl-lysine binding modules. We show that these target classes are generally less druggable than bromodomains, but that some proteins stand as notable exceptions. PMID:22146969

  12. Acetylation curtails nucleosome binding, not stable nucleosome remodeling, by FoxO1

    SciTech Connect

    Hatta, M.; Liu, F.; Cirillo, L.A.

    2009-02-20

    Transcriptional activity of FoxO factors is controlled through the actions of multiple growth factors signaling through protein kinase B, whereby phosphorylation of FoxO factors inhibits FoxO-mediated transactivation by promoting nuclear export. Phosphorylation of FoxO factors is enhanced by p300-mediated acetylation, which decreases their affinity for DNA. The negative effect of acetylation on FoxO DNA binding, together with nuclear FoxO mobility, is eliminated by over-expression of the de-acetylase Sirt1, suggesting that acetylation mobilizes FoxO factors in chromatin for inducible gene expression. Here, we show that acetylation significantly curtails the affinity of FoxO1 for its binding sites in nucleosomal DNA but has no effect on either stable nucleosome binding or remodeling by this factor. We suggest that, while acetylation provides a first, essential step toward mobilizing FoxO factors for inducible gene repression, additional mechanisms exist for overcoming their inherent capacity to stably bind and remodel nuclear chromatin.

  13. IDENTIFICATION OF HISTONE H3 LYSINE 36 ACETYLATION AS A HIGHLY CONSERVED HISTONE MODIFICATION*

    PubMed Central

    Morris, Stephanie A.; Rao, Bhargavi; Garcia, Benjamin A.; Hake, Sandra B.; Diaz, Robert L.; Shabanowitz, Jeffrey; Hunt, Donald F.; Allis, C. David; Lieb, Jason D.; Strahl, Brian D.

    2010-01-01

    Histone lysine (K) acetylation is a major mechanism by which cells regulate the structure and function of chromatin, and new sites of acetylation continue to be discovered. Here we identify and characterize histone H3K36 acetylation (H3K36ac). By mass spectrometric analyses of H3 purified from Tetrahymena thermophila and Saccharomyces cerevisiae (yeast), we find that H3K36 can be acetylated or methylated. Using an antibody specific to H3K36ac, we show that this modification is conserved in mammals. In yeast, genome-wide ChIP-chip experiments show that H3K36ac is localized predominantly to the promoters of RNA polymerase II-transcribed genes, a pattern inversely related to that of H3K36 methylation. The pattern of H3K36ac localization is similar to that of other sites of H3 acetylation, including H3K9ac and H3K14ac. Using histone acetyltransferase complexes purified from yeast, we show that the Gcn5-containing SAGA complex that regulates transcription specifically acetylates H3K36 in vitro. Deletion of GCN5 completely abolishes H3K36ac in vivo. These data expand our knowledge of the genomic targets of Gcn5, show H3K36ac is highly conserved, and raise the intriguing possibility that the transition between H3K36ac and H3K36me acts as an “acetyl/methyl switch” governing chromatin function along transcription units. PMID:17189264

  14. Nucleosome competition reveals processive acetylation by the SAGA HAT module.

    PubMed

    Ringel, Alison E; Cieniewicz, Anne M; Taverna, Sean D; Wolberger, Cynthia

    2015-10-01

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator complex hyperacetylates histone tails in vivo in a manner that depends upon histone 3 lysine 4 trimethylation (H3K4me3), a histone mark enriched at promoters of actively transcribed genes. SAGA contains a separable subcomplex known as the histone acetyltransferase (HAT) module that contains the HAT, Gcn5, bound to Sgf29, Ada2, and Ada3. Sgf29 contains a tandem Tudor domain that recognizes H3K4me3-containing peptides and is required for histone hyperacetylation in vivo. However, the mechanism by which H3K4me3 recognition leads to lysine hyperacetylation is unknown, as in vitro studies show no effect of the H3K4me3 modification on histone peptide acetylation by Gcn5. To determine how H3K4me3 binding by Sgf29 leads to histone hyperacetylation by Gcn5, we used differential fluorescent labeling of histones to monitor acetylation of individual subpopulations of methylated and unmodified nucleosomes in a mixture. We find that the SAGA HAT module preferentially acetylates H3K4me3 nucleosomes in a mixture containing excess unmodified nucleosomes and that this effect requires the Tudor domain of Sgf29. The H3K4me3 mark promotes processive, multisite acetylation of histone H3 by Gcn5 that can account for the different acetylation patterns established by SAGA at promoters versus coding regions. Our results establish a model for Sgf29 function at gene promoters and define a mechanism governing crosstalk between histone modifications. PMID:26401015

  15. 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. PMID:26660885

  16. The regulation of p53 by phosphorylation: a model for how distinct signals integrate into the p53 pathway.

    PubMed

    Maclaine, Nicola J; Hupp, Ted R

    2009-05-01

    The tumour suppressor p53 is a transcription factor that has evolved the ability to integrate distinct environmental signals including DNA damage, virus infection, and cytokine signaling into a common biological outcome that maintains normal cellular control. Mutations in p53 switch the cellular transcription program resulting in deregulation of the stress responses that normally maintain cell and tissue integrity. Transgenic studies in mice have indicated that changes in the specific activity of p53 can have profound effects not only on cancer development, but also on organism aging. As the specific activity of p53 is regulated at a post-translational level by sets of enzymes that mediate phosphorylation, acetylation, methylation, and ubiquitin-like modifications, it is likely that physiological modifiers of the aging function of p53 would be enzymes that catalyze such covalent modifications. We demonstrate that distinct stress-activated kinases, including ataxia telangiectasia mutated (ATM), casein kinase 1 (CK1) and AMP-activated protein kinase (AMPK), mediate phosphorylation of a key phospho-acceptor site in the p53 transactivation domain in response to diverse stresses including ionizing radiation, DNA virus infection, and elevation in the intracellular AMP/ATP ratio. As diseases linked to aging can involve activation of p53-dependent changes in cellular protective pathways, the development of specific physiological models might further shed light on the role of p53 kinases in modifying age-related diseases. PMID:20157532

  17. KLF4 mediates the link between TGF-β1-induced gene transcription and H3 acetylation in vascular smooth muscle cells.

    PubMed

    He, Ming; Zheng, Bin; Zhang, Yu; Zhang, Xin-Hua; Wang, Chang; Yang, Zhan; Sun, Yan; Wu, Xiao-Li; Wen, Jin-Kun

    2015-09-01

    Transcriptional activation by transcription factors is coupled with histone acetylation and chromatin remodeling. However, the relationship between TGF-β1-induced gene transcription by Krüppel-like factor (KLF)-4 and histone acetylation remains unknown. In our study, KLF4 overexpression or knockdown, respectively increased or decreased H3 acetylation and p300 occupancy, which is concentrated in the region containing TGF-β1 control elements (TCEs) of the genes by TGF-β1 regulation during vascular smooth muscle cell (VSMC) differentiation. Coimmunoprecipitation and glutathione S-transferase pull-down assays showed that phosphatase and tensin homolog (PTEN) formed a complex with KLF4 to inhibit the phosphorylation of the latter in basal conditions. After TGF-β1 signaling activation, PTEN was phosphorylated by p38 MAPK or PI3K/Akt signaling, phosphorylated PTEN lost its ability to dephosphorylate KLF4, and the cofactors interacting with KLF4 switched from PTEN to p300. Then, KLF4-p300 complexes were recruited to KLF4-binding sites of the gene promoter of VSMCs, to acetylate histone H3 and activate transcription. In addition, phosphorylated KLF4 enhanced p300 histone acetyltransferase (HAT) activity via the p38 MAPK pathway, which may be responsible for H3 acetylation. Taken together, the results of our study reveal a novel mechanism whereby KLF4 mediates the link between TGF-β1-induced gene transcription activation and H3 acetylation during VSMC differentiation. PMID:26082460

  18. Acetylation in vitro of constituent polypeptides by smooth endoplasmic reticulum (SER) and Golgi membrane fractions

    SciTech Connect

    Sambasivam, H.; Murray, R.K.

    1986-05-01

    Many polypeptides of the membranes of the ER are phosphorylated. To determine if any such polypeptides are acetylated, microsomal and other classical subcellular fractions were incubated with (/sup 3/H) acetyl-CoA; the specific activity of the microsomal fraction (MF) was the greatest. SDS-PAGE revealed that some 20 polypeptides of the MF were acetylated; 2-D electrophoretograms extended this number to approximately 60. Separation of the MF into smooth (S) and rough (R) fractions showed that the great majority of the labelled polypeptides belonged to the former. Isolation of a Golgi fraction revealed that its acetylation activity was approximately 3-fold greater than the SER fraction. Extensive proteolytic digestion of the MF followed by radiochromatography disclosed some 9 components whose precise nature (acetylated amino acids and/or sialic acids, etc.) is under study. Assuming that the majority of the radioactivity is in the former components and that a similar process occurs in vivo, the authors suggest that the Golgi apparatus may be a major site of acetylation of membrane and possibly other proteins.

  19. Autoregulation of the Rsc4 Tandem Bromodomain by Gcn5 Acetylation

    SciTech Connect

    VanDemark,A.; Kasten, M.; Ferris, E.; Heroux, A.; Hill, C.; Cairns, B.

    2007-01-01

    An important issue for chromatin remodeling complexes is how their bromodomains recognize particular acetylated lysine residues in histones. The Rsc4 subunit of the yeast remodeler RSC contains an essential tandem bromodomain (TBD) that binds acetylated K14 of histone H3 (H3K14ac). We report a series of crystal structures that reveal a compact TBD that binds H3K14ac in the second bromodomain and, remarkably, binds acetylated K25 of Rsc4 itself in the first bromodomain. Endogenous Rsc4 is acetylated only at K25, and Gcn5 is identified as necessary and sufficient for Rsc4 K25 acetylation in vivo and in vitro. Rsc4 K25 acetylation inhibits binding to H3K14ac, and mutation of Rsc4 K25 results in altered growth rates. These data suggest an autoregulatory mechanism in which Gcn5 performs both the activating (H3K14ac) and inhibitory (Rsc4 K25ac) modifications, perhaps to provide temporal regulation. Additional regulatory mechanisms are indicated as H3S10 phosphorylation inhibits Rsc4 binding to H3K14ac peptides.

  20. Acetylation of Lysine 201 Inhibits the DNA-Binding Ability of PhoP to Regulate Salmonella Virulence.

    PubMed

    Ren, Jie; Sang, Yu; Tan, Yongcong; Tao, Jing; Ni, Jinjing; Liu, Shuting; Fan, Xia; Zhao, Wei; Lu, Jie; Wu, Wenjuan; Yao, Yu-Feng

    2016-03-01

    The two-component system PhoP-PhoQ is highly conserved in bacteria and regulates virulence in response to various signals for bacteria within the mammalian host. Here, we demonstrate that PhoP could be acetylated by Pat and deacetylated by deacetylase CobB enzymatically in vitro and in vivo in Salmonella Typhimurium. Specifically, the conserved lysine residue 201(K201) in winged helix-turn-helix motif at C-terminal DNA-binding domain of PhoP could be acetylated, and its acetylation level decreases dramatically when bacteria encounter low magnesium, acid stress or phagocytosis of macrophages. PhoP has a decreased acetylation and increased DNA-binding ability in the deletion mutant of pat. However, acetylation of K201 does not counteract PhoP phosphorylation, which is essential for PhoP activity. In addition, acetylation of K201 (mimicked by glutamine substitute) in S. Typhimurium causes significantly attenuated intestinal inflammation as well as systemic infection in mouse model, suggesting that deacetylation of PhoP K201 is essential for Salmonella pathogenesis. Therefore, we propose that the reversible acetylation of PhoP K201 may ensure Salmonella promptly respond to different stresses in host cells. These findings suggest that reversible lysine acetylation in the DNA-binding domain, as a novel regulatory mechanism of gene expression, is involved in bacterial virulence across microorganisms. PMID:26943369

  1. Acetylation of Lysine 201 Inhibits the DNA-Binding Ability of PhoP to Regulate Salmonella Virulence

    PubMed Central

    Tan, Yongcong; Tao, Jing; Ni, Jinjing; Liu, Shuting; Fan, Xia; Zhao, Wei; Lu, Jie; Wu, Wenjuan; Yao, Yu-Feng

    2016-01-01

    The two-component system PhoP-PhoQ is highly conserved in bacteria and regulates virulence in response to various signals for bacteria within the mammalian host. Here, we demonstrate that PhoP could be acetylated by Pat and deacetylated by deacetylase CobB enzymatically in vitro and in vivo in Salmonella Typhimurium. Specifically, the conserved lysine residue 201(K201) in winged helix–turn–helix motif at C-terminal DNA-binding domain of PhoP could be acetylated, and its acetylation level decreases dramatically when bacteria encounter low magnesium, acid stress or phagocytosis of macrophages. PhoP has a decreased acetylation and increased DNA-binding ability in the deletion mutant of pat. However, acetylation of K201 does not counteract PhoP phosphorylation, which is essential for PhoP activity. In addition, acetylation of K201 (mimicked by glutamine substitute) in S. Typhimurium causes significantly attenuated intestinal inflammation as well as systemic infection in mouse model, suggesting that deacetylation of PhoP K201 is essential for Salmonella pathogenesis. Therefore, we propose that the reversible acetylation of PhoP K201 may ensure Salmonella promptly respond to different stresses in host cells. These findings suggest that reversible lysine acetylation in the DNA-binding domain, as a novel regulatory mechanism of gene expression, is involved in bacterial virulence across microorganisms. PMID:26943369

  2. Roles of subunit phosphorylation in regulating glutamate receptor function

    PubMed Central

    Wang, John Q.; Guo, Ming-Lei; Jin, Dao-Zhong; Xue, Bing; Fibuch, Eugene E.; Mao, Li-Min

    2014-01-01

    Protein phosphorylation is an important mechanism for regulating ionotropic glutamate receptors (iGluRs). Early studies have established that major iGluR subtypes, including α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and N-methyl-D-aspartate (NMDA) receptors, are subject to phosphorylation. Multiple serine, threonine, and tyrosine residues predominantly within the C-terminal regions of AMPA receptor and NMDA receptor subunits have been identified as sensitive phosphorylation sites. These distinct sites undergo either constitutive phosphorylation or activity-dependent phosphorylation induced by changing cellular and synaptic inputs as reversible events. An increasing number of synapse-enriched protein kinases have been found to phosphorylate iGluR. The common kinases include protein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, Src/Fyn non-receptor tyrosine kinases, and cyclin dependent kinase-5. Regulated phosphorylation plays a well-documented role in modulating the biochemical, biophysical, and functional properties of the receptor. In the future, identifying the precise mechanisms how phosphorylation regulates iGluR activities and finding the link between iGluR phosphorylation and the pathogenesis of various brain diseases, including psychiatric and neurodegenerative diseases, chronic pain, stroke, Alzheimer’s disease and substance addiction, will be hot topics and could contribute to the development of novel pharmacotherapies, by targeting the defined phosphorylation process, for suppressing iGluR-related disorders. PMID:24291102

  3. An isotope labeling strategy for quantifying the degree of phosphorylation at multiple sites in proteins.

    PubMed

    Hegeman, Adrian D; Harms, Amy C; Sussman, Michael R; Bunner, Anne E; Harper, Jeffrey F

    2004-05-01

    A procedure for determining the extent of phosphorylation at individual sites of multiply phosphorylated proteins was developed and applied to two polyphosphorylated proteins. The protocol, using simple chemical (Fischer methyl-esterification) and enzymatic (phosphatase) modification steps and an accessible isotopic labeling reagent (methyl alcohol-d(4)), is described in detail. Site-specific phosphorylation stoichiometries are derived from the comparison of chemically identical but isotopically distinct peptide species analyzed by microspray liquid chromatography-mass spectrometry (microLC-MS) using a Micromass Q-TOF2 mass spectrometer. Ten phosphorylation sites were unambiguously identified in tryptic digests of both proteins, and phosphorylation stoichiometries were determined for eight of the ten sites using the isotope-coded strategy. The extent of phosphorylation was also estimated from the mass spectral peak areas for the phosphorylated and unmodified peptides, and these estimates, when compared with stoichiometries determined using the isotope-coded technique, differed only marginally (within approximately 20%). PMID:15121193

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

    PubMed Central

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

    2016-01-01

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

  5. Exposure to 3,3',5-triiodothyronine affects histone and RNA polymerase II modifications, but not DNA methylation status, in the regulatory region of the Xenopus laevis thyroid hormone receptor βΑ gene.

    PubMed

    Kasai, Kentaro; Nishiyama, Norihito; Izumi, Yushi; Otsuka, Shunsuke; Ishihara, Akinori; Yamauchi, Kiyoshi

    2015-11-01

    Thyroid hormones (THs) play a critical role in amphibian metamorphosis, during which the TH receptor (TR) gene, thrb, is upregulated in a tissue-specific manner. The Xenopus laevis thrb gene has 3 TH response elements (TREs) in the 5' flanking regulatory region and 1 TRE in the exon b region, around which CpG sites are highly distributed. To clarify whether exposure to 3,3',5-triiodothyronine (T3) affects histone and RNA polymerase II (RNAPII) modifications and the level of DNA methylation in the 5' regulatory region, we conducted reverse transcription-quantitative polymerase chain reaction, bisulfite sequencing and chromatin immunoprecipitation assay using X. laevis cultured cells and premetamorphic tadpoles treated with or without 2 nM T3. Exposure to T3 increased the amount of the thrb transcript, in parallel with enhanced histone H4 acetylation and RNAPII recruitment, and probably phosphorylation of RNAPII at serine 5, in the 5' regulatory and exon b regions. However, the 5' regulatory region remained hypermethylated even with exposure to T3, and there was no significant difference in the methylation status between DNAs from T3-untreated and -treated cultured cells or tadpole tissues. Our results demonstrate that exposure to T3 induced euchromatin-associated epigenetic marks by enhancing histone acetylation and RNAPII recruitment, but not by decreasing the level of DNA methylation, in the 5' regulatory region of the X. laevis thrb gene. PMID:26417689

  6. Poly(ADP-Ribosyl)ation Affects Histone Acetylation and Transcription

    PubMed Central

    Verdone, Loredana; La Fortezza, Marco; Ciccarone, Fabio; Caiafa, Paola; Zampieri, Michele; Caserta, Micaela

    2015-01-01

    Poly(ADP-ribosyl)ation (PARylation) is a posttranslational protein modification catalyzed by members of the poly(ADP-ribose) polymerase (PARP) enzyme family. PARylation regulates a wide variety of biological processes in most eukaryotic cells including energy metabolism and cell death, maintenance of genomic stability, chromatin structure and transcription. Inside the nucleus, cross-talk between PARylation and other epigenetic modifications, such as DNA and histone methylation, was already described. In the present work, using PJ34 or ABT888 to inhibit PARP activity or over-expressing poly(ADP-ribose) glycohydrolase (PARG), we show decrease of global histone H3 and H4 acetylation. This effect is accompanied by a reduction of the steady state mRNA level of p300, Pcaf, and Tnfα, but not of Dnmt1. Chromatin immunoprecipitation (ChIP) analyses, performed at the level of the Transcription Start Site (TSS) of these four genes, reveal that changes in histone acetylation are specific for each promoter. Finally, we demonstrate an increase of global deacetylase activity in nuclear extracts from cells treated with PJ34, whereas global acetyltransferase activity is not affected, suggesting a role for PARP in the inhibition of histone deacetylases. Taken together, these results show an important link between PARylation and histone acetylation regulated transcription. PMID:26636673

  7. Phosphatase inhibitor 2 promotes acetylation of tubulin in the primary cilium of human retinal epithelial cells

    PubMed Central

    Wang, Weiping; Brautigan, David L

    2008-01-01

    Background Primary cilia are flagella-like projections from the centriole of mammalian cells that have a key role in cell signaling. Human diseases are linked to defects in primary cilia. Microtubules make up the axoneme of cilia and are selectively acetylated and this is thought to contribute to the stability of the structure. However, mechanisms to regulate tubulin acetylation in cilia are poorly understood. Results Endogenous phosphatase inhibitor-2 (I-2) was found concentrated in cilia of human epithelial cells, and was localized to cilia early in the process of formation, prior to the full acetylation of microtubules. Knockdown of I-2 by siRNA significantly reduced the acetylation of microtubules in cilia, without a net decrease in whole cell tubulin acetylation. There was a reduction in the percentage of I-2 knockdown cells with a primary cilium, but no apparent alteration in the cilium length, suggesting no change in microtubule-based transport processes. Inhibition of either histone deacetylases with trichostatin A, or protein phosphatase-1 with calyculin A in I-2 knockdown cells partially rescued the acetylation of microtubules in cilia and the percentage of cells with a primary cilium. Conclusion The regulatory protein I-2 localizes to the primary cilium where it affects both Ser/Thr phosphorylation and is required for full tubulin acetylation. Rescue of tubulin acetylation in I-2 knockdown cells by different chemical inhibitors shows that deacetylases and phosphatases are functionally interconnected to regulate microtubules. As a multifunctional protein, I-2 may link cell cycle progression to structure and stability of the primary cilium. PMID:19036150

  8. Phosphorylation and RLK signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant genomes encode hundreds of receptor-like kinases (RLKs) with an organization of functional domains similar to that of animal receptor kinases. Ligand-dependent phosphorylation has now been demonstrated for several plant RLKs and identification of specific phosphorylation sites followed by thei...

  9. Investigating Histone Acetylation Stoichiometry and Turnover Rate.

    PubMed

    Fan, J; Baeza, J; Denu, J M

    2016-01-01

    Histone acetylation is a dynamic epigenetic modification that functions in the regulation of DNA-templated reactions, such as transcription. This lysine modification is reversibly controlled by histone (lysine) acetyltransferases and deacetylases. Here, we present methods employing isotopic labeling and mass spectrometry (MS) to comprehensively investigate histone acetylation dynamics. Turnover rates of histone acetylation are determined by measuring the kinetics of labeling from (13)C-labeled precursors of acetyl-CoA, which incorporates (13)C-carbon onto histones via the acetyltransferase reaction. Overall histone acetylation states are assessed from complete protease digestion to single amino acids, which is followed by MS analysis. Determination of site-specific acetylation stoichiometry is achieved by chemically acetylating endogenous histones with isotopic acetic anhydride, followed by trypsin digestion and LC-MS analysis. Combining metabolic labeling with stoichiometric analysis permits determination of both acetylation level and acetylation dynamics. When comparing genetic, diet, or environmental perturbations, these methods permit both a global and site-specific evaluation of how histone acetylation is dynamically regulated. PMID:27423860

  10. HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism.

    PubMed

    Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

    2016-01-01

    Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

  11. HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism

    PubMed Central

    Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

    2016-01-01

    Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

  12. Systematic profiling of the bacterial phosphoproteome reveals bacterium-specific features of phosphorylation.

    PubMed

    Lin, Miao-Hsia; Sugiyama, Naoyuki; Ishihama, Yasushi

    2015-09-15

    Protein phosphorylation is a crucial posttranslational modification for regulating cellular processes in bacteria; however, it has not been extensively studied because of technical difficulties in the enrichment of phosphopeptides. We devised an enrichment protocol that enabled the identification of >1000 phosphopeptides from a single bacterial sample. We discovered three high-confidence serine and threonine phosphorylation motifs, as well as 29 other motifs at various levels of confidence, from three distinct bacterial phosphoproteomes. We found that the proline-directed and basophilic phosphorylation motifs that are commonly enriched in eukaryotes were not observed in bacteria. Unlike eukaryotes, bacteria had a low occurrence of both phosphorylation and acetylation in N-terminal phosphopeptides. Because infection of host cells by bacterial pathogens is often accompanied by kinase-mediated phosphorylation events, the differences in phosphorylation preferences between bacteria and eukaryotes revealed by this study could be useful in identifying bacterial-specific targets for future therapies. PMID:26373674

  13. Mitochondrial Acetylation and Diseases of Aging

    PubMed Central

    Wagner, Gregory R.; Payne, R. Mark

    2011-01-01

    In recent years, protein lysine acetylation has emerged as a prominent and conserved regulatory posttranslational modification that is abundant on numerous enzymes involved in the processes of intermediary metabolism. Well-characterized mitochondrial processes of carbon utilization are enriched in acetyl-lysine modifications. Although seminal discoveries have been made in the basic biology of mitochondrial acetylation, an understanding of how acetylation states influence enzyme function and metabolic reprogramming during pathological states remains largely unknown. This paper will examine our current understanding of eukaryotic acetate metabolism and present recent findings in the field of mitochondrial acetylation biology. The implications of mitochondrial acetylation for the aging process will be discussed, as well as its potential implications for the unique and localized metabolic states that occur during the aging-associated conditions of heart failure and cancer growth. PMID:21437190

  14. Acetylation of Aurora B by TIP60 ensures accurate chromosomal segregation

    PubMed Central

    Mo, Fei; Zhuang, Xiaoxuan; Liu, Xing; Yao, Phil Y.; Qin, Bo; Su, Zeqi; Zang, Jianye; Wang, Zhiyong; Zhang, Jiancun; Dou, Zhen; Tian, Changlin; Teng, Maikun; Niu, Liwen; Hill, Donald L.; Fang, Guowei; Ding, Xia; Fu, Chuanhai; Yao, Xuebiao

    2015-01-01

    Faithful chromosome segregation in mammalian cells requires the bi-orientation of sister chromatids which relies on sensing correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying cyclin-dependent kinase 1 (CDK1) activation that triggers mitotic entry is extensively studied, the regulatory mechanisms that couple CDK1-cyclin B activity to chromosome stability are not well understood. Here, we identified a signaling axis in which Aurora B activity is modulated by CDK1-cyclin B via acetyltransferase TIP60 (Tat-interactive protein 60 kDa) in human cell division. CDK1-cyclin B phosphorylated Ser90 of TIP60, which elicited TIP60-dependent acetylation of Aurora B and promoted accurate chromosome segregation in mitosis. Mechanistically, TIP60 acetylation of Aurora B at Lys215 protected the phosphorylation of its activation loop from PP2A reactivation-elicited dephosphorylation to ensure a robust, error-free metaphase-anaphase transition. These findings delineated a conserved signaling cascade that integrates protein phosphorylation and acetylation to cell cycle progression for maintenance of genomic stability. PMID:26829474

  15. 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.

  16. Stress-induced gene expression and behavior are controlled by DNA methylation and methyl donor availability in the dentate gyrus.

    PubMed

    Saunderson, Emily A; Spiers, Helen; Mifsud, Karen R; Gutierrez-Mecinas, Maria; Trollope, Alexandra F; Shaikh, Abeera; Mill, Jonathan; Reul, Johannes M H M

    2016-04-26

    Stressful events evoke long-term changes in behavioral responses; however, the underlying mechanisms in the brain are not well understood. Previous work has shown that epigenetic changes and immediate-early gene (IEG) induction in stress-activated dentate gyrus (DG) granule neurons play a crucial role in these behavioral responses. Here, we show that an acute stressful challenge [i.e., forced swimming (FS)] results in DNA demethylation at specific CpG (5'-cytosine-phosphate-guanine-3') sites close to the c-Fos (FBJ murine osteosarcoma viral oncogene homolog) transcriptional start site and within the gene promoter region of Egr-1 (early growth response protein 1) specifically in the DG. Administration of the (endogenous) methyl donor S-adenosyl methionine (SAM) did not affect CpG methylation and IEG gene expression at baseline. However, administration of SAM before the FS challenge resulted in an enhanced CpG methylation at the IEG loci and suppression of IEG induction specifically in the DG and an impaired behavioral immobility response 24 h later. The stressor also specifically increased the expression of the de novo DNA methyltransferase Dnmt3a [DNA (cytosine-5-)-methyltransferase 3 alpha] in this hippocampus region. Moreover, stress resulted in an increased association of Dnmt3a enzyme with the affected CpG loci within the IEG genes. No effects of SAM were observed on stress-evoked histone modifications, including H3S10p-K14ac (histone H3, phosphorylated serine 10 and acetylated lysine-14), H3K4me3 (histone H3, trimethylated lysine-4), H3K9me3 (histone H3, trimethylated lysine-9), and H3K27me3 (histone H3, trimethylated lysine-27). We conclude that the DNA methylation status of IEGs plays a crucial role in FS-induced IEG induction in DG granule neurons and associated behavioral responses. In addition, the concentration of available methyl donor, possibly in conjunction with Dnmt3a, is critical for the responsiveness of dentate neurons to environmental stimuli in

  17. ERK2-mediated phosphorylation of transcriptional coactivator binding protein PIMT/NCoA6IP at Ser298 augments hepatic gluconeogenesis.

    PubMed

    Kapadia, Bandish; Viswakarma, Navin; Parsa, Kishore V L; Kain, Vasundhara; Behera, Soma; Suraj, Sashidhara Kaimal; Babu, Phanithi Prakash; Kar, Anand; Panda, Sunanda; Zhu, Yi-jun; Jia, Yuzhi; Thimmapaya, Bayar; Reddy, Janardan K; Misra, Parimal

    2013-01-01

    PRIP-Interacting protein with methyl transferase domain (PIMT) serves as a molecular bridge between CREB-binding protein (CBP)/ E1A binding protein p300 (Ep300) -anchored histone acetyl transferase and the Mediator complex sub-unit1 (Med1) and modulates nuclear receptor transcription. Here, we report that ERK2 phosphorylates PIMT at Ser(298) and enhances its ability to activate PEPCK promoter. We observed that PIMT is recruited to PEPCK promoter and adenoviral-mediated over-expression of PIMT in rat primary hepatocytes up-regulated expression of gluconeogenic genes including PEPCK. Reporter experiments with phosphomimetic PIMT mutant (PIMT(S298D)) suggested that conformational change may play an important role in PIMT-dependent PEPCK promoter activity. Overexpression of PIMT and Med1 together augmented hepatic glucose output in an additive manner. Importantly, expression of gluconeogenic genes and hepatic glucose output were suppressed in isolated liver specific PIMT knockout mouse hepatocytes. Furthermore, consistent with reporter experiments, PIMT(S298D) but not PIMT(S298A) augmented hepatic glucose output via up-regulating the expression of gluconeogenic genes. Pharmacological blockade of MAPK/ERK pathway using U0126, abolished PIMT/Med1-dependent gluconeogenic program leading to reduced hepatic glucose output. Further, systemic administration of T4 hormone to rats activated ERK1/2 resulting in enhanced PIMT ser(298) phosphorylation. Phosphorylation of PIMT led to its increased binding to the PEPCK promoter, increased PEPCK expression and induction of gluconeogenesis in liver. Thus, ERK2-mediated phosphorylation of PIMT at Ser(298) is essential in hepatic gluconeogenesis, demonstrating an important role of PIMT in the pathogenesis of hyperglycemia. PMID:24358311

  18. ERK2-Mediated Phosphorylation of Transcriptional Coactivator Binding Protein PIMT/NCoA6IP at Ser298 Augments Hepatic Gluconeogenesis

    PubMed Central

    Parsa, Kishore V. L.; Kain, Vasundhara; Behera, Soma; Suraj, Sashidhara Kaimal; Babu, Phanithi Prakash; Kar, Anand; Panda, Sunanda; Zhu, Yi-jun; Jia, Yuzhi; Thimmapaya, Bayar; Reddy, Janardan K.; Misra, Parimal

    2013-01-01

    PRIP-Interacting protein with methyl transferase domain (PIMT) serves as a molecular bridge between CREB-binding protein (CBP)/ E1A binding protein p300 (Ep300) -anchored histone acetyl transferase and the Mediator complex sub-unit1 (Med1) and modulates nuclear receptor transcription. Here, we report that ERK2 phosphorylates PIMT at Ser298 and enhances its ability to activate PEPCK promoter. We observed that PIMT is recruited to PEPCK promoter and adenoviral-mediated over-expression of PIMT in rat primary hepatocytes up-regulated expression of gluconeogenic genes including PEPCK. Reporter experiments with phosphomimetic PIMT mutant (PIMTS298D) suggested that conformational change may play an important role in PIMT-dependent PEPCK promoter activity. Overexpression of PIMT and Med1 together augmented hepatic glucose output in an additive manner. Importantly, expression of gluconeogenic genes and hepatic glucose output were suppressed in isolated liver specific PIMT knockout mouse hepatocytes. Furthermore, consistent with reporter experiments, PIMTS298D but not PIMTS298A augmented hepatic glucose output via up-regulating the expression of gluconeogenic genes. Pharmacological blockade of MAPK/ERK pathway using U0126, abolished PIMT/Med1-dependent gluconeogenic program leading to reduced hepatic glucose output. Further, systemic administration of T4 hormone to rats activated ERK1/2 resulting in enhanced PIMT ser298 phosphorylation. Phosphorylation of PIMT led to its increased binding to the PEPCK promoter, increased PEPCK expression and induction of gluconeogenesis in liver. Thus, ERK2-mediated phosphorylation of PIMT at Ser298 is essential in hepatic gluconeogenesis, demonstrating an important role of PIMT in the pathogenesis of hyperglycemia. PMID:24358311

  19. Activation of hepatic acetyl-CoA carboxylase by glutamate and Mg2+ is mediated by protein phosphatase-2A.

    PubMed Central

    Gaussin, V; Hue, L; Stalmans, W; Bollen, M

    1996-01-01

    The activation of hepatic acetyl-CoA carboxylase by Na(+)-cotransported amino acids such as glutamine has been attributed mainly to the stimulation of its dephosphorylation by accumulating dicarboxylic acids, e.g. glutamate. We report here on a hepatic species of protein phosphatase-2A that activates acetyl-CoA carboxylase in the presence of physiological concentrations of glutamate or Mg2+ and, under these conditions, accounts for virtually all the hepatic acetyl-CoA carboxylase phosphatase activity. Glutamate also stimulated the dephosphorylation of a synthetic pentadecapeptide encompassing the Ser-79 phosphorylation site of rat acetyl-CoA carboxylase, but did not affect the dephosphorylation of other substrates such as phosphorylase. Conversely, protamine, which stimulated the dephosphorylation of phosphorylase, inhibited the activation of acetyl-CoA carboxylase. A comparison with various species of muscle protein phosphatase-2A showed that the stimulatory effects of glutamate and Mg2+ on the acetyl-CoA carboxylase phosphatase activity are largely mediated by the regulatory A subunit. Glutamate and Mg2+ emerge from our study as novel regulators of protein phosphatase-2A when acting on acetyl-CoA carboxylase. PMID:8645208

  20. Acetylation of C/EBPε is a prerequisite for terminal neutrophil differentiation.

    PubMed

    Bartels, Marije; Govers, Anita M; Fleskens, Veerle; Lourenço, Ana Rita; Pals, Cornelieke E; Vervoort, Stephin J; van Gent, Rogier; Brenkman, Arjan B; Bierings, Marc B; Ackerman, Steven J; van Loosdregt, Jorg; Coffer, Paul J

    2015-03-12

    C/EBPε, a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, is exclusively expressed in myeloid cells and regulates transition from the promyelocytic stage to the myelocytic stage of neutrophil development, being indispensable for secondary and tertiary granule formation. Knowledge concerning the functional role of C/EBPε posttranslational modifications is limited to studies concerning phosphorylation and sumoylation. In the current study, using ectopic expression and ex vivo differentiation of CD34(+) hematopoietic progenitor cells, we demonstrate that C/EBPε is acetylated, which was confirmed by mass spectrometry analysis, identifying 4 acetylated lysines in 3 distinct functional domains. Regulation of C/EBPε acetylation levels by the p300 acetyltransferase and the sirtuin 1 deacetylase controls transcriptional activity, which can at least in part be explained by modulation of DNA binding. During neutrophil development, acetylation of lysines 121 and 198 were found to be crucial for terminal neutrophil differentiation and the expression of neutrophil-specific granule proteins, including lactoferrin and collagenase. Taken together, our data illustrate a critical role for acetylation in the functional regulation of C/EBPε activity during terminal neutrophil development. PMID:25568349

  1. Regulation of Plant Acetyl-CoA Carboxylase by Adenylate Nucleotides 1

    PubMed Central

    Eastwell, Kenneth C.; Stumpf, Paul K.

    1983-01-01

    The assay of acetyl-CoA carboxylase (EC 6.4.1.2) does not follow ideal zero-order kinetics when assayed in a crude extract from wheat (Triticum aestivum L.) germ. Our results show that the lack of ideality is the consequence of contamination by ATPase and adenylate kinase. These enzyme activities generate significant amounts of ADP and AMP in the assay mixture, thus limiting the availability of ATP for the carboxylase reaction. Moreover, ADP and AMP are competitive inhibitors, with respect to ATP, of acetyl-CoA carboxylase. Similar relationships between adenylate nucleotides and acetyl-CoA carboxylase are found in isolated chloroplasts. There is no evidence that acetyl-CoA carboxylase activity in the extracts of the plant systems examined is altered by covalent modification, such as a phosphorylation-dephosphorylation cycle. A scheme is presented that illustrates the dependency of acetyl-CoA carboxylase and fatty acid synthesis on the energy demands of the chloroplasts in vivo. PMID:16662980

  2. NUCLEOPHOSMIN/B23 NEGATIVELY REGULATES GCN5-DEPENDENT HISTONE ACETYLATION AND TRANSACTIVATION

    SciTech Connect

    Zou, Yonglong; Wu, Jun; Giannone, Richard J; Boucher, Lorrie; Du, Hansen; Huang, Ying; Johnson, Dabney K; Liu, Yie; Wang, Yisong

    2007-01-01

    Nucleophosmin/B23 is a multifunctional phosphoprotein that is overexpressed in cancer cells and has been shown to be involved in both positive and negative regulation of transcription. In this study, we first identified GCN5 acetyltransferase as a B23-interacting protein by mass spectrometry, which was then confirmed by in vivo co-immunoprecipitation. In vitro assay demonstrated that B23 bound the PCAF-N domain of GCN5 and inhibited GCN5-mediated acetylation of both free and mononucleosomal histones, probably through interfering with GCN5 and masking histones from being acetylated. Mitotic B23 exhibited higher inhibitory activity on GCN5-mediated histone acetylation than interphase B23. Immunodepletion experiments of mitotic extracts revealed that phosphorylation of B23 at Thr199 enhanced the inhibition of GCN5-mediated histone acetylation. Moreover, luciferase reporter and microarray analyses suggested that B23 attenuated GCN5-mediated transactivation in vivo. Taken together, our studies suggest a molecular mechanism of B23 in the mitotic inhibition of GCN5-mediated histone acetylation and transactivation.

  3. Obesity-induced lysine acetylation increases cardiac fatty acid oxidation and impairs insulin signalling

    PubMed Central

    Alrob, Osama Abo; Sankaralingam, Sowndramalingam; Ma, Cary; Wagg, Cory S.; Fillmore, Natasha; Jaswal, Jagdip S.; Sack, Michael N.; Lehner, Richard; Gupta, Mahesh P.; Michelakis, Evangelos D.; Padwal, Raj S.; Johnstone, David E.; Sharma, Arya M.; Lopaschuk, Gary D.

    2014-01-01

    Aims Lysine acetylation is a novel post-translational pathway that regulates the activities of enzymes involved in both fatty acid and glucose metabolism. We examined whether lysine acetylation controls heart glucose and fatty acid oxidation in high-fat diet (HFD) obese and SIRT3 knockout (KO) mice. Methods and results C57BL/6 mice were placed on either a HFD (60% fat) or a low-fat diet (LFD; 4% fat) for 16 or 18 weeks. Cardiac fatty acid oxidation rates were significantly increased in HFD vs. LFD mice (845 ± 76 vs. 551 ± 87 nmol/g dry wt min, P < 0.05). Activities of the fatty acid oxidation enzymes, long-chain acyl-CoA dehydrogenase (LCAD), and β-hydroxyacyl-CoA dehydrogenase (β-HAD) were increased in hearts from HFD vs. LFD mice, and were associated with LCAD and β-HAD hyperacetylation. Cardiac protein hyperacetylation in HFD-fed mice was associated with a decrease in SIRT3 expression, while expression of the mitochondrial acetylase, general control of amino acid synthesis 5 (GCN5)-like 1 (GCN5L1), did not change. Interestingly, SIRT3 deletion in mice also led to an increase in cardiac fatty acid oxidation compared with wild-type (WT) mice (422 ± 29 vs. 291 ± 17 nmol/g dry wt min, P < 0.05). Cardiac lysine acetylation was increased in SIRT3 KO mice compared with WT mice, including increased acetylation and activity of LCAD and β-HAD. Although the HFD and SIRT3 deletion decreased glucose oxidation, pyruvate dehydrogenase acetylation was unaltered. However, the HFD did increase Akt acetylation, while decreasing its phosphorylation and activity. Conclusion We conclude that increased cardiac fatty acid oxidation in response to high-fat feeding is controlled, in part, via the down-regulation of SIRT3 and concomitant increased acetylation of mitochondrial β-oxidation enzymes. PMID:24966184

  4. Mining Conditional Phosphorylation Motifs.

    PubMed

    Liu, Xiaoqing; Wu, Jun; Gong, Haipeng; Deng, Shengchun; He, Zengyou

    2014-01-01

    Phosphorylation motifs represent position-specific amino acid patterns around the phosphorylation sites in the set of phosphopeptides. Several algorithms have been proposed to uncover phosphorylation motifs, whereas the problem of efficiently discovering a set of significant motifs with sufficiently high coverage and non-redundancy still remains unsolved. Here we present a novel notion called conditional phosphorylation motifs. Through this new concept, the motifs whose over-expressiveness mainly benefits from its constituting parts can be filtered out effectively. To discover conditional phosphorylation motifs, we propose an algorithm called C-Motif for a non-redundant identification of significant phosphorylation motifs. C-Motif is implemented under the Apriori framework, and it tests the statistical significance together with the frequency of candidate motifs in a single stage. Experiments demonstrate that C-Motif outperforms some current algorithms such as MMFPh and Motif-All in terms of coverage and non-redundancy of the results and efficiency of the execution. The source code of C-Motif is available at: https://sourceforge. net/projects/cmotif/. PMID:26356863

  5. Roles of dynamic and reversible histone acetylation in plant development and polyploidy

    PubMed Central

    Chen, Z. Jeffrey; Tian, Lu

    2007-01-01

    Transcriptional regulation in eukaryotes is not simply determined by the DNA sequence, but rather mediated through dynamic chromatin modifications and remodeling. Recent studies have shown that reversible and rapid changes in histone acetylation play an essential role in chromatin modification, induce genome-wide and specific changes in gene expression, and affect a variety of biological processes in response to internal and external signals, such as cell differentiation, growth, development, light, temperature, and abiotic and biotic stresses. Moreover, histone acetylation and deacetylation are associated with RNA interference and other chromatin modifications including DNA and histone methylation. The reversible changes in histone acetylation also contribute to cell cycle regulation and epigenetic silencing of rDNA and redundant genes in response to interspecific hybridization and polyploidy. PMID:17556080

  6. Catalytic Depolymerization of Chitin with Retention of N-Acetyl Group.

    PubMed

    Yabushita, Mizuho; Kobayashi, Hirokazu; Kuroki, Kyoichi; Ito, Shogo; Fukuoka, Atsushi

    2015-11-01

    Chitin, a polymer of N-acetylglucosamine units with β-1,4-glycosidic linkages, is the most abundant marine biomass. Chitin monomers containing N-acetyl groups are useful precursors to various fine chemicals and medicines. However, the selective conversion of robust chitin to N-acetylated monomers currently requires a large excess of acid or a long reaction time, which limits its application. We demonstrate a fast catalytic transformation of chitin to monomers with retention of N-acetyl groups by combining mechanochemistry and homogeneous catalysis. Mechanical-force-assisted depolymerization of chitin with a catalytic amount of H2SO4 gave soluble short-chain oligomers. Subsequent hydrolysis of the ball-milled sample provided N-acetylglucosamine in 53% yield, and methanolysis afforded 1-O-methyl-N-acetylglucosamine in yields of up to 70%. Our process can greatly reduce the use of acid compared to the conventional process. PMID:26538108

  7. Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation

    NASA Astrophysics Data System (ADS)

    Cheng, Jingdong; Yang, Huirong; Fang, Jian; Ma, Lixiang; Gong, Rui; Wang, Ping; Li, Ze; Xu, Yanhui

    2015-05-01

    DNMT1 is an important epigenetic regulator that plays a key role in the maintenance of DNA methylation. Here we determined the crystal structure of DNMT1 in complex with USP7 at 2.9 Å resolution. The interaction between the two proteins is primarily mediated by an acidic pocket in USP7 and Lysine residues within DNMT1's KG linker. This intermolecular interaction is required for USP7-mediated stabilization of DNMT1. Acetylation of the KG linker Lysine residues impair DNMT1-USP7 interaction and promote the degradation of DNMT1. Treatment with HDAC inhibitors results in an increase in acetylated DNMT1 and decreased total DNMT1 protein. This negative correlation is observed in differentiated neuronal cells and pancreatic cancer cells. Our studies reveal that USP7-mediated stabilization of DNMT1 is regulated by acetylation and provide a structural basis for the design of inhibitors, targeting the DNMT1-USP7 interaction surface for therapeutic applications.

  8. Inhibition of IKKα by BAY61-3606 Reveals IKKα-Dependent Histone H3 Phosphorylation in Human Cytomegalovirus Infected Cells

    PubMed Central

    Ho, Catherine M. K.; Donovan-Banfield, I’ah Z.; Tan, Li; Zhang, Tinghu; Gray, Nathanael S.; Strang, Blair L.

    2016-01-01

    Protein kinase inhibitors can be used as tools to identify proteins and pathways required for virus replication. Using virus replication assays and western blotting we found that the widely used protein kinase inhibitor BAY61-3606 inhibits replication of human cytomegalovirus (HCMV) strain AD169 and the accumulation of HCMV immediate-early proteins in AD169 infected cells, but has no effect on replication of HCMV strain Merlin. Using in vitro kinase assays we found that BAY61-3606 is a potent inhibitor of the cellular kinase IKKα. Infection of cells treated with siRNA targeting IKKα indicated IKKα was required for efficient AD169 replication and immediate-early protein production. We hypothesized that IKKα was required for AD169 immediate-early protein production as part of the canonical NF-κB signaling pathway. However, although BAY61-3606 inhibited phosphorylation of the IKKα substrate IκBα, we found no canonical or non-canonical NF-κB signaling in AD169 infected cells. Rather, we observed that treatment of cells with BAY61-3606 or siRNA targeting IKKα decreased phosphorylation of histone H3 at serine 10 (H3S10p) in western blotting assays. Furthermore, we found treatment of cells with BAY61-3606, but not siRNA targeting IKKα, inhibited the accumulation of histone H3 acetylation (H3K9ac, H3K18ac and H3K27ac) and tri-methylation (H3K27me3 and H3K36me3) modifications. Therefore, the requirement for IKKα in HCMV replication was strain-dependent and during replication of an HCMV strain requiring IKKα, IKKα-dependent H3S10 phosphorylation was associated with efficient HCMV replication and immediate-early protein production. Plus, inhibition of HCMV replication by BAY61-3606 is associated with acetylation and tri-methylation modifications of histone H3 that do not involve IKKα. PMID:26930276

  9. Inhibition of IKKα by BAY61-3606 Reveals IKKα-Dependent Histone H3 Phosphorylation in Human Cytomegalovirus Infected Cells.

    PubMed

    Ho, Catherine M K; Donovan-Banfield, I'ah Z; Tan, Li; Zhang, Tinghu; Gray, Nathanael S; Strang, Blair L

    2016-01-01

    Protein kinase inhibitors can be used as tools to identify proteins and pathways required for virus replication. Using virus replication assays and western blotting we found that the widely used protein kinase inhibitor BAY61-3606 inhibits replication of human cytomegalovirus (HCMV) strain AD169 and the accumulation of HCMV immediate-early proteins in AD169 infected cells, but has no effect on replication of HCMV strain Merlin. Using in vitro kinase assays we found that BAY61-3606 is a potent inhibitor of the cellular kinase IKKα. Infection of cells treated with siRNA targeting IKKα indicated IKKα was required for efficient AD169 replication and immediate-early protein production. We hypothesized that IKKα was required for AD169 immediate-early protein production as part of the canonical NF-κB signaling pathway. However, although BAY61-3606 inhibited phosphorylation of the IKKα substrate IκBα, we found no canonical or non-canonical NF-κB signaling in AD169 infected cells. Rather, we observed that treatment of cells with BAY61-3606 or siRNA targeting IKKα decreased phosphorylation of histone H3 at serine 10 (H3S10p) in western blotting assays. Furthermore, we found treatment of cells with BAY61-3606, but not siRNA targeting IKKα, inhibited the accumulation of histone H3 acetylation (H3K9ac, H3K18ac and H3K27ac) and tri-methylation (H3K27me3 and H3K36me3) modifications. Therefore, the requirement for IKKα in HCMV replication was strain-dependent and during replication of an HCMV strain requiring IKKα, IKKα-dependent H3S10 phosphorylation was associated with efficient HCMV replication and immediate-early protein production. Plus, inhibition of HCMV replication by BAY61-3606 is associated with acetylation and tri-methylation modifications of histone H3 that do not involve IKKα. PMID:26930276

  10. Methylation of arginine by PRMT1 regulates Nrf2 transcriptional activity during the antioxidative response.

    PubMed

    Liu, Xin; Li, Hongyuan; Liu, Lingxia; Lu, Yang; Gao, Yanyan; Geng, Pengyu; Li, Xiaoxue; Huang, Baiqu; Zhang, Yu; Lu, Jun

    2016-08-01

    The cap 'n' collar (CNC) family of transcription factors play important roles in resistance of oxidative and electrophilic stresses. Among the CNC family members, NF-E2-related factor 2 (Nrf2) is critical for regulating the antioxidant and phase II enzymes through antioxidant response element (ARE)-mediated transactivation. The activity of Nrf2 is controlled by a variety of post-translational modifications, including phosphorylation, ubiquitination, acetylation and sumoylation. Here we demonstrate that the arginine methyltransferase-1 (PRMT1) methylates Nrf2 protein at a single residue of arginine 437, both in vitro and in vivo. Using the heme oxygenase-1 (HO-1) as a model of phase II enzyme gene, we found that methylation of Nrf2 by PRMT1 led to a moderate increase of its DNA-binding activity and transactivation, which subsequently protected cells against the tBHP-induced glutathione depletion and cell death. Collectively, our results define a novel modification of Nrf2, which operates as a fine-tuning mechanism for the transcriptional activity of Nrf2 under the oxidative stress. PMID:27183873

  11. Histone acetylation is involved in TCDD‑induced cleft palate formation in fetal mice.

    PubMed

    Yuan, Xingang; Qiu, Lin; Pu, Yalan; Liu, Cuiping; Zhang, Xuan; Wang, Chen; Pu, Wei; Fu, Yuexian

    2016-08-01

    The aim of the present was to evaluate the effects of DNA methylation and histone acetylation on 2,3,7,8‑tetrachlorodibenzo‑p‑dioxin (TCDD)‑induced cleft palate in fetal mice. Pregnant mice (n=10) were randomly divided into two groups: i) TCDD group, mice were treated with 28 µg/kg TCDD on gestation day (GD) 10 by oral gavage; ii) control group, mice were treated with an equal volume of corn oil. On GD 16.5, the fetal mice were evaluated for the presence of a cleft palate. An additional 36 pregnant mice were divided into the control and TCDD groups, and palate samples were collected on GD 13.5, GD 14.5 and GD 15.5, respectively. Transforming growth factor‑β3 (TGF‑β3) mRNA expression, TGF‑β3 promoter methylation, histone acetyltransferase (HAT) activity and histone H3 (H3) acetylation in the palates were evaluated in the two groups. The incidence of a cleft palate in the TCDD group was 93.55%, and no cases of cleft palate were identified in the control group. On GD 13.5 and GD 14.5, TGF‑β3 mRNA expression, HAT activity and acetylated H3 levels were significantly increased in the TCDD group compared with the control. Methylated bands were not observed in the TCDD or control groups. In conclusion, at the critical period of palate fusion (GD 13.5‑14.5), TCDD significantly increased TGF‑β3 gene expression, HAT activity and H3 acetylation. Therefore, histone acetylation may be involved in TCDD‑induced cleft palate formation in fetal mice. PMID:27279340

  12. Histone acetylation is involved in TCDD-induced cleft palate formation in fetal mice

    PubMed Central

    Yuan, Xingang; Qiu, Lin; Pu, Yalan; Liu, Cuiping; Zhang, Xuan; Wang, Chen; Pu, Wei; Fu, Yuexian

    2016-01-01

    The aim of the present was to evaluate the effects of DNA methylation and histone acetylation on 2,3,7,8-tetrachlo-rodibenzo-p-dioxin (TCDD)-induced cleft palate in fetal mice. Pregnant mice (n=10) were randomly divided into two groups: i) TCDD group, mice were treated with 28 µg/kg TCDD on gestation day (GD) 10 by oral gavage; ii) control group, mice were treated with an equal volume of corn oil. On GD 16.5, the fetal mice were evaluated for the presence of a cleft palate. An additional 36 pregnant mice were divided into the control and TCDD groups, and palate samples were collected on GD 13.5, GD 14.5 and GD 15.5, respectively. Transforming growth factor-β3 (TGF-β3) mRNA expression, TGF-β3 promoter methylation, histone acetyltransferase (HAT) activity and histone H3 (H3) acetylation in the palates were evaluated in the two groups. The incidence of a cleft palate in the TCDD group was 93.55%, and no cases of cleft palate were identified in the control group. On GD 13.5 and GD 14.5, TGF-β3 mRNA expression, HAT activity and acetylated H3 levels were significantly increased in the TCDD group compared with the control. Methylated bands were not observed in the TCDD or control groups. In conclusion, at the critical period of palate fusion (GD 13.5–14.5), TCDD significantly increased TGF-β3 gene expression, HAT activity and H3 acetylation. Therefore, histone acetylation may be involved in TCDD-induced cleft palate formation in fetal mice. PMID:27279340

  13. Proteomic profiling of lysine acetylation in Pseudomonas aeruginosa reveals the diversity of acetylated proteins.

    PubMed

    Ouidir, Tassadit; Cosette, Pascal; Jouenne, Thierry; Hardouin, Julie

    2015-07-01

    Protein lysine acetylation is a reversible and highly regulated post-translational modification with the well demonstrated physiological relevance in eukaryotes. Recently, its important role in the regulation of metabolic processes in bacteria was highlighted. Here, we reported the lysine acetylproteome of Pseudomonas aeruginosa using a proteomic approach. We identified 430 unique peptides corresponding to 320 acetylated proteins. In addition to the proteins involved in various metabolic pathways, several enzymes contributing to the lipopolysaccharides biosynthesis were characterized as acetylated. This data set illustrated the abundance and the diversity of acetylated lysine proteins in P. aeruginosa and opens opportunities to explore the role of the acetylation in the bacterial physiology. PMID:25900529

  14. A Method to Determine Lysine Acetylation Stoichiometries

    DOE PAGESBeta

    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.; et al

    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

  15. 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.

  16. Histone Acetylation in Fungal Pathogens of Plants

    PubMed Central

    Jeon, Junhyun; Kwon, Seomun; Lee, Yong-Hwan

    2014-01-01

    Acetylation of histone lysine residues occurs in different organisms ranging from yeast to plants and mammals for the regulation of diverse cellular processes. With the identification of enzymes that create or reverse this modification, our understanding on histone acetylation has expanded at an amazing pace during the last two decades. In fungal pathogens of plants, however, the importance of such modification has only just begun to be appreciated in the recent years and there is a dearth of information on how histone acetylation is implicated in fungal pathogenesis. This review covers the current status of research related to histone acetylation in plant pathogenic fungi and considers relevant findings in the interaction between fungal pathogens and host plants. We first describe the families of histone acetyltransferases and deacetylases. Then we provide the cases where histone acetylation was investigated in the context of fungal pathogenesis. Finally, future directions and perspectives in epigenetics of fungal pathogenesis are discussed. PMID:25288980

  17. Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development

    PubMed Central

    Loponte, Sara; Segré, Chiara V.; Senese, Silvia; Miccolo, Claudia; Santaguida, Stefano; Deflorian, Gianluca; Citro, Simona; Mattoscio, Domenico; Pisati, Federica; Moser, Mirjam A.; Visintin, Rosella; Seiser, Christian; Chiocca, Susanna

    2016-01-01

    Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology. PMID:27458029

  18. Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development.

    PubMed

    Loponte, Sara; Segré, Chiara V; Senese, Silvia; Miccolo, Claudia; Santaguida, Stefano; Deflorian, Gianluca; Citro, Simona; Mattoscio, Domenico; Pisati, Federica; Moser, Mirjam A; Visintin, Rosella; Seiser, Christian; Chiocca, Susanna

    2016-01-01

    Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology. PMID:27458029

  19. Methyl Iodide

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methyl iodide (MeI, iodomethane, CH3I) was reported as a potential alternative to the stratospheric ozone-depleting fumigant methyl bromide (MeBr) in the mid-1990s (Sims et al., 1995; Ohr et al., 1996). It has since received significant research attention to determine its environmental fate and tran...

  20. Methyl chloroform

    SciTech Connect

    Wray, T.K.

    1994-04-01

    Methyl chloroform is identified as a Class 1 ozone-depleting substance under Title VI of the CAA Amendments. On Nov. 30, 1993, EPA ordered the phaseout of Class 1 ozone-depleting substances -- chlorofluorocarbons (CFCs), halons, carbon tetrachloride and methyl chloroform -- by Jan. 1, 1996. Methyl chloroform and other Class 1 substances may be used after the dead-line if sources can be found through recycling or existing inventories. Methyl chloroform is listed as a hazardous air pollutant under CAA. It also is a SARA Title III, Sec. 313 compound with a reportable quantity of 1,000 pounds. OSHA and the American Conference of Government Industrial Hygienists have set 350 ppm as the time-weighted average airborne exposure level for methyl chloroform. NIOSH lists its immediately dangerous to life or health'' concentration as 1,000 parts per million. DOT identifies the substance as a hazardous material, Class 6.1 (poison).

  1. Autotrophic acetyl coenzyme A biosynthesis in Methanococcus maripaludis.

    PubMed Central

    Shieh, J; Whitman, W B

    1988-01-01

    To detect autotrophic CO2 assimilation in cell extracts of Methanococcus maripaludis, lactate dehydrogenase and NADH were added to convert pyruvate formed from autotrophically synthesized acetyl coenzyme A to lactate. The lactate produced was determined spectrophotometrically. When CO2 fixation was pulled in the direction of lactate synthesis, CO2 reduction to methane was inhibited. Bromoethanesulfonate (BES), a potent inhibitor of methanogenesis, enhanced lactate synthesis, and methyl coenzyme M inhibited it in the absence of BES. Lactate synthesis was dependent on CO2 and H2, but H2 + CO2-independent synthesis was also observed. In cell extracts, the rate of lactate synthesis was about 1.2 nmol min-1 mg of protein-1. When BES was added, the rate of lactate synthesis increased to 2.3 nmol min-1 mg of protein-1. Because acetyl coenzyme A did not stimulate lactate synthesis, pyruvate synthase may have been the limiting activity in these assays. Radiolabel from 14CO2 was incorporated into lactate. The percentages of radiolabel in the C-1, C-2, and C-3 positions of lactate were 73, 33, and 11%, respectively. Both carbon monoxide and formaldehyde stimulated lactate synthesis. 14CH2O was specifically incorporated into the C-3 of lactate, and 14CO was incorporated into the C-1 and C-2 positions. Low concentrations of cyanide also inhibited autotrophic growth, CO dehydrogenase activity, and autotrophic lactate synthesis. These observations are in agreement with the acetogenic pathway of autotrophic CO2 assimilation. PMID:3133359

  2. Acetylation phenotypes in patients with bladder carcinoma.

    PubMed

    Bicho, M P; Breitenfeld, L; Carvalho, A A; Manso, C F

    1988-01-01

    The present study was done to evaluate the possible association of bladder carcinoma with the slow acetylator phenotype in a portuguese population. 49 patients with bladder carcinoma were compared to a normal control group of 84 individuals. No statistically significant association was detected. But when subdividing the group of slow acetylators it is found that in the subgroup with 12-36% acetylation there is a higher percentage of patients, which is statistically significant. These results are in agreement with two other studies, using populations of similar ethnic origin. PMID:3265609

  3. Involvement of histamine receptors in SAPK/JNK phosphorylation.

    PubMed

    Dandekar, Radhika D; Khan, Manzoor M

    2012-06-01

    Histamine is a mediator of inflammation in allergic disease and asthma. Stress activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) are involved in asthma. This study examined the role of histamine receptors on the phosphorylation of SAPK/JNK in splenocytes. C57BL/6 mice splenocytes were treated with histamine (10⁻⁴ M to 10⁻¹¹ M), and its selective receptor agonists, phorbol 12 myristate 13-acetate (PMA) was used as a positive control, and phosphorylation of SAPK/JNK was determined. Histamine (10⁻⁴ M-10⁻⁸ M) inhibited phosphorylation of SAPK/JNK. H1R agonist betahistine (10⁻⁵ M) decreased SAPK/JNK phosphorylation and H2R agonist amthamine (10⁻⁵ M) did not show any significant effect. However, H3R agonist methimepip (10⁻⁶ M) and H4R agonist 4-methyl histamine (10⁻⁶ M), increased SAPK/JNK phosphorylation. We used TNFα knockout mice to determine if histamine regulated SAPK/JNK phosphorylation via TNFα. While the effects of histamine and H1 agonists were similar to that of wild type mice in inhibiting the phosphorylation of SAPK/JNK, the effects of H3 and H4 agonists differed in TNFα knockout mice splenocytes. Activation of H3 receptors decreased SAPK/JNK phosphorylation in TNFα knockout mice, as opposed to an increase in wild type mice, whereas H4 agonist did not show any significant effect on the phosphorylation of SAPK/JNK. This data showed that histamine acting through H4 receptors caused the phosphorylation of SAPK/JNK via TNFα. The role of H4 receptors in pro-inflammatory response is intriguing. PMID:22487127

  4. Acetylation of Aurora B by TIP60 ensures accurate chromosomal segregation.

    PubMed

    Mo, Fei; Zhuang, Xiaoxuan; Liu, Xing; Yao, Phil Y; Qin, Bo; Su, Zeqi; Zang, Jianye; Wang, Zhiyong; Zhang, Jiancun; Dou, Zhen; Tian, Changlin; Teng, Maikun; Niu, Liwen; Hill, Donald L; Fang, Guowei; Ding, Xia; Fu, Chuanhai; Yao, Xuebiao

    2016-04-01

    Faithful segregation of chromosomes in mammalian cells requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying cyclin-dependent kinase 1 (CDK1) activation, which triggers mitotic entry, have been extensively studied, the regulatory mechanisms that couple CDK1-cyclin B activity to chromosome stability are not well understood. Here, we identified a signaling axis in which Aurora B activity is modulated by CDK1-cyclin B via the acetyltransferase TIP60 in human cell division. CDK1-cyclin B phosphorylates Ser90 of TIP60, which elicits TIP60-dependent acetylation of Aurora B and promotes accurate chromosome segregation in mitosis. Mechanistically, TIP60 acetylation of Aurora B at Lys215 protects Aurora B's activation loop from dephosphorylation by the phosphatase PP2A to ensure a robust, error-free metaphase-anaphase transition. These findings delineate a conserved signaling cascade that integrates protein phosphorylation and acetylation with cell cycle progression for maintenance of genomic stability. PMID:26829474

  5. Combination of the dipeptidyl peptidase IV inhibitor LAF237 [(S)-1-[(3-hydroxy-1-adamantyl)ammo]acetyl-2-cyanopyrrolidine] with the angiotensin II type 1 receptor antagonist valsartan [N-(1-oxopentyl)-N-[[2'-(1H-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl]methyl]-L-valine] enhances pancreatic islet morphology and function in a mouse model of type 2 diabetes.

    PubMed

    Cheng, Qianni; Law, Pui Ki; de Gasparo, Marc; Leung, Po Sing

    2008-12-01

    LAF237 [(S)-1-[(3-hydroxy-1-adamantyl)ammo]acetyl-2-cyanopyrrolidine] is an inhibitor of dipeptidyl peptidase IV that delays the degradation of glucagon-like peptide-1 (GLP-1). Valsartan [N-(1-oxopentyl)-N-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-l-valine] is an antagonist of the angiotensin II type 1 receptor (AT1R) that reduces the incidence of type 2 diabetes mellitus. LAF237 and valsartan act on a common target through separate pathways to improve pancreatic islet cell function. We hypothesize that the combination of these two drugs acts in a synergistic or additive manner on islet function and structure. To test this hypothesis, we performed in vitro and in vivo studies. To measure the acute effect of the treatment, pancreatic islets of db/db mice were isolated and stimulated in vitro with glucose in the presence of valsartan (1 microM) and exendin-4 (100 nM), a GLP-1 receptor agonist. Combination treatment with valsartan and exendin-4 significantly enhanced glucose-stimulated insulin secretion from isolated islets. For studies of chronic effect, db/db mice received LAF237 (1 mg/kg/day) and/or valsartan (10 mg/kg/day). Islet cell reactive oxygen species (ROS), proliferation, apoptosis, fibrosis, beta-cell area, and glucose homeostasis were evaluated after 8 weeks of treatment, which showed that combination treatment resulted in a significant increase in pancreatic islet beta-cell area compared with monotherapy. This beneficial effect correlated with an increase in beta-cell proliferation and a decrease in ROS-induced islet apoptosis and fibrosis. These in vitro and in vivo data indicate that combination treatment with LAF237 and valsartan has significant beneficial additive effects on pancreatic beta-cell structure and function compared with their respective monotherapeutic effects. PMID:18787107

  6. Struvite and prebiotic phosphorylation.

    NASA Technical Reports Server (NTRS)

    Handschuh, G. J.; Orgel, L. E.

    1973-01-01

    Struvite rather than apatite or amorphous calcium phosphate is precipitated when phosphate is added to seawater containing more than 0.01M NH4+ ions. Struvite may have precipitated from evaporating seawater on the primitive earth, and may have been important for prebiotic phosphorylation.

  7. Phosphorylation of the chromatin remodeling factor DPF3a induces cardiac hypertrophy through releasing HEY repressors from DNA.

    PubMed

    Cui, Huanhuan; Schlesinger, Jenny; Schoenhals, Sophia; Tönjes, Martje; Dunkel, Ilona; Meierhofer, David; Cano, Elena; Schulz, Kerstin; Berger, Michael F; Haack, Timm; Abdelilah-Seyfried, Salim; Bulyk, Martha L; Sauer, Sascha; Sperling, Silke R

    2016-04-01

    DPF3 (BAF45c) is a member of the BAF chromatin remodeling complex. Two isoforms have been described, namely DPF3a and DPF3b. The latter binds to acetylated and methylated lysine residues of histones. Here, we elaborate on the role of DPF3a and describe a novel pathway of cardiac gene transcription leading to pathological cardiac hypertrophy. Upon hypertrophic stimuli, casein kinase 2 phosphorylates DPF3a at serine 348. This initiates the interaction of DPF3a with the transcriptional repressors HEY, followed by the release of HEY from the DNA. Moreover, BRG1 is bound by DPF3a, and is thus recruited to HEY genomic targets upon interaction of the two components. Consequently, the transcription of downstream targets such asNPPAandGATA4is initiated and pathological cardiac hypertrophy is established. In human, DPF3a is significantly up-regulated in hypertrophic hearts of patients with hypertrophic cardiomyopathy or aortic stenosis. Taken together, we show that activation of DPF3a upon hypertrophic stimuli switches cardiac fetal gene expression from being silenced by HEY to being activated by BRG1. Thus, we present a novel pathway for pathological cardiac hypertrophy, whose inhibition is a long-term therapeutic goal for the treatment of the course of heart failure. PMID:26582913

  8. Phosphorylation of the chromatin remodeling factor DPF3a induces cardiac hypertrophy through releasing HEY repressors from DNA

    PubMed Central

    Cui, Huanhuan; Schlesinger, Jenny; Schoenhals, Sophia; Tönjes, Martje; Dunkel, Ilona; Meierhofer, David; Cano, Elena; Schulz, Kerstin; Berger, Michael F.; Haack, Timm; Abdelilah-Seyfried, Salim; Bulyk, Martha L.; Sauer, Sascha; Sperling, Silke R.

    2016-01-01

    DPF3 (BAF45c) is a member of the BAF chromatin remodeling complex. Two isoforms have been described, namely DPF3a and DPF3b. The latter binds to acetylated and methylated lysine residues of histones. Here, we elaborate on the role of DPF3a and describe a novel pathway of cardiac gene transcription leading to pathological cardiac hypertrophy. Upon hypertrophic stimuli, casein kinase 2 phosphorylates DPF3a at serine 348. This initiates the interaction of DPF3a with the transcriptional repressors HEY, followed by the release of HEY from the DNA. Moreover, BRG1 is bound by DPF3a, and is thus recruited to HEY genomic targets upon interaction of the two components. Consequently, the transcription of downstream targets such as NPPA and GATA4 is initiated and pathological cardiac hypertrophy is established. In human, DPF3a is significantly up-regulated in hypertrophic hearts of patients with hypertrophic cardiomyopathy or aortic stenosis. Taken together, we show that activation of DPF3a upon hypertrophic stimuli switches cardiac fetal gene expression from being silenced by HEY to being activated by BRG1. Thus, we present a novel pathway for pathological cardiac hypertrophy, whose inhibition is a long-term therapeutic goal for the treatment of the course of heart failure. PMID:26582913

  9. Impact of acetylation on tumor metabolism

    PubMed Central

    Zhao, Di; Li, Fu-Long; Cheng, Zhou-Li; Lei, Qun-Ying

    2014-01-01

    Acetylation of protein lysine residues is a reversible and dynamic process that is controlled by histone acetyltransferases (HATs) and deacetylases (HDACs and SIRTs). Recent studies have revealed that acetylation modulates not only nuclear proteins but also cytoplasmic or mitochondrial proteins, including many metabolic enzymes. In tumors, cellular metabolism is reprogrammed to provide intermediates for biosynthesis such as nucleotides, fatty acids, and amino acids, and thereby favor the rapid proliferation of cancer cells and tumor development. An increasing number of investigations have indicated that acetylation plays an important role in tumor metabolism. Here, we summarize the substrates that are modified by acetylation, especially oncogenes, tumor suppressor genes, and enzymes that are implicated in tumor metabolism. PMID:27308346

  10. Acetylator phenotypes in Papua New Guinea

    PubMed Central

    Penketh, R J A; Gibney, S F A; Nurse, G T; Hopkinson, D A

    1983-01-01

    Acetylator phenotypes have been determined in 139 unrelated subjects from the hitherto untested populations of Papua New Guinea, and their relevance to current antituberculous isoniazid chemotherapy is discussed. PMID:6842533

  11. Chronic ethanol consumption induces mitochondrial protein acetylation and oxidative stress in the kidney

    PubMed Central

    Harris, Peter S.; Roy, Samantha R.; Coughlan, Christina; Orlicky, David J.; Liang, Yongliang; Shearn, Colin T.; Roede, James R.; Fritz, Kristofer S.

    2015-01-01

    In this study, we present the novel findings that chronic ethanol consumption induces mitochondrial protein hyperacetylation in the kidney and correlates with significantly increased renal oxidative stress. A major proteomic footprint of alcoholic liver disease (ALD) is an increase in hepatic mitochondrial protein acetylation. Protein hyperacetylation has been shown to alter enzymatic function of numerous proteins and plays a role in regulating metabolic processes. Renal mitochondrial targets of hyperacetylation include numerous metabolic and antioxidant pathways, such as lipid metabolism, oxidative phosphorylation, and amino acid metabolism, as well as glutathione and thioredoxin pathways. Disruption of protein lysine acetylation has the potential to impair renal function through metabolic dysregulation and decreased antioxidant capacity. Due to a significant elevation in ethanol-mediated renal oxidative stress, we highlight the acetylation of superoxide dismutase, peroxiredoxins, glutathione reductase, and glutathione transferase enzymes. Since oxidative stress is a known factor in ethanol-induced nephrotoxicity, we examined biochemical markers of protein hyperacetylation and oxidative stress. Our results demonstrate increased protein acetylation concurrent with depleted glutathione, altered Cys redox potential, and the presence of 4-HNE protein modifications in our 6-week model of early-stage alcoholic nephrotoxicity. These findings support the hypothesis that ethanol metabolism causes an influx of mitochondrial metabolic substrate, resulting in mitochondrial protein hyperacetylation with the potential to impact mitochondrial metabolic and antioxidant processes. PMID:26177469

  12. CheY’s acetylation sites responsible for generating clockwise flagellar rotation in Escherichia coli

    PubMed Central

    Fraiberg, Milana; Afanzar, Oshri; Cassidy, C. Keith; Gabashvili, Alexandra; Schulten, Klaus; Levin, Yishai; Eisenbach, Michael

    2015-01-01

    Summary Stimulation of Escherichia coli with acetate elevates the acetylation level of the chemotaxis response regulator CheY. This elevation, in an unknown mechanism, activates CheY to generate clockwise rotation. Here, using quantitative selective reaction monitoring mass spectrometry and high-resolution targeted mass spectrometry, we identified K91 and K109 as the major sites whose acetylation level in vivo increases in response to acetate. Employing single and multiple lysine replacements in CheY, we found that K91 and K109 are also the sites mainly responsible for acetate-dependent clockwise generation. Furthermore, we showed that clockwise rotation is repressed when residue K91 is non-modified, as evidenced by an increased ability of CheY to generate clockwise rotation when K91 was acetylated or replaced by specific amino acids. Using molecular dynamics simulations we show that K91 repression is manifested in the conformational dynamics of the β4α4 loop, shifted towards an active state upon mutation. Removal of β4α4 loop repression may represent a general activation mechanism in CheY, pertaining also to the canonical phosphorylation activation pathway as suggested by crystal structures of active and inactive CheY from Thermotoga maritima. By way of elimination we further suggest that K109 acetylation is actively involved in generating clockwise rotation. PMID:25388160

  13. 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.

  14. Acetyl-L-carnitine increases mitochondrial protein acetylation in the aged rat heart.

    PubMed

    Kerner, Janos; Yohannes, Elizabeth; Lee, Kwangwon; Virmani, Ashraf; Koverech, Aleardo; Cavazza, Claudio; Chance, Mark R; Hoppel, Charles

    2015-01-01

    Previously we showed that in vivo treatment of elderly Fisher 344 rats with acetylcarnitine abolished the age-associated defect in respiratory chain complex III in interfibrillar mitochondria and improved the functional recovery of the ischemic/reperfused heart. Herein, we explored mitochondrial protein acetylation as a possible mechanism for acetylcarnitine's effect. In vivo treatment of elderly rats with acetylcarnitine restored cardiac acetylcarnitine content and increased mitochondrial protein lysine acetylation and increased the number of lysine-acetylated proteins in cardiac subsarcolemmal and interfibrillar mitochondria. Enzymes of the tricarboxylic acid cycle, mitochondrial β-oxidation, and ATP synthase of the respiratory chain showed the greatest acetylation. Acetylation of isocitrate dehydrogenase, long-chain acyl-CoA dehydrogenase, complex V, and aspartate aminotransferase was accompanied by decreased catalytic activity. Several proteins were found to be acetylated only after treatment with acetylcarnitine, suggesting that exogenous acetylcarnitine served as the acetyl-donor. Two-dimensional fluorescence difference gel electrophoresis analysis revealed that acetylcarnitine treatment also induced changes in mitochondrial protein amount; a two-fold or greater increase/decrease in abundance was observed for thirty one proteins. Collectively, our data provide evidence for the first time that in the aged rat heart in vivo administration of acetylcarnitine provides acetyl groups for protein acetylation and affects the amount of mitochondrial proteins. PMID:25660059

  15. Levels of histone acetylation in thyroid tumors.

    PubMed

    Puppin, Cinzia; Passon, Nadia; Lavarone, Elisa; Di Loreto, Carla; Frasca, Francesco; Vella, Veronica; Vigneri, Riccardo; Damante, Giuseppe

    2011-08-12

    Histone acetylation is a major mechanism to regulate gene transcription. This post-translational modification is modified in cancer cells. In various tumor types the levels of acetylation at several histone residues are associated to clinical aggressiveness. By using immunohistochemistry we show that acetylated levels of lysines at positions 9-14 of H3 histone (H3K9-K14ac) are significantly higher in follicular adenomas (FA), papillary thyroid carcinomas (PTC), follicular thyroid carcinomas (FTC) and undifferentiated carcinomas (UC) than in normal tissues (NT). Similar data have been obtained when acetylated levels of lysine 18 of H3 histone (H3K18ac) were evaluated. In this case, however, no difference was observed between NT and UC. When acetylated levels of lysine 12 of H4 histone (H4K12ac) were evaluated, only FA showed significantly higher levels in comparison with NT. These data indicate that modification histone acetylation is an early event along thyroid tumor progression and that H3K18 acetylation is switched off in the transition between differentiated and undifferentiated thyroid tumors. By using rat thyroid cell lines that are stably transfected with doxycyclin-inducible oncogenes, we show that the oncoproteins RET-PTC, RAS and BRAF increase levels of H3K9-K14ac and H3K18ac. In the non-tumorigenic rat thyroid cell line FRTL-5, TSH increases levels of H3K18ac. However, this hormone decreases levels of H3K9-K14ac and H4K12ac. In conclusion, our data indicate that neoplastic transformation and hormonal stimulation can modify levels of histone acetylation in thyroid cells. PMID:21763277

  16. Protein acetylation in metabolism - metabolites and cofactors.

    PubMed

    Menzies, Keir J; Zhang, Hongbo; Katsyuba, Elena; Auwerx, Johan

    2016-01-01

    Reversible acetylation was initially described as an epigenetic mechanism regulating DNA accessibility. Since then, this process has emerged as a controller of histone and nonhistone acetylation that integrates key physiological processes such as metabolism, circadian rhythm and cell cycle, along with gene regulation in various organisms. The widespread and reversible nature of acetylation also revitalized interest in the mechanisms that regulate lysine acetyltransferases (KATs) and deacetylases (KDACs) in health and disease. Changes in protein or histone acetylation are especially relevant for many common diseases including obesity, diabetes mellitus, neurodegenerative diseases and cancer, as well as for some rare diseases such as mitochondrial diseases and lipodystrophies. In this Review, we examine the role of reversible acetylation in metabolic control and how changes in levels of metabolites or cofactors, including nicotinamide adenine dinucleotide, nicotinamide, coenzyme A, acetyl coenzyme A, zinc and butyrate and/or β-hydroxybutyrate, directly alter KAT or KDAC activity to link energy status to adaptive cellular and organismal homeostasis. PMID:26503676

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

    PubMed Central

    2011-01-01

    Background The involvement of histone acetylation in facilitating gene expression is well-established, particularly in the case of histones H3 and H4. It was previously shown in Saccharomyces cerevisiae that gene expression was significantly down-regulated and chromatin more condensed in stationary phase compared to exponential phase. We were therefore interested in establishing the acetylation state of histone H3 and H4 in stationary and in exponential phase, since the regulation of this modification could contribute to transcriptional shut-down and chromatin compaction during semi-quiescence. Results We made use of nano-spray tandem mass spectrometry to perform a precursor ion scan to detect an m/z 126 immonium ion, diagnostic of an Nε-acetylated lysine residue that allowed unambiguous identification of acetylated as opposed to tri-methylated lysine. The fragmentation spectra of peptides thus identified were searched with Mascot against the Swiss-Prot database, and the y-ion and b-ion fragmentation series subsequently analyzed for mass shifts compatible with acetylated lysine residues. We found that K9, K14 and K36 of histone H3 and K12 and K16 of histone H4 were acetylated in exponential phase (bulk histones), but could not detect these modifications in histones isolated from stationary phase cells at the sensitivity level of the mass spectrometer. The corresponding un-acetylated peptides were, however, observed. A significantly higher level of acetylation of these residues in exponential phase was confirmed by immuno-blotting. Conclusion H4K16 acetylation was previously shown to disrupt formation of condensed chromatin in vitro. We propose that de-acetylation of H4K16 allowed formation of condensed chromatin in stationary phase, and that acetylation of H3K9, H3K14, H3K36, and H4K12 reflected the active transcriptional state of the yeast genome in exponential phase. PMID:21726436

  18. DNA Methylation

    PubMed Central

    Marinus, M.G.; Løbner-Olesen, A.

    2014-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential and in C. crescentus, it is important for temporal gene expression which, in turn, is required for coordinating chromosome initiation, replication and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage; decrease transformation frequency in certain bacteria; decrease the stability of short direct repeats; are necessary for site-directed mutagenesis; and to probe eukaryotic structure and function. PMID:26442938

  19. Infrared spectroscopy of the acetyl cation and its protonated ketene isomer

    NASA Astrophysics Data System (ADS)

    Mosley, J. D.; Young, J. W.; Duncan, M. A.

    2014-07-01

    [C2,H3,O]+ ions are generated with a pulsed discharge in a supersonic expansion containing methyl acetate or acetone. These ions are mass selected and their infrared spectra are recorded via laser photodissociation and the method of argon tagging. Computational chemistry is employed to investigate structural isomers and their spectra. The acetyl cation (CH3CO+) is the global minimum and protonated ketene (CH2COH+) is the next lowest energy isomer (+176.2 kJ/mol). When methyl acetate is employed as the precursor, the infrared spectrum reveals that only the acetyl cation is formed. Partially resolved rotational structure reveals rotation about the C3 axis. When acetone is used as the precursor, acetyl is still the most abundant cation, but there is also a minor component of protonated ketene. Computations reveal a significant barrier to interconversion between the two isomers (+221 kJ/mol), indicating that protonated ketene must be obtained via kinetic trapping. Both isomers may be present in interstellar environments, and their implications for astrochemistry are discussed.

  20. Infrared spectroscopy of the acetyl cation and its protonated ketene isomer

    SciTech Connect

    Mosley, J. D.; Young, J. W.; Duncan, M. A.

    2014-07-14

    [C{sub 2},H{sub 3},O]{sup +} ions are generated with a pulsed discharge in a supersonic expansion containing methyl acetate or acetone. These ions are mass selected and their infrared spectra are recorded via laser photodissociation and the method of argon tagging. Computational chemistry is employed to investigate structural isomers and their spectra. The acetyl cation (CH{sub 3}CO{sup +}) is the global minimum and protonated ketene (CH{sub 2}COH{sup +}) is the next lowest energy isomer (+176.2 kJ/mol). When methyl acetate is employed as the precursor, the infrared spectrum reveals that only the acetyl cation is formed. Partially resolved rotational structure reveals rotation about the C{sub 3} axis. When acetone is used as the precursor, acetyl is still the most abundant cation, but there is also a minor component of protonated ketene. Computations reveal a significant barrier to interconversion between the two isomers (+221 kJ/mol), indicating that protonated ketene must be obtained via kinetic trapping. Both isomers may be present in interstellar environments, and their implications for astrochemistry are discussed.

  1. Histone Acetylation Regulates Intracellular pH

    PubMed Central

    McBrian, Matthew A.; Behbahan, Iman Saramipoor; Ferrari, Roberto; Su, Trent; Huang, Ta-Wei; Li, Kunwu; Hong, Candice S.; Christofk, Heather R.; Vogelauer, Maria; Seligson, David B.; Kurdistani, Siavash K.

    2014-01-01

    SUMMARY Differences in global levels of histone acetylation occur in normal and cancer cells, although the reason why cells regulate these levels has been unclear. Here we demonstrate a role for histone acetylation in regulating intracellular pH (pHi). As pHi decreases, histones are globally deacetylated by histone deacetylases (HDACs), and the released acetate anions are coexported with protons out of the cell by monocarboxylate transporters (MCTs), preventing further reductions in pHi. Conversely, global histone acetylation increases as pHi rises, such as when resting cells are induced to proliferate. Inhibition of HDACs or MCTs decreases acetate export and lowers pHi, particularly compromising pHi maintenance in acidic environments. Global deacetylation at low pH is reflected at a genomic level by decreased abundance and extensive redistribution of acetylation throughout the genome. Thus, acetylation of chromatin functions as a rheostat to regulate pHi with important implications for mechanism of action and therapeutic use of HDAC inhibitors. PMID:23201122

  2. Proteomic analysis of acetylation in thermophilic Geobacillus kaustophilus.

    PubMed

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

    2013-08-01

    Recent analysis of prokaryotic N(ε)-lysine-acetylated proteins highlights the posttranslational regulation of a broad spectrum of cellular proteins. However, the exact role of acetylation remains unclear due to a lack of acetylated proteome data in prokaryotes. Here, we present the N(ε)-lysine-acetylated proteome of gram-positive thermophilic Geobacillus kaustophilus. Affinity enrichment using acetyl-lysine-specific antibodies followed by LC-MS/MS analysis revealed 253 acetylated peptides representing 114 proteins. These acetylated proteins include not only common orthologs from mesophilic Bacillus counterparts, but also unique G. kaustophilus proteins, indicating that lysine acetylation is pronounced in thermophilic bacteria. These data complement current knowledge of the bacterial acetylproteome and provide an expanded platform for better understanding of the function of acetylation in cellular metabolism. PMID:23696451

  3. Gestational choline supplementation normalized fetal alcohol-induced alterations in histone modifications, DNA methylation and POMC gene expression in β-endorphin-producing POMC neurons of the hypothalamus

    PubMed Central

    Bekdash, Rola A.; Zhang, Changqing; Sarkar, Dipak K.

    2013-01-01

    Background Prenatal exposure to ethanol reduces the expression of hypothalamic proopiomelanocortin (POMC) gene, known to control various physiological functions including the organismal stress response. In this study, we determined whether the changes in POMC neuronal functions are associated with altered expressions of histone-modifying and DNA-methylating enzymes in POMC-producing neurons, since these enzymes are known to be involved in regulation of gene expression. In addition, we tested whether gestational choline supplementation prevents the adverse effects of ethanol on these neurons. Methods Pregnant rat dams were fed with alcohol-containing liquid diet or control diet during gestational days 7 and 21 with or without choline, and their male offspring rats were used during the adult period. Using double-immunohistochemistry, real-time reverse transcription polymerase chain reaction (RT-PCR) and methylation specific RT-PCR, we determined protein and mRNA levels of histone-modifying and DNA-methylating enzymes, and the changes in POMC gene methylation and expression in the hypothalamus of adult male offspring rats. Additionally, we measured the basal and lipopolysaccharide (LPS)-induced corticosterone levels in plasma by enzyme-linked immunoabsorbent assay. Results Prenatal ethanol treatment suppressed hypothalamic levels of protein and mRNA of histone activation marks (H3K4me3, Set7/9, acetylated H3K9, phosphorylated H3S10) increased the repressive marks (H3K9me2, G9a, Setdb1) and DNA methylating enzyme (Dnmt1) and the methyl-CpG-binding protein (MeCP2). The treatment also elevated the level of POMC gene methylation, while it reduced levels of POMC mRNA and β-EP, and elevated corticosterone response to LPS. Gestational choline normalized the ethanol-altered protein and the mRNA levels of H3K4me3, Set7/9, H3K9me2, G9a, Setdb1, Dnmt1 and MeCP2. It also normalizes the changes in POMC gene methylation and gene expression, β-EP production and the corticosterone

  4. Synaptic plasticity and phosphorylation

    PubMed Central

    Lee, Hey-Kyoung

    2009-01-01

    A number of neuronal functions, including synaptic plasticity, depend on proper regulation of synaptic proteins, many of which can be rapidly regulated by phosphorylation. Neuronal activity controls the function of these synaptic proteins by exquisitely regulating the balance of various protein kinase and protein phosphatase activity. Recent understanding of synaptic plasticity mechanisms underscores important roles that these synaptic phosphoproteins play in regulating both pre- and post-synaptic functions. This review will focus on key postsynaptic phosphoproteins that have been implicated to play a role in synaptic plasticity. PMID:16904750

  5. Investigation of the quantitative metabolic fate and urinary excretion of 3-methyl-4-trifluoromethylaniline and 3-methyl-4-trifluoromethylacetanilide in the rat.

    PubMed

    Scarfe, G B; Lindon, J C; Nicholson, J K; Wright, B; Clayton, E; Wilson, I D

    1999-10-01

    The urinary metabolites of 3-methyl-4-trifluoromethylaniline in the rat were characterized and quantified using a combination of (19)F NMR, HPLC-NMR ((1)H and (19)F), and HPLC-mass spectrometry techniques. The major routes of metabolism were amine N-acetylation and methyl group C-oxidation to the benzyl alcohol (with subsequent glucuronide conjugation) and further to the corresponding benzoic acid derivative. Quantitatively only a small proportion of the urinary metabolites contained the free amino group, and these were products of ortho-hydroxylation (2 and 6 position) with additional conjugation to form the ether sulfates and glucuronides. An N-glucuronide of the parent compound was also identified. 3-Methyl-4-trifluoromethylacetanilide ((13)C-labeled in the acetyl group) gave virtually the same overall metabolite profile as 3-methyl-4-trifluoromethylaniline; however, a significant level of futile N-deacetylation and reacetylation occurred as ca. 50% of the excreted N-acetylated major metabolites contained no (13)C-label at the acetyl, having been replaced by an endogenous (12)C-acetyl source. This level of futile deacetylation is the highest yet reported for a substituted aniline/acetanilide and indicates a high degree of electronic activation of the amino group toward the acetyltransferase enzymes in vivo. PMID:10497144

  6. Determination of GPCR Phosphorylation Status: Establishing a Phosphorylation Barcode.

    PubMed

    Prihandoko, Rudi; Bradley, Sophie J; Tobin, Andrew B; Butcher, Adrian J

    2015-01-01

    G protein-coupled receptors (GPCRs) are rapidly phosphorylated following agonist occupation in a process that mediates receptor uncoupling from its cognate G protein, a process referred to as desensitization. In addition, this process provides a mechanism by which receptors can engage with arrestin adaptor molecules and couple to downstream signaling pathways. The importance of this regulatory process has been highlighted recently by the understanding that ligands can direct receptor signaling along one pathway in preference to another, the phenomenon of signaling bias that is partly mediated by the phosphorylation status or phosphorylation barcode of the receptor. Methods to determine the phosphorylation status of a GPCR in vitro and in vivo are necessary to understand not only the physiological mechanisms involved in GPCR signaling, but also to fully examine the signaling properties of GPCR ligands. This unit describes detailed methods for determining the overall phosphorylation pattern on a receptor (the phosphorylation barcode), as well as mass spectrometry approaches that can define the precise sites that become phosphorylated. These techniques, coupled with the generation and characterization of receptor phosphorylation-specific antibodies, provide a full palate of techniques necessary to determine the phosphorylation status of any given GPCR subtype. PMID:26344213

  7. Methyl chloride

    Integrated Risk Information System (IRIS)

    Methyl chloride ; CASRN 74 - 87 - 3 ( 07 / 17 / 2001 ) 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

  8. Methyl acrylate

    Integrated Risk Information System (IRIS)

    Methyl acrylate ; CASRN 96 - 33 - 3 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

  9. Methyl isocyanate

    Integrated Risk Information System (IRIS)

    Methyl isocyanate ; CASRN 624 - 83 - 9 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

  10. Methyl iodide

    Integrated Risk Information System (IRIS)

    Methyl iodide ; CASRN 74 - 88 - 4 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 Effe

  11. Methyl parathion

    Integrated Risk Information System (IRIS)

    Methyl parathion ; CASRN 298 - 00 - 0 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

  12. Methyl chlorocarbonate

    Integrated Risk Information System (IRIS)

    Methyl chlorocarbonate ; CASRN 79 - 22 - 1 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 Noncarcinog

  13. Methyl methacrylate

    Integrated Risk Information System (IRIS)

    Methyl methacrylate ; CASRN 80 - 62 - 6 ( 03 / 02 / 98 ) 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 f

  14. Acetylation of bleached Kraft pulp: effect of xylan content on properties of acetylated compounds.

    PubMed

    Peredo, Karol; Reyes, Herna; Escobar, Danilo; Vega-Lara, Johana; Berg, Alex; Pereira, Miguel

    2015-03-01

    Bleached Kraft pulp (BKP) from Eucalyptus globulus and cotton xylan blends (CXB) was acetylated. The effects of xylan content on cellulose acetylation and the properties of the acetylated material were studied. An increase in xylan content caused a slight decrease in the degree of substitution (2.98 to 2.68 for CXB; 2.93 to 2.84 for BKP). Thermal analysis showed that the melting temperature also decreases from 268.0 to 188.8 °C for CXB and from 221.4 to 212.8 °C for BKP. Moreover, the solubility decreased due to the partial dissolution of acetylated xylans. The presence of xylans during Kraft pulp acetylation does not have a significant negative effect on the physical properties of the acetylated material, but the decrease in melting temperature was beneficial for the application of acetylated polymer as a natural internal plasticizer. This is considered to be an important argument for BKP utilization in the cellulose acetate manufacturing process. PMID:25498729

  15. Uncouplers of oxidative phosphorylation.

    PubMed

    Terada, H

    1990-07-01

    Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling between the electron transport and phosphorylation reactions and thus inhibit ATP synthesis without affecting the respiratory chain and ATP synthase (H(+)-ATPase). Miscellaneous compounds are known to be uncouplers, but weakly acidic uncouplers are representative because they show very potent activities. The most potent uncouplers discovered so far are the hindered phenol SF 6847, and hydrophobic salicylanilide S-13, which are active in vitro at concentrations in the 10 nM range. For induction of uncoupling, an acid dissociable group, bulky hydrophobic moiety and strong electron-withdrawing group are required. Weakly acidic uncouplers are considered to produce uncoupling by their protonophoric action in the H(+)-impermeable mitochondrial membrane. For exerting these effects, the stability of the respective uncoupler anions in the hydrophobic membrane is very important. High stability is achieved by delocalization of the polar ionic charge through uncoupler (chemical)-specific mechanisms. Such an action of weakly acidic uncouplers is characteristic of the highly efficient membrane targeting action of a nonsite-specific type of bioactive compound. PMID:2176586

  16. Uncouplers of oxidative phosphorylation.

    PubMed Central

    Terada, H

    1990-01-01

    Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling between the electron transport and phosphorylation reactions and thus inhibit ATP synthesis without affecting the respiratory chain and ATP synthase (H(+)-ATPase). Miscellaneous compounds are known to be uncouplers, but weakly acidic uncouplers are representative because they show very potent activities. The most potent uncouplers discovered so far are the hindered phenol SF 6847, and hydrophobic salicylanilide S-13, which are active in vitro at concentrations in the 10 nM range. For induction of uncoupling, an acid dissociable group, bulky hydrophobic moiety and strong electron-withdrawing group are required. Weakly acidic uncouplers are considered to produce uncoupling by their protonophoric action in the H(+)-impermeable mitochondrial membrane. For exerting these effects, the stability of the respective uncoupler anions in the hydrophobic membrane is very important. High stability is achieved by delocalization of the polar ionic charge through uncoupler (chemical)-specific mechanisms. Such an action of weakly acidic uncouplers is characteristic of the highly efficient membrane targeting action of a nonsite-specific type of bioactive compound. PMID:2176586

  17. Measuring N-acetyl aspartate synthesis in vivo using proton magnetic resonance spectroscopy

    PubMed Central

    Xu, Su; Yang, Jehoon; Shen, Jun

    2008-01-01

    N-acetyl aspartate (NAA) is an important marker of neuronal function and viability that can be measured using magnetic resonance spectroscopy (MRS). In this paper, we proposed a method to measure NAA synthesis using proton MRS with infusion of uniformly 13C-labeled glucose, and demonstrated its feasibility in an in vivo study of the rat brain. The rate of 13C-label incorporation into the acetyl group of NAA was measured using a localized, long echo-time proton MRS method. Signals from the 13C satellites of the main NAA methyl protons at 2.02 ppm were continuously monitored for 10 hours. Quantification of the data based on a linear kinetic model showed that NAA synthesis rate in isoflurane-anesthetized rats was 0.19 ± 0.02 µmol/g/h (mean ± standard deviation, n = 12). PMID:18486230

  18. (E)-(25S)-23-Acetyl-5β-furost-22-ene-3β,26-diol

    PubMed Central

    Hernández Linares, María-Guadalupe; Sandoval Ramírez, Jesús; Meza Reyes, Socorro; Montiel Smith, Sara; Bernès, Sylvain

    2008-01-01

    The title steroid, C29H46O4, is a furostene derivative with a C=C double-bond length of 1.353 (3) Å and an E configuration. The side chain is oriented toward the α face of the A–E steroidal nucleus and presents a disordered terminal CH2—OH group [occupancies for resolved sites are 0.591 (9) and 0.409 (9)]. The methyl group at C20 attached to ring E is also oriented toward the α face, avoiding steric hindrance with the carbonyl O atom of the acetyl group. The furostene and acetyl functionalities form an α,β-unsaturated ketone system, with an s-cis configuration. All hydr­oxy and carbonyl groups are involved in weak inter­molecular hydrogen bonds. The absolute configuration was assigned from the synthesis. PMID:21201948

  19. Non-enzymatic protein acetylation detected by NAPPA protein arrays*

    PubMed Central

    Olia, Adam S.; Barker, Kristi; McCullough, Cheryl E.; Tang, Hsin-Yao; Speicher, David W.; Qiu, Ji; LaBaer, Joshua; Marmorstein, Ronen

    2015-01-01

    Acetylation is a post-translational modification that occurs on thousands of proteins located in many cellular organelles. This process mediates many protein functions and modulates diverse biological processes. In mammalian cells, where acetyl-CoA is the primary acetyl donor, acetylation in the mitochondria is thought to occur by chemical means due to the relatively high concentration of acetyl-CoA located in this organelle. In contrast, acetylation outside of the mitochondria is thought to be mediated predominantly by acetyltransferase enzymes. Here we address the possibility that non-enzymatic chemical acetylation outside of the mitochondria may be more common than previously appreciated. We employed the Nucleic Acid Programmable Protein Array platform to perform an unbiased screen for human proteins that undergo chemical acetylation, which resulted in the identification of a multitude of proteins with diverse functions and cellular localization. Mass spectrometry analysis revealed that basic residues typically precede the acetylated lysine in the −7 to −3 position, and we show by mutagenesis that these basic residues contribute to chemical acetylation capacity. We propose that these basic residues lower the pKa of the substrate lysine for efficient chemical acetylation. Many of the identified proteins reside outside of the mitochondria, and have been previously demonstrated to be acetylated in vivo. As such, our studies demonstrate that chemical acetylation occurs more broadly throughout the eukaryotic cell than previously appreciated, and suggests that this post-translational protein modification may have more diverse roles in protein function and pathway regulation. PMID:26083674

  20. Nucleosome structure incorporated histone acetylation site prediction in arabidopsis thaliana

    PubMed Central

    2010-01-01

    Abstract Background Acetylation is a crucial post-translational modification for histones, and plays a key role in gene expression regulation. Due to limited data and lack of a clear acetylation consensus sequence, a few researches have focused on prediction of lysine acetylation sites. Several systematic prediction studies have been conducted for human and yeast, but less for Arabidopsis thaliana. Results Concerning the insufficient observation on acetylation site, we analyzed contributions of the peptide-alignment-based distance definition and 3D structure factors in acetylation prediction. We found that traditional structure contributes little to acetylation site prediction. Identified acetylation sites of histones in Arabidopsis thaliana are conserved and cross predictable with that of human by peptide based methods. However, the predicted specificity is overestimated, because of the existence of non-observed acetylable site. Here, by performing a complete exploration on the factors that affect the acetylability of lysines in histones, we focused on the relative position of lysine at nucleosome level, and defined a new structure feature to promote the performance in predicting the acetylability of all the histone lysines in A. thaliana. Conclusion We found a new spacial correlated acetylation factor, and defined a ε-N spacial location based feature, which contains five core spacial ellipsoid wired areas. By incorporating the new feature, the performance of predicting the acetylability of all the histone lysines in A. Thaliana was promoted, in which the previous mispredicted acetylable lysines were corrected by comparing to the peptide-based prediction. PMID:21047388

  1. Protein phosphorylation in stomatal movement

    PubMed Central

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  2. Phosphorylation site prediction in plants.

    PubMed

    Yao, Qiuming; Schulze, Waltraud X; Xu, Dong

    2015-01-01

    Protein phosphorylation events on serine, threonine, and tyrosine residues are the most pervasive protein covalent bond modifications in plant signaling. Both low and high throughput studies reveal the importance of phosphorylation in plant molecular biology. Although becoming more and more common, the proteome-wide screening on phosphorylation by experiments remains time consuming and costly. Therefore, in silico prediction methods are proposed as a complementary analysis tool to enhance the phosphorylation site identification, develop biological hypothesis, or help experimental design. These methods build statistical models based on the experimental data, and they do not have some of the technical-specific bias, which may have advantage in proteome-wide analysis. More importantly computational methods are very fast and cheap to run, which makes large-scale phosphorylation identifications very practical for any types of biological study. Thus, the phosphorylation prediction tools become more and more popular. In this chapter, we will focus on plant specific phosphorylation site prediction tools, with essential illustration of technical details and application guidelines. We will use Musite, PhosPhAt and PlantPhos as the representative tools. We will present the results on the prediction of the Arabidopsis protein phosphorylation events to give users a general idea of the performance range of the three tools, together with their strengths and limitations. We believe these prediction tools will contribute more and more to the plant phosphorylation research community. PMID:25930706

  3. Protein phosphorylation in stomatal movement.

    PubMed

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  4. Structure Elucidation of New Acetylated Saponins, Lessoniosides A, B, C, D, and E, and Non-Acetylated Saponins, Lessoniosides F and G, from the Viscera of the Sea Cucumber Holothuria lessoni

    PubMed Central

    Bahrami, Yadollah; Franco, Christopher M. M.

    2015-01-01

    Sea cucumbers produce numerous compounds with a wide range of chemical structural diversity. Among these, saponins are the most diverse and include sulfated, non-sulfated, acetylated and methylated congeners with different aglycone and sugar moieties. In this study, MALDI and ESI tandem mass spectrometry, in the positive ion mode, were used to elucidate the structure of new saponins extracted from the viscera of H. lessoni. Fragmentation of the aglycone provided structural information on the presence of the acetyl group. The presence of the O-acetyl group was confirmed by observing the mass transition of 60 u corresponding to the loss of a molecule of acetic acid. Ion fingerprints from the glycosidic cleavage provided information on the mass of the aglycone (core), and the sequence and type of monosaccharides that constitute the sugar moiety. The tandem mass spectra of the saponin precursor ions [M + Na]+ provided a wealth of detailed structural information on the glycosidic bond cleavages. As a result, and in conjunction with existing literature, we characterized the structure of five new acetylated saponins, Lessoniosides A–E, along with two non-acetylated saponins Lessoniosides F and G at m/z 1477.7, which are promising candidates for future drug development. The presented strategy allows a rapid, reliable and complete analysis of native saponins. PMID:25603350

  5. The effect of N-acetyl-aspartyl-glutamate and N-acetyl-aspartate on white matter oligodendrocytes.

    PubMed

    Kolodziejczyk, Karolina; Hamilton, Nicola B; Wade, Anna; Káradóttir, Ragnhildur; Attwell, David

    2009-06-01

    Elevations of the levels of N-acetyl-aspartyl-glutamate (NAAG) and N-acetyl-aspartate (NAA) are associated with myelin loss in the leucodystrophies Canavan's disease and Pelizaeus-Merzbacher-like disease. NAAG and NAA can activate and antagonize neuronal N-methyl-D-aspartate (NMDA) receptors, and also act on group II metabotropic glutamate receptors. Oligodendrocytes and their precursors have recently been shown to express NMDA receptors, and activation of these receptors in ischaemia leads to the death of oligodendrocyte precursors and the loss of myelin. This raises the possibility that the failure to develop myelin, or demyelination, occurring in the leucodystrophies could reflect an action of NAAG or NAA on oligodendrocyte NMDA receptors. However, since the putative subunit composition of NMDA receptors on oligodendrocytes differs from that of neuronal NMDA receptors, the effects of NAAG and NAA on them are unknown. We show that NAAG, but not NAA, evokes an inward membrane current in cerebellar white matter oligodendrocytes, which is reduced by NMDA receptor block (but not by block of metabotropic glutamate receptors). The size of the current evoked by NAAG, relative to that evoked by NMDA, was much smaller in oligodendrocytes than in neurons, and NAAG induced a rise in [Ca(2+)](i) in neurons but not in oligodendrocytes. These differences in the effect of NAAG on oligodendrocytes and neurons may reflect the aforementioned difference in receptor subunit composition. In addition, as a major part of the response in oligodendrocytes was blocked by tetrodotoxin (TTX), much of the NAAG-evoked current in oligodendrocytes is a secondary consequence of activating neuronal NMDA receptors. Six hours exposure to 1 mM NAAG did not lead to the death of cells in the white matter. We conclude that an action of NAAG on oligodendrocyte NMDA receptors is unlikely to be a major contributor to white matter damage in the leucodystrophies. PMID:19383832

  6. Insulin stimulates the dephosphorylation and activation of acetyl-CoA carboxylase

    SciTech Connect

    Witters, L.A.; Watts, T.D.; Daniels, D.L.; Evans, J.L. )

    1988-08-01

    The mechanism underlying the ability of insulin to acutely activate acetyl-CoA carboxylase has been examined in Fao Reuber hepatoma cells. Insulin promotes the rapid activation of AcCoACase, as measured in cell lysates, and this stimulation persists to the same degree after isolation of AcCoACase by avidin-Sepharose chromatography. The insulin-stimulated enzyme, as compared with control enzyme, exhibits an increase in both citrate-independent and -dependent activity and a decrease in the K{sub a} for citrate. Direct examination of the phosphorylation state of isolated {sup 32}P-labeled AcCoACase after insulin exposure reveals a marked decrease in total enzyme phosphorylation coincident with activation. The dephosphorylation due to insulin appears to be restricted to the phosphorylation sites previously shown to regulate AcCoACase activity. All of these effects of insulin are mimicked by a low molecular weight autocrine factor, tentatively identified as an oligosaccharide, present in conditioned medium of hepatoma cells. These data suggest that insulin may activate AcCoACase by inhibiting the activity of protein kinase(s) or stimulating the activity of protein phosphatase(s) that control the phosphorylation state of the enzyme.

  7. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, P.G.; Ohlrogge, J.B.

    1996-09-24

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.

  8. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... molecular distillation or by steam stripping; or (2) The direct acetylation of edible monoglycerides with acetic anhydride without the use of catalyst or molecular distillation, and with the removal by vacuum distillation, if necessary, of the acetic acid, acetic anhydride, and triacetin. (b) The food additive has...

  9. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... molecular distillation or by steam stripping; or (2) The direct acetylation of edible monoglycerides with acetic anhydride without the use of catalyst or molecular distillation, and with the removal by vacuum distillation, if necessary, of the acetic acid, acetic anhydride, and triacetin. (b) The food additive has...

  10. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... molecular distillation or by steam stripping; or (2) The direct acetylation of edible monoglycerides with acetic anhydride without the use of catalyst or molecular distillation, and with the removal by vacuum distillation, if necessary, of the acetic acid, acetic anhydride, and triacetin. (b) The food additive has...

  11. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... molecular distillation or by steam stripping; or (2) The direct acetylation of edible monoglycerides with acetic anhydride without the use of catalyst or molecular distillation, and with the removal by vacuum distillation, if necessary, of the acetic acid, acetic anhydride, and triacetin. (b) The food additive has...

  12. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, Paul G.; Ohlrogge, John B.

    1996-01-01

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives thereof which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides.

  13. Phosphorylated. beta. -dicarbonyl compounds

    SciTech Connect

    Liorber, B.G.; Tarzivolova, T.A.; Pavlov, V.A.; Zykova, T.V.; Kisilev, V.V.; Tumasheva, N.A.; Slizkii, A.Yu.; Shagvaleev, F.S.

    1987-08-20

    The reaction of trialkyl phosphites with alkyl malonyl chlorides leads to alkyl 3-dialkoxyphosphoryl-3-oxopropionates, which exist in the stable E-enol form. Depending on the basicities of the bases, the reactions of alkyl 3-dialkoxyphosphoryl-3-oxopropionates with nitrogen bases proceed with retention of the C-P bond and the formation of phosphorylated azomethine derivatives or with cleavage of the C-P bond and the liberation of nitrogen-containing derivatives of malonic acid. The /sup 1/H, /sup 13/C, and /sup 13/P NMR spectra were recorded with a Bruker WP-80 NMR spectrometer. The chemical shifts of the protons and carbon atoms are presented relative to tetramethylsilane (TMS). The chemical shifts of the /sup 31/P nuclei were determined relative to H/sub 3/PO/sub 4/.

  14. Lectin-induced activation of platelets may require only limited phosphorylation of the 47K protein

    SciTech Connect

    Ganguly, C.; Chelladurai, M.; Ganguly, P.

    1986-05-01

    Wheat germ agglutinin (WGA) is an N-acetylglucosamine (Glc-NAc) specific lectin which can activate platelets. Like thrombin, stimulation of platelets by WGA is accompanied by enhanced phosphorylation of two polypeptides of M/sub r/ 47K and 20K. Addition of GlcNAc at different time intervals arrested that aggregation of platelets by WGA and paralleled the modification of phosphorylation of the 47K polypeptide. So, the phosphorylation of the 47K polypeptide may regulate the WGA-receptor mediated stimulation of platelets. However, the ratio of phosphoserine to phosphothreonine in the 47K protein was markedly different in WGA-activated than thrombin-stimulated platelets. Thus, the molecular mechanism of action of thrombin and WGA could be different. To explore this idea, /sup 32/P/sub i/-labeled platelets were stimulated with WGA and the activation arrested with N-acetyl-glucosamine at different times. Two-dimensional gel electrophoresis of total protein at 5s showed only two phosphorylated species of 47K protein. At 60s, maximally four phosphorylated species were noted. In contrast, with thrombin using the same technique, seven to nine phosphorylated components have been reported. These results suggest that the different activators of platelets may act by different mechanisms. In addition, activation of platelets may require only limited levels of phosphorylation of the 47K polypeptide.

  15. Investigation of Mercury Methylation Pathways in Biofilm versus Planktonic Cultures of Desulfovibrio desulfuricans

    PubMed Central

    Lin, Tiffany Y.; Kampalath, Rita A.; Lin, Chu-Ching; Zhang, Ming; Chavarria, Karina; Lacson, Jessica; Jay, Jennifer A.

    2014-01-01

    Biofilms can methylate mercury (Hg) at higher rates than unattached bacteria and are increasingly recognized as important Hg methylation sites in the environment. Our previous study showed that methylation rates in biofilm cultures were up to 1 order of magnitude greater than those in planktonic cultures of a sulfate-reducing bacterium. To probe whether the differential Hg methylation rates resulted from metabolic differences between these two cultures, Hg methylation assays following molybdate or chloroform inhibition (a specific inhibitor of the acetyl-CoA pathway) were conducted on biofilm and planktonic cultures of Desulfovibrio desulfuricans strains M8 and ND132. Molybdate was as effective in inhibiting Hg methylation as well as growth in both planktonic and biofilm cultures. The addition of chloroform only impacted Hg methylation in biofilm cultures, suggesting that different pathways are used for methylation in biofilm compared to planktonic cultures. To investigate this further, expression of the cooS gene, which encodes for carbon monoxide dehydrogenase, a key enzyme in the acetyl-CoA pathway, was compared in biofilm and planktonic cultures of ND132. Biofilm cultures showed up to 4 times higher expression of cooS than planktonic cultures. On the basis of these results, the acetyl-CoA pathway appears to play an important role in methylation in biofilm cultures of this organism, possibly by supplying the methyl group to Hg methylating enzymes; methylation in planktonic cultures appears to be independent of this pathway. This observation has important implications, particularly in developing reliable models to predict Hg methylation rates in different environments and perhaps eventually in being able to control this undesirable chemical transformation. PMID:23634937

  16. [3H]Indole-3-acetyl-myo-inositol hydrolysis by extracts of Zea mays L. vegetative tissue

    NASA Technical Reports Server (NTRS)

    Hall, P. J.; Bandurski, R. S.

    1986-01-01

    [3H]Indole-3-acetyl-myo-inositol was hydrolyzed by buffered extracts of acetone powders prepared from 4 day shoots of dark grown Zea mays L. seedlings. The hydrolytic activity was proportional to the amount of extract added and was linear for up to 6 hours at 37 degrees C. Boiled or alcohol denatured extracts were inactive. Analysis of reaction mixtures by high performance liquid chromatography demonstrated that not all isomers of indole-3-acetyl-myo-inositol were hydrolyzed at the same rate. Buffered extracts of acetone powders were prepared from coleoptiles and mesocotyls. The rates of hydrolysis observed with coleoptile extracts were greater than those observed with mesocotyl extracts. Active extracts also catalyzed the hydrolysis of esterase substrates such as alpha-naphthyl acetate and the methyl esters of indoleacetic acid and naphthyleneacetic acid. Attempts to purify the indole-3-acetyl-myo-inositol hydrolyzing activity by chromatographic procedures resulted in only slight purification with large losses of activity. Chromatography over hydroxylapatite allowed separation of two enzymically active fractions, one of which catalyzed the hydrolysis of both indole-3-acetyl-myo-inositol and esterase substrates. With the other enzymic hydrolysis of esterase substrates was readily demonstrated, but no hydrolysis of indole-3-acetyl-myo-inositol was ever detected.

  17. Isolation, purification and structural characterization of an acetylated heteroglycan from the unripe fruits of Manilkara zapota L.

    PubMed

    Mondal, Subhas; Das, Debsankar; Roy, Sadhan K; Islam, Syed S

    2012-06-01

    A water soluble polysaccharide isolated from the hot water extract of the unripe fruits of Manilkara zapota L. was found to consist of 3-O-acyl-L-rhamnose, L-arabinose, 3-O-acetyl-D-methyl galacturonate in a molar proportion of nearly 1:1:1. Structural investigation of the polysaccharide was carried out using total hydrolysis, methylation analysis; periodate oxidation followed by GLC-MS, and NMR experiments. On the basis of the above experiments it is concluded that the following repeating unit is present in the polysaccharide. PMID:22560630

  18. Quantitative Proteomic Atlas of Ubiquitination and Acetylation in the DNA Damage Response

    PubMed Central

    Elia, Andrew E.H.; Boardman, Alexander P.; Wang, David C.; Huttlin, Edward L.; Everley, Robert A.; Dephoure, Noah; Zhou, Chunshui; Koren, Itay; Gygi, Steven P.; Elledge, Stephen J.

    2015-01-01

    Summary Execution of the DNA damage response (DDR) relies upon a dynamic array of protein modifications. Using quantitative proteomics, we have globally profiled ubiquitination, acetylation, and phosphorylation in response to ultraviolet and ionizing radiation. To improve acetylation site profiling, we developed the strategy FACET-IP. Our datasets of 33,500 ubiquitination and 16,740 acetylation sites provide valuable insight into DDR remodeling of the proteome. We find that K6- and K33-linked polyubiquitination undergo bulk increases in response to DNA damage, raising the possibility that these linkages are largely dedicated to DDR function. We also show that Cullin Ring Ligases mediate 10% of DNA damage induced ubiquitination events and that EXO1 is an SCF-Cyclin F substrate in the response to UV radiation. Our extensive datasets uncover additional regulated sites on known DDR players such as PCNA and identify previously unknown DDR targets such as CENPs, underscoring the broad impact of the DDR on cellular physiology. PMID:26051181

  19. Inhibition of DNA Methylation Impairs Synaptic Plasticity during an Early Time Window in Rats

    PubMed Central

    Díaz, Paula; Ardiles, Álvaro O.

    2016-01-01

    Although the importance of DNA methylation-dependent gene expression to neuronal plasticity is well established, the dynamics of methylation and demethylation during the induction and expression of synaptic plasticity have not been explored. Here, we combined electrophysiological, pharmacological, molecular, and immunohistochemical approaches to examine the contribution of DNA methylation and the phosphorylation of Methyl-CpG-binding protein 2 (MeCP2) to synaptic plasticity. We found that, at twenty minutes after theta burst stimulation (TBS), the DNA methylation inhibitor 5-aza-2-deoxycytidine (5AZA) impaired hippocampal long-term potentiation (LTP). Surprisingly, after two hours of TBS, when LTP had become a transcription-dependent process, 5AZA treatment had no effect. By comparing these results to those in naive slices, we found that, at two hours after TBS, an intergenic region of the RLN gene was hypomethylated and that the phosphorylation of residue S80 of MeCP2 was decreased, while the phosphorylation of residue S421 was increased. As expected, 5AZA affected only the methylation of the RLN gene and exerted no effect on MeCP2 phosphorylation patterns. In summary, our data suggest that tetanic stimulation induces critical changes in synaptic plasticity that affects both DNA methylation and the phosphorylation of MeCP2. These data also suggest that early alterations in DNA methylation are sufficient to impair the full expression of LTP. PMID:27493805

  20. Modulation of 14-3-3 interaction with phosphorylated histone H3 by combinatorial modification patterns

    PubMed Central

    Winter, Stefan; Fischle, Wolfgang; Seiser, Christian

    2011-01-01

    Post-translational modifications of histones are determining factors in the global and local regulation of genome activity. Phosphorylation of histone H3 is globally associated with mitotic chromatin compaction but occurs in a much more restricted manner during interphase transcriptional regulation of a limited subset of genes. In the course of gene regulation, serine 10 phosphorylation at histone H3 is targeted to a very small fraction of nucleosomes that is highly susceptible to additional acetylation events. Recently, we and others have identified 14-3-3 as a binding protein that recognizes both phosphorylated serine 10 and phosphorylated serine 28 on histone H3. In vitro, the affinity of 14-3-3 for phosphoserine 10 is weak but becomes significantly increased by additional acetylation of either lysine 9 or lysine 14 on the same histone tail. In contrast, the histone H3S28 site matches elements of 14-3-3 high affinity consensus motifs. This region mediates an initial stronger interaction that is less susceptible to modulation by “auxiliary” modifications. Here we discuss the binding of 14-3-3 proteins to histone H3 in detail and putative biological implications of these interactions. PMID:18418070

  1. Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.

    PubMed

    Chen, Chongguang; Chiu, Yi-Ting; Wu, Wenman; Huang, Peng; Mann, Anika; Schulz, Stefan; Liu-Chen, Lee-Yuan

    2016-02-15

    Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated

  2. Methyl gallate.

    PubMed

    Bebout, Deborah; Pagola, Silvina

    2009-01-01

    THE CRYSTAL STRUCTURE OF THE TITLE COMPOUND (SYSTEMATIC NAME: methyl 3,4,5-trihydroxy-benzoate), C(8)H(8)O(5), is composed of essentially planar mol-ecules [maximum departures from the mean carbon and oxygen skeleton plane of 0.0348 (10) Å]. The H atoms of the three hydroxyl groups, which function as hydrogen-bond donors and acceptors simultaneously, are oriented in the same direction around the aromatic ring. In addition to two intra-molecular hydrogen bonds, each mol-ecule is hydrogen bonded to six others, creating a three-dimensional hydrogen-bonded network. PMID:21581923

  3. Methyl gallate

    PubMed Central

    Bebout, Deborah; Pagola, Silvina

    2009-01-01

    The crystal structure of the title compound (systematic name: methyl 3,4,5-trihydroxy­benzoate), C8H8O5, is composed of essentially planar mol­ecules [maximum departures from the mean carbon and oxygen skeleton plane of 0.0348 (10) Å]. The H atoms of the three hydroxyl groups, which function as hydrogen-bond donors and acceptors simultaneously, are oriented in the same direction around the aromatic ring. In addition to two intra­molecular hydrogen bonds, each mol­ecule is hydrogen bonded to six others, creating a three-dimensional hydrogen-bonded network. PMID:21581923

  4. 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

  5. [Epigenetics and drug addiction: a focus on MeCP2 and on histone acetylation].

    PubMed

    Zwiller, Jean

    2015-04-01

    Chronic drug exposure alters gene expression in the brain, which is believed to underlie compulsive drug seeking and drug taking behavior. Recent evidence shows that drug-induced long-term neuroadaptations in the brain are mediated in part by epigenetic mechanisms. By remodeling chromatin, this type of regulation contributes to drug-induced synaptic plasticity that translates into behavioral modifications. How drug-induced alterations in DNA methylation regulate gene expression is reviewed here, with a focus on MeCP2, a protein binding methylated DNA. The importance of histone modifications, especially acetylation is also discussed, with an emphasis on the effects of inhibitors of histone deacetylases on drug-induced behavioral changes. The precise identification of the epigenetic mechanisms that are under the control of drugs of abuse may help to uncover novel targets for the treatment of drug seeking and relapse. PMID:25958763

  6. The neurobiology of acetyl-L-carnitine.

    PubMed

    Traina, Giovanna

    2016-01-01

    A large body of evidence points to the positive effects of dietary supplementation of acetyl-L-carnitine (ALC). Its use has shown health benefits in neuroinflammation, which is a common denominator in a host of neurodegenerative diseases. ALC is the principal acetyl ester of L-Carnitine (LC), and it plays an essential role in intermediary metabolism, acting as a donor of acetyl groups and facilitating the transfer of fatty acids from cytosol to mitochondria during beta-oxidation. Dietary supplementation of ALC exerts neuroprotective, neurotrophic, antidepressive and analgesic effects in painful neuropathies. ALC also has antioxidant and anti-apoptotic activity. Moreover, ALC exhibits positive effects on mitochondrial metabolism, and shows promise in the treatment of aging and neurodegenerative pathologies by slowing the progression of mental deterioration. In addition, ALC plays neuromodulatory effects on both synaptic morphology and synaptic transmission. These effects are likely due to affects of ALC through modulation of gene expression on several targets in the central nervous system. Here, we review the current state of knowledge on effects of ALC in the nervous system. PMID:27100509

  7. Enhancement of lysine acetylation accelerates wound repair

    PubMed Central

    Spallotta, Francesco; Cencioni, Chiara; Straino, Stefania; Sbardella, Gianluca; Castellano, Sabrina; Capogrossi, Maurizio C; Martelli, Fabio; Gaetano, Carlo

    2013-01-01

    In physiopathological conditions, such as diabetes, wound healing is significantly compromised and chronic complications, including ulcers, may occur. In a mouse model of skin repair, we recently reported that wound treatment with Sirtuin activators and class I HDAC inhibitors induced keratinocyte proliferation and enhanced healing via a nitric oxide (NO) dependent mechanism. We observed an increase in total protein acetylation in the wound area, as determined by acetylation of α-tubulin and histone H3 Lysine 9. We reasoned that this process activated cell function as well as regulated gene expression to foster tissue repair. We report here that the direct activation of P300/CBP-associated factor (PCAF) by the histone acetylase activator pentadecylidenemalonate 1b (SPV-106) induced Lysine acetylation in the wound area. This intervention was sufficient to enhance repair process by a NO-independent mechanism. Hence, an impairment of PCAF and/or other GCN5 family acetylases may delay skin repair in physiopathological conditions. PMID:24265859

  8. Fragrance material review on acetyl cedrene.

    PubMed

    Scognamiglio, J; Letizia, C S; Politano, V T; Api, A M

    2013-12-01

    A toxicologic and dermatologic review of acetyl cedrene when used as a fragrance ingredient is presented. Acetyl cedrene is a member of the fragrance structural group Alkyl Cyclic Ketones. The generic formula for this group can be represented as (R1)(R2)CO. These fragrances can be described as being composed of an alkyl, R1, and various substituted and bicyclic saturated or unsaturated cyclic hydrocarbons, R2, in which one of the rings may include up to 12 carbons. Alternatively, R2 may be a carbon bridge of C2-C4 carbon chain length between the ketone and cyclic hydrocarbon. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for acetyl cedrene were evaluated then summarized and includes physical properties, acute toxicity, skin irritation, mucous membrane (eye) irritation, skin sensitization, elicitation, phototoxicity, photoallergy, toxicokinetics, repeated dose, reproductive toxicity, and genotoxicity data. A safety assessment of the entire Alkyl Cyclic Ketones will be published simultaneously with this document; please refer to Belsito et al. (2013) (Belsito, D., Bickers, D., Bruze, M., Calow, P., Dagli, M., Fryer, A.D., Greim, H., Miyachi, Y., Saurat, J.H., Sipes, I.G., 2013. A Toxicologic and Dermatologic Assessment of Alkyl Cyclic Ketones When Used as Fragrance Ingredients. Submitted with this manuscript.) for an overall assessment of the safe use of this material and all Alkyl Cyclic Ketones in fragrances. PMID:23907023

  9. Loss of BubR1 acetylation causes defects in spindle assembly checkpoint signaling and promotes tumor formation

    PubMed Central

    Park, Inai; Lee, Hae-ock; Choi, Eunhee; Lee, Yoo-Kyung; Kwon, Mi-Sun; Min, Jaewon; Park, Pil-Gu; Lee, Seonju; Kong, Young-Yun; Gong, Gyungyub

    2013-01-01

    BubR1 acetylation is essential in mitosis. Mice heterozygous for the acetylation-deficient BubR1 allele (K243R/+) spontaneously developed tumors with massive chromosome missegregations. K243R/+ mouse embryonic fibroblasts (MEFs) exhibited a weakened spindle assembly checkpoint (SAC) with shortened mitotic timing. The generation of the SAC signal was intact, as Mad2 localization to the unattached kinetochore (KT) was unaltered; however, because of the premature degradation of K243R-BubR1, the mitotic checkpoint complex disassociated prematurely in the nocodazole-treated condition, suggesting that maintenance of the SAC is compromised. BubR1 acetylation was also required to counteract excessive Aurora B activity at the KT for stable chromosome–spindle attachments. The association of acetylation-deficient BubR1 with PP2A-B56α phosphatase was reduced, and the phosphorylated Ndc80 at the KT was elevated in K243R/+ MEFs. In relation, there was a marked increase of micronuclei and p53 mutation was frequently detected in primary tumors of K243R/+ mice. Collectively, the combined effects of failure in chromosome–spindle attachment and weakened SAC cause genetic instability and cancer in K243R/+ mice. PMID:23878276

  10. Reduction and Acetylation of 2,4-Dinitrotoluene by a Pseudomonas aeruginosa Strain

    PubMed Central

    Noguera, D. R.; Freedman, D. L.

    1996-01-01

    Aerobic and anoxic biotransformation of 2,4-dinitrotoluene (DNT) was examined by using a Pseudomonas aeruginosa strain isolated from a plant treating propellant manufacturing wastewater. DNT biotransformation in the presence and absence of oxygen was mostly reductive and was representative of the type of cometabolic transformations that occur when a high concentration of an easily degradable carbon source is present. P. aeruginosa reduced both nitro groups on DNT, with the formation of mainly 4-amino-2-nitrotoluene and 2-amino-4-nitrotoluene and small quantities of 2,4-diaminotoluene. Acetylation of the arylamines was a significant reaction. 4-Acetamide-2-nitrotoluene and the novel compounds 2-acetamide-4-nitrotoluene, 4-acetamide-2-aminotoluene, and 2,4-diacetamidetoluene were identified as DNT metabolites. The biotransformation of 2,4-diaminotoluene to 4-acetamide-2-aminotoluene was 24 times faster than abiotic transformation. 2-Nitrotoluene and 4-nitrotoluene were also reduced to their corresponding toluidines and then acetylated. However, the yield of 4-acetamidetoluene was much higher than that of 2-acetamidetoluene, demonstrating that acetylation at the position para to the methyl group was favored. PMID:16535348

  11. MOF Acetylates the Histone Demethylase LSD1 to Suppress Epithelial-to-Mesenchymal Transition.

    PubMed

    Luo, Huacheng; Shenoy, Anitha K; Li, Xuehui; Jin, Yue; Jin, Lihua; Cai, Qingsong; Tang, Ming; Liu, Yang; Chen, Hao; Reisman, David; Wu, Lizi; Seto, Edward; Qiu, Yi; Dou, Yali; Casero, Robert A; Lu, Jianrong

    2016-06-21

    The histone demethylase LSD1 facilitates epithelial-to-mesenchymal transition (EMT) and tumor progression by repressing epithelial marker expression. However, little is known about how its function may be modulated. Here, we report that LSD1 is acetylated in epithelial but not mesenchymal cells. Acetylation of LSD1 reduces its association with nucleosomes, thus increasing histone H3K4 methylation at its target genes and activating transcription. The MOF acetyltransferase interacts with LSD1 and is responsible for its acetylation. MOF is preferentially expressed in epithelial cells and is downregulated by EMT-inducing signals. Expression of exogenous MOF impedes LSD1 binding to epithelial gene promoters and histone demethylation, thereby suppressing EMT and tumor invasion. Conversely, MOF depletion enhances EMT and tumor metastasis. In human cancer, high MOF expression correlates with epithelial markers and a favorable prognosis. These findings provide insight into the regulation of LSD1 and EMT and identify MOF as a critical suppressor of EMT and tumor progression. PMID:27292636

  12. Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation

    PubMed Central

    Cheng, Jingdong; Yang, Huirong; Fang, Jian; Ma, Lixiang; Gong, Rui; Wang, Ping; Li, Ze; Xu, Yanhui

    2015-01-01

    DNMT1 is an important epigenetic regulator that plays a key role in the maintenance of DNA methylation. Here we determined the crystal structure of DNMT1 in complex with USP7 at 2.9 Å resolution. The interaction between the two proteins is primarily mediated by an acidic pocket in USP7 and Lysine residues within DNMT1's KG linker. This intermolecular interaction is required for USP7-mediated stabilization of DNMT1. Acetylation of the KG linker Lysine residues impair DNMT1–USP7 interaction and promote the degradation of DNMT1. Treatment with HDAC inhibitors results in an increase in acetylated DNMT1 and decreased total DNMT1 protein. This negative correlation is observed in differentiated neuronal cells and pancreatic cancer cells. Our studies reveal that USP7-mediated stabilization of DNMT1 is regulated by acetylation and provide a structural basis for the design of inhibitors, targeting the DNMT1–USP7 interaction surface for therapeutic applications. PMID:25960197

  13. Protein kinase C-mediated phosphorylation and functional regulation of dopamine transporters in striatal synaptosomes.

    PubMed

    Vaughan, R A; Huff, R A; Uhl, G R; Kuhar, M J

    1997-06-13

    Dopamine transporters (DATs) are members of a family of Na+- and Cl--dependent neurotransmitter transporters responsible for the rapid clearance of dopamine from synaptic clefts. The predicted primary sequence of DAT contains numerous consensus phosphorylation sites. In this report we demonstrate that DATs undergo endogenous phosphorylation in striatal synaptosomes that is regulated by activators of protein kinase C. Rat striatal synaptosomes were metabolically labeled with [32P]orthophosphate, and solubilized homogenates were subjected to immunoprecipitation with an antiserum specific for DAT. Basal phosphorylation occurred in the absence of exogenous treatments, and the phosphorylation level was rapidly increased when synaptosomes were treated with the phosphatase inhibitors okadaic acid or calyculin. Treatment of synaptosomes with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also increased the level of phosphate incorporation. This occurred within 10 min and was dosedependent between 0.1 and 1 microM PMA. DAT phosphorylation was also significantly increased by two other protein kinase C activators, (-)-indolactam V and 1-oleoyl-2-acetyl-sn-glycerol. The inactive phorbol ester 4alpha-phorbol 12,13-didecanoate at 10 microM was without effect, and PMA-induced phosphorylation was blocked by treatment of synaptosomes with the protein kinase C inhibitors staurosporine and bisindoylmaleimide. These results indicate that DATs undergo rapid in vivo phosphorylation in response to protein kinase C activation and that a robust mechanism exists in synaptosomes for DAT dephosphorylation. Dopamine transport activity in synaptosomes was reduced by all treatments that promoted DAT phosphorylation, with comparable dose, time, and inhibitor characteristics. The change in transport activity was produced by a reduction in Vmax with no significant effect on the Km for dopamine. These results suggest that synaptosomal dopamine transport activity is regulated by

  14. Fenofibrate activates AMPK and increases eNOS phosphorylation in HUVEC

    SciTech Connect

    Murakami, Hisashi; Murakami, Ryuichiro . E-mail: ryuichi@med.nagoya-u.ac.jp; Kambe, Fukushi; Cao, Xia; Takahashi, Ryotaro; Asai, Toru; Hirai, Toshihisa; Numaguchi, Yasushi; Okumura, Kenji; Seo, Hisao; Murohara, Toyoaki

    2006-03-24

    Fenofibrate improves endothelial function by lipid-lowering and anti-inflammatory effects. Additionally, fenofibrate has been demonstrated to upregulate endothelial nitric oxide synthase (eNOS). AMP-activated protein kinase (AMPK) has been reported to phosphorylate eNOS at Ser-1177 and stimulate vascular endothelium-derived nitric oxide (NO) production. We report here that fenofibrate activates AMPK and increases eNOS phosphorylation and NO production in human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with fenofibrate increased the phosphorylation of AMPK and acetyl-CoA carboxylase. Fenofibrate simultaneously increased eNOS phosphorylation and NO production. Inhibitors of protein kinase A and phosphatidylinositol 3-kinase failed to suppress the fenofibrate-induced eNOS phosphorylation. Neither bezafibrate nor WY-14643 activated AMPK in HUVEC. Furthermore, fenofibrate activated AMPK without requiring any transcriptional activities. These results indicate that fenofibrate stimulates eNOS phosphorylation and NO production through AMPK activation, which is suggested to be a novel characteristic of this agonist and unrelated to its effects on peroxisome proliferator-activated receptor {alpha}.

  15. Oxidative and Photosynthetic Phosphorylation Mechanisms

    ERIC Educational Resources Information Center

    Wang, Jui H.

    1970-01-01

    Proposes a molecular mechanism for the coupling of phosphorylation to electron transport in both mitochondria and chloroplasts. Justifies the proposed reaction schemes in terms of thermodynamics and biochemical data. Suggests how areobic respiration could have evolved. (EB)

  16. Regulation of Sulfate Assimilation by Light and O-Acetyl-l-Serine in Lemna minor L. 1

    PubMed Central

    Neuenschwander, Urs; Suter, Marianne; Brunold, Christian

    1991-01-01

    The effect of 0.5 millimolar O-acetyl-l-serine added to the nutrient solution on sulfate assimilation of Lemna minor L., cultivated in the light or in the dark, or transferred from light to the dark, was examined. During 24 hours after transfer from light to the dark the extractable activity of adenosine 5′-phosphosulfate sulfotransferase, a key enzyme of sulfate assimilation, decreased to 10% of the light control. Nitrate reductase (EC 1.7.7.1.) activity, measured for comparison, decreased to 40%. Adenosine 5′-triphosphate (ATP) sulfurylase (EC 2.7.7.4.) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8.) activities were not affected by the transfer. When O-acetyl-l-serine was added to the nutrient solution at the time of transfer to the dark, adenosine 5′-phosphosulfate sulfotransferase activity was still at 50% of the light control after 24 hours, ATP sulfurylase and O-acetyl-l-serine sulfhydrylase activity were again not affected, and nitrate reductase activity decreased as before. Addition of O-acetyl-l-serine at the time of the transfer caused a 100% increase in acid-soluble SH compounds after 24 hours in the dark. In continuous light the corresponding increase was 200%. During 24 hours after transfer to the dark the assimilation of 35SO42− into organic compounds decreased by 80% without O-acetyl-l-serine but was comparable to light controls in its presence. The addition of O-acetyl-l-serine to Lemna minor precultivated in the dark for 24 hours induced an increase in adenosine 5′-phosphosulfate sulfotransferase activity so that a constant level of 50% of the light control was reached after an additional 9 hours. Cycloheximide as well as 6-methyl-purine inhibited this effect. In the same type of experiment O-acetyl-l-serine induced a 100-fold increase in the incorporation of label from 35SO42− into cysteine after additional 24 hours in the dark. Taken together, these results show that exogenous O-acetyl-l-serine has a regulatory effect on

  17. Properties of phosphorylated thymidylate synthase.

    PubMed

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Paweł; Rogowska-Wrzesinska, Adelina; Cieśla, Joanna; Zieliński, Zbigniew; Nizioł, Joanna; Jarmuła, Adam; Maj, Piotr; Gołos, Barbara; Wińska, Patrycja; Ostafil, Sylwia; Wałajtys-Rode, Elżbieta; Shugar, David; Rode, Wojciech

    2015-12-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent. PMID:26315778

  18. Apoptosis and autophagy induced by pyropheophorbide-α methyl ester-mediated photodynamic therapy in human osteosarcoma MG-63 cells.

    PubMed

    Huang, Qiu; Ou, Yun-Sheng; Tao, Yong; Yin, Hang; Tu, Ping-Hua

    2016-06-01

    Pyropheophorbide-α methyl ester (MPPa) was a second-generation photosensitizer with many potential applications. Here, we explored the impact of MPPa-mediated photodynamic therapy (MPPa-PDT) on the apoptosis and autophagy of human osteosarcoma (MG-63) cells as well as the relationships between apoptosis and autophagy of the cells, and investigated the related molecular mechanisms. We found that MPPa-PDT demonstrated the ability to inhibit MG-63 cell viability in an MPPa concentration- and light dose-dependent manner, and to induce apoptosis via the mitochondrial apoptosis pathway. Additionally, MPPa-PDT could also induce autophagy of MG-63 cell. Meanwhile, the ROS scavenger N-acetyl-L-cysteine (NAC) and the Jnk inhibitor SP600125 were found to inhibit the MPPa-PDT-induced autophagy, and NAC could also inhibit Jnk phosphorylation. Furthermore, pretreatment with the autophagy inhibitor 3-methyladenine or chloroquine showed the potential in reducing the apoptosis rate induced by MPPa-PDT in MG-63 cells. Our results indicated that the mitochondrial pathway was involved in MPPa-PDT-induced apoptosis of MG-63 cells. Meanwhile the ROS-Jnk signaling pathway was involved in MPPa-PDT-induced autophagy, which further promoted the apoptosis in MG-63 cells. PMID:27108344

  19. Active, phosphorylated fingolimod inhibits histone deacetylases and facilitates fear extinction memory

    PubMed Central

    Hait, Nitai C; Wise, Laura E; Allegood, Jeremy C; O’Brien, Megan; Avni, Dorit; Reeves, Thomas M; Knapp, Pamela E; Lu, Junyan; Luo, Cheng; Miles, Michael F; Milstien, Sheldon; Lichtman, Aron H; Spiegel, Sarah

    2014-01-01

    FTY720 (fingolimod), an FDA-approved drug for treatment of multiple sclerosis, has beneficial effects in the CNS that are not yet well understood, independent of its effects on immune cell trafficking. We show that FTY720 enters the nucleus, where it is phosphorylated by sphingosine kinase 2 (SphK2), and that nuclear FTY720-P binds and inhibits class I histone deacetylases (HDACs), enhancing specific histone acetylations. FTY720 is also phosphorylated in mice and accumulates in the brain, including the hippocampus, inhibits HDACs and enhances histone acetylation and gene expression programs associated with memory and learning, and rescues memory deficits independently of its immunosuppressive actions. Sphk2−/− mice have lower levels of hippocampal sphingosine-1-phosphate, an endogenous HDAC inhibitor, and reduced histone acetylation, and display deficits in spatial memory and impaired contextual fear extinction. Thus, sphingosine-1-phosphate and SphK2 play specific roles in memory functions and FTY720 may be a useful adjuvant therapy to facilitate extinction of aversive memories. PMID:24859201

  20. Acetylated tau neuropathology in sporadic and hereditary tauopathies.

    PubMed

    Irwin, David J; Cohen, Todd J; Grossman, Murray; Arnold, Steven E; McCarty-Wood, Elisabeth; Van Deerlin, Vivianna M; Lee, Virginia M-Y; Trojanowski, John Q

    2013-08-01

    We have recently shown acetylation of tau at lysine residue 280 (AC-K280) to be a disease-specific modification in Alzheimer disease (AD), corticobasal degeneration, and progressive supranuclear palsy, likely representing a major regulatory tau modification. Herein, we extend our observations using IHC with a polyclonal antibody specific for AC-K280. Thirty brain regions were examined in argyrophilic grain disease (AGD; n = 5), tangle-predominant senile dementia (TPSD; n = 5), Pick disease (n = 4), familial AD (FAD; n = 2; PSEN1 p.G206A and p.S170P), and frontotemporal dementia with parkinsonism linked to chromosome-17 (FTDP-17; n = 2; MAPT p.P301L and IVS10 + 16). All AGD, TPSD, FAD, and FTDP-17 cases had significant AC-K280 reactivity that was similar in severity and distribution to phosphorylated tau. AC-K280 robustly labeled grain pathological characteristics in AGD and was predominantly associated with thioflavin-S-positive neurofibrillary tangles and less reactive in neuropil threads and extracellular tangles in TPSD and FAD. Thioflavin-S-negative neuronal and glial inclusions of patients with FTDP-17 were robustly AC-K280 reactive. A low degree of AC-K280 was found in a subset of 4-repeat tau-containing lesions in Pick disease. AC-K280 is a prominent feature of both neuronal and glial tau aggregations in tauopathies of various etiologies. The close association of AC-K280 with amyloid and pre-amyloid conformations of tau suggests a potential role in tangle maturation and, thus, could serve as a useful biomarker or therapeutic target in a variety of tauopathies. PMID:23885714

  1. Alkylating agent methyl methanesulfonate (MMS) induces a wave of global protein hyperacetylation: Implications in cancer cell death

    SciTech Connect

    Lee, Min-Young; Kim, Myoung-Ae; Kim, Hyun-Ju; Bae, Yoe-Sik; Park, Joo-In; Kwak, Jong-Young; Chung, Jay H.; Yun, Jeanho . E-mail: yunj@dau.ac.kr

    2007-08-24

    Protein acetylation modification has been implicated in many cellular processes but the direct evidence for the involvement of protein acetylation in signal transduction is very limited. In the present study, we found that an alkylating agent methyl methanesulfonate (MMS) induces a robust and reversible hyperacetylation of both cytoplasmic and nuclear proteins during the early phase of the cellular response to MMS. Notably, the acetylation level upon MMS treatment was strongly correlated with the susceptibility of cancer cells, and the enhancement of MMS-induced acetylation by histone deacetylase (HDAC) inhibitors was shown to increase the cellular susceptibility. These results suggest protein acetylation is important for the cell death signal transduction pathway and indicate that the use of HDAC inhibitors for the treatment of cancer is relevant.

  2. Acetylation and characterization of spruce (Picea abies) galactoglucomannans.

    PubMed

    Xu, Chunlin; Leppänen, Ann-Sofie; Eklund, Patrik; Holmlund, Peter; Sjöholm, Rainer; Sundberg, Kenneth; Willför, Stefan

    2010-04-19

    Acetylated galactoglucomannans (GGMs) are the main hemicellulose type in most softwood species and can be utilized as, for example, bioactive polymers, hydrocolloids, papermaking chemicals, or coating polymers. Acetylation of spruce GGM using acetic anhydride with pyridine as catalyst under different conditions was conducted to obtain different degrees of acetylation on a laboratory scale, whereas, as a classic method, it can be potentially transferred to the industrial scale. The effects of the amount of catalyst and acetic anhydride, reaction time, temperature and pretreatment by acetic acid were investigated. A fully acetylated product was obtained by refluxing GGM for two hours. The structures of the acetylated GGMs were determined by SEC-MALLS/RI, (1)H and (13)C NMR and FTIR spectroscopy. NMR studies also indicated migration of acetyl groups from O-2 or O-3 to O-6 after a heating treatment in a water bath. The thermal stability of the products was investigated by DSC-TGA. PMID:20144827

  3. Lunasin sensitivity in non-small cell lung cancer cells is linked to suppression of integrin signaling and changes in histone acetylation.

    PubMed

    Inaba, Junichi; McConnell, Elizabeth J; Davis, Keith R

    2014-01-01

    Lunasin is a plant derived bioactive peptide with both cancer chemopreventive and therapeutic activity. We recently showed lunasin inhibits non-small cell lung cancer (NSCLC) cell proliferation in a cell-line-specific manner. We now compared the effects of lunasin treatment of lunasin-sensitive (H661) and lunasin-insensitive (H1299) NSCLC cells with respect to lunasin uptake, histone acetylation and integrin signaling. Both cell lines exhibited changes in histone acetylation, with H661 cells showing a unique increase in H4K16 acetylation. Proximity ligation assays demonstrated lunasin interacted with integrins containing αv, α5, β1 and β3 subunits to a larger extent in the H661 compared to H1299 cells. Moreover, lunasin specifically disrupted the interaction of β1 and β3 subunits with the downstream signaling components phosphorylated Focal Adhesion Kinase (pFAK), Kindlin and Intergrin Linked Kinase in H661 cells. Immunoblot analyses demonstrated lunasin treatment of H661 resulted in reduced levels of pFAK, phosphorylated Akt and phosphorylated ERK1/2 whereas no changes were observed in H1299 cells. Silencing of αv expression in H661 cells confirmed signaling through integrins containing αv is essential for proliferation. Moreover, lunasin was unable to further inhibit proliferation in αv-silenced H661 cells. This indicates antagonism of integrin signaling via αv-containing integrins is an important component of lunasin's mechanism of action. PMID:25530619

  4. Protein phosphorylation in chloroplasts - a survey of phosphorylation targets.

    PubMed

    Baginsky, Sacha

    2016-06-01

    The development of new software tools, improved mass spectrometry equipment, a suite of optimized scan types, and better-quality phosphopeptide affinity capture have paved the way for an explosion of mass spectrometry data on phosphopeptides. Because phosphoproteomics achieves good sensitivity, most studies use complete cell extracts for phosphopeptide enrichment and identification without prior enrichment of proteins or subcellular compartments. As a consequence, the phosphoproteome of cell organelles often comes as a by-product from large-scale studies and is commonly assembled from these in meta-analyses. This review aims at providing some guidance on the limitations of meta-analyses that combine data from analyses with different scopes, reports on the current status of knowledge on chloroplast phosphorylation targets, provides initial insights into phosphorylation site conservation in different plant species, and highlights emerging information on the integration of gene expression with metabolism and photosynthesis by means of protein phosphorylation. PMID:26969742

  5. Phosphorylation releases constraints to domain motion in ERK2

    PubMed Central

    Xiao, Yao; Lee, Thomas; Latham, Michael Parker; Warner, Lisa Rose; Tanimoto, Akiko; Pardi, Arthur; Ahn, Natalie G.

    2014-01-01

    Protein motions control enzyme catalysis through mechanisms that are incompletely understood. Here NMR 13C relaxation dispersion experiments were used to monitor changes in side-chain motions that occur in response to activation by phosphorylation of the MAP kinase ERK2. NMR data for the methyl side chains on Ile, Leu, and Val residues showed changes in conformational exchange dynamics in the microsecond-to-millisecond time regime between the different activity states of ERK2. In inactive, unphosphorylated ERK2, localized conformational exchange was observed among methyl side chains, with little evidence for coupling between residues. Upon dual phosphorylation by MAP kinase kinase 1, the dynamics of assigned methyls in ERK2 were altered throughout the conserved kinase core, including many residues in the catalytic pocket. The majority of residues in active ERK2 fit to a single conformational exchange process, with kex ≈ 300 s−1 (kAB ≈ 240 s−1/kBA ≈ 60 s−1) and pA/pB ≈ 20%/80%, suggesting global domain motions involving interconversion between two states. A mutant of ERK2, engineered to enhance conformational mobility at the hinge region linking the N- and C-terminal domains, also induced two-state conformational exchange throughout the kinase core, with exchange properties of kex ≈ 500 s−1 (kAB ≈ 15 s−1/kBA ≈ 485 s−1) and pA/pB ≈ 97%/3%. Thus, phosphorylation and activation of ERK2 lead to a dramatic shift in conformational exchange dynamics, likely through release of constraints at the hinge. PMID:24550275

  6. Determination of amphetamine by HPLC after acetylation.

    PubMed

    Veress, T

    2000-01-01

    An analytical procedure has been developed for the HPLC determination of amphetamine by off-line pre-column derivatization. The proposed procedure consists of sample preparation by acetylation of amphetamine with acetic anhydride and a subsequent reversed-phase HPLC separation on an octadecyl silica stationary phase with salt-free mobile phase (tetrahydrofuran, acetonitrile, 0.1% triethylamine in water, 15:15:70 v/v) applying UV-detection. The applicability of the elaborated procedure is demonstrated with results obtained by analysis of real samples seized in the Hungarian black market. PMID:10641931

  7. Phosphorylation of Ser-204 and Tyr-405 in human malonyl-CoA decarboxylase expressed in silkworm Bombyx mori regulates catalytic decarboxylase activity.

    PubMed

    Hwang, In-Wook; Makishima, Yu; Suzuki, Tomohiro; Kato, Tatsuya; Park, Sungjo; Terzic, Andre; Chung, Shin-Kyo; Park, Enoch Y

    2015-11-01

    Decarboxylation of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (MCD; EC 4.1.1.9) is a vital catalytic reaction of lipid metabolism. While it is established that phosphorylation of MCD modulates the enzymatic activity, the specific phosphorylation sites associated with the catalytic function have not been documented due to lack of sufficient production of MCD with proper post-translational modifications. Here, we used the silkworm-based Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system to express human MCD (hMCD) and mapped phosphorylation effects on enzymatic function. Purified MCD from silkworm displayed post-translational phosphorylation and demonstrated coherent enzymatic activity with high yield (-200 μg/silkworm). Point mutations in putative phosphorylation sites, Ser-204 or Tyr-405 of hMCD, identified by bioinformatics and proteomics analyses reduced the catalytic activity, underscoring the functional significance of phosphorylation in modulating decarboxylase-based catalysis. Identified phosphorylated residues are distinct from the decarboxylation catalytic site, implicating a phosphorylation-induced global conformational change of MCD as responsible in altering catalytic function. We conclude that phosphorylation of Ser-204 and Tyr-405 regulates the decarboxylase function of hMCD leveraging the silkworm-based BmNPV bacmid expression system that offers a fail-safe eukaryotic production platform implementing proper post-translational modification such as phosphorylation. PMID:26004805

  8. Determination of the degree of acetylation and the distribution of acetyl groups in chitosan by HPLC analysis of nitrous acid degraded and PMP labeled products.

    PubMed

    Han, Zhangrun; Zeng, Yangyang; Lu, Hong; Zhang, Lijuan

    2015-09-01

    Chitin is one of the most abundant polysaccharides on earth. It consists of repeating β-1,4 linked N-acetylated glucosamine (A) units. Chitosan is an N-deacetylated product of chitin. Chitosan and its derivatives have broad medical applications as drugs, nutraceuticals, or drug delivery agents. However, a reliable analytical method for quality control of medically used chitosans is still lacking. In current study, nitrous acid was used to cleave all glucosamine residues in chitosan into 2,5-anhydromannose (M) or M at the reducing end of di-, tri-, and oligosaccharides. PMP, i.e. 1-phenyl-3-methyl-5-pyrazolone, was used to label all the Ms. Online UV detection allowed quantification of all M-containing UV peaks whereas online MS analysis directly identified 11 different kinds of mono-, di-, tri-, and oligosaccharides that correlated each oligosaccharide with specific UV peak after HPLC separation. The DA (degree of acetylation) for chitosans was calculated based on the A/(A+M) value derived from the UV data. This newly developed method had several advantages for quality control of chitosan: 1. the experimental procedures were extensively optimized; 2. the reliability of the method was confirmed by online LC-MS analysis; 3. the DA value was obtainable based on the UV data after HPLC analysis, which was comparableto that of (1)H NMR and conductometric titration analyses; 4. finally and most importantly, this method could be used to obtain the DA as well as chemical acetylation/deacetylation mechanisms for chitosan by any laboratory equipped with a HPLC and an online UV detector. PMID:26114886

  9. Lipase-catalyzed synthesis of acetylated EGCG and antioxidant properties of the acetylated derivatives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    (-)-Epigallocatechin-3-O-gallate (EGCG) acetylated derivatives were prepared by lipase catalyzed acylation of EGCG with vinyl acetate to improve its lipophilicity and expand its application in lipophilic media. The immobilized lipase, Lipozyme RM IM, was found to be the optimum catalyst. The optimiz...

  10. Structure, morphology and functionality of acetylated and oxidised barley starches.

    PubMed

    El Halal, Shanise Lisie Mello; Colussi, Rosana; Pinto, Vânia Zanella; Bartz, Josiane; Radunz, Marjana; Carreño, Neftali Lenin Villarreal; Dias, Alvaro Renato Guerra; Zavareze, Elessandra da Rosa

    2015-02-01

    Acetylation and oxidation are chemical modifications which alter the properties of starch. The degree of modification of acetylated and oxidized starches is dependent on the catalyst and active chlorine concentrations, respectively. The objective of this study was to evaluate the effect of acetylation and oxidation on the structural, morphological, physical-chemical, thermal and pasting properties of barley starch. Barley starches were acetylated at different catalyst levels (11%, 17%, and 23% of NaOH solution) and oxidized at different sodium hypochlorite concentrations (1.0%, 1.5%, and 2.0% of active chlorine). Fourier-transformed infrared spectroscopy (FTIR), X-ray diffractograms, thermal, morphological, and pasting properties, swelling power and solubility of starches were evaluated. The degree of substitution (DS) of the acetylated starches increased with the rise in catalyst concentration. The percentage of carbonyl (CO) and carboxyl (COOH) groups in oxidized starches also increased with the rise of active chlorine level. The presence of hydrophobic acetyl groups, carbonyl and carboxyl groups caused a partial disorganization and depolymerization of starch granules. The structural, morphological and functional changes in acetylated and oxidized starches varied according to reaction conditions. Acetylation makes barley starch more hydrophobic by the insertion of acetyl groups. Also the oxidation promotes low retrogradation and viscosity. All these characteristics are important for biodegradable film production. PMID:25172707

  11. Protein lysine acetylation in bacteria: Current state of the art.

    PubMed

    Ouidir, Tassadit; Kentache, Takfarinas; Hardouin, Julie

    2016-01-01

    Post-translational modifications of proteins are key events in cellular metabolism and physiology regulation. Lysine acetylation is one of the best studied protein modifications in eukaryotes, but, until recently, ignored in bacteria. However, proteomic advances have highlighted the diversity of bacterial lysine-acetylated proteins. The current data support the implication of lysine acetylation in various metabolic pathways, adaptation and virulence. In this review, we present a broad overview of the current knowledge of lysine acetylation in bacteria. We emphasize particularly the significant contribution of proteomics in this field. PMID:26390373

  12. Probing the acetylation code of histone H4.

    PubMed

    Lang, Diana; Schümann, Michael; Gelato, Kathy; Fischle, Wolfgang; Schwarzer, Dirk; Krause, Eberhard

    2013-10-01

    Histone modifications play crucial roles in genome regulation with lysine acetylation being implicated in transcriptional control. Here we report a proteome-wide investigation of the acetylation-dependent protein-protein interactions of the N-terminal tail of histone H4. Quantitative peptide-based affinity MS experiments using the SILAC approach determined the interactomes of H4 tails monoacetylated at the four known acetylation sites K5, K8, K12, and K16, bis-acetylated at K5/K12, triple-acetylated at K8/12/16 and fully tetra-acetylated. A set of 29 proteins was found enriched on the fully acetylated H4 tail while specific binders of the mono and bis-acetylated tails were barely detectable. These observations are in good agreement with earlier reports indicating that the H4 acetylation state establishes its regulatory effects in a cumulative manner rather than via site-specific recruitment of regulatory proteins. PMID:23970329

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

    PubMed Central

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

    2016-01-01

    Lysine acetylation has emerged as one of the major post-translational modifications, as indicated by its roles in chromatin remodeling, activation of transcription factors and, most recently, regulation of metabolic enzymes. Identification of acetylation sites in a protein is the first essential step for functional characterization of acetylation in physiological regulation. However, the study of the acetylome is hindered by the lack of suitable physical and biochemical properties of the acetyl group and existence of high-abundance acetylated histones in the cell, and needs a robust method to overcome these problems. Here we present protocols for (i) using chemically acetylated ovalbumin and synthetic acetylated peptide to generate a pan-acetyllysine antibody and a site-specific antibody to Lys288-acetylated argininosuccinate lyase, respectively; (ii) using subcellular fractionation to reduce highly abundant acetylated histones; and (iii) using acetyllysine antibody affinity purification and mass spectrometry to characterize acetylome of human liver tissue. The entire characterization procedure takes ~2–3 d to complete. PMID:21085124

  14. Interphase phosphorylation of lamin A.

    PubMed

    Kochin, Vitaly; Shimi, Takeshi; Torvaldson, Elin; Adam, Stephen A; Goldman, Anne; Pack, Chan-Gi; Melo-Cardenas, Johanna; Imanishi, Susumu Y; Goldman, Robert D; Eriksson, John E

    2014-06-15

    Nuclear lamins form the major structural elements that comprise the nuclear lamina. Loss of nuclear structural integrity has been implicated as a key factor in the lamin A/C gene mutations that cause laminopathies, whereas the normal regulation of lamin A assembly and organization in interphase cells is still undefined. We assumed phosphorylation to be a major determinant, identifying 20 prime interphase phosphorylation sites, of which eight were high-turnover sites. We examined the roles of these latter sites by site-directed mutagenesis, followed by detailed microscopic analysis - including fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and nuclear extraction techniques. The results reveal three phosphorylation regions, each with dominant sites, together controlling lamin A structure and dynamics. Interestingly, two of these interphase sites are hyper-phosphorylated in mitotic cells and one of these sites is within the sequence that is missing in progerin of the Hutchinson-Gilford progeria syndrome. We present a model where different phosphorylation combinations yield markedly different effects on the assembly, subunit turnover and the mobility of lamin A between, and within, the lamina, the nucleoplasm and the cytoplasm of interphase cells. PMID:24741066

  15. Regulation and function of stimulus-induced phosphorylation of MeCP2

    PubMed Central

    Li, Hongda; Chang, Qiang

    2014-01-01

    DNA methylation-dependent epigenetic regulation plays important roles in the development and function of the mammalian nervous system. MeCP2 is a key player in recognizing methylated DNA and interpreting the epigenetic information encoded in different DNA methylation patterns. Mutations in the MECP2 gene cause Rett syndrome, a devastating neurological disease that shares many features with autism. One interesting aspect of MeCP2 function is that it can be phosphorylated in response to diverse stimuli. Insights into the regulation and function of MeCP2 phosphorylation will help improve our understanding of how MeCP2 integrates environmental stimuli in neuronal nuclei to generate adaptive responses and may eventually lead to treatments for patients. PMID:25568644

  16. Metabolic Pathways Leading to Mercury Methylation in Desulfovibrio desulfuricans LS †

    PubMed Central

    Choi, Sung-Chan; Chase, Theodore; Bartha, Richard

    1994-01-01

    The synthesis of methylmercury by Desulfovibrio desulfuricans LS was investigated on the basis of 14C incorporation from precursors and the measurement of relevant enzyme activities in cell extracts. The previously observed incorporation of C-3 from serine into methylmercury was confirmed by measurement of relatively high activities of serine hydroxymethyltransferase and other enzymes of this pathway. High rates of label incorporation into methylmercury from H14COO- and H14CO3- prompted the assay of enzymes of the acetyl coenzyme A (CoA) synthase pathway. These enzymes were found to be present but at activity levels much lower than those reported for acetogens. Propyl iodide inhibited methylmercury and acetyl-CoA syntheses to similar extents, and methylmercury synthesis was found to compete with acetyl-CoA synthesis for methyl groups. On the basis of these findings, we propose that in methylmercury synthesis by D. desulfuricans LS the methyl group is transferred from CH3-tetrahydrofolate via methylcobalamin. The methyl group may originate from C-3 of serine or from formate via the acetyl-CoA synthase pathway. These pathways are not unique to D. desulfuricans LS, and thus the ability of this bacterium to methylate mercury is most likely associated with the substrate specificity of its enzymes. PMID:16349435

  17. RpoS proteolysis is regulated by a mechanism that does not require the SprE (RssB) response regulator phosphorylation site.

    PubMed

    Peterson, Celeste N; Ruiz, Natividad; Silhavy, Thomas J

    2004-11-01

    In Escherichia coli the response regulator SprE (RssB) facilitates degradation of the sigma factor RpoS by delivering it to the ClpXP protease. This process is regulated: RpoS is degraded in logarithmic phase but becomes stable upon carbon starvation, resulting in its accumulation. Because SprE contains a CheY domain with a conserved phosphorylation site (D58), the prevailing model posits that this control is mediated by phosphorylation. To test this model, we mutated the conserved response regulator phosphorylation site (D58A) of the chromosomal allele of sprE and monitored RpoS levels in response to carbon starvation. Though phosphorylation contributed to the SprE basal activity, we found that RpoS proteolysis was still regulated upon carbon starvation. Furthermore, our results indicate that phosphorylation of wild-type SprE occurs by a mechanism that is independent of acetyl phosphate. PMID:15489452

  18. Deep, Quantitative Coverage of the Lysine Acetylome Using Novel Anti-acetyl-lysine Antibodies and an Optimized Proteomic Workflow.

    PubMed

    Svinkina, Tanya; Gu, Hongbo; Silva, Jeffrey C; Mertins, Philipp; Qiao, Jana; Fereshetian, Shaunt; Jaffe, Jacob D; Kuhn, Eric; Udeshi, Namrata D; Carr, Steven A

    2015-09-01

    Introduction of antibodies specific for acetylated lysine has significantly improved the detection of endogenous acetylation sites by mass spectrometry. Here, we describe a new, commercially available mixture of anti-lysine acetylation (Kac) antibodies and show its utility for in-depth profiling of the acetylome. Specifically, seven complementary monoclones with high specificity for Kac were combined into a final anti-Kac reagent which results in at least a twofold increase in identification of Kac peptides over a commonly used Kac antibody. We outline optimal antibody usage conditions, effective offline basic reversed phase separation, and use of state-of-the-art LC-MS technology for achieving unprecedented coverage of the acetylome. The methods were applied to quantify acetylation sites in suberoylanilide hydroxamic acid-treated Jurkat cells. Over 10,000 Kac peptides from over 3000 Kac proteins were quantified from a single stable isotope labeling by amino acids in cell culture labeled sample using 7.5 mg of peptide input per state. This constitutes the deepest coverage of acetylation sites in quantitative experiments obtained to-date. The approach was also applied to breast tumor xenograft samples using isobaric mass tag labeling of peptides (iTRAQ4, TMT6 and TMT10-plex reagents) for quantification. Greater than 6700 Kac peptides from over 2300 Kac proteins were quantified using 1 mg of tumor protein per iTRAQ 4-plex channel. The novel reagents and methods we describe here enable quantitative, global acetylome analyses with depth and sensitivity approaching that obtained for other well-studied post-translational modifications such as phosphorylation and ubiquitylation, and should have widespread application in biological and clinical studies employing mass spectrometry-based proteomics. PMID:25953088

  19. CRY Drives Cyclic CK2-Mediated BMAL1 Phosphorylation to Control the Mammalian Circadian Clock

    PubMed Central

    Tamaru, Teruya; Hattori, Mitsuru; Honda, Kousuke; Nakahata, Yasukazu; Sassone-Corsi, Paolo; van der Horst, Gijsbertus T. J.; Ozawa, Takeaki; Takamatsu, Ken

    2015-01-01

    Intracellular circadian clocks, composed of clock genes that act in transcription-translation feedback loops, drive global rhythmic expression of the mammalian transcriptome and allow an organism to anticipate to the momentum of the day. Using a novel clock-perturbing peptide, we established a pivotal role for casein kinase (CK)-2-mediated circadian BMAL1-Ser90 phosphorylation (BMAL1-P) in regulating central and peripheral core clocks. Subsequent analysis of the underlying mechanism showed a novel role of CRY as a repressor for protein kinase. Co-immunoprecipitation experiments and real-time monitoring of protein–protein interactions revealed that CRY-mediated periodic binding of CK2β to BMAL1 inhibits BMAL1-Ser90 phosphorylation by CK2α. The FAD binding domain of CRY1, two C-terminal BMAL1 domains, and particularly BMAL1-Lys537 acetylation/deacetylation by CLOCK/SIRT1, were shown to be critical for CRY-mediated BMAL1–CK2β binding. Reciprocally, BMAL1-Ser90 phosphorylation is prerequisite for BMAL1-Lys537 acetylation. We propose a dual negative-feedback model in which a CRY-dependent CK2-driven posttranslational BMAL1–P-BMAL1 loop is an integral part of the core clock oscillator. PMID:26562092

  20. 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

  1. N-acetylaspartate catabolism determines cytosolic acetyl-CoA levels and histone acetylation in brown adipocytes

    PubMed Central

    Prokesch, A.; Pelzmann, H. J.; Pessentheiner, A. R.; Huber, K.; Madreiter-Sokolowski, C. T.; Drougard, A.; Schittmayer, M.; Kolb, D.; Magnes, C.; Trausinger, G.; Graier, W. F.; Birner-Gruenberger, R.; Pospisilik, J. A.; Bogner-Strauss, J. G.

    2016-01-01

    Histone acetylation depends on the abundance of nucleo-cytoplasmic acetyl-CoA. Here, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. N-acetylaspartate (NAA) is a highly abundant brain metabolite catabolized by aspartoacylase yielding aspartate and acetate. The latter can be further used for acetyl-CoA production. Prior to this work, the presence of NAA has not been described in adipocytes. Here, we show that accumulation of NAA decreases the brown adipocyte phenotype. We increased intracellular NAA concentrations in brown adipocytes via media supplementation or knock-down of aspartoacylase and measured reduced lipolysis, thermogenic gene expression, and oxygen consumption. Combinations of approaches to increase intracellular NAA levels showed additive effects on lipolysis and gene repression, nearly abolishing the expression of Ucp1, Cidea, Prdm16, and Ppara. Transcriptome analyses of aspartoacylase knock-down cells indicate deficiencies in acetyl-CoA and lipid metabolism. Concordantly, cytoplasmic acetyl-CoA levels and global histone H3 acetylation were decreased. Further, activating histone marks (H3K27ac and H3K9ac) in promoters/enhancers of brown marker genes showed reduced acetylation status. Taken together, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. Thereby, we mechanistically connect the NAA pathway to the epigenomic regulation of gene expression, modulating the phenotype of brown adipocytes. PMID:27045997

  2. [A short path to synthesis of 4-deoxy-4-fluoroglucosaminides: methylumbelliferyl N-acetyl-4-deoxy-4-fluoro-beta-D-glucosaminide].

    PubMed

    Markina, N A; Voznyĭ, Ia V

    2008-01-01

    A convenient method of synthesis of 1,6-anhydro-4-deoxy-2-O-tosyl-4-fluoro-beta-D-glucopyranose by fusion of 1,6;3,4-dianhydro-2-O-tosyl-beta-D-galactopyranose with 2,4,6-trimethylpyridinium fluoride was found. By successive action of ammonia, methyl trifluoroacetate, and acetic anhydride, the resulting compound was transformed into 1,6-anhydro-3-O-acetyl-2,4-dideoxy-2-trifluoroacetamido-4-fluoro-beta-D-glucopyranose, which was converted into 3,6-di-O-acetyl-2,4-dideoxy-2-trifluoroacetamido-4-fluoro-alpha-D-glucopyranosyl fluoride by the reaction with HF/Py. The resulting fluoride was further used as a glycosyl donor in the synthesis of methylumbelliferyl N-acetyl-4-deoxy-4-fluoro-beta-D-glucosaminide. PMID:18695726

  3. MOZ increases p53 acetylation and premature senescence through its complex formation with PML.

    PubMed

    Rokudai, Susumu; Laptenko, Oleg; Arnal, Suzzette M; Taya, Yoichi; Kitabayashi, Issay; Prives, Carol

    2013-03-01

    Monocytic leukemia zinc finger (MOZ)/KAT6A is a MOZ, Ybf2/Sas3, Sas2, Tip60 (MYST)-type histone acetyltransferase that functions as a coactivator for acute myeloid leukemia 1 protein (AML1)- and Ets family transcription factor PU.1-dependent transcription. We previously reported that MOZ directly interacts with p53 and is essential for p53-dependent selective regulation of p21 expression. We show here that MOZ is an acetyltransferase of p53 at K120 and K382 and colocalizes with p53 in promyelocytic leukemia (PML) nuclear bodies following cellular stress. The MOZ-PML-p53 interaction enhances MOZ-mediated acetylation of p53, and this ternary complex enhances p53-dependent p21 expression. Moreover, we identified an Akt/protein kinase B recognition sequence in the PML-binding domain of MOZ protein. Akt-mediated phosphorylation of MOZ at T369 has a negative effect on complex formation between PML and MOZ. As a result of PML-mediated suppression of Akt, the increased PML-MOZ interaction enhances p21 expression and induces p53-dependent premature senescence upon forced PML expression. Our research demonstrates that MOZ controls p53 acetylation and transcriptional activity via association with PML. PMID:23431171

  4. Thermochemical characteristics of cellulose acetates with different degrees of acetylation

    NASA Astrophysics Data System (ADS)

    Larina, V. N.; Ur'yash, V. F.; Kushch, D. S.

    2012-12-01

    The standard enthalpies of combustion and formation of cellulose acetates with different degrees of acetylation are determined. It is established that there is a proportional dependence of these thermochemical characteristics vs. the degree of acetylation, weight fraction of bonded acetic acid, and molar mass of the repeating unit of cellulose acetates.

  5. Emerging Functions for N-Terminal Protein Acetylation in Plants.

    PubMed

    Gibbs, Daniel J

    2015-10-01

    N-terminal (Nt-) acetylation is a widespread but poorly understood co-translational protein modification. Two reports now shed light onto the proteome-wide dynamics and protein-specific consequences of Nt-acetylation in relation to plant development, stress-response, and protein stability, identifying this modification as a key regulator of diverse aspects of plant growth and behaviour. PMID:26319188

  6. Effect of acetaminophen on sulfamethazine acetylation in male volunteers.

    PubMed

    Tahir, I M; Iqbal, T; Saleem, S; Mehboob, H; Akhter, N; Riaz, M

    2016-03-01

    The effect of acetaminophen on sulfamethazine N-acetylation by human N-acetyltrasferase-2 (NAT2) was studied in 19 (n=19) healthy male volunteers in two different phases. In the first phase of the study the volunteers were given an oral dose of sulfamethazine 500 mg alone and blood and urine samples were collected. After the 10-day washout period the same selected volunteers were again administered sulfamethazine 500 mg along with 1000 mg acetaminophen. The acetylation of sulfamethazine by human NAT2 in both phases with and without acetaminophen was determined by HPLC to establish their respective phenotypes. In conclusion obtained statistics of present study revealed that acetaminophen significantly (P<0.0001) decreased sulfamethazine acetylation in plasma of both slow and fast acetylator male volunteers. A highly significant (P<0.0001) decrease in plasma-free and total sulfamethazine concentration was also observed when acetaminophen was co-administered. Urine acetylation status in both phases of the study was found not to be in complete concordance with that of plasma. Acetaminophen significantly (P<0.0001) increased the acetyl, free and total sulfamethazine concentration in urine of both slow and fast acetylators. Urine acetylation analysis has not been found to be a suitable approach for phenotypic studies. PMID:26519524

  7. 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. PMID:27606599

  8. Medial temporal N-acetyl aspartate in pediatric major depression

    PubMed Central

    MacMaster, Frank P.; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S. Preeya; Buhagiar, Christian; Rosenberg, David R.

    2008-01-01

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD-case control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  9. Medial temporal N-acetyl-aspartate in pediatric major depression.

    PubMed

    MacMaster, Frank P; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S Preeya; Buhagiar, Christian; Rosenberg, David R

    2008-10-30

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD case-control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in the left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  10. 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

  11. SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

    SciTech Connect

    JOHN C WALKER

    2011-11-01

    Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

  12. Autophagy proteins regulate ERK phosphorylation

    PubMed Central

    Martinez-Lopez, Nuria; Athonvarangkul, Diana; Mishall, Priti; Sahu, Srabani; Singh, Rajat

    2013-01-01

    Autophagy is a conserved pathway that maintains cellular quality control. Extracellular signal-regulated kinase (ERK) controls various aspects of cell physiology including proliferation. Multiple signalling cascades, including ERK, have been shown to regulate autophagy, however whether autophagy proteins (ATG) regulate cell signalling is unknown. Here we show that growth factor exposure increases the interaction of ERK cascade components with ATG proteins in the cytosol and nucleus. ERK and its upstream kinase MEK localize to the extra-luminal face of autophagosomes. ERK2 interacts with ATG proteins via its substrate-binding domains. Deleting Atg7 or Atg5 or blocking LC3 lipidation or ATG5–ATG12 conjugation decreases ERK phosphorylation. Conversely, increasing LC3-II availability by silencing the cysteine protease ATG4B or acute trehalose exposure increases ERK phosphorylation. Decreased ERK phosphorylation in Atg5−/− cells does not occur from overactive phosphatases. Our findings thus reveal an unconventional function of ATG proteins as cellular scaffolds in the regulation of ERK phosphorylation. PMID:24240988

  13. Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells

    SciTech Connect

    Li, Lingzhi; Qiu, Ping; Chen, Bailing; Lu, Yongju; Wu, Kai; Thakur, Chitra; Chang, Qingshan; Sun, Jiaying; Chen, Fei

    2014-05-01

    Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-L-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H{sub 2}O{sub 2}, the most important form of ROS in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H{sub 2}O{sub 2}. Furthermore, both arsenic and H{sub 2}O{sub 2} were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation. - Highlights:: • Arsenic (As{sup 3+}) induces EZH phosphorylation. • JNK, STAT3, and Akt contribute to EZH2 phosphorylation. • Oxidative stress is involved in As{sup 3+}-induced EZH2 phosphorylation. • As{sup 3+} induces direct interaction of Akt and EZH2. • Phosphorylated EZH2 localized in cytoplasm.

  14. 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. PMID:26920270

  15. The Arabidopsis acetylated histone-binding protein BRAT1 forms a complex with BRP1 and prevents transcriptional silencing

    PubMed Central

    Zhang, Cui-Jun; Hou, Xiao-Mei; Tan, Lian-Mei; Shao, Chang-Rong; Huang, Huan-Wei; Li, Yong-Qiang; Li, Lin; Cai, Tao; Chen, She; He, Xin-Jian

    2016-01-01

    Transposable elements and other repetitive DNA sequences are usually subject to DNA methylation and transcriptional silencing. However, anti-silencing mechanisms that promote transcription in these regions are not well understood. Here, we describe an anti-silencing factor, Bromodomain and ATPase domain-containing protein 1 (BRAT1), which we identified by a genetic screen in Arabidopsis thaliana. BRAT1 interacts with an ATPase domain-containing protein, BRP1 (BRAT1 Partner 1), and both prevent transcriptional silencing at methylated genomic regions. Although BRAT1 mediates DNA demethylation at a small set of loci targeted by the 5-methylcytosine DNA glycosylase ROS1, the involvement of BRAT1 in anti-silencing is largely independent of DNA demethylation. We also demonstrate that the bromodomain of BRAT1 binds to acetylated histone, which may facilitate the prevention of transcriptional silencing. Thus, BRAT1 represents a potential link between histone acetylation and transcriptional anti-silencing at methylated genomic regions, which may be conserved in eukaryotes. PMID:27273316

  16. Phosphorylation in halobacterial signal transduction.

    PubMed Central

    Rudolph, J; Tolliday, N; Schmitt, C; Schuster, S C; Oesterhelt, D

    1995-01-01

    Regulated phosphorylation of proteins has been shown to be a hallmark of signal transduction mechanisms in both Eubacteria and Eukarya. Here we demonstrate that phosphorylation and dephosphorylation are also the underlying mechanism of chemo- and phototactic signal transduction in Archaea, the third branch of the living world. Cloning and sequencing of the region upstream of the cheA gene, known to be required for chemo- and phototaxis in Halobacterium salinarium, has identified cheY and cheB analogs which appear to form part of an operon which also includes cheA and the following open reading frame of 585 nucleotides. The CheY and CheB proteins have 31.3 and 37.5% sequence identity compared with the known signal transduction proteins CheY and CheB from Escherichia coli, respectively. The biochemical activities of both CheA and CheY were investigated following their expression in E.coli, isolation and renaturation. Wild-type CheA could be phosphorylated in a time-dependent manner in the presence of [gamma-32P]ATP and Mg2+, whereas the mutant CheA(H44Q) remained unlabeled. Phosphorylated CheA was dephosphorylated rapidly by the addition of wild-type CheY. The mutant CheY(D53A) had no effect on phosphorylated CheA. The mechanism of chemo- and phototactic signal transduction in the Archaeon H.salinarium, therefore, is similar to the two-component signaling system known from chemotaxis in the eubacterium E.coli. Images PMID:7556066

  17. 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.

  18. SUV420H1 enhances the phosphorylation and transcription of ERK1 in cancer cells

    PubMed Central

    Saloura, Vassiliki; Cho, Hyun-Soo; Suzuki, Takehiro; Dohmae, Naoshi; Alachkar, Houda; Nakamura, Yusuke; Hamamoto, Ryuji

    2015-01-01

    The oncogenic protein ERK, a member of the extracellular signal-regulated kinase (ERK) cascade, is a well characterized signaling molecule involved in tumorigenesis. The ERK signaling pathway is activated in a large proportion of cancers and plays a critical role in tumor development. Functional regulation by phosphorylation of kinases in the ERK pathway has been extensively studied, however methylation of the ERK protein has not been reported to date. Here, we demonstrated that the protein lysine methyltransferase SUV420H1 tri-methylated ERK1 at lysines 302 and 361, and that substitution of methylation sites diminished phosphorylation levels of ERK1. Concordantly, knockdown of SUV420H1 reduced phosphorylated ERK1 and total ERK1 proteins, and interestingly suppressed ERK1 at the transcriptional level. Our results indicate that overexpression of SUV420H1 may result in activation of the ERK signaling pathway through enhancement of ERK phosphorylation and transcription, thereby providing new insights in the regulation of the ERK cascade in human cancer. PMID:26586479

  19. Glutaredoxin 1 regulates cigarette smoke-mediated lung inflammation through differential modulation of IκB kinases in mice: impact on histone acetylation

    PubMed Central

    Chung, Sangwoon; Sundar, Isaac Kirubakaran; Yao, Hongwei; Ho, Ye-Shih

    2010-01-01

    Glutaredoxin 1 (Glrx1) is a small dithiol protein that regulates the cellular redox state and redox-dependent signaling pathways via modulation of protein glutathionylation. IκB kinase (IKK), an essential enzyme for NF-κB activation, can be subjected to S-glutathionylation leading to alteration of its activity. However, the role of Glrx1 in cigarette smoke (CS)-induced lung inflammation and chromatin modifications are not known. We hypothesized that Glrx1 regulates the CS-induced lung inflammation and chromatin modifications via differential regulation of IKKs by S-glutathionylation in mouse lung. Glrx1 knockout (KO) and wild-type (WT) mice were exposed to CS for 3 days and determined the role of Glrx1 in regulation of proinflammatory response in the lung. Neutrophil influx in bronchoalveolar lavage fluid and proinflammatory cytokine release in lung were increased in Glrx1 KO mice compared with WT mice exposed to CS, which was associated with augmented nuclear translocation of RelA/p65 and its phospho-acetylation. Interestingly, phosphorylated and total levels of IKKα, but not total and phosphorylated IKKβ levels, were increased in lungs of Glrx1 KO mice compared with WT mice exposed to CS. Ablation of Glrx1 leads to increased CS-induced IKKβ glutathionylation rendering it inactive, whereas IKKα was activated resulting in increased phospho-acetylation of histone H3 in mouse lung. Thus, targeted disruption of Glrx1 regulates the lung proinflammatory response via histone acetylation specifically by activation of IKKα in response to CS exposure. Overall, our study suggests that S-glutathionylation and phosphorylation of IKKα plays an important role in histone acetylation on proinflammatory gene promoters and NF-κB-mediated abnormal and sustained lung inflammation in pathogenesis of chronic inflammatory lung diseases. PMID:20472709

  20. `Up-regulation of histone acetylation induced by social defeat mediates the conditioned rewarding effects of cocaine.

    PubMed

    Montagud-Romero, S; Montesinos, J; Pascual, M; Aguilar, M A; Roger-Sanchez, C; Guerri, C; Miñarro, J; Rodríguez-Arias, M

    2016-10-01

    Social defeat (SD) induces a long-lasting increase in the rewarding effects of psychostimulants measured using the self-administration and conditioned place procedures (CPP). However, little is known about the epigenetic changes induced by social stress and about their role in the increased response to the rewarding effects of psychostimulants. Considering that histone acetylation regulates transcriptional activity and contributes to drug-induced behavioral changes, we addressed the hypothesis that SD induces transcriptional changes by histone modifications associated with the acquisition of place conditioning. After a fourth defeat, H3(K9) acetylation was decreased in the hippocampus, while there was an increase of HAT and a decrease of HDAC levels in the cortex. Three weeks after the last defeat, mice displayed an increase in histone H4(K12) acetylation and an upregulation of histone acetyl transferase (HAT) activity in the hippocampus. In addition, H3(K4)me3, which is closely associated with transcriptional initiation, was also augmented in the hippocampus three weeks after the last defeat. Inhibition of HAT by curcumin (100mg/kg) before each SD blocked the increase in the conditioned reinforcing effects of 1mg/kg of cocaine, while inhibition of HDAC by valproic acid (500mg/kg) before social stress potentiated cocaine-induced CPP. Preference was reinstated when animals received a priming dose of 0.5mg/kg of cocaine, an effect that was absent in untreated defeated mice. These results suggest that the experience of SD induces chromatin remodeling, alters histone acetylation and methylation, and modifies the effects of cocaine on place conditioning. They also point to epigenetic mechanisms as potential avenues leading to new treatments for the long-term effects of social stress on drug addiction. PMID:27180319

  1. Simultaneous Identification of Tyrosine Phosphorylation and Sulfation Sites Utilizing Tyrosine-Specific Bromination

    NASA Astrophysics Data System (ADS)

    Kim, Jong-Seo; Song, Si-Uk; Kim, Hie-Joon

    2011-11-01

    Tyrosine phosphorylation and sulfation play many key roles in the cell. Isobaric phosphotyrosine and sulfotyrosine residues in peptides were determined by mass spectrometry using phosphatase or sulfatase to remove the phosphate or the sulfate group. Unique Br signature was introduced to the resulting tyrosine residues by incubation with 32% HBr at -20 °C for 20 min. MS/MS analysis of the brominated peptide enabled unambiguous determination of the phosphotyrosine and the sulfotyrosine sites. When phosphotyrosine and sulfotyrosine as well as free tyrosine were present in the same peptide, they could be determined simultaneously using either phosphatase or sulfatase following acetylation of the free tyrosine.

  2. Systematic Mapping of Posttranslational Modifications in Human Estrogen Receptor-α with Emphasis on Novel Phosphorylation Sites*S⃞

    PubMed Central

    Atsriku, Christian; Britton, David J.; Held, Jason M.; Schilling, Birgit; Scott, Gary K.; Gibson, Bradford W.; Benz, Christopher C.; Baldwin, Michael A.

    2009-01-01

    A systematic study of posttranslational modifications of the estrogen receptor isolated from the MCF-7 human breast cancer cell line is reported. Proteolysis with multiple enzymes, mass spectrometry, and tandem mass spectrometry achieved very high sequence coverage for the full-length 66-kDa endogenous protein from estradiol-treated cell cultures. Nine phosphorylated serine residues were identified, three of which were previously unreported and none of which were previously observed by mass spectrometry by any other laboratory. Two additional modified serine residues were identified in recombinant protein, one previously reported but not observed here in endogenous protein and the other previously unknown. Although major emphasis was placed on identifying new phosphorylation sites, N-terminal loss of methionine accompanied by amino acetylation and a lysine side chain acetylation (or possibly trimethylation) were also detected. The use of both HPLC-ESI and MALDI interfaced to different mass analyzers gave higher sequence coverage and identified more sites than could be achieved by either method alone. The estrogen receptor is critical in the development and progression of breast cancer. One previously unreported phosphorylation site identified here was shown to be strongly dependent on estradiol, confirming its potential significance to breast cancer. Greater knowledge of this array of posttranslational modifications of estrogen receptor, particularly phosphorylation, will increase our understanding of the processes that lead to estradiol-induced activation of this protein and may aid the development of therapeutic strategies for management of hormone-dependent breast cancer. PMID:18984578

  3. (αR,4R,4aR,6aS,7R,8S,10R,11S)-Methyl α-acet-oxy-4-(3-furan-yl)-10-hy-droxy-4a,7,9,9-tetra-methyl-2,13-dioxo-1,4,4a,5,6,6a,7,8,9,10,11,12-dodeca-hydro-7,11-methano-2H-cyclo-octa-[f][2]benzopyran-8-acetate (6-O-acetyl-swietenolide) from the seeds of Swietenia macrophylla.

    PubMed

    Goh, Bey Hing; Abdul Kadir, Habsah; Abdul Malek, Sri Nurestri; Ng, Seik Weng

    2010-01-01

    The mol-ecule of O-acetyl-swietenolide, C(29)H(36)O(9), isolated from the seeds of Swietenia macrophylla, features four six-membered rings connected together in the shape of a bowl; one of the inner rings adopts a twisted chair conformation owing to the C=C double bond. The furyl substitutent is connected to an outer ring, and it points away from the bowl cavity. The hy-droxy group is connected to a carbonyl O atom of an adjacent mol-ecule by an O-H⋯O hydrogen bond, generating a chain running along the b axis. PMID:21588997

  4. Methyl salicylate overdose

    MedlinePlus

    Methyl salicylate (oil of wintergreen) is a chemical that smells like wintergreen. It is used in many over- ... muscle ache creams. It is related to aspirin. Methyl salicylate overdose occurs when someone swallows a dangerous amount ...

  5. Characterization of O-Acetylation of N-Acetylglucosamine

    PubMed Central

    Bernard, Elvis; Rolain, Thomas; Courtin, Pascal; Guillot, Alain; Langella, Philippe; Hols, Pascal; Chapot-Chartier, Marie-Pierre

    2011-01-01

    Peptidoglycan (PG) N-acetyl muramic acid (MurNAc) O-acetylation is widely spread in Gram-positive bacteria and is generally associated with resistance against lysozyme and endogenous autolysins. We report here the presence of O-acetylation on N-acetylglucosamine (GlcNAc) in Lactobacillus plantarum PG. This modification of glycan strands was never described in bacteria. Fine structural characterization of acetylated muropeptides released from L. plantarum PG demonstrated that both MurNAc and GlcNAc are O-acetylated in this species. These two PG post-modifications rely on two dedicated O-acetyltransferase encoding genes, named oatA and oatB, respectively. By analyzing the resistance to cell wall hydrolysis of mutant strains, we showed that GlcNAc O-acetylation inhibits N-acetylglucosaminidase Acm2, the major L. plantarum autolysin. In this bacterial species, inactivation of oatA, encoding MurNAc O-acetyltransferase, resulted in marked sensitivity to lysozyme. Moreover, MurNAc over-O-acetylation was shown to activate autolysis through the putative N-acetylmuramoyl-l-alanine amidase LytH enzyme. Our data indicate that in L. plantarum, two different O-acetyltransferases play original and antagonistic roles in the modulation of the activity of endogenous autolysins. PMID:21586574

  6. Chitosan Molecular Structure as a Function of N-Acetylation

    SciTech Connect

    Franca, Eduardo F.; Freitas, Luiz C.; Lins, Roberto D.

    2011-07-01

    Molecular dynamics simulations have been carried out to characterize the structure and solubility of chitosan nanoparticle-like structures as a function of the deacetylation level (0, 40, 60, and 100%) and the spatial distribution of the N-acetyl groups in the particles. The polysaccharide chains of highly N-deacetylated particles where the N-acetyl groups are uniformly distributed present a high flexibility and preference for the relaxed two-fold helix and five-fold helix motifs. When these groups are confined to a given region of the particle, the chains adopt preferentially a two-fold helix with f and w values close to crystalline chitin. Nanoparticles with up to 40% acetylation are moderately soluble, forming stable aggregates when the N-acetyl groups are unevenly distributed. Systems with 60% or higher N-acetylation levels are insoluble and present similar degrees of swelling regardless the distribution of their N-acetyl groups. Overall particle solvation is highly affected by electrostatic forces resulting from the degree of acetylation. The water mobility and orientation around the polysaccharide chains affects the stability of the intramolecular O3- HO3(n) ... O5(n+ 1) hydrogen bond, which in turn controls particle aggregation.

  7. Role of Histone Acetylation in Cell Cycle Regulation.

    PubMed

    Koprinarova, Miglena; Schnekenburger, Michael; Diederich, Marc

    2016-01-01

    Core histone acetylation is a key prerequisite for chromatin decondensation and plays a pivotal role in regulation of chromatin structure, function and dynamics. The addition of acetyl groups disturbs histone/DNA interactions in the nucleosome and alters histone/histone interactions in the same or adjacent nucleosomes. Acetyl groups can also provide binding sites for recruitment of bromodomain (BRD)-containing non-histone readers and regulatory complexes to chromatin allowing them to perform distinct downstream functions. The presence of a particular acetylation pattern influences appearance of other histone modifications in the immediate vicinity forming the "histone code". Although the roles of the acetylation of particular lysine residues for the ongoing chromatin functions is largely studied, the epigenetic inheritance of histone acetylation is a debated issue. The dynamics of local or global histone acetylation is associated with fundamental cellular processes such as gene transcription, DNA replication, DNA repair or chromatin condensation. Therefore, it is an essential part of the epigenetic cell response to processes related to internal and external signals. PMID:26303420

  8. Cellular regulation by protein phosphorylation.

    PubMed

    Fischer, Edmond H

    2013-01-11

    A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca(2+) and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert. PMID:23058924

  9. Dynamics of histone H3 acetylation in the nucleosome core during mouse pre-implantation development.

    PubMed

    Ziegler-Birling, Céline; Daujat, Sylvain; Schneider, Robert; Torres-Padilla, Maria-Elena

    2016-08-01

    In mammals, the time period that follows fertilization is characterized by extensive chromatin remodeling, which enables epigenetic reprogramming of the gametes. Major changes in chromatin structure persist until the time of implantation, when the embryo develops into a blastocyst, which comprises the inner cell mass and the trophectoderm. Changes in DNA methylation, histone variant incorporation, and covalent modifications of the histones tails have been intensively studied during pre-implantation development. However, modifications within the core of the nucleosomes have not been systematically analyzed. Here, we report the first characterization and temporal analysis of 3 key acetylated residues in the core of the histone H3: H3K64ac, H3K122ac, and H3K56ac, all located at structurally important positions close to the DNA. We found that all 3 acetylations occur during pre-implantation development, but with different temporal kinetics. Globally, H3K64ac and H3K56ac were detected throughout cleavage stages, while H3K122ac was only weakly detectable during this time. Our work contributes to the understanding of the contribution of histone modifications in the core of the nucleosome to the "marking" of the newly established embryonic chromatin and unveils new modification pathways potentially involved in epigenetic reprogramming. PMID:26479850

  10. Acetylated histones contribute to the immunostimulatory potential of neutrophil extracellular traps in systemic lupus erythematosus

    PubMed Central

    Pieterse, E; Hofstra, J; Berden, J; Herrmann, M; Dieker, J; van der Vlag, J

    2015-01-01

    In addition to disturbed apoptosis and insufficient clearance of apoptotic cells, there is recent evidence for a role of neutrophils in the aetiopathogenesis of systemic lupus erythematosus (SLE). In response to various stimuli, neutrophils can rapidly release DNA fibres decorated with citrullinated histones and anti-microbial peptides. These structures are referred to as neutrophil extracellular traps (NETs). In addition to apoptotic cell-derived microparticles, these NETs may comprise a further source of autoantigens, able to drive the autoimmune response in SLE. Our group recently identified specific histone modifications occurring during apoptosis that play an important role in the autoimmune response in SLE. In the current study, we evaluated the presence and immunostimulatory potential of these previously identified histone modifications in NETs. Compared to NETs from healthy donors, the histones present in NETs formed by SLE-derived neutrophils contain increased amounts of acetylated and methylated residues, which we previously observed to be associated with apoptosis and SLE. Treatment of neutrophils with histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), prior to induction of NETosis, induced NETs containing hyperacetylated histones, endowed with an increased capacity to activate macrophages. This implies that specific histone modifications, in particular acetylation, might enhance the immunostimulatory potential of NETs in SLE. PMID:24758196

  11. Resveratrol, Acetyl-Resveratrol, and Polydatin Exhibit Antigrowth Activity against 3D Cell Aggregates of the SKOV-3 and OVCAR-8 Ovarian Cancer Cell Lines

    PubMed Central

    Hogg, Simon J.; Chitcholtan, Kenny; Hassan, Wafaa; Sykes, Peter H.; Garrill, Ashley

    2015-01-01

    Resveratrol has aroused significant scientific interest as it has been claimed that it exhibits a spectrum of health benefits. These include effects as an anti-inflammatory and an antitumour compound. The purpose of this study was to investigate and compare any potential antigrowth effects of resveratrol and two of its derivatives, acetyl-resveratrol and polydatin, on 3D cell aggregates of the EGFR/Her-2 positive and negative ovarian cancer cell lines SKOV-3 and OVCAR-8, respectively. Results showed that resveratrol and acetyl-resveratrol reduced cell growth in the SKOV-3 and OVCAR-8 in a dose-dependant manner. The growth reduction was mediated by the induction of apoptosis via the cleavage of poly(ADP-ribose) polymerase (PARP-1). At lower concentrations, 5 and 10 µM, resveratrol, acetyl-resveratrol, and polydatin were less effective than higher concentrations, 50 and 100 µM. In SKOV-3 line, at higher concentrations, resveratrol and polydatin significantly reduced the phosphorylation of Her-2 and EGFR and the expression of Erk. Acetyl-resveratrol, on the other hand, did not change the activation of Her-2 and EGFR. Resveratrol, acetyl-resveratrol, and polydatin suppressed the secretion of VEGF in a dose-dependant fashion. In the OVCAR-8 cell line, resveratrol and acetyl-resveratrol at 5 and 10 µM increased the activation of Erk. Above these concentrations they decreased activation. Polydatin did not produce this effect. This study demonstrates that resveratrol and its derivatives may inhibit growth of 3D cell aggregates of ovarian cancer cell lines via different signalling molecules. Resveratrol and its derivatives, therefore, warrant further in vivo evaluation to assess their potential clinical utility. PMID:26617640

  12. ERK2 phosphorylates Krüppel-like factor 8 protein at serine 48 to maintain its stability.

    PubMed

    Lahiri, Satadru K; Lu, Heng; Mukherjee, Debarati; Yu, Lin; Zhao, Jihe

    2016-01-01

    Krüppel-like factor 8 (KLF8) plays important roles in cancer and is strictly regulated by various post-translational modifications such as sumoylation, acetylation, ubiquitylation and PARylation. Here we report a novel phosphorylation of KLF8 by ERK2 responsible and critical for the stability of KLF8 protein. The full-length KLF8 protein displays a doublet in SDS-PAGE gel. The upper band of the doublet, however, disappeared when the N-terminal 50 amino acids were deleted. In its full-length the upper band disappeared upon phosphatase treatment or mutation of the serine 48 (S48) to alanine whereas the lower band was lost when the S48 was mutated to aspartic acid that mimics phosphorylated S48. These results suggest that S48 phosphorylation is responsible for the motility up-shift of KLF8 protein. Pharmacological and genetic manipulations of various potential kinases identified ERK2 as the likely one that phosphorylates KLF8 at S48. Functional studies indicated that this phosphorylation is crucial for protecting KLF8 protein from degradation in the nucleus and promoting cell migration. Taken together, this study identifies a novel mechanism of phosphorylation critical for KLF8 protein stabilization and function. PMID:27293988

  13. ERK2 phosphorylates Krüppel-like factor 8 protein at serine 48 to maintain its stability

    PubMed Central

    Lahiri, Satadru K; Lu, Heng; Mukherjee, Debarati; Yu, Lin; Zhao, Jihe

    2016-01-01

    Krüppel-like factor 8 (KLF8) plays important roles in cancer and is strictly regulated by various post-translational modifications such as sumoylation, acetylation, ubiquitylation and PARylation. Here we report a novel phosphorylation of KLF8 by ERK2 responsible and critical for the stability of KLF8 protein. The full-length KLF8 protein displays a doublet in SDS-PAGE gel. The upper band of the doublet, however, disappeared when the N-terminal 50 amino acids were deleted. In its full-length the upper band disappeared upon phosphatase treatment or mutation of the serine 48 (S48) to alanine whereas the lower band was lost when the S48 was mutated to aspartic acid that mimics phosphorylated S48. These results suggest that S48 phosphorylation is responsible for the motility up-shift of KLF8 protein. Pharmacological and genetic manipulations of various potential kinases identified ERK2 as the likely one that phosphorylates KLF8 at S48. Functional studies indicated that this phosphorylation is crucial for protecting KLF8 protein from degradation in the nucleus and promoting cell migration. Taken together, this study identifies a novel mechanism of phosphorylation critical for KLF8 protein stabilization and function. PMID:27293988

  14. Evidence for N----O acetyl migration as the mechanism for O acetylation of peptidoglycan in Proteus mirabilis.

    PubMed Central

    Dupont, C; Clarke, A J

    1991-01-01

    O-acetylated peptidoglycan was purified from Proteus mirabilis grown in the presence of specifically radiolabelled glucosamine derivatives, and the migration of the radiolabel was monitored. Mild-base hydrolysis of the isolated peptidoglycan (to release ester-linked acetate) from cells grown in the presence of 40 microM [acetyl-3H]N-acetyl-D-glucosamine resulted in the release of [3H]acetate, as detected by high-pressure liquid chromatography. The inclusion of either acetate, pyruvate, or acetyl phosphate, each at 1 mM final concentration, did not result in a diminution of mild-base-released [3H]acetate levels. No such release of [3H]acetate was observed with peptidoglycan isolated from either Escherichia coli incubated with the same radiolabel or P. mirabilis grown with [1,6-3H]N-acetyl-D-glucosamine or D-[1-14C]glucosamine. These observations support a hypothesis that O acetylation occurs by N----O acetyl transfer within the sacculus. A decrease in [3H]acetate release by mild-base hydrolysis was observed with the peptidoglycan of P. mirabilis cultures incubated in the presence of antagonists of peptidoglycan biosynthesis, penicillin G and D-cycloserine. The absence of free-amino sugars in the peptidoglycan of P. mirabilis but the detection of glucosamine in spent culture broths implies that N----O transacetylation is intimately associated with peptidoglycan turnover. PMID:2066331

  15. 9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate

    PubMed Central

    Baumann, Anna-Maria T.; Bakkers, Mark J. G.; Buettner, Falk F. R.; Hartmann, Maike; Grove, Melanie; Langereis, Martijn A.; de Groot, Raoul J.; Mühlenhoff, Martina

    2015-01-01

    Sialic acids, terminal sugars of glycoproteins and glycolipids, play important roles in development, cellular recognition processes and host–pathogen interactions. A common modification of sialic acids is 9-O-acetylation, which has been implicated in sialoglycan recognition, ganglioside biology, and the survival and drug resistance of acute lymphoblastic leukaemia cells. Despite many functional implications, the molecular basis of 9-O-acetylation has remained elusive thus far. Following cellular approaches, including selective gene knockout by CRISPR/Cas genome editing, we here show that CASD1—a previously identified human candidate gene—is essential for sialic acid 9-O-acetylation. In vitro assays with the purified N-terminal luminal domain of CASD1 demonstrate transfer of acetyl groups from acetyl-coenzyme A to CMP-activated sialic acid and formation of a covalent acetyl-enzyme intermediate. Our study provides direct evidence that CASD1 is a sialate O-acetyltransferase and serves as key enzyme in the biosynthesis of 9-O-acetylated sialoglycans. PMID:26169044

  16. Application of reverse-phase HPLC to quantify oligopeptide acetylation eliminates interference from unspecific acetyl CoA hydrolysis

    PubMed Central

    Evjenth, Rune; Hole, Kristine; Ziegler, Mathias; Lillehaug, Johan R

    2009-01-01

    Protein acetylation is a common modification that plays a central role in several cellular processes. The most widely used methods to study these modifications are either based on the detection of radioactively acetylated oligopetide products or an enzyme-coupled reaction measuring conversion of the acetyl donor acetyl CoA to the product CoASH. Due to several disadvantages of these methods, we designed a new method to study oligopeptide acetylation. Based on reverse phase HPLC we detect both reaction products in a highly robust and reproducible way. The method reported here is also fully compatible with subsequent product analysis, e.g. by mass spectroscopy. The catalytic subunit, hNaa30p, of the human NatC protein N-acetyltransferase complex was used for N-terminal oligopeptide acetylation. We show that unacetylated and acetylated oligopeptides can be efficiently separated and quantified by the HPLC-based analysis. The method is highly reproducible and enables reliable quantification of both substrates and products. It is therefore well-suited to determine kinetic parameters of acetyltransferases. PMID:19660098

  17. 9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate.

    PubMed

    Baumann, Anna-Maria T; Bakkers, Mark J G; Buettner, Falk F R; Hartmann, Maike; Grove, Melanie; Langereis, Martijn A; de Groot, Raoul J; Mühlenhoff, Martina

    2015-01-01

    Sialic acids, terminal sugars of glycoproteins and glycolipids, play important roles in development, cellular recognition processes and host-pathogen interactions. A common modification of sialic acids is 9-O-acetylation, which has been implicated in sialoglycan recognition, ganglioside biology, and the survival and drug resistance of acute lymphoblastic leukaemia cells. Despite many functional implications, the molecular basis of 9-O-acetylation has remained elusive thus far. Following cellular approaches, including selective gene knockout by CRISPR/Cas genome editing, we here show that CASD1--a previously identified human candidate gene--is essential for sialic acid 9-O-acetylation. In vitro assays with the purified N-terminal luminal domain of CASD1 demonstrate transfer of acetyl groups from acetyl-coenzyme A to CMP-activated sialic acid and formation of a covalent acetyl-enzyme intermediate. Our study provides direct evidence that CASD1 is a sialate O-acetyltransferase and serves as key enzyme in the biosynthesis of 9-O-acetylated sialoglycans. PMID:26169044

  18. Mycobacterium tuberculosis supports protein tyrosine phosphorylation

    PubMed Central

    Kusebauch, Ulrike; Ortega, Corrie; Ollodart, Anja; Rogers, Richard S.; Sherman, David R.; Moritz, Robert L.; Grundner, Christoph

    2014-01-01

    Reversible protein phosphorylation determines growth and adaptive decisions in Mycobacterium tuberculosis (Mtb). At least 11 two-component systems and 11 Ser/Thr protein kinases (STPKs) mediate phosphorylation on Asp, His, Ser, and Thr. In contrast, protein phosphorylation on Tyr has not been described previously in Mtb. Here, using a combination of phospho-enrichment and highly sensitive mass spectrometry, we show extensive protein Tyr phosphorylation of diverse Mtb proteins, including STPKs. Several STPKs function as dual-specificity kinases that phosphorylate Tyr in cis and in trans, suggesting that dual-specificity kinases have a major role in bacterial phospho-signaling. Mutation of a phosphotyrosine site of the essential STPK PknB reduces its activity in vitro and in live Mtb, indicating that Tyr phosphorylation has a functional role in bacterial growth. These data identify a previously unrecognized phosphorylation system in a human pathogen that claims ∼1.4 million lives every year. PMID:24927537

  19. Techniques in protein methylation.

    PubMed

    Lee, Jaeho; Cheng, Donghang; Bedford, Mark T

    2004-01-01

    Proteins can be methylated on the side-chain nitrogens of arginine and lysine residues or on carboxy-termini. Protein methylation is a way of subtly changing the primary sequence of a peptide so that it can encode more information. This common posttranslational modification is implicated in the regulation of a variety of processes including protein trafficking, transcription and protein-protein interactions. In this chapter, we will use the arginine methyltransferases to illustrate different approaches that have been developed to assess protein methylation. Both in vivo and in vitro methylation techniques are described, and the use of small molecule inhibitors of protein methylation will be demonstrated. PMID:15173617

  20. Molecular mechanism of APC/C activation by mitotic phosphorylation.

    PubMed

    Zhang, Suyang; Chang, Leifu; Alfieri, Claudio; Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

    2016-05-12

    In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our

  1. Tumor Necrosis Factor alpha (TNF{alpha}) regulates CD40 expression through SMAR1 phosphorylation

    SciTech Connect

    Singh, Kamini; Sinha, Surajit; Malonia, Sunil Kumar; Chattopadhyay, Samit

    2010-01-08

    CD40 plays an important role in mediating inflammatory response and is mainly induced by JAK/STAT phosphorylation cascade. TNF{alpha} is the key cytokine that activates CD40 during inflammation and tumorigenesis. We have earlier shown that SMAR1 can repress the transcription of Cyclin D1 promoter by forming a HDAC1 dependent repressor complex. In this study, we show that SMAR1 regulates the transcription of NF-{kappa}B target gene CD40. SMAR1 recruits HDAC1 and forms a repressor complex on CD40 promoter and keeps its basal transcription in check. Further, we show that TNF{alpha} stimulation induces SMAR1 phosphorylation at Ser-347 and promotes its cytoplasmic translocation, thus releasing its negative effect. Concomitantly, TNF{alpha} induced phosphorylation of STAT1 at Tyr-701 by JAK1 facilitates its nuclear translocation and activation of CD40 through p300 recruitment and core Histone-3 acetylation. Thus, TNF{alpha} mediated regulation of CD40 expression occurs by dual phosphorylation of SMAR1 and STAT1.

  2. Structural and functional characterization of the phosphorylation-dependent interaction between PML and SUMO1.

    PubMed

    Cappadocia, Laurent; Mascle, Xavier H; Bourdeau, Véronique; Tremblay-Belzile, Samuel; Chaker-Margot, Malik; Lussier-Price, Mathieu; Wada, Junya; Sakaguchi, Kazuyasu; Aubry, Muriel; Ferbeyre, Gerardo; Omichinski, James G

    2015-01-01

    PML and several other proteins localizing in PML-nuclear bodies (PML-NB) contain phosphoSIMs (SUMO-interacting motifs), and phosphorylation of this motif plays a key role in their interaction with SUMO family proteins. We examined the role that phosphorylation plays in the binding of the phosphoSIMs of PML and Daxx to SUMO1 at the atomic level. The crystal structures of SUMO1 bound to unphosphorylated and tetraphosphorylated PML-SIM peptides indicate that three phosphoserines directly contact specific positively charged residues of SUMO1. Surprisingly, the crystal structure of SUMO1 bound to a diphosphorylated Daxx-SIM peptide indicate that the hydrophobic residues of the phosphoSIM bind in a manner similar to that seen with PML, but important differences are observed when comparing the phosphorylated residues. Together, the results provide an atomic level description of how specific acetylation patterns within different SUMO family proteins can work together with phosphorylation of phosphoSIM's regions of target proteins to regulate binding specificity. PMID:25497731

  3. Mitogen-activated Protein Kinase Signaling Mediates Phosphorylation of Polycomb Ortholog Cbx7*

    PubMed Central

    Wu, Hsan-au; Balsbaugh, Jeremy L.; Chandler, Hollie; Georgilis, Athena; Zullow, Hayley; Shabanowitz, Jeffrey; Hunt, Donald F.; Gil, Jesus; Peters, Gordon; Bernstein, Emily

    2013-01-01

    Cbx7 is one of five mammalian orthologs of the Drosophila Polycomb. Cbx7 recognizes methylated lysine residues on the histone H3 tail and contributes to gene silencing in the context of the Polycomb repressive complex 1 (PRC1). However, our knowledge of Cbx7 post-translational modifications remains limited. Through combined biochemical and mass spectrometry approaches, we report a novel phosphorylation site on mouse Cbx7 at residue Thr-118 (Cbx7T118ph), near the highly conserved Polycomb box. The generation of a site-specific antibody to Cbx7T118ph demonstrates that Cbx7 is phosphorylated via MAPK signaling. Furthermore, we find Cbx7T118 phosphorylation in murine mammary carcinoma cells, which can be blocked by MEK inhibitors. Upon EGF stimulation, Cbx7 interacts robustly with other members of PRC1. To test the role of Cbx7T118 phosphorylation in gene silencing, we employed a RAS-induced senescence model system. We demonstrate that Cbx7T118 phosphorylation moderately enhances repression of its target gene p16. In summary, we have identified and characterized a novel MAPK-mediated phosphorylation site on Cbx7 and propose that mitogen signaling to the chromatin template regulates PRC1 function. PMID:24194518

  4. 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

  5. Acetylation of C/EBPα inhibits its granulopoietic function.

    PubMed

    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

  6. Data detailing the platelet acetyl-lysine proteome

    PubMed Central

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

    2015-01-01

    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. PMID:26904711

  7. 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. PMID:26904711

  8. Partially Acetylated Sugarcane Bagasse For Wicking Oil From Contaminated Wetlands

    EPA Science Inventory

    Sugarcane bagasse was partially acetylated to enhance its oil-wicking ability in saturated environments while holding moisture for hydrocarbon biodegradation. The water sorption capacity of raw bagasse was reduced fourfold after treatment, which indicated considerably increased ...

  9. Phytogenic biosynthesis and emission of methyl acetate.

    PubMed

    Jardine, Kolby; Wegener, Frederik; Abrell, Leif; van Haren, Joost; Werner, Christiane

    2014-02-01

    Acetylation of plant metabolites fundamentally changes their volatility, solubility and activity as semiochemicals. Here we present a new technique termed dynamic (13) C-pulse chasing to track the fate of C1-3 carbon atoms of pyruvate into the biosynthesis and emission of methyl acetate (MA) and CO2 . (13) C-labelling of MA and CO2 branch emissions respond within minutes to changes in (13) C-positionally labelled pyruvate solutions fed through the transpiration stream. Strong (13) C-labelling of MA emissions occurred only under pyruvate-2-(13) C and pyruvate-2,3-(13) C feeding, but not pyruvate-1-(13) C feeding. In contrast, strong (13) CO2 emissions were only observed under pyruvate-1-(13) C feeding. These results demonstrate that MA (and other volatile and non-volatile metabolites) derive from the C2,3 atoms of pyruvate while the C1 atom undergoes decarboxylation. The latter is a non-mitochondrial source of CO2 in the light generally not considered in studies of CO2 sources and sinks. Within a tropical rainforest mesocosm, we also observed atmospheric concentrations of MA up to 0.6 ppbv that tracked light and temperature conditions. Moreover, signals partially attributed to MA were observed in ambient air within and above a tropical rainforest in the Amazon. Our study highlights the potential importance of acetyl coenzyme A (CoA) biosynthesis as a source of acetate esters and CO2 to the atmosphere. PMID:23862653

  10. Cross Talk Mechanism among EMT, ROS, and Histone Acetylation in Phorbol Ester-Treated Human Breast Cancer MCF-7 Cells

    PubMed Central

    Kamiya, Tetsuro; Goto, Aki; Kurokawa, Eri; Hara, Hirokazu; Adachi, Tetsuo

    2016-01-01

    Epithelial-mesenchymal transition (EMT) plays a pivotal role in the progression of cancer, and some transcription factors including Slug and Snail are known to be involved in EMT processes. It has been well established that the excess production of reactive oxygen species (ROS) and epigenetics such as DNA methylation and histone modifications participate in carcinogenesis; however, the cross talk mechanism among EMT, ROS, and epigenetics remains unclear. In the present study, we demonstrated that the treatment of human breast cancer MCF-7 cells with phorbol ester (TPA), a protein kinase C activator, significantly induced cell proliferation and migration, and these were accompanied by the significant induction of Slug expression. Moreover, the TPA-elicited induction of Slug expression was regulated by histone H3 acetylation and NADPH oxidase (NOX) 2-derived ROS signaling, indicating that ROS and histone acetylation are involved in TPA-elicited EMT processes. We herein determined the cross talk mechanism among EMT, ROS, and histone acetylation, and our results provide an insight into the progression of cancer metastasis. PMID:27127545

  11. Structure and Reactivity of the N-Acetyl-Cysteine Radical Cation and Anion: Does Radical Migration Occur?

    NASA Astrophysics Data System (ADS)

    Osburn, Sandra; Berden, Giel; Oomens, Jos; O'Hair, Richard A. J.; Ryzhov, Victor

    2011-10-01

    The structure and reactivity of the N-acetyl-cysteine radical cation and anion were studied using ion-molecule reactions, infrared multi-photon dissociation (IRMPD) spectroscopy, and density functional theory (DFT) calculations. The radical cation was generated by first nitrosylating the thiol of N-acetyl-cysteine followed by the homolytic cleavage of the S-NO bond in the gas phase. IRMPD spectroscopy coupled with DFT calculations revealed that for the radical cation the radical migrates from its initial position on the sulfur atom to the α-carbon position, which is 2.5 kJ mol-1 lower in energy. The radical migration was confirmed by time-resolved ion-molecule reactions. These results are in contrast with our previous study on cysteine methyl ester radical cation (Osburn et al., Chem. Eur. J. 2011, 17, 873-879) and the study by Sinha et al. for cysteine radical cation ( Phys. Chem. Chem. Phys. 2010, 12, 9794-9800) where the radical was found to stay on the sulfur atom as formed. A similar approach allowed us to form a hydrogen-deficient radical anion of N-acetyl-cysteine, (M - 2H) •- . IRMPD studies and ion-molecule reactions performed on the radical anion showed that the radical remains on the sulfur, which is the initial and more stable (by 63.6 kJ mol-1) position, and does not rearrange.

  12. CXCL8 histone H3 acetylation is dysfunctional in airway smooth muscle in asthma: regulation by BET

    PubMed Central

    Clifford, Rachel L.; Patel, Jamie K.; John, Alison E.; Tatler, Amanda L.; Mazengarb, Lisa; Brightling, Christopher E.

    2015-01-01

    Asthma is characterized by airway inflammation and remodeling and CXCL8 is a CXC chemokine that drives steroid-resistant neutrophilic airway inflammation. We have shown that airway smooth muscle (ASM) cells isolated from asthmatic individuals secrete more CXCL8 than cells from nonasthmatic individuals. Here we investigated chromatin modifications at the CXCL8 promoter in ASM cells from nonasthmatic and asthmatic donors to further understand how CXCL8 is dysregulated in asthma. ASM cells from asthmatic donors had increased histone H3 acetylation, specifically histone H3K18 acetylation, and increased binding of histone acetyltransferase p300 compared with nonasthmatic donors but no differences in CXCL8 DNA methylation. The acetylation reader proteins Brd3 and Brd4 were bound to the CXCL8 promoter and Brd inhibitors inhibited CXCL8 secretion from ASM cells by disrupting Brd4 and RNA polymerase II binding to the CXCL8 promoter. Our results show a novel dysregulation of CXCL8 transcriptional regulation in asthma characterized by a promoter complex that is abnormal in ASM cells isolated from asthmatic donors and can be modulated by Brd inhibitors. Brd inhibitors may provide a new therapeutic strategy for steroid-resistant inflammation. PMID:25713319

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

    PubMed Central

    2012-01-01

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

  14. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false N-Acetyl-L-methionine. 172.372 Section 172.372 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.372 N-Acetyl-L-methionine....

  15. 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

  16. Nucleosome Dancing at the Tempo of Histone Tail Acetylation

    PubMed Central

    Galvani, Angélique; Thiriet, Christophe

    2015-01-01

    The impact of histone acetylation on transcription was revealed over 50 years ago by Allfrey and colleagues. However, it took decades for an understanding of the fine mechanism by which this posttranslational modification affects chromatin structure and promotes transcription. Here, we review breakthroughs linking histone tail acetylation, histone dynamics, and transcription. We also discuss the histone exchange during transcription and highlight the important function of a pool of non-chromatinized histones in chromatin dynamics. PMID:26184324

  17. Synthesis of trisaccharide methyl glycosides related to fragments of the capsular polysaccharide of Streptococcus pneumoniae type 18C.

    PubMed

    van Steijn, A M; Kamerling, J P; Vliegenthart, J F

    1992-03-01

    The synthesis is reported of methyl 3-O-(4-O-beta-D-galactopyranosyl-alpha-D- glucopyranosyl)-alpha-L-rhamnopyranoside (1), methyl 2-O-alpha-D-glucopyranosyl-4-O-beta-D-glucopyranosyl-beta-D- galactopyranoside (3), methyl 3-O-(4-O-beta-D-galactopyranosyl-alpha-D-glucopyranosyl)-alpha-L- rhamnopyranoside 3"-(sn-glycer-3-yl sodium phosphate) (2), and methyl 2-O-alpha-D-glucopyranosyl-4-O-beta-D- glucopyranosyl-beta-D-galactopyranoside 3-(sn-glycer-3-yl sodium phosphate) (4), which are trisaccharide methyl glycosides related to fragments of the capsular polysaccharide of Streptococcus pneumoniae type 18C ([----4)-beta-D- Glcp-(1----4)-[alpha-D-Glcp-(1----2)]-[Glycerol-(1-P----3)]-beta-D-Galp - (1----4)-alpha-D-Glcp-(1----3)-alpha-L-Rhap-(1----]n). Ethyl 4-O-acetyl-2,3,6-tri-O-benzyl-1-thio-beta-D-glucopyranoside (10) was coupled with benzyl 2,4-di-O-benzyl-alpha-L-rhamnopyranoside (6). Deacetylation of the product, followed by condensation with 2,4,6-tri-O-acetyl-3-O-allyl-alpha-D-galactopyranosyl trichloroacetimidate (18), gave benzyl 2,4-di-O-benzyl-3-O-[2,3,6-tri-O- benzyl-4-O-(2,4,6-tri-O-acetyl-3-O-allyl-beta-D-galactopyranosyl)-alpha- D- glucopyranosyl]-alpha-L-rhamnopyranoside (19). Acetolysis of 19, followed by methylation, deallylation (----22), and further deprotection afforded 1. Condensation of methyl 2,4-di-O-benzyl-3-O-[2,3,6-tri-O-benzyl-4-O-(2,4,6-tri- O-acetyl-beta-D-galactopyranosyl)-alpha-D-glucopyranosyl]-alpha-L- rhamnopyranoside (22) with 1,2-di-O-benzyl-sn-glycerol 3-(triethyl-ammonium phosphonate) (24), followed by oxidation and deprotection, yielded 2. Condensation of ethyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-glucopyranoside (27) with methyl 3-O-allyl-4,6-O-benzylidene-beta-D-galactopyranoside (28), selective benzylidene ring-opening of the product, coupling with 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (31), and deallylation afforded methyl 6-O-benzyl-4-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-2-O- (2

  18. 6-Methyl-2-pyridyl N-acetyl-1-thio-β-d-glucosa-minide methanol monosolvate.

    PubMed

    Chen, Bo; Guo, Miao; Jin, Wei-Hua; Wang, Yan-Wei; Liang, Hong-Ze

    2010-01-01

    In the title compound, C(14)H(20)N(2)O(5)S·CH(4)O, the pyran-ose and pyridine rings are linked through an S atom. The pyran-ose ring has a normal chair conformation. An intra-molecular O-H⋯N hydrogen bond occurs. Inter-molecular O-H⋯O, N-H⋯O, O-H⋯N and weak C-H⋯O hydrogen bonding is present in the crystal structure. PMID:21587547

  19. Tyr-phosphorylation of PDP1 toggles recruitment between ACAT1 and SIRT3 to regulate pyruvate dehydrogenase complex

    PubMed Central

    Fan, Jun; Shan, Changliang; Kang, Hee-Bum; Elf, Shannon; Xie, Jianxin; Tucker, Meghan; Gu, Ting-Lei; Aguiar, Mike; Lonning, Scott; Chen, Huaibin; Mohammadi, Moosa; Britton, Laura-Mae P.; Garcia, Benjamin A.; Aleckovic, Masa; Kang, Yibin; Kaluz, Stefan; Devi, Narra; Van Meir, Erwin; Hitosugi, Taro; Seo, Jae Ho; Lonial, Sagar; Gaddh, Manila; Arellano, Martha; Khoury, Hanna J.; Khuri, Fadlo R.; Boggon, Titus J.; Kang, Sumin; Chen, Jing

    2014-01-01

    SUMMARY Mitochondrial pyruvate dehydrogenase complex (PDC) is crucial for glucose homoeostasis in mammalian cells. The current understanding of PDC regulation involves inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) by PDH kinase (PDK), whereas dephosphorylation of PDH by PDH phosphatase (PDP) activates PDC. Here we report that lysine acetylation of PDHA1 and PDP1 is common in EGF-stimulated cells and diverse human cancer cells. K321 acetylation inhibits PDHA1 by recruiting PDK1 and K202 acetylation inhibits PDP1 by dissociating its substrate PDHA1, both of which are important to promote glycolysis in cancer cells and consequent tumor growth. Moreover, we identified mitochondrial ACAT1 and SIRT3 as the upstream acetyltransferase and deacetylase, respectively, of PDHA1 and PDP1, while knockdown of ACAT1 attenuates tumor growth. Furthermore, Y381 phosphorylation of PDP1 dissociates SIRT3 and recruits ACAT1 to PDC. Together, hierarchical, distinct post-translational modifications act in concert to control molecular composition of PDC and contribute to the Warburg effect. PMID:24486017

  20. UV Damage-Induced Phosphorylation of HBO1 Triggers CRL4DDB2-Mediated Degradation To Regulate Cell Proliferation.

    PubMed

    Matsunuma, Ryoichi; Niida, Hiroyuki; Ohhata, Tatsuya; Kitagawa, Kyoko; Sakai, Satoshi; Uchida, Chiharu; Shiotani, Bunsyo; Matsumoto, Masaki; Nakayama, Keiichi I; Ogura, Hiroyuki; Shiiya, Norihiko; Kitagawa, Masatoshi

    2016-02-01

    Histone acetyltransferase binding to ORC-1 (HBO1) is a critically important histone acetyltransferase for forming the prereplicative complex (pre-RC) at the replication origin. Pre-RC formation is completed by loading of the MCM2-7 heterohexameric complex, which functions as a helicase in DNA replication. HBO1 recruited to the replication origin by CDT1 acetylates histone H4 to relax the chromatin conformation and facilitates loading of the MCM complex onto replication origins. However, the acetylation status and mechanism of regulation of histone H3 at replication origins remain elusive. HBO1 positively regulates cell proliferation under normal cell growth conditions. Whether HBO1 regulates proliferation in response to DNA damage is poorly understood. In this study, we demonstrated that HBO1 was degraded after DNA damage to suppress cell proliferation. Ser50 and Ser53 of HBO1 were phosphorylated in an ATM/ATR DNA damage sensor-dependent manner after UV treatment. ATM/ATR-dependently phosphorylated HBO1 preferentially interacted with DDB2 and was ubiquitylated by CRL4(DDB2). Replacement of endogenous HBO1 in Ser50/53Ala mutants maintained acetylation of histone H3K14 and impaired cell cycle regulation in response to UV irradiation. Our findings demonstrate that HBO1 is one of the targets in the DNA damage checkpoint. These results show that ubiquitin-dependent control of the HBO1 protein contributes to cell survival during UV irradiation. PMID:26572825

  1. Effects of peptide acetylation and dimethylation on electrospray ionization efficiency.

    PubMed

    Cho, Kyung-Cho; Kang, Jeong Won; Choi, Yuri; Kim, Tae Woo; Kim, Kwang Pyo

    2016-02-01

    Peptide acetylation and dimethylation have been widely used to derivatize primary amino groups (peptide N-termini and the ε-amino group of lysines) for chemical isotope labeling of quantitative proteomics or for affinity tag labeling for selection and enrichment of labeled peptides. However, peptide acetylation results in signal suppression during electrospray ionization (ESI) due to charge neutralization. In contrast, dimethylated peptides show increased ionization efficiency after derivatization, since dimethylation increases hydrophobicity and maintains a positive charge on the peptide under common LC conditions. In this study, we quantitatively compared the ESI efficiencies of acetylated and dimethylated model peptides and tryptic peptides of BSA. Dimethylated peptides showed higher ionization efficiency than acetylated peptides for both model peptides and tryptic BSA peptides. At the proteome level, peptide dimethylation led to better protein identification than peptide acetylation when tryptic peptides of mouse brain lysate were analyzed with LC-ESI-MS/MS. These results demonstrate that dimethylation of tryptic peptides enhanced ESI efficiency and provided up to two-fold improved protein identification sensitivity in comparison with acetylation. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26889926

  2. Acetyl radical generation in cigarette smoke: Quantification and simulations

    NASA Astrophysics Data System (ADS)

    Hu, Na; Green, Sarah A.

    2014-10-01

    Free radicals are present in cigarette smoke and can have a negative effect on human health. However, little is known about their formation mechanisms. Acetyl radicals were quantified in tobacco smoke and mechanisms for their generation were investigated by computer simulations. Acetyl radicals were trapped from the gas phase using 3-amino-2, 2, 5, 5-tetramethyl-proxyl (3AP) on solid support to form stable 3AP adducts for later analysis by high-performance liquid chromatography (HPLC), mass spectrometry/tandem mass spectrometry (MS-MS/MS) and liquid chromatography-mass spectrometry (LC-MS). Simulations were performed using the Master Chemical Mechanism (MCM). A range of 10-150 nmol/cigarette of acetyl radical was measured from gas phase tobacco smoke of both commercial and research cigarettes under several different smoking conditions. More radicals were detected from the puff smoking method compared to continuous flow sampling. Approximately twice as many acetyl radicals were trapped when a glass fiber particle filter (GF/F specifications) was placed before the trapping zone. Simulations showed that NO/NO2 reacts with isoprene, initiating chain reactions to produce hydroxyl radical, which abstracts hydrogen from acetaldehyde to generate acetyl radical. These mechanisms can account for the full amount of acetyl radical detected experimentally from cigarette smoke. Similar mechanisms may generate radicals in second hand smoke.

  3. Acetyl Radical Generation in Cigarette Smoke: Quantification and Simulations

    PubMed Central

    Hu, Na; Green, Sarah A.

    2014-01-01

    Free radicals are present in cigarette smoke and can have a negative effect on human health. However, little is known about their formation mechanisms. Acetyl radicals were quantified in tobacco smoke and mechanisms for their generation were investigated by computer simulations. Acetyl radicals were trapped from the gas phase using 3-amino-2, 2, 5, 5-tetramethyl-proxyl (3AP) on solid support to form stable 3AP adducts for later analysis by high performance liquid chromatography (HPLC), mass spectrometry/tandem mass spectrometry (MS-MS/MS) and liquid chromatography–mass spectrometry (LC-MS). Simulations were performed using the Master Chemical Mechanism (MCM). A range of 10–150 nmol/cigarette of acetyl radical was measured from gas phase tobacco smoke of both commerial and research cigarettes under several different smoking conditions. More radicals were detected from the puff smoking method compared to continuous flow sampling. Approximately twice as many acetyl radicals were trapped when a glass filber particle filter (GF/F specifications) was placed before the trapping zone. Simulations showed that NO/NO2 reacts with isoprene, initiating chain reactions to produce hydroxyl radical, which abstracts hydrogen from acealdehyde to generate acetyl radical. These mechanisms can account for the full amount of acetyl radical detected experimentally from cigarette smoke. Similar mechanisms may generate radicals in second hand smoke. PMID:25253993

  4. Phosphorylated nano-diamond/ Polyimide Nanocomposites

    NASA Astrophysics Data System (ADS)

    Beyler-Çiǧil, Asli; Çakmakçi, Emrah; Vezir Kahraman, Memet

    2014-08-01

    In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased.

  5. mTOR complex 2 controls glycolytic metabolism in glioblastoma through FoxO acetylation and upregulation of c-Myc.

    PubMed

    Masui, Kenta; Tanaka, Kazuhiro; Akhavan, David; Babic, Ivan; Gini, Beatrice; Matsutani, Tomoo; Iwanami, Akio; Liu, Feng; Villa, Genaro R; Gu, Yuchao; Campos, Carl; Zhu, Shaojun; Yang, Huijun; Yong, William H; Cloughesy, Timothy F; Mellinghoff, Ingo K; Cavenee, Webster K; Shaw, Reuben J; Mischel, Paul S

    2013-11-01

    Aerobic glycolysis (the Warburg effect) is a core hallmark of cancer, but the molecular mechanisms underlying it remain unclear. Here, we identify an unexpected central role for mTORC2 in cancer metabolic reprogramming where it controls glycolytic metabolism by ultimately regulating the cellular level of c-Myc. We show that mTORC2 promotes inactivating phosphorylation of class IIa histone deacetylases, which leads to the acetylation of FoxO1 and FoxO3, and this in turn releases c-Myc from a suppressive miR-34c-dependent network. These central features of activated mTORC2 signaling, acetylated FoxO, and c-Myc levels are highly intercorrelated in clinical samples and with shorter survival of GBM patients. These results identify a specific, Akt-independent role for mTORC2 in regulating glycolytic metabolism in cancer. PMID:24140020

  6. Starch phosphorylation: insights and perspectives.

    PubMed

    Mahlow, Sebastian; Orzechowski, Sławomir; Fettke, Joerg

    2016-07-01

    During starch metabolism, the phosphorylation of glucosyl residues of starch, to be more precise of amylopectin, is a repeatedly observed process. This phosphorylation is mediated by dikinases, the glucan, water dikinase (GWD) and the phosphoglucan, water dikinase (PWD). The starch-related dikinases utilize ATP as dual phosphate donor transferring the terminal γ-phosphate group to water and the β-phosphate group selectively to either C6 position or C3 position of a glucosyl residue within amylopectin. By the collaborative action of both enzymes, the initiation of a transition of α-glucans from highly ordered, water-insoluble state to a less order state is realized and thus the initial process of starch degradation. Consequently, mutants lacking either GWD or PWD reveal a starch excess phenotype as well as growth retardation. In this review, we focus on the increased knowledge collected over the last years related to enzymatic properties, the precise definition of the substrates, the physiological implications, and discuss ongoing questions. PMID:27147464

  7. Phosphorylation Sites Identified in the NEIL1 DNA Glycosylase Are Potential Targets for the JNK1 Kinase

    PubMed Central

    Prakash, Aishwarya; Cao, Vy Bao; Doublié, Sylvie

    2016-01-01

    The NEIL1 DNA glycosylase is one of eleven mammalian DNA glycosylases that partake in the first step of the base excision repair (BER) pathway. NEIL1 recognizes and cleaves mainly oxidized pyrimidines from DNA. The past decade has witnessed the identification of an increasing number of post-translational modifications (PTMs) in BER enzymes including phosphorylation, acetylation, and sumoylation, which modulate enzyme function. In this work, we performed the first comprehensive analysis of phosphorylation sites in human NEIL1 expressed in human cells. Mass spectrometry (MS) analysis revealed phosphorylation at three serine residues: S207, S306, and a third novel site, S61. We expressed, purified, and characterized phosphomimetic (glutamate) and phosphoablating (alanine) mutants of the three phosphorylation sites in NEIL1 revealed by the MS analysis. All mutant enzymes were active and bound tightly to DNA, indicating that phosphorylation does not affect DNA binding and enzyme activity at these three serine sites. We also characterized phosphomimetic mutants of two other sites of phosphorylation, Y263 and S269, reported previously, and observed that mutation of Y263 to E yielded a completely inactive enzyme. Furthermore, based on sequence motifs and kinase prediction algorithms, we identified the c-Jun N-terminal kinase 1 (JNK1) as the kinase involved in the phosphorylation of NEIL1. JNK1, a member of the mitogen activated protein kinase (MAPK) family, was detected in NEIL1 immunoprecipitates, interacted with NEIL1 in vitro, and was able to phosphorylate the enzyme at residues S207, S306, and S61. PMID:27518429

  8. Hepatitis B virus basal core promoter mutations show lower replication fitness associated with cccDNA acetylation status.

    PubMed

    Koumbi, Lemonica; Pollicino, Teresa; Raimondo, Giovanni; Stampoulis, Dimitrios; Khakoo, Salim; Karayiannis, Peter

    2016-07-15

    In chronic hepatitis B virus (HBV) infection, variants with mutations in the basal core promoter (BCP) and precore region predominate and associate with more severe disease forms. Studies on their effect on viral replication remain controversial. Increasing evidence shows that epigenetic modifications of cccDNA regulate HBV replication and disease outcome. Here we determined the transcription and viral replication efficiency of well-defined BCP and precore mutations and their effect on cccDNA epigenetic control. HBV monomers bearing BCP mutations A1762T/G1764A and A1762T/G1764A/C1766T, and precore mutations G1896A, G1899A and G1896A/G1899A, were transfected into HepG2 cells using a plasmid-free approach. Viral RNA transcripts were detected by Northern blot hybridization and RT PCR, DNA replicative intermediates by Southern blotting and RT PCR, and viral release was measured by ELISA. Acetylation of cccDNA-bound histones was assessed by Chromatin ImmunoPrecipitation (ChIP) assay and methylation of cccDNA by bisulfite sequencing. BCP mutations resulted in low viral release, mRNA transcription and pgRNA/cccDNA ratios that paralleled the acetylation of cccDNA-bound H4 histone and inversely correlated with the HDAC1 recruitment onto cccDNA. Independently of the mutations, cccDNA was a target for methylation, accompanied by the upregulation of DNMT1 expression and DNMT1 recruitment onto cccDNA. Our results suggest that BCP mutations decrease viral replication capacity possibly by modulating the acetylation and deacetylation of cccDNA-bound histones while precore mutations do not have a significant effect on viral replication. These data provide evidence that epigenetic factors contribute to the regulation of HBV viral replication. PMID:27132039

  9. Histone tyrosine phosphorylation comes of age

    PubMed Central

    Singh, Rakesh Kumar

    2011-01-01

    Histones were discovered over a century ago and have since been found to be the most extensively post-translationally modified proteins, although tyrosine phosphorylation of histones had remained elusive until recently. The year 2009 proved to be a landmark year for histone tyrosine (Y) phosphorylation as five research groups independently discovered this modification. Three groups describe phosphorylation of Y142 in the variant histone H2A.X, where it may be involved in the cellular decision making process to either undergo DNA repair or apoptosis in response to DNA damage. Further, one group suggests that phosphorylation of histone H3 on Y99 is crucial for its regulated proteolysis in yeast, while another found that Y41 phosphorylation modulates chromatin architecture and oncogenesis in mammalian cells. These pioneering studies provide the initial conceptual framework for further analyses of the diverse roles of tyrosine phosphorylation on different histones, with far reaching implications for human health and disease. PMID:20935492

  10. Prebiotic phosphorylation of nucleosides in formamide

    NASA Technical Reports Server (NTRS)

    Schoffstall, A. M.

    1976-01-01

    Results are presented for an experimental study intended to assess phosphorylation under neither aqueous nor dry thermal conditions. Instead, phosphorylations were attempted in possible nonaqueous prebiotic solvents. Formamide appeared to be the most obvious candidate for phosphorylation studies. Three main classes of phosphorylated products were formed in formamide solution: adenosine monophosphates, cyclic adenosine phosphate, and adenosine diphosphates. Experiments were designed to investigate the extent of phosphorylation of nucleosides in formamide, the relative amounts of nucleoside monophosphate, diphosphates and cyclic phosphate formed and the relative effectiveness of different sources of phosphate as phosphorylating agents in formamide. Reaction variables were temperature, nature of the phosphate or condensed phosphate, nucleoside, concentration of reactants and possible effects of additives. Product identification was based on qualitative and quantitative thin layer chromatography.

  11. The Rotational Spectrum and Conformational Structures of Methyl Valerate

    NASA Astrophysics Data System (ADS)

    Nguyen, Ha Vinh Lam; Stahl, Wolfgang

    2015-06-01

    Methyl valerate, C4H9COOCH3, belongs to the class of fruit esters, which play an important role in nature as odorants of different fruits, flowers, and wines. A sufficient explanation for the structure-odor relation of is not available. It is known that predicting the odor of a substance is not possible by knowing only its chemical formula. A typical example is the blueberry- or pine apple-like odor of ethyl isovalerate while its isomers ethyl valerate and isoamyl acetate smell like green apple and banana, respectively. Obviously, not only the composition but also the molecular structures are not negligible by determining the odor of a substance. Gas phase structures of fruit esters are thus important for a first step towards the determination of structure-odor relation since the sense of smell starts from gas phase molecules. For this purpose, a combination of microwave spectroscopy and quantum chemical calculations (QCCs) is an excellent tool. Small esters often have sufficient vapor pressure to be transferred easily in the gas phase for a rotational study but already contain a large number of atoms which makes them too big for classical structure determination by isotopic substitution and requires nowadays a comparison with the structures optimized by QCCs. On the other hand, the results from QCCs have to be validated by the experimental values. About the internal dynamics, the methoxy methyl group -COOCH3 of methyl acetate shows internal rotation with a barrier of 424.581(56) wn. A similar barrier height of 429.324(23) wn was found in methyl propionate, where the acetyl group is extended to the propionyl group. The investigation on methyl valerate fits well in this series of methyl alkynoates. In this talk, the structure of the most energetic favorable conformer as well as the internal rotation shown by the methoxy methyl group will be reported. It could be confirmed that the internal rotation barrier of the methoxy methyl group remains by longer alkyl chain.

  12. Identification of cellular factors binding to acetylated HIV-1 integrase.

    PubMed

    Allouch, Awatef; Cereseto, Anna

    2011-11-01

    The viral protein integrase (IN) catalyzes the integration of the HIV-1 cDNA into the host cellular genome. We have recently demonstrated that IN is acetylated by a cellular histone acetyltransferase, p300, which modifies three lysines located in the C-terminus of the viral factor (Cereseto et al. in EMBO J 24:3070-3081, 2005). This modification enhances IN catalytic activity, as demonstrated by in vitro assays. Consistently, mutations introduced in the targeted lysines greatly decrease the efficiency of HIV-1 integration. Acetylation was proven to regulate protein functions by modulating protein-protein interactions. HIV-1 to efficiently complete its replication steps, including the integration reaction, requires interacting with numerous cellular factors. Therefore, we sought to investigate whether acetylation might modulate the interaction between IN and the cellular factors. To this aim we performed a yeast two-hybrid screening that differs from the screenings so far performed (Rain et al. in Methods 47:291-297, 2009; Studamire and Goff in Retrovirology 5:48, 2008) for using as bait IN constitutively acetylated. From this analysis we have identified thirteen cellular factors involved in transcription, chromatin remodeling, nuclear transport, RNA binding, protein synthesis regulation and microtubule organization. To validate these interactions, binding assays were performed showing that acetylation increases the affinity of IN with specific factors. Nevertheless, few two-hybrid hits bind with the same affinity the acetylated and the unmodified IN. These results further underlie the relevance of IN post-translational modification by acetylation in HIV-1 replication cycle. PMID:20016921

  13. [Sugar phosphorylation activities in acetogenic bacteria].

    PubMed

    Jiang, W; Patterson, J A

    1999-12-01

    Seven acetogenic bacteria (Acetitomaculum ruminis, Acetobacterium woodii, Eubacterium limosum as well as isolates A2, A4, A10 and H3HH) were tested for PEP- and ATP-dependent phosphorylation of glucose and 2-deoxyglucose. Although all organisms had detectable phosphorylation activity, substantial variation existed in the rates of both PEP- and ATP-dependent phosphorylation. Isolate Alo had the highest rate of PEP-dependent phosphorylation of 11.62 nmol.L-1.mg-1.min-1. Isolate A10, H3HH as well as E. limosum most likely have a glucose phosphotransferase system(PTS). In contrast, A ruminis, A. woodii and isolate A2, A4 had PEP-dependent glucose phosphorylation rates very similar to control rates, suggesting the lack of PTS activity. The rates of ATP-dependent glucose phosphorylation were higher than PEP-dependent phosphorylation in all organisms surveyed. However, substantial variation existed in the rates of ATP-dependent glucose phosphorylation. The glucose PTS of isolates A10 and H3HH were induced by the presence of extracellular glucose. Moreover, the specific activity of the glucose PTS of both isolates increased as cultures progressed from the early log to late log phase of growth. ATP- and PEP-dependent maltose and sucrose phosphorylation was detected in isolates A10 and H3HH. Although activity was detected in both isolates(A10 and H3HH), the rate of activity varied considerably, depending on the sugar and organism tested. PMID:12555560

  14. Hydroxyl radical formation via iron-mediated Fenton chemistry is inhibited by methylated catechols.

    PubMed

    Nappi, A J; Vass, E

    1998-09-16

    The differing effects of O-methylated catecholamines and their dihydroxyphenyl precursors on the production of *OH were quantified using a previously established specific salicylate hydroxylation assay in conjunction with a sensitive electrochemical detection system. The production of *OH by the Fenton reaction was diminished significantly by O-methylated catecholamines (O-methyldopa, O-methyldopamine, O-methyltyrosine, and N-acetyl-O-methyldopamine), whereas radical production was augmented by dihydroxyphenyls (DOPA, dopamine, and N-acetyldopamine), including those with methylated side chains (N-methyldopamine and alpha-methyldopa). Monohydroxyphenyls such as octopamine, tyramine, tyrosine, and alpha-methyltyrosine had little or no effect on radical production. These data show that a methyl group positioned on the side chain of a catecholamine does not alter its pro-oxidant behavior, while a methyl group positioned on the aromatic ring renders the catecholamine sterically or kinetically unfavorable for coordination with transition metals, thus preventing the promotion of Fenton chemistry. These results highlight the importance of O-methylation in forming catechols that are less reactive than their dihydroxyphenyl precursors. Thus, factors regulating the methylation of brain catecholamines may play a crucial role in mediating neuronal integrity during aging and in the pathogenesis of certain neurodegenerative disorders. Competitive side-chain methylation reactions may sustain or perpetuate some dihydroxyphenyls, creating an oxidatively less favorable environment for cells than would result from compounds formed by O-methylation. PMID:9813302

  15. Arabidopsis and Brachypodium distachyon transgenic plants expressing Aspergillus nidulans acetylesterases have decreased degree of polysaccharide acetylation and increased resistance to pathogens.

    PubMed

    Pogorelko, Gennady; Lionetti, Vincenzo; Fursova, Oksana; Sundaram, Raman M; Qi, Mingsheng; Whitham, Steven A; Bogdanove, Adam J; Bellincampi, Daniela; Zabotina, Olga A

    2013-05-01

    The plant cell wall has many significant structural and physiological roles, but the contributions of the various components to these roles remain unclear. Modification of cell wall properties can affect key agronomic traits such as disease resistance and plant growth. The plant cell wall is composed of diverse polysaccharides often decorated with methyl, acetyl, and feruloyl groups linked to the sugar subunits. In this study, we examined the effect of perturbing cell wall acetylation by making transgenic Arabidopsis (Arabidopsis thaliana) and Brachypodium (Brachypodium distachyon) plants expressing hemicellulose- and pectin-specific fungal acetylesterases. All transgenic plants carried highly expressed active Aspergillus nidulans acetylesterases localized to the apoplast and had significant reduction of cell wall acetylation compared with wild-type plants. Partial deacetylation of polysaccharides caused compensatory up-regulation of three known acetyltransferases and increased polysaccharide accessibility to glycosyl hydrolases. Transgenic plants showed increased resistance to the fungal pathogens Botrytis cinerea and Bipolaris sorokiniana but not to the bacterial pathogens Pseudomonas syringae and Xanthomonas oryzae. These results demonstrate a role, in both monocot and dicot plants, of hemicellulose and pectin acetylation in plant defense against fungal pathogens. PMID:23463782

  16. Thyroid-stimulating hormone decreases HMG-CoA reductase phosphorylation via AMP-activated protein kinase in the liver

    PubMed Central

    Zhang, Xiujuan; Song, Yongfeng; Feng, Mei; Zhou, Xinli; Lu, Yingli; Gao, Ling; Yu, Chunxiao; Jiang, Xiuyun; Zhao, Jiajun

    2015-01-01

    Cholesterol homeostasis is strictly regulated through the modulation of HMG-CoA reductase (HMGCR), the rate-limiting enzyme of cholesterol synthesis. Phosphorylation of HMGCR inactivates it and dephosphorylation activates it. AMP-activated protein kinase (AMPK) is the major kinase phosphorylating the enzyme. Our previous study found that thyroid-stimulating hormone (TSH) increased the hepatocytic HMGCR expression, but it was still unclear whether TSH affected hepatic HMGCR phosphorylation associated with AMPK. We used bovine TSH (bTSH) to treat the primary mouse hepatocytes and HepG2 cells with or without constitutively active (CA)-AMPK plasmid or protein kinase A inhibitor (H89), and set up the TSH receptor (Tshr)-KO mouse models. The p-HMGCR, p-AMPK, and related molecular expression were tested. The ratios of p-HMGCR/HMGCR and p-AMPK/AMPK decreased in the hepatocytes in a dose-dependent manner following bTSH stimulation. The changes above were inversed when the cells were treated with CA-AMPK plasmid or H89. In Tshr-KO mice, the ratios of liver p-HMGCR/HMGCR and p-AMPK/AMPK were increased relative to the littermate wild-type mice. Consistently, the phosphorylation of acetyl-CoA carboxylase, a downstream target molecule of AMPK, increased. All results suggested that TSH could regulate the phosphorylation of HMGCR via AMPK, which established a potential mechanism for hypercholesterolemia involved in a direct action of the TSH in the liver. PMID:25713102

  17. Phosphorylation of human link proteins

    SciTech Connect

    Oester, D.A.; Caterson, B.; Schwartz, E.R.

    1986-06-13

    Three link proteins of 48, 44 and 40 kDa were purified from human articular cartilage and identified with monoclonal anti-link protein antibody 8-A-4. Two sets of lower molecular weight proteins of 30-31 kDa and 24-26 kDa also contained link protein epitopes recognized by the monoclonal antibody and were most likely degradative products of the intact link proteins. The link proteins of 48 and 40 kDa were identified as phosphoproteins while the 44 kDa link protein did not contain /sup 32/P. The phosphorylated 48 and 40 kDa link proteins contained approximately 2 moles PO/sub 4//mole link protein.

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

    PubMed Central

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

    2016-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first reaction in the pentose phosphate pathway, and generates ribose sugars, which are required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosphate (NADPH), which is important for neutralization of oxidative stress. The expression of G6PD is elevated in several types of tumor, including colon, breast and lung cancer, and has been implicated in cancer cell growth. Our previous study demonstrated that exposure of HCT 116 human colorectal cancer cells to aspirin caused acetylation of G6PD, and this was associated with a decrease in its enzyme activity. In the present study, this observation was expanded to HT-29 colorectal cancer cells, in order to compare aspirin-mediated acetylation of G6PD and its activity between HCT 116 and HT-29 cells. In addition, the present study aimed to determine the acetylation targets of aspirin on recombinant G6PD to provide an insight into the mechanisms of inhibition. The results demonstrated that the extent of G6PD acetylation was significantly higher in HCT 116 cells compared with in HT-29 cells; accordingly, a greater reduction in G6PD enzyme activity was observed in the HCT 116 cells. Mass spectrometry analysis of aspirin-acetylated G6PD (isoform a) revealed that aspirin acetylated a total of 14 lysine residues, which were dispersed throughout the length of the G6PD protein. One of the important amino acid targets of aspirin included lysine 235 (K235, in isoform a) and this corresponds to K205 in isoform b, which has previously been identified as being important for catalysis. Acetylation of G6PD at several sites, including K235 (K205 in isoform b), may mediate inhibition of G6PD activity, which may contribute to the ability of aspirin to exert anticancer effects through decreased synthesis of ribose sugars and NADPH. PMID:27356773

  19. Olig1 Acetylation and Nuclear Export Mediate Oligodendrocyte Development.

    PubMed

    Dai, Jinxiang; Bercury, Kathryn K; Jin, Weilin; Macklin, Wendy B

    2015-12-01

    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. PMID:26631469

  20. Acetyl group availability influences phosphocreatine degradation even during intense muscle contraction.

    PubMed

    Timmons, James A; Constantin-Teodosiu, Dumitru; Poucher, Simon M; Greenhaff, Paul L

    2004-12-15

    We previously established that activation of the pyruvate dehydrogenase complex (PDC) using dichloroacetate (DCA) reduced the reliance on substrate-level phosphorylation (SLP) at the onset of exercise, with normal and reduced blood flow. PDC activation also reduced fatigue development during contraction with reduced blood flow. Since these observations, several studies have re-evaluated our observations. One study demonstrated a performance benefit without a reduction in SLP, raising a question mark over PDC's role in the regulation of ATP regeneration and our interpretation of fatigue mechanisms. Using a model of muscle contraction similar to the conflicting study (i.e. tetanic rather than twitch stimulation), we re-examined this question. Using canine skeletal muscle, one group was infused with saline while the other was pretreated with 300 mg (kg body mass)(-1) DCA. Muscle biopsies were taken at rest, peak tension (1 min) and after 6 min of tetanic electrical stimulation (75 ms on-925 ms off per second) and blood flow was limited to 25% of normal values observed during contraction. DCA reduced phosphocreatine (PCr) degradation by 40% during the first minute of contraction, but did not prevent the almost complete depletion of PCr stores at 6 min, while muscle fatigue did not differ between the two groups. During intermittent tetanic stimulation PCr degradation was 75% greater than with our previous 3 Hz twitch contraction protocol, despite a similar rate of oxygen consumption at 6 min. Thus, in the present study enhanced acetyl group availability altered the time course of PCr utilization but did not prevent the decline towards depletion. Consistent with our earlier conclusions, DCA pretreatment reduces muscle fatigue only when SLP is attenuated. The present study and our met-analysis indicates that enhanced acetyl group availability results in a readily measurable reduction in SLP when the initial rate of PCr utilization is approximately 1 mmol (kg dry mass)(-1

  1. Reactive Oxygen Species Mediate Epstein-Barr Virus Reactivation by N-Methyl-N’-Nitro-N-Nitrosoguanidine

    PubMed Central

    Huang, Sheng-Yen; Fang, Chih-Yeu; Wu, Chung-Chun; Tsai, Ching-Hwa; Lin, Su-Fang; Chen, Jen-Yang

    2013-01-01

    N-nitroso compounds (NOCs) and Epstein-Barr virus (EBV) reactivation have been suggested to play a role in the development of nasopharyngeal carcinoma (NPC). Although chemicals have been shown to be a risk factor contributing to the carcinogenesis of NPC, the underlying mechanism is not fully understood. We demonstrated recently that N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) enhances the genomic instability and tumorigenicity of NPC cells via induction of EBV reactivation. However, the mechanisms that trigger EBV reactivation from latency remain unclear. Here, we address the role of ROS in induction of EBV reactivation under MNNG treatment. EBV reactivation was induced in over 70% of EBV-positive NA cells and the promoter of Rta (Rp) was activated after MNNG treatment. Inhibitor experiments revealed ATM, p38 MAPK and JNK were activated by ROS and involved in MNNG-induced EBV reactivation. Significantly, ROS scavengers N-acetyl-L-cysteine (NAC), catalase and reduced glutathione inhibited EBV reactivation under MNNG and H2O2 treatment, suggesting ROS mediate EBV reactivation. The p53 was essential for EBV reactivation and the Rp activation by MNNG. Moreover, the p53 was phosphorylated, translocated into nucleus, and bound to Rp following ROS stimulation. The results suggest ROS play an important role in initiation of EBV reactivation by MNNG through a p53-dependent mechanism. Our findings demonstrate novel signaling mechanisms used by NOCs to induce EBV reactivation and provide a novel insight into NOCs link the EBV reactivation in the contribution to the development of NPC. Notably, this study indicates that antioxidants might be effective for inhibiting N-nitroso compound-induced EBV reactivation and therefore could be promising preventive and therapeutic agents for EBV reactivation-associated malignancies. PMID:24376853

  2. Phosphorylation of the multidrug resistance associated glycoprotein

    SciTech Connect

    Mellado, W.; Horwitz, S.B.

    1987-11-03

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistant phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl/sub 2/ was enhanced a minimum of 2-fold by 10 ..mu..M cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by (..gamma..-/sup 32/P)ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance.

  3. Phosphorylation of the multidrug resistance associated glycoprotein.

    PubMed

    Mellado, W; Horwitz, S B

    1987-11-01

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 microM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [gamma-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance. PMID:3427052

  4. [Accidental methyl alcohol poisoning].

    PubMed

    Xiao, J H

    1990-05-01

    An accidental poisoning due to drinking methyl alcohol in Chaoyang county is reported, analysing the accident. The poison came from the "retail white spirit" which was contaminated with methyl alcohol. Twenty-nine persons drank the wine, fourteen of them died, two of them became blind. After drinking this "retail white spirit" the drinkers showed symptoms of vertigo, headache, weakness, vomiting, night sweat, dyspnea and blurring of vision etc. within 6-120 hours. On examining the remaining spirit, we found the content of methyl alcohol to be between 16.6 and 40.69 g/100 ml. Some of the patients' urine and blood also contained methyl alcohol. We reckoned that each one of the twenty patients had taken more than 27 g of methyl alcohol and each of the ten dead drank more than 40 ml of the alcohol. PMID:2253526

  5. Determination of NAT2 acetylation status in the Greenlandic population.

    PubMed

    Geller, Frank; Soborg, Bolette; Koch, Anders; Michelsen, Sascha Wilk; Bjorn-Mortensen, Karen; Carstensen, Lisbeth; Birch, Emilie; Nordholm, Anne Christine; Johansen, Marie Mila Broby; Børresen, Malene Landbo; Feenstra, Bjarke; Melbye, Mads

    2016-04-01

    N-acetyltransferase 2 (NAT2) is a well-studied phase II xenobiotic metabolizing enzyme relevant in drug metabolism and cancerogenesis. NAT2 activity is largely determined by genetic polymorphisms in the coding region of the corresponding gene. We investigated NAT2 acetylation status in 1556 individuals from Greenland based on four different single nucleotide polymorphism (SNP) panels and the tagging SNP rs1495741. There was good concordance between the NAT2 status inferred by the different SNP combinations. Overall, the fraction of slow acetylators was low with 17.5 % and varied depending on the degree of Inuit ancestry; in individuals with <50 % Inuit ancestry, we observed more than 25 % slow acetylators reflecting European ancestry. Greenland has a high incidence of tuberculosis, and individual dosing of isoniazid according to NAT2 status has been shown to improve treatment and reduce side effects. Our findings could be a first step in pharmacogenetics-based tuberculosis therapy in Greenland. PMID:25794903

  6. Synthetic biology for engineering acetyl coenzyme A metabolism in yeast.

    PubMed

    Nielsen, Jens

    2014-01-01

    The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels, chemicals, and pharmaceuticals. The use of this cell factory for cost-efficient production of novel fuels and chemicals requires high yields and low by-product production. Many industrially interesting chemicals are biosynthesized from acetyl coenzyme A (acetyl-CoA), which serves as a central precursor metabolite in yeast. To ensure high yields in production of these chemicals, it is necessary to engineer the central carbon metabolism so that ethanol production is minimized (or eliminated) and acetyl-CoA can be formed from glucose in high yield. Here the perspective of generating yeast platform strains that have such properties is discussed in the context of a major breakthrough with expression of a functional pyruvate dehydrogenase complex in the cytosol. PMID:25370498

  7. Cisplatin stimulates protein tyrosine phosphorylation in macrophages.

    PubMed

    Kumar, R; Shrivastava, A; Sodhi, A

    1995-03-01

    Cisplatin [cis-dichlorodiamine platinum (II)], a potent anti-tumor compound, stimulates immune responses by activating monocyte-macrophages and other cells of the immune system. The mechanism by which cisplatin activates these cells is poorly characterized. Since protein tyrosine phosphorylation appears to be a major intracellular signalling event that mediates cellular responses, we examined whether cisplatin alters tyrosine phosphorylation in macrophages. We found that cisplatin increased tyrosine phosphorylation of several proteins in peritoneal macrophages and in P388D1 and IC-21 macrophage cell lines. Treatment of macrophages with tyrosine kinase inhibitors, genestein and lavendustin A, inhibited cisplatin-stimulated protein tyrosine phosphorylation in macrophages. Macrophages treated with cisplatin also exhibit increased fluorescence with anti-phosphotyrosine-FITC antibody. These data indicate that protein tyrosine phosphorylation plays a role in cisplatin-induced activation of macrophages. PMID:7539662

  8. Synthesis of polyrotaxanes from acetyl-β-cyclodextrin

    NASA Astrophysics Data System (ADS)

    Ristić, I. S.; Nikolić, L.; Nikolić, V.; Ilić, D.; Budinski-Simendić, J.

    2011-12-01

    Polyrotaxanes are intermediary products in the synthesis of topological gels. They are created by inclusion complex formation of hydrophobic linear macromolecules with cyclodextrins or their derivatives. Then, pairs of cyclodextrin molecules with covalently linkage were practically forming the nodes of the semi-flexible polymer network. Such gels are called topological gels and they can absorb huge quantities of water due to the net flexibility allowing the poly(ethylene oxide) chains to slide through the cyclodextrin cavities, without being pulled out altogether. For polyrotaxane formation poly(ethylene oxide) was used like linear macromolecules. There are hydroxyl groups at poly(ethylene oxide) chains, whereby the linking of the voluminous molecules should be made. To avoid the reaction of cyclodextrin OH groups with stoppers, they should be protected by, e.g., acetylation. In this work, the acetylation of the OH groups of β-cyclodextrin was performed by acetic acid anhydride with iodine as the catalyst. The acetylation reaction was assessed by the FTIR and HPLC method. By the HPLC analysis was found that the acetylation was completed in 20 minutes. Inserting of poly(ethylene oxide) with 4000 g/mol molecule mass into acetyl-β-cyclodextrin with 2:1 poly(ethylene oxide) monomer unit to acetyl-β-cyclodextrin ratio was also monitored by FTIR, and it was found that the process was completed in 12 h at the temperature of 10°C. If the process is performed at temperatures above 10°C, or for periods longer than 12 hours, the process of uncontrolled hydrolysis of acetate groups was initiated.

  9. Phosphorylation of NMDA NR1 subunits in the myenteric plexus during TNBS induced colitis.

    PubMed

    Zhou, QiQi; Caudle, Robert M; Moshiree, Baharak; Price, Donald D; Verne, G Nicholas

    2006-10-01

    N-Methyl-d-aspartic acid (NMDA) receptors are known to function in the mediation of pain and have a significant role in the development of hyperalgesia following inflammation. Serine phosphorylation regulation of NMDA receptor function occurs in a variety of conditions. No studies have demonstrated a change in phosphorylation of enteric NMDA receptors following colonic inflammation. We examined the levels of NMDA NR1 phosphorylation in trinitrobenzene sulfonic acid (TNBS) induced colitis in rats and compared it to protein translation and the development of visceral hypersensitivity. We have previously, demonstrated an increase in the C1 cassette of NR1 mRNA expression at 14, 21, and 28 days following TNBS administration. In this study, we examined the NR1 serine phosphorylation at 14 days following TNBS injection. Male Sprague-Dawley rats (200-250 g) were treated with TNBS (20mg per rat) diluted in 50% ethanol (n=3) and vehicle controls of 50% ethanol (n=3). TNBS and vehicle controls were administered with a 24 gauge catheter inserted into the lumen of the rat colon. The animals were sacrificed at 14 days after induction of the colitis and their distal colon was retrieved for two-dimensional (2D) western blot analysis. Serine phosphorylation of the NR1 subunit with C1 cassette appears at 14 days after TNBS injection. In contrast, there was no NR1-C1 expression in the vehicle controls and untreated normal controls. These results suggest a role for colonic-NMDA receptor phosphorylation in the development of neuronal plasticity following colonic inflammation. Phosphorylation of NR1 may partially explain visceral hypersensitivity present during colonic inflammation. PMID:16942839

  10. Phosphorylation decreases ubiquitylation of the thiazide-sensitive cotransporter NCC and subsequent clathrin-mediated endocytosis.

    PubMed

    Rosenbaek, Lena L; Kortenoeven, Marleen L A; Aroankins, Takwa S; Fenton, Robert A

    2014-05-01

    The thiazide-sensitive sodium chloride cotransporter, NCC, is the major NaCl transport protein in the distal convoluted tubule (DCT). The transport activity of NCC can be regulated by phosphorylation, but knowledge of modulation of NCC trafficking by phosphorylation is limited. In this study, we generated novel tetracycline-inducible Madin-Darby canine kidney type I (MDCKI) cell lines expressing NCC to examine the role of NCC phosphorylation and ubiquitylation on NCC endocytosis. In MDCKI-NCC cells, NCC was highly glycosylated at molecular weights consistent with NCC monomers and dimers. NCC constitutively cycles to the apical plasma membrane of MDCKI-NCC cells, with 20-30% of the membrane pool of NCC internalized within 30 min. The use of dynasore, PitStop2, methyl-β-cyclodextrin, nystatin, and filipin (specific inhibitors of either clathrin-dependent or -independent endocytosis) demonstrated that NCC is internalized via a clathrin-mediated pathway. Reduction of endocytosis resulted in greater levels of NCC in the plasma membrane. Immunogold electron microscopy confirmed the association of NCC with the clathrin-mediated internalization pathway in rat DCT cells. Compared with controls, inducing phosphorylation of NCC via low chloride treatment or mimicking phosphorylation by replacing Thr-53, Thr-58, and Ser-71 residues with Asp resulted in increased membrane abundance and reduced rates of NCC internalization. NCC ubiquitylation was lowest in the conditions with greatest NCC phosphorylation, thus providing a mechanism for the reduced endocytosis. In conclusion, our data support a model where NCC is constitutively cycled to the plasma membrane, and upon stimulation, it can be phosphorylated to both increase NCC activity and decrease NCC endocytosis, together increasing NaCl transport in the DCT. PMID:24668812

  11. Ca(2+)-calmodulin-dependent phosphorylation of islet secretory granule proteins

    SciTech Connect

    Watkins, D.T. )

    1991-08-01

    The effect of Ca2+ and calmodulin on phosphorylation of islet secretory granule proteins was studied. Secretory granules were incubated in a phosphorylation reaction mixture containing (32P)ATP and test reagents. The 32P-labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 32P content was visualized by autoradiography, and the relative intensities of specific bands were quantitated. When the reaction mixture contained EGTA and no added Ca2+, 32P was incorporated into two proteins with molecular weights of 45,000 and 13,000. When 10(-4) M Ca2+ was added without EGTA, two additional proteins (58,000 and 48,000 Mr) were phosphorylated, and the 13,000-Mr protein was absent. The addition of 2.4 microM calmodulin markedly enhanced the phosphorylation of the 58,000- and 48,000-Mr proteins and resulted in the phosphorylation of a major protein whose molecular weight (64,000 Mr) is identical to that of one of the calmodulin binding proteins located on the granule surface. Calmodulin had no effect on phosphorylation in the absence of Ca2+ but was effective in the presence of calcium between 10 nM and 50 microM. Trifluoperazine and calmidazolium, calmodulin antagonists, produced a dose-dependent inhibition of the calmodulin effect. 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester that activates protein kinase C, produced no increase in phosphorylation, and 1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride, an inhibitor of protein kinase C, had no effect. These results indicate that Ca(2+)-calmodulin-dependent protein kinases and endogenous substrates are present in islet secretory granules.

  12. Identification of a bacterial pectin acetyl esterase in Erwinia chrysanthemi 3937.

    PubMed

    Shevchik, V E; Hugouvieux-Cotte-Pattat, N

    1997-06-01

    Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. The structural complexity of pectin requires the combined action of several pectinases for its efficient breakdown. Three types of pectinases have so far been identified in E. chrysanthemi: two pectin methyl esterases (PemA, PemB), a polygalacturonase (PehX), and eight pectate lyases (PelA, PelB, PelC, PelD, PelE, PelL, PelZ, PelX). We report in this paper the analysis of a novel enzyme, the pectin acetyl esterase encoded by the paeY gene. No bacterial form of pectin acetyl esterases has been described previously, while plant tissues and some pectinolytic fungi were found to produce similar enzymes. The paeY gene is present in a cluster of five pectinase-encoding genes, pelA-pelE-pelD-paeY-pemA. The paeY open reading frame is 1650 bases long and encodes a 551-residue precursor protein of 60704Da, including a 25-amino-acid signal peptide. PaeY shares one region of homology with a rhamnogalacturonan acetyl esterase of Aspergillus aculeatus. To characterize the enzyme, the paeY gene was overexpressed and its protein product was purified. PaeY releases acetate from sugar-beet pectin and from various synthetic substrates. Moreover, the enzyme was shown to act in synergy with other pectinases. The de-esterification rate by PaeY increased after previous demethylation of the pectins by PemA and after depolymerization of the pectin by pectate lyases. In addition, the degradation of sugar-beet pectin by pectate lyases is favoured after the removal of methyl and acetyl groups by PemA and PaeY, respectively. The paeY gene was first identified on the basis of its regulation, which shares several characteristics with that of other pectinases. Analysis of the paeY transcription, using gene fusions, revealed that it is induced by pectic catabolic products and is affected by growth phase, oxygen limitation and catabolite repression. Regulation of pae

  13. VEZF1 Elements Mediate Protection from DNA Methylation

    PubMed Central

    Strogantsev, Ruslan; Gaszner, Miklos; Hair, Alan; Felsenfeld, Gary; West, Adam G.

    2010-01-01

    There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm β-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state. PMID:20062523

  14. Interaction of RNA polymerase II with acetylated nucleosomal core particles

    SciTech Connect

    Pineiro, M.; Gonzalez, P.J.; Hernandez, F.; Palacian, E. )

    1991-05-31

    Chemical acetylation of nucleosomal cores is accompanied by an increase in their efficiency as in vitro transcription templates. Low amounts of acetic anhydride cause preferential modification of the amino-terminal tails of core histones. Modification of these domains, which causes moderate structural effects, is apparently correlated with the observed stimulation of RNA synthesis. In contrast, extensive modification of the globular regions of core histones, which is accompanied by a large structural relaxation of the particle, causes little additional effect on transcription. Acetylation of the amino-terminal domains of histones might stimulate transcription by changing the interaction of the histone tails with components of the transcriptional machinery.

  15. Histone acetylation: a switch between repressive and permissive chromatin

    PubMed Central

    Eberharter, Anton; Becker, Peter B.

    2002-01-01

    The organization of eukaryotic chromatin has a major impact on all nuclear processes involving DNA substrates. Gene expression is affected by the positioning of individual nucleosomes relative to regulatory sequence elements, by the folding of the nucleosomal fiber into higher-order structures and by the compartmentalization of functional domains within the nucleus. Because site-specific acetylation of nucleosomal histones influences all three aspects of chromatin organization, it is central to the switch between permissive and repressive chromatin structure. The targeting of enzymes that modulate the histone acetylation status of chromatin, in synergy with the effects mediated by other chromatin remodeling factors, is central to gene regulation. PMID:11882541

  16. Involvement of phosphorylated Apis mellifera CREB in gating a honeybee's behavioral response to an external stimulus.

    PubMed

    Gehring, Katrin B; Heufelder, Karin; Feige, Janina; Bauer, Paul; Dyck, Yan; Ehrhardt, Lea; Kühnemund, Johannes; Bergmann, Anja; Göbel, Josefine; Isecke, Marlene; Eisenhardt, Dorothea

    2016-05-01

    The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees(Apis mellifera)we recently demonstrated a particular high abundance of the phosphorylated honeybee CREB homolog (pAmCREB) in the central brain and in a subpopulation of mushroom body neurons. We hypothesize that these high pAmCREB levels are related to learning and memory formation. Here, we tested this hypothesis by analyzing brain pAmCREB levels in classically conditioned bees and bees experiencing unpaired presentations of conditioned stimulus (CS) and unconditioned stimulus (US). We demonstrate that both behavioral protocols display differences in memory formation but do not alter the level of pAmCREB in bee brains directly after training. Nevertheless, we report that bees responding to the CS during unpaired stimulus presentations exhibit higher levels of pAmCREB than nonresponding bees. In addition, Trichostatin A, a histone deacetylase inhibitor that is thought to enhance histone acetylation by CREB-binding protein, increases the bees' CS responsiveness. We conclude that pAmCREB is involved in gating a bee's behavioral response driven by an external stimulus. PMID:27084927

  17. DNA methylation in plants.

    PubMed

    Vanyushin, B F

    2006-01-01

    DNA in plants is highly methylated, containing 5-methylcytosine (m5C) and N6-methyladenine (m6A); m5C is located mainly in symmetrical CG and CNG sequences but it may occur also in other non-symmetrical contexts. m6A but not m5C was found in plant mitochondrial DNA. DNA methylation in plants is species-, tissue-, organelle- and age-specific. It is controlled by phytohormones and changes on seed germination, flowering and under the influence of various pathogens (viral, bacterial, fungal). DNA methylation controls plant growth and development, with particular involvement in regulation of gene expression and DNA replication. DNA replication is accompanied by the appearance of under-methylated, newly formed DNA strands including Okazaki fragments; asymmetry of strand DNA methylation disappears until the end of the cell cycle. A model for regulation of DNA replication by methylation is suggested. Cytosine DNA methylation in plants is more rich and diverse compared with animals. It is carried out by the families of specific enzymes that belong to at least three classes of DNA methyltransferases. Open reading frames (ORF) for adenine DNA methyltransferases are found in plant and animal genomes, and a first eukaryotic (plant) adenine DNA methyltransferase (wadmtase) is described; the enzyme seems to be involved in regulation of the mitochondria replication. Like in animals, DNA methylation in plants is closely associated with histone modifications and it affects binding of specific proteins to DNA and formation of respective transcription complexes in chromatin. The same gene (DRM2) in Arabidopsis thaliana is methylated both at cytosine and adenine residues; thus, at least two different, and probably interdependent, systems of DNA modification are present in plants. Plants seem to have a restriction-modification (R-M) system. RNA-directed DNA methylation has been observed in plants; it involves de novo methylation of almost all cytosine residues in a region of si

  18. Biosynthetic pathways of the osmolytes N epsilon-acetyl-beta-lysine, beta-glutamine, and betaine in Methanohalophilus strain FDF1 suggested by nuclear magnetic resonance analyses.

    PubMed Central

    Roberts, M F; Lai, M C; Gunsalus, R P

    1992-01-01

    Methanohalophilus strain FDF1 synthesizes beta-glutamine, betaine, and N epsilon-acetyl-beta-lysine as osmoprotective agents when the cells are grown in high external concentrations of NaCl. Nuclear magnetic resonance spectroscopic analyses of 13CH3OH-12CO2 label incorporation by the cells provide information on the biosynthetic pathways of these organic osmolytes. The labeling studies indicate that Methanohalophilus strain FDF1 produces glutamate and beta-glutamine via a partial oxidative Krebs pathway. 13C labeling of betaine is consistent with methylation of glycine generated from serine (via serine hydroxymethyltransferase). The labeling pattern for N epsilon-acetyl-beta-lysine is consistent with the synthesis of its precursor alpha-lysine occurring by the diaminopimelate pathway in these cells. PMID:1400220

  19. ATRA transcriptionally induces nSMase2 through CBP/p300-mediated histone acetylation.

    PubMed

    Clarke, Christopher J; Shamseddine, Achraf A; Jacob, Joseph J; Khalife, Gabrielle; Burns, Tara A; Hannun, Yusuf A

    2016-05-01

    Neutral sphingomyelinase-2 (nSMase2) is a key ceramide-producing enzyme in cellular stress responses. While many posttranslational regulators of nSMase2 are known, emerging evidence suggests a more protracted regulation of nSMase2 at the transcriptional level. Previously, we reported that nSMase2 is induced by all-trans retinoic acid (ATRA) in MCF7 cells and implicated nSMase2 in ATRA-induced growth arrest. Here, we further investigated how ATRA regulates nSMase2. We find that ATRA regulates nSMase2 transcriptionally through the retinoic acid receptor-α, but this is independent of previously identified transcriptional regulators of nSMase2 (Sp1, Sp3, Runx2) and is not through increased promoter activity. Epigenetically, the nSMase2 gene is not repressively methylated in MCF7 cells. However, inhibition of histone deacetylases (HDACs) with trichostatin A (TSA) induced nSMase2 comparably to ATRA; furthermore, combined ATRA and TSA treatment was not additive, suggesting ATRA regulates nSMase2 through direct modulation of histone acetylation. Confirming this, the histone acetyltransferases CREB-binding protein and p300 were required for ATRA induction of nSMase2. Finally, use of class-specific HDAC inhibitors suggested that HDAC4 and/or HDAC5 are negative regulators of nSMase2 expression. Collectively, these results identify a novel pathway of nSMase2 regulation and suggest that physiological or pharmacological modulation of histone acetylation can directly affect nSMase2 levels. PMID:27013100

  20. Copper-catalyzed aerobic oxidation and cleavage/formation of C-S bond: a novel synthesis of aryl methyl sulfones from aryl halides and DMSO.

    PubMed

    Yuan, Gaoqing; Zheng, Junhua; Gao, Xiaofang; Li, Xianwei; Huang, Liangbin; Chen, Huoji; Jiang, Huanfeng

    2012-08-01

    With atmospheric oxygen as the oxidant, a novel copper(I)-catalyzed synthesis of aryl methyl sulfones from aryl halides and widely available DMSO is described. The procedure tolerates aryl halides with various functional groups (such as methoxy, acetyl, chloro, fluoro and nitro groups), which could afford aryl methyl sulfones in moderate to high yields. The copper-catalyzed aerobic oxidation and the cleavage/formation of C-S bond are the key steps for this transformation. PMID:22728918

  1. Melatonin inhibits TPA-induced oral cancer cell migration by suppressing matrix metalloproteinase-9 activation through the histone acetylation

    PubMed Central

    Yeh, Chia-Ming; Lin, Chiao-Wen; Yang, Jia-Sin; Yang, Wei-En; Su, Shih-Chi; Yang, Shun-Fa

    2016-01-01

    Melatonin exerts antimetastatic effects on liver and breast cancer and also inhibits matrix metalloproteinase (MMP) activity. However, the detailed impacts and underlying mechanisms of melatonin on oral cancer cell metastasis are still unclear. This study showed that melatonin attenuated the 12-O-tetradecanoylphorbol-13-acetate-induced migration of oral cancer cell lines, HSC-3 and OECM-1. Zymography, quantitative real-time PCR, and Western blotting analyses revealed that melatonin lessened MMP-9 enzyme activity as well as the expression of MMP-9 mRNA and protein. Furthermore, melatonin suppressed the phosphorylation of the ERK1/2 signalling pathway, which dampened MMP-9 gene transcription by affecting the expression of transcriptional coactivators, such as CREB-binding protein (CREBBP) and E1A binding protein p300 (EP300), and decreasing histone acetylation in HSC-3 and OECM-1 cells. Examinations on clinical samples exhibited that MMP-9, CREBBP, and EP300 were significantly increased in oral cancer tissues. Moreover, the relative level of CREBBP was positively correlated with the expression of MMP-9 and EP300. In conclusion, we demonstrated that melatonin inhibits the motility of HSC-3 and OECM-1 cells in vitro through a molecular mechanism that involves attenuation of MMP-9 expression and activity mediated by decreased histone acetylation. PMID:26980735

  2. Phosphorylation by the c-Abl protein tyrosine kinase inhibits parkin's ubiquitination and protective function

    PubMed Central

    Ko, Han Seok; Lee, Yunjong; Shin, Joo-Ho; Karuppagounder, Senthilkumar S.; Gadad, Bharathi Shrikanth; Koleske, Anthony J.; Pletnikova, Olga; Troncoso, Juan C.; Dawson, Valina L.; Dawson, Ted M.

    2010-01-01

    Mutations in PARK2/Parkin, which encodes a ubiquitin E3 ligase, cause autosomal recessive Parkinson disease (PD). Here we show that the nonreceptor tyrosine kinase c-Abl phosphorylates tyrosine 143 of parkin, inhibiting parkin's ubiquitin E3 ligase activity and protective function. c-Abl is activated by dopaminergic stress and by dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium (MPP+) in vitro and in vivo by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), leading to parkin inactivation, accumulation of the parkin substrates aminoacyl-tRNA synthetase-interacting multifunctional protein type 2 (AIMP2) (p38/JTV-1) and fuse-binding protein 1 (FBP1), and cell death. STI-571, a c-Abl-family kinase inhibitor, prevents the phosphorylation of parkin, maintaining parkin in a catalytically active and protective state. STI-571’s protective effects require parkin, as shRNA knockdown of parkin prevents STI-571 protection. Conditional knockout of c-Abl in the nervous system also prevents the phosphorylation of parkin, the accumulation of its substrates, and subsequent neurotoxicity in response to MPTP intoxication. In human postmortem PD brain, c-Abl is active, parkin is tyrosine-phosphorylated, and AIMP2 and FBP1 accumulate in the substantia nigra and striatum. Thus, tyrosine phosphorylation of parkin by c-Abl is a major posttranslational modification that inhibits parkin function, possibly contributing to pathogenesis of sporadic PD. Moreover, inhibition of c-Abl may be a neuroprotective approach in the treatment of PD. PMID:20823226

  3. Oxidative phosphorylation and lacunar stroke

    PubMed Central

    Anderson, Christopher D.; Hurford, Robert; Bevan, Steve; Markus, Hugh S.

    2016-01-01

    Objective: We investigated whether oxidative phosphorylation (OXPHOS) abnormalities were associated with lacunar stroke, hypothesizing that these would be more strongly associated in patients with multiple lacunar infarcts and leukoaraiosis (LA). Methods: In 1,012 MRI-confirmed lacunar stroke cases and 964 age-matched controls recruited from general practice surgeries, we investigated associations between common genetic variants within the OXPHOS pathway and lacunar stroke using a permutation-based enrichment approach. Cases were phenotyped using MRI into those with multiple infarcts or LA (MLI/LA) and those with isolated lacunar infarcts (ILI) based on the number of subcortical infarcts and degree of LA, using the Fazekas grading. Using gene-level association statistics, we tested for enrichment of genes in the OXPHOS pathway with all lacunar stroke and the 2 subtypes. Results: There was a specific association with strong evidence of enrichment in the top 1% of genes in the MLI/LA (subtype p = 0.0017) but not in the ILI subtype (p = 1). Genes in the top percentile for the all lacunar stroke analysis were not significantly enriched (p = 0.07). Conclusions: Our results implicate the OXPHOS pathway in the pathogenesis of lacunar stroke, and show the association is specific to patients with the MLI/LA subtype. They show that MRI-based subtyping of lacunar stroke can provide insights into disease pathophysiology, and imply that different radiologic subtypes of lacunar stroke subtypes have distinct underlying pathophysiologic processes. PMID:26674331

  4. Molecular characterization of a new acetyl xylan esterase (AXEII) from edible straw mushroom Volvariella volvacea with both de-O-acetylation and de-N-acetylation activity.

    PubMed

    Liu, Xiufeng; Ding, Shaojun

    2009-06-01

    A new Volvariella volvacea gene encoding a carbohydrate esterase (CE) family 4 acetyl xylan esterase (AXE) (designated as VvaxeII) was cloned and characterized. The coded polypeptide had 253 amino acid residues, with the first 19 serving as a secretion signal peptide. The VvaxeII transcript levels were high when the fungus was grown on oat spelt xylan, cellobiose, microcrystalline cellulose, carboxymethyl-cellulose, lactose, galactose, and chitin from crab as carbon sources. The recombinant VvAXEII produced by expression of VvaxeII in Pichia pastoris exhibited activity toward acetylated oat spelt xylan and various chitinous substrates, but was totally inactive against artificial aromatic acetates such as beta-nitrophenyl, 4-methylumbelliferyl, and alpha-naphthyl acetates. Enzyme-catalyzed hydrolysis was maximal at pH 7.0 and 60 degrees C, and reciprocal plots revealed an apparent K(m) value of 1.42 mg mL(-1) and a V(max) value of 833 IU micromol(-1) protein using glycol chitin as a substrate. The recombinant VvAXEII requires activation by bivalent cations such as Co2+ and Mg2+. Interestingly, the recombinant VvAXEII showed no deacetylation activity to fully acetylated monosaccharides such as xylose tetraacetate. PMID:19473250

  5. N-acetyl-cysteine attenuates neuropathic pain by suppressing matrix metalloproteinases.

    PubMed

    Li, Jiajie; Xu, Lujie; Deng, Xueting; Jiang, Chunyi; Pan, Cailong; Chen, Lu; Han, Yuan; Dai, Wenling; Hu, Liang; Zhang, Guangqin; Cheng, Zhixiang; Liu, Wentao

    2016-08-01

    The treatment of neuropathic pain remains a clinical challenge because of its unclear mechanisms and broad clinical morbidity. Matrix metalloproteinase (MMP)-9 and MMP-2 have previously been described as key components in neuropathic pain because of their facilitation of inflammatory cytokine maturation and induction of neural inflammation. Therefore, the inhibition of MMPs may represent a novel therapeutic approach to the treatment of neuropathic pain. In this study, we report that N-acetyl-cysteine (NAC), which is a broadly used respiratory drug, significantly attenuates neuropathic pain through a unique mechanism of MMP inhibition. Both the in vitro (0.1 mM) and in vivo application of NAC significantly suppressed the activity of MMP-9/2. Orally administered NAC (50, 100, and 200 mg/kg) not only postponed the occurrence but also inhibited the maintenance of chronic constrictive injury (CCI)-induced neuropathic pain in rats. The administration of NAC blocked the maturation of interleukin-1β, which is a critical substrate of MMPs, and markedly suppressed the neuronal activation induced by CCI, including inhibiting the phosphorylation of protein kinase Cγ, NMDAR1, and mitogen-activated protein kinases. Finally, NAC significantly inhibited CCI-induced microglia activation but elicited no notable effects on astrocytes. These results demonstrate an effective and safe approach that has been used clinically to alleviate neuropathic pain through the powerful inhibition of the activation of MMPs. PMID:27075430

  6. Improving polyketide and fatty acid synthesis by engineering of the yeast acetyl-CoA carboxylase.

    PubMed

    Choi, Jin Wook; Da Silva, Nancy A

    2014-10-10

    Polyketides and fatty acids are important in the production of pharmaceuticals, industrial chemicals, and biofuels. The synthesis of the malonyl-CoA building block, catalyzed by acetyl-CoA carboxylase (Acc1), is considered a limiting step to achieving high titers of polyketides and fatty acids in Saccharomyces cerevisiae. Acc1 is deactivated by AMP-activated serine/threonine protein kinase (Snf1) when glucose is depleted. To prevent this deactivation, the enzyme was aligned with the Rattus norvegicus (rat) Acc1 to identify a critical amino acid (Ser-1157) for phosphorylation and deactivation. Introduction of a S1157A mutation into Acc1 resulted in 9-fold higher specific activity following glucose depletion. The enzyme was tested in yeast engineered to produce the polyketide 6-methylsalisylic acid (6-MSA). Both 6-MSA and native fatty acid levels increased by 3-fold. Utilization of this modified Acc1 enzyme will also be beneficial for other products built from malonyl-CoA. PMID:25078432

  7. Long-term effects of early-life caregiving experiences on brain-derived neurotrophic factor histone acetylation in the adult rat mPFC

    PubMed Central

    Blaze, Jennifer; Asok, Arun; Roth, Tania L.

    2016-01-01

    Infant-caregiver experiences are major contributing factors to neural and behavioral development. Research continues to indicate that epigenetic mechanisms provide a way in which infant-caregiver experiences affect gene activity and other downstream processes in the brain that influence behavioral development. Our laboratory previously demonstrated in a rodent model that exposure to maltreatment alters methylation of DNA associated with the brain derived neurotrophic factor (bdnf) and reelin genes as well as mRNA of key epigenetic regulatory genes in the medial prefrontal cortex (mPFC). In the current study we characterized patterns of histone acetylation at bdnf and reelin gene loci after our caregiver manipulations. Using a within-litter design (n=8–10/group from 8 litters), pups were exposed to adverse (maltreatment condition: exposure to a stressed caregiver) or nurturing (cross-foster condition: exposure to a nurturing caregiver) caregiving environments outside the home cage for 30 minutes each day during the first postnatal week. Remaining pups in a litter were left with the biological mother during each session (providing normal care controls). We then used chromatin immunoprecipitation (ChIP) and quantitative RT-PCR to measure histone 3 lysine 9/14 acetylation at bdnf exons I and IV and the reelin promoter in the adult mPFC. Maltreated females had decreased acetylation at bdnf IV, while neither males nor females exhibited histone acetylation alterations at bdnf I or the reelin promoter. These data demonstrate the ability of maltreatment to have long-term consequences on histone acetylation in the mPFC, and provide further evidence of the epigenetic susceptibility of bdnf IV to the quality of infant-caregiver experiences. PMID:26305287

  8. Tubulin acetylation: responsible enzymes, biological functions and human diseases.

    PubMed

    Li, Lin; Yang, Xiang-Jiao

    2015-11-01

    Microtubules have important functions ranging from maintenance of cell morphology to subcellular transport, cellular signaling, cell migration, and formation of cell polarity. At the organismal level, microtubules are crucial for various biological processes, such as viral entry, inflammation, immunity, learning and memory in mammals. Microtubules are subject to various covalent modifications. One such modification is tubulin acetylation, which is associated with stable microtubules and conserved from protists to humans. In the past three decades, this reversible modification has been studied extensively. In mammals, its level is mainly governed by opposing actions of α-tubulin acetyltransferase 1 (ATAT1) and histone deacetylase 6 (HDAC6). Knockout studies of the mouse enzymes have yielded new insights into biological functions of tubulin acetylation. Abnormal levels of this modification are linked to neurological disorders, cancer, heart diseases and other pathological conditions, thereby yielding important therapeutic implications. This review summarizes related studies and concludes that tubulin acetylation is important for regulating microtubule architecture and maintaining microtubule integrity. Together with detyrosination, glutamylation and other modifications, tubulin acetylation may form a unique 'language' to regulate microtubule structure and function. PMID:26227334

  9. Mass spectrometry-based detection of protein acetylation

    PubMed Central

    Li, Yu; Silva, Jeffrey C.; Skinner, Mary E.; Lombard, David B.

    2014-01-01

    Summary Improved sample preparation techniques and increasingly sensitive mass spectrometry (MS) analysis have revolutionized the study of protein post-translational modifications (PTMs). Here, we describe a general approach for immunopurification and MS-based identification of acetylated proteins in biological samples. This approach is useful characterizing changes in the acetylome in response to biological interventions (1). PMID:24014401

  10. Prebiotically plausible oligoribonucleotide ligation facilitated by chemoselective acetylation

    NASA Astrophysics Data System (ADS)

    Bowler, Frank R.; Chan, Christopher K. W.; Duffy, Colm D.; Gerland, Béatrice; Islam, Saidul; Powner, Matthew W.; Sutherland, John D.; Xu, Jianfeng

    2013-05-01

    The recent synthesis of pyrimidine ribonucleoside-2‧,3‧-cyclic phosphates under prebiotically plausible conditions has strengthened the case for the involvement of ribonucleic acid (RNA) at an early stage in the origin of life. However, a prebiotic conversion of these weakly activated monomers, and their purine counterparts, to the 3‧,5‧-linked RNA polymers of extant biochemistry has been lacking (previous attempts led only to short oligomers with mixed linkages). Here we show that the 2‧-hydroxyl group of oligoribonucleotide-3‧-phosphates can be chemoselectively acetylated in water under prebiotically credible conditions, which allows rapid and efficient template-directed ligation. The 2‧-O-acetyl group at the ligation junction of the product RNA strand can be removed under conditions that leave the internucleotide bonds intact. Remarkably, acetylation of mixed oligomers that possess either 2‧- or 3‧-terminal phosphates is selective for the 2‧-hydroxyl group of the latter. This newly discovered chemistry thus suggests a prebiotic route from ribonucleoside-2‧,3‧-cyclic phosphates to predominantly 3‧,5‧-linked RNA via partially 2‧-O-acetylated RNA.

  11. Monitoring sterol uptake, acetylation, and export in yeast.

    PubMed

    Choudhary, Vineet; Schneiter, Roger

    2009-01-01

    Sterols are essential lipid components of eukaryotic membranes. They are synthesized in the endoplasmatic reticulum (ER) from where they are efficiently transported to the plasma membrane, which harbors ~90% of the free sterol pool of the cell. The molecular mechanisms that govern this lipid transport, however, are not well characterized and are challenging to analyze. Saccharomyces cerevisiae offers the opportunity to circumvent some of the technical limitations associated with studying this forward transport of sterols from the ER to the plasma membrane, because the organism can also take up sterols from the environment, incorporate them into the plasma membrane and transport them back to the ER, where the free sterol is converted to steryl esters. This reverse sterol transport, however, occurs only under anaerobic conditions, where the cells become sterol auxotroph, or in mutant cells that cannot synthesize heme. The reverse sterol transport pathway, however, is more amenable to experimental studies, because arrival of the sterol in the ER membrane can be monitored unambiguously by following the formation of steryl esters. Apart from sterol acylation, we have recently described a reversible sterol acetylation cycle that is operating in the lumen of the ER. Acetylation occurs on both cholesterol and pregnenolone, a steroid precursor, and serves as a signal for export of the acetylated sterols into the culture media. The time-dependent appearance of acetylated sterols in the culture supernatant thus provides a new means to monitor the forward transport of chemically modified sterols out of the ER. PMID:19784602

  12. Acetylation regulates DNA repair mechanisms in human cells.

    PubMed

    Piekna-Przybylska, Dorota; Bambara, Robert A; Balakrishnan, Lata

    2016-06-01

    The p300-mediated acetylation of enzymes involved in DNA repair and replication has been previously shown to stimulate or inhibit their activities in reconstituted systems. To explore the role of acetylation on DNA repair in cells we constructed plasmid substrates carrying inactivating damages in the EGFP reporter gene, which should be repaired in cells through DNA mismatch repair (MMR) or base excision repair (BER) mechanisms. We analyzed efficiency of repair within these plasmid substrates in cells exposed to deacetylase and acetyltransferase inhibitors, and also in cells deficient in p300 acetyltransferase. Our results indicate that protein acetylation improves DNA mismatch repair in MMR-proficient HeLa cells and also in MMR-deficient HCT116 cells. Moreover, results suggest that stimulated repair of mismatches in MMR-deficient HCT116 cells is done though a strand-displacement synthesis mechanism described previously for Okazaki fragments maturation and also for the EXOI-independent pathway of MMR. Loss of p300 reduced repair of mismatches in MMR-deficient cells, but did not have evident effects on BER mechanisms, including the long patch BER pathway. Hypoacetylation of the cells in the presence of acetyltransferase inhibitor, garcinol generally reduced efficiency of BER of 8-oxoG damage, indicating that some steps in the pathway are stimulated by acetylation. PMID:27104361

  13. An Acetylation Switch Regulates SUMO-Dependent Protein Interaction Networks

    PubMed Central

    Ullmann, Rebecca; Chien, Christopher D.; Avantaggiati, Maria Laura; Muller, Stefan

    2013-01-01

    SUMMARY The attachment of the SUMO modifier to proteins controls cellular signaling pathways through noncovalent binding to SUMO-interaction motifs (SIMs). Canonical SIMs contain a core of hydrophobic residues that bind to a hydrophobic pocket on SUMO. Negatively charged residues of SIMs frequently contribute to binding by interacting with a basic surface on SUMO. Here we define acetylation within this basic interface as a central mechanism for the control of SUMO-mediated interactions. The acetyl-mediated neutralization of basic charges on SUMO prevents binding to SIMs in PML, Daxx, and PIAS family members but does not affect the interaction between RanBP2 and SUMO. Acetylation is controlled by HDACs and attenuates SUMO- and PIAS-mediated gene silencing. Moreover, it affects the assembly of PML nuclear bodies and restrains the recruitment of the corepressor Daxx to these structures. This acetyl-dependent switch thus expands the regulatory repertoire of SUMO signaling and determines the selectivity and dynamics of SUMO-SIM interactions. PMID:22578841

  14. Protein Acetylation Is Involved in Salmonella enterica Serovar Typhimurium Virulence.

    PubMed

    Sang, Yu; Ren, Jie; Ni, Jinjing; Tao, Jing; Lu, Jie; Yao, Yu-Feng

    2016-06-01

    Salmonella causes a range of diseases in different hosts, including enterocolitis and systemic infection. Lysine acetylation regulates many eukaryotic cellular processes, but its function in bacteria is largely unexplored. The acetyltransferase Pat and NAD(+)-dependent deacetylase CobB are involved in the reversible protein acetylation in Salmonella Typhimurium. Here, we used cell and animal models to evaluate the virulence of pat and cobB deletion mutants in S. Typhimurium and found that pat is critical for bacterial intestinal colonization and systemic infection. Next, to understand the underlying mechanism, genome-wide transcriptome was analyzed. RNA sequencing data showed that the expression of Salmonella pathogenicity island 1 (SPI-1) is partially dependent on pat In addition, we found that HilD, a key transcriptional regulator of SPI-1, is a substrate of Pat. The acetylation of HilD by Pat maintained HilD stability and was essential for the transcriptional activation of HilA. Taken together, these results suggest that a protein acetylation system regulates SPI-1 expression by controlling HilD in a posttranslational manner to mediate S. Typhimurium virulence. PMID:26810370

  15. Cyclical DNA Methylation and Histone Changes Are Induced by LPS to Activate COX-2 in Human Intestinal Epithelial Cells

    PubMed Central

    Brancaccio, Mariarita; Coretti, Lorena; Florio, Ermanno; Pezone, Antonio; Calabrò, Viola; Falco, Geppino; Keller, Simona; Lembo, Francesca; Avvedimento, Vittorio Enrico; Chiariotti, Lorenzo

    2016-01-01

    Bacterial lipopolysaccharide (LPS) induces release of inflammatory mediators both in immune and epithelial cells. We investigated whether changes of epigenetic marks, including selected histone modification and DNA methylation, may drive or accompany the activation of COX-2 gene in HT-29 human intestinal epithelial cells upon exposure to LPS. Here we describe cyclical histone acetylation (H3), methylation (H3K4, H3K9, H3K27) and DNA methylation changes occurring at COX-2 gene promoter overtime after LPS stimulation. Histone K27 methylation changes are carried out by the H3 demethylase JMJD3 and are essential for COX-2 induction by LPS. The changes of the histone code are associated with cyclical methylation signatures at the promoter and gene body of COX-2 gene. PMID:27253528

  16. 40 CFR 721.6920 - Butyl acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... substituted methyl styrene, methyl methacrylate, and substituted silane. 721.6920 Section 721.6920 Protection... acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted silane. (a... butyl acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted...

  17. 40 CFR 721.6920 - Butyl acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... substituted methyl styrene, methyl methacrylate, and substituted silane. 721.6920 Section 721.6920 Protection... acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted silane. (a... butyl acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted...

  18. 40 CFR 721.6920 - Butyl acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... substituted methyl styrene, methyl methacrylate, and substituted silane. 721.6920 Section 721.6920 Protection... acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted silane. (a... butyl acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted...

  19. In the Beginning, There Was Protein Phosphorylation

    PubMed Central

    Kyriakis, John M.

    2014-01-01

    The importance of reversible protein phosphorylation to cellular regulation cannot be overstated. In eukaryotic cells, protein kinase/phosphatase signaling pathways regulate a staggering number of cellular processes, including cell proliferation, cell death (apoptosis, necroptosis, necrosis), metabolism (at both the cellular and organismal levels), behavior and neurological function, development, and pathogen resistance. Although protein phosphorylation as a mode of eukaryotic cell regulation is familiar to most biochemists, many are less familiar with protein kinase/phosphatase signaling networks that function in prokaryotes. In this thematic minireview series, we present four minireviews that cover the important field of prokaryotic protein phosphorylation. PMID:24554697

  20. ENZYMOLOGY OF ARSENIC METHYLATION

    EPA Science Inventory

    Enzymology of Arsenic Methylation

    David J. Thomas, Pharmacokinetics Branch, Experimental Toxicology Division, National
    Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park...

  1. Methyl salicylate overdose

    MedlinePlus

    Deep heating rubs overdose; Oil of wintergreen overdose ... These products contain methyl salicylate: Deep-heating creams used to relieve sore muscles and joints (Ben Gay, Icy Hot) Oil of wintergreen Solutions for vaporizers Other products ...

  2. Inheritance of Cytosine Methylation.

    PubMed

    Tillo, Desiree; Mukherjee, Sanjit; Vinson, Charles

    2016-11-01

    There are numerous examples of parental transgenerational inheritance that is epigenetic. The informational molecules include RNA, chromatin modifications, and cytosine methylation. With advances in DNA sequencing technologies, the molecular and epigenetic mechanisms mediating these effects are now starting to be uncovered. This mini-review will highlight some of the examples of epigenetic inheritance, the establishment of cytosine methylation in sperm, and recent genomic studies linking sperm cytosine methylation to epigenetic effects on offspring. A recent paper examining changes in diet and sperm cytosine methylation from pools of eight animals each, found differences between a normal diet, a high fat diet, and a low protein diet. However, epivariation between individuals within a group was greater than the differences between groups obscuring any potential methylation changes linked to diet. Learning more about epivariation may help unravel the mechanisms that regulate cytosine methylation. In addition, other experimental and genetic systems may also produce more dramatic changes in the sperm methylome, making it easier to unravel potential transgenerational phenomena. J. Cell. Physiol. 231: 2346-2352, 2016. © 2016 Wiley Periodicals, Inc. PMID:26910768

  3. The structure of putative N-acetyl glutamate kinase from Thermus thermophilus reveals an intermediate active site conformation of the enzyme.

    PubMed

    Sundaresan, Ramya; Ragunathan, Preethi; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Kumarevel, Thirumananseri; Ponnuraj, Karthe

    2012-04-13

    The de novo biosynthesis of arginine in microorganisms and plants is accomplished via several enzymatic steps. The enzyme N-acetyl glutamate kinase (NAGK) catalyzes the phosphorylation of the γ-COO(-) group of N-acetyl-L-glutamate (NAG) by adenosine triphosphate (ATP) which is the second rate limiting step in arginine biosynthesis pathway. Here we report the crystal structure of putative N-acetyl glutamate kinase (NAGK) from Thermus thermophilus HB8 (TtNAGK) determined at 1.92Å resolution. The structural analysis of TtNAGK suggests that the dimeric quaternary state of the enzyme and arginine insensitive nature are similar to mesophilic Escherichia coli NAGK. These features are significantly different from its thermophilic homolog Thermatoga maritima NAGK which is hexameric and arginine-sensitive. TtNAGK is devoid of its substrates but contains two sulfates at the active site. Very interestingly the active site of the enzyme adopts a conformation which is not completely open or closed and likely represents an intermediate stage in the catalytic cycle unlike its structural homologs, which all exist either in the open or closed conformation. Engineering arginine biosynthesis pathway enzymes for the production of l-arginine is an important industrial application. The structural comparison of TtNAGK with EcNAGK revealed the structural basis of thermostability of TtNAGK and this information could be very useful to generate mutants of NAGK with increased overall stability. PMID:22452987

  4. Structural and biochemical analyses reveal how ornithine acetyl transferase binds acidic and basic amino acid substrates.

    PubMed

    Iqbal, Aman; Clifton, Ian J; Chowdhury, Rasheduzzaman; Ivison, David; Domene, Carmen; Schofield, Christopher J

    2011-09-21

    Structural and biochemical analyses reveal how ornithine acetyl-transferases catalyse the reversible transfer of an acetyl-group from a basic (ornithine) to an acidic (glutamate) amino acid by employing a common mechanism involving an acetyl-enzyme intermediate but using different side chain binding modes. PMID:21796301

  5. mTOR complex-2 stimulates acetyl-CoA and de novo lipogenesis through ATP citrate lyase in HER2/PIK3CA-hyperactive breast cancer.

    PubMed

    Chen, Yaqing; Qian, Jianchang; He, Qun; Zhao, Hui; Toral-Barza, Lourdes; Shi, Celine; Zhang, Xuesai; Wu, Jiang; Yu, Ker

    2016-05-01

    The mechanistic target of rapamycin (mTOR) is a major regulator of cell growth and is frequently dysregulated in cancer. While mTOR complex-1 (mTORC1) is a validated cancer target, the role of mTOR complex-2 (mTORC2) remains less defined. Here, we reveal mTORC2 as a critical regulator of breast cancer metabolism. We showed that hyperphosphorylation in ATP citrate lyase (ACL) occurs frequently in human breast tumors and correlates well with HER2+ and/or PIK3CA-mutant (HER2+/PIK3CAmut) status in breast tumor cell lines. In HER2+/PIK3CAmut cells, mTORC2 controls Ser-455 phosphorylation of ACL thereby promoting acetyl-CoA production, de novo lipogenesis and mitochondrial physiology, all of which were inhibited by an mTORC1/mTORC2 kinase inhibitor (mTOR-KI) or cellular depletion of mTORC2 or ACL. mTOR-KI but not rapamycin blocked the IGF-1-induced ACL phosphorylation and glucose to lipid conversion. Depletion of mTORC2 but not mTORC1 specifically inhibited the ACL-dependent acetyl-CoA production. In the HER2+/PIK3CAmut MDA361, MDA453, BT-474 and T47D cells, depletion of mTORC2 or ACL led to growth inhibition and mitochondrial hyperpolarization, which were partially rescued by an alternate source of acetyl-CoA. These same changes were not apparent in mTORC2- or ACL-depleted HER2-/PIK3CAwt MDA231 and HCC1806 cells, highlighting a differential dependence of mTORC2-ACL for survival in these two cell types. Moreover, ACL Ser-455 mutants S455E (phosphomimetic) and S455A (non-phosphorylatable) each increased or decreased, respectively, the acetyl-CoA production, mitochondrial homeostasis and survival in ACL-depleted MDA453 cells. These studies define a new and rapamycin-resistant mechanism of mTORC2-ACL in lipogenesis and acetyl-CoA biology and provide a rationale for targeting of mTORC1 and mTORC2 in HER2+/PIK3CAmut breast cancer. PMID:27015560

  6. 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

  7. The Chemical Biology of Protein Phosphorylation

    PubMed Central

    Tarrant, Mary Katherine; Cole, Philip A.

    2011-01-01

    The explosion of scientific interest in protein kinase-mediated signaling networks has led to the infusion of new chemical methods and their applications related to the analysis of phosphorylation pathways. We highlight some of these chemical biology approaches across three areas. First, we discuss the development of chemical tools to modulate the activity of protein kinases to explore kinase mechanisms and their contributions to phosphorylation events and cellular processes. Second, we describe chemical techniques developed in the past few years to dissect the structural and functional effects of phosphate modifications at specific sites in proteins. Third, we cover newly developed molecular imaging approaches to elucidate the spatiotemporal aspects of phosphorylation cascades in live cells. Exciting advances in our understanding of protein phosphorylation have been obtained with these chemical biology approaches, but continuing opportunities for technological innovation remain. PMID:19489734

  8. The reductive acetyl coenzyme A pathway: sequence and heterologous expression of active methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase from Clostridium thermoaceticum.

    PubMed Central

    Roberts, D L; Zhao, S; Doukov, T; Ragsdale, S W

    1994-01-01

    The methyltransferase (MeTr) from Clostridium thermoaceticum transfers the N5-methyl group of (6S)-methyltetrahydrofolate to the cobalt center of a corrinoid/iron-sulfur protein in the acetyl coenzyme A pathway. MeTr was purified to homogeneity and shown to lack metals. The acsE gene encoding MeTr was sequenced and actively expressed in Escherichia coli at a level of 9% of cell protein. Regions in the sequence of MeTr and the E. coli cobalamin-dependent methionine synthase were found to share significant homology, suggesting that they may represent tetrahydrofolate-binding domains. PMID:7928975

  9. Search for Deuterated methyl acetate in the ISM

    NASA Astrophysics Data System (ADS)

    Gorai, Prasanta; Chakrabarti, Sandip Kumar; Das, Ankan; Majumdar, Liton; Sahu, Dipen; Sivaraman, Bhalamurugan

    2016-07-01

    Methyl acetate (CH_3COOCH_3 ) has been recently observed by IRAM 30 m radio telescope in Orion. But the existence of its deuterated form are yet to be confirmed. Here, we study the properties of methyl acetate and its singly deuterated forms (CH_3COOCH_3, CH_2DCOOCH_3 and CH_3COOCH_2D). Our simulation results reveal that deuterated forms of methyl acetate could efficiently be produced both in gas as well as in ice phase. Production of methyl acetate could follow radical-radical reaction between acetyl (CH_3CO) and methoxy (CH_3O) radicals. To predict abundances of CH_3COOCH_3 along with its two singly deuterated isotopomers and its two isomers (ethyl formate and hydroxy acetone), we prepare a large gas-grain chemical network to study chemical evolution of these molecules. Since gas phase rate coefficients of our newly adopted network for methyl acetate and its related species were unknown, in our simulation, either we consider similar rate coefficients for similar types of reactions (by following existing data bases) or we carry out quantum chemical calculations to estimate the unknown rate coefficients. For the surface reactions, we use adsorption energies of reactants from some earlier studies. Moreover, we perform quantum chemical calculations to find out various spectral properties of various forms of methyl acetate in infrared, ultraviolet and sub-millimeter regions. We prepare two catalog files for the rotational transitions of CH_2DCOOCH_3 and CH_3COOCH_2D in JPL format, which might be useful for its detection in regions of interstellar media where CH_3COOCH_3 has already been observed.

  10. An Adenosine Triphosphate-Phosphate Exchange Catalyzed by a Soluble Enzyme Couple Inhibited by Uncouplers of Oxidative Phosphorylation

    PubMed Central

    Allison, William S.; Benitez, Lita V.

    1972-01-01

    The sulfenic acid form of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), which is an acyl phosphatase, will catalyze an acetyl phosphate-Pi exchange reaction. This exchange reaction is reversibly inhibited by the uncouplers of oxidative phosphorylation, 2,4-dinitrophenol, m-Cl carbonylcyanide-phenylhydrazone, pentachlorophenol, and 5-chloro-3-tert-butyl-2′-chloro-4′-nitrosalicylanalide, and is irreversibly inhibited by cyanide and dicumarol. An ATP-Pi exchange reaction similar to that catalyzed by mitochondria can be simulated by a system composed of oxidized glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase (EC 2.7.1.28), 3-phosphoglycerate, ATP, 32Pi, and appropriate cofactors. The ATP-Pi exchange is inhibited by uncouplers of oxidative phosphorylation. Higher concentrations of uncouplers will also inhibit the ATPase reaction catalyzed by the coupled enzyme system. The exchange reactions catalyzed by the sulfenic acid form of glyceraldehyde-3-phosphate are consistent with a sulfenyl carboxylate intermediate. On the basis of these observations, a reaction scheme has been postulated for covalent coupling in oxidative phosphorylation that includes a sulfenyl carboxylate as a nonphosphorylated, high energy intermediate and an acyl phosphate as a phosphorylated, high energy intermediate. PMID:4507619

  11. Protein phosphorylation: Localization in regenerating optic axons

    SciTech Connect

    Larrivee, D. )

    1990-09-01

    A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration. (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of (3H)proline and 32P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the (3H)proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the 32P label, suggesting that phosphorylation was largely independent of synthesis. (2) To determine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced (3H)proline labeling of total protein by 88% and 32P labeling by 49%. Among the individual proteins (3H)proline labeling was reduced by 90% or more in 18 cases but 32P labeling was reduced only by 50% or less. (3) When 32P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.

  12. Phosphorylation of human skeletal muscle myosin

    SciTech Connect

    Houston, M.E.; Lingley, M.D.; Stuart, D.S.; Hoffman-Goetz, L.

    1986-03-01

    Phosphorylation of the P-light chains (phosphorylatable light chains) in human skeletal muscle myosin was studied in vitro and in vivo under resting an d contracted conditions. biopsy samples from rested vastus lateralis muscle of male and female subjects were incubated in oxygenated physiological solution at 30/sup 0/C. Samples frozen following a quiescent period showed the presence of only unphosphorylated P-light chains designated LC2f (light chain two of fast myosin) CL2s and LC2s'(light chains two of slow myosin). Treatment with caffeine (10 mM) or direct electrical stimulation resulted in the appearance of three additional bands which were identified as the phosphorylated forms of the P-light chains i.e. LC2f-P, LC2s-P and LC2s'-P. The presence of phosphate was confirmed by prior incubation with (/sup 30/P) orthophosphate. Muscle samples rapidly frozen from resting vastus lateralis muscle revealed the presence of unphosphorylated and phosphorylated P-light chains in approximately equal ratios. Muscle samples rapidly frozen following a maximal 10 second isometric contraction showed virtually only phosphorylated fast and slow P-light chains. These results reveal that the P-light chains in human fast and slow myosin may be rapidly phosphorylated, but the basal level of phosphorylation in rested human muscle considerably exceeds that observed in animal muscles studied in vitro or in situ.

  13. Protein phosphorylation in neurodegeneration: friend or foe?

    PubMed Central

    Tenreiro, Sandra; Eckermann, Katrin; Outeiro, Tiago F.

    2014-01-01

    Protein misfolding and aggregation is a common hallmark in neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and fronto-temporal dementia (FTD). In these disorders, the misfolding and aggregation of specific proteins occurs alongside neuronal degeneration in somewhat specific brain areas, depending on the disorder and the stage of the disease. However, we still do not fully understand the mechanisms governing protein aggregation, and whether this constitutes a protective or detrimental process. In PD, alpha-synuclein (aSyn) forms protein aggregates, known as Lewy bodies, and is phosphorylated at serine 129. Other residues have also been shown to be phosphorylated, but the significance of phosphorylation in the biology and pathophysiology of the protein is still controversial. In AD and in FTD, hyperphosphorylation of tau protein causes its misfolding and aggregation. Again, our understanding of the precise consequences of tau phosphorylation in the biology and pathophysiology of the protein is still limited. Through the use of a variety of model organisms and technical approaches, we are now gaining stronger insight into the effects of phosphorylation in the behavior of these proteins. In this review, we cover recent findings in the field and discuss how targeting phosphorylation events might be used for therapeutic intervention in these devastating diseases of the nervous system. PMID:24860424

  14. Protein phosphorylation during Plasmodium berghei gametogenesis.

    PubMed

    Alonso-Morales, Alberto; González-López, Lorena; Cázares-Raga, Febe Elena; Cortés-Martínez, Leticia; Torres-Monzón, Jorge Aurelio; Gallegos-Pérez, José Luis; Rodríguez, Mario Henry; James, Anthony A; Hernández-Hernández, Fidel de la Cruz

    2015-09-01

    Plasmodium gametogenesis within the mosquito midgut is a complex differentiation process involving signaling mediated by phosphorylation, which modulate metabolic routes and protein synthesis required to complete this development. However, the mechanisms leading to gametogenesis activation are poorly understood. We analyzed protein phosphorylation during Plasmodium berghei gametogenesis in vitro in serum-free medium using bidimensional electrophoresis (2-DE) combined with immunoblotting (IB) and antibodies specific to phosphorylated serine, threonine and tyrosine. Approximately 75 protein exhibited phosphorylation changes, of which 23 were identified by mass spectrometry. These included components of the cytoskeleton, heat shock proteins, and proteins involved in DNA synthesis and signaling pathways among others. Novel phosphorylation events support a role for these proteins during gametogenesis. The phosphorylation sites of six of the identified proteins, HSP70, WD40 repeat protein msi1, enolase, actin-1 and two isoforms of large subunit of ribonucleoside reductase were investigated using TiO2 phosphopeptides enrichment and tandem mass spectrometry. In addition, transient exposure to hydroxyurea, an inhibitor of ribonucleoside reductase, impaired male gametocytes exflagellation in a dose-dependent manner, and provides a resource for functional studies. PMID:26008612

  15. Fibronectin phosphorylation by ecto-protein kinase

    SciTech Connect

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru )

    1988-12-01

    The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with ({gamma}-{sup 32})ATP for 10 min at 37{degree}C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with ({gamma}-{sup 32}P)ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation.

  16. Long-term dynamics of multisite phosphorylation.

    PubMed

    Rubinstein, Boris Y; Mattingly, Henry H; Berezhkovskii, Alexander M; Shvartsman, Stanislav Y

    2016-07-15

    Multisite phosphorylation cycles are ubiquitous in cell regulation systems and are studied at multiple levels of complexity, from molecules to organisms, with the ultimate goal of establishing predictive understanding of the effects of genetic and pharmacological perturbations of protein phosphorylation in vivo. Achieving this goal is essentially impossible without mathematical models, which provide a systematic framework for exploring dynamic interactions of multiple network components. Most of the models studied to date do not discriminate between the distinct partially phosphorylated forms and focus on two limiting reaction regimes, distributive and processive, which differ in the number of enzyme-substrate binding events needed for complete phosphorylation or dephosphorylation. Here we use a minimal model of extracellular signal-related kinase regulation to explore the dynamics of a reaction network that includes all essential phosphorylation forms and arbitrary levels of reaction processivity. In addition to bistability, which has been studied extensively in distributive mechanisms, this network can generate periodic oscillations. Both bistability and oscillations can be realized at high levels of reaction processivity. Our work provides a general framework for systematic analysis of dynamics in multisite phosphorylation systems. PMID:27226482

  17. Compartment-Specific Phosphorylation of Squid Neurofilaments.

    PubMed

    Grant, Philip; Pant, Harish C

    2016-01-01

    Studies of the giant axon and synapse of third-order neurons in the squid stellate ganglion have provided a vast literature on neuronal physiology and axon transport. Large neuronal size also lends itself to comparative biochemical studies of cell body versus axon. These have focused on the regulation of synthesis, assembly, posttranslational modification and function of neuronal cytoskeletal proteins (microtubules (MTs) and neurofilaments (NFs)), the predominant proteins in axoplasm. These contribute to axonal organization, stability, transport, and impulse transmission responsible for rapid contractions of mantle muscles underlying jet propulsion. Studies of vertebrate NFs have established an extensive literature on NF structure, organization, and function; studies of squid NFs, however, have made it possible to compare compartment-specific regulation of NF synthesis, assembly, and function in soma versus axoplasm. Since NFs contain over 100 eligible sites for phosphorylation by protein kinases, the compartment-specific patterns of phosphorylation have been a primary focus of biochemical studies. We have learned that NF phosphorylation is tightly compartmentalized; extensive phosphorylation occurs only in the axonal compartment in squid and in vertebrate neurons. This extensive phosphorylation plays a key role in organizing NFs, in association with microtubules (MTs), into a stable, dynamic functional lattice that supports axon growth, diameter, impulse transmission, and synaptic activity. To understand how cytoskeletal phosphorylation is topographically regulated, the kinases and phosphatases, bound to NFs isolated from cell bodies and axoplasm, have also been studied. PMID:26795486

  18. Phosphorylation meets nuclear import: a review

    PubMed Central

    2010-01-01

    Phosphorylation is the most common and pleiotropic modification in biology, which plays a vital role in regulating and finely tuning a multitude of biological pathways. Transport across the nuclear envelope is also an essential cellular function and is intimately linked to many degeneration processes that lead to disease. It is therefore not surprising that phosphorylation of cargos trafficking between the cytoplasm and nucleus is emerging as an important step to regulate nuclear availability, which directly affects gene expression, cell growth and proliferation. However, the literature on phosphorylation of nucleocytoplasmic trafficking cargos is often confusing. Phosphorylation, and its mirror process dephosphorylation, has been shown to have opposite and often contradictory effects on the ability of cargos to be transported across the nuclear envelope. Without a clear connection between attachment of a phosphate moiety and biological response, it is difficult to fully understand and predict how phosphorylation regulates nucleocytoplasmic trafficking. In this review, we will recapitulate clue findings in the field and provide some general rules on how reversible phosphorylation can affect the nuclear-cytoplasmic localization of substrates. This is only now beginning to emerge as a key regulatory step in biology. PMID:21182795

  19. Complex O-acetylation in non-typeable Haemophilus influenzae lipopolysaccharide: evidence for a novel site of O-acetylation.

    PubMed

    Yildirim, Håkan H; Li, Jianjun; Richards, James C; Hood, Derek W; Moxon, E Richard; Schweda, Elke K H

    2005-12-12

    The structure of the lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae strain 723 has been elucidated using NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESI-MSn on permethylated dephosphorylated OS. It was found that the LPS contains the common structural element of H. influenzae, l-alpha-D-Hepp-(1-->2)-[PEtn-->6]-l-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-l-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdo-(2-->6)-Lipid A, in which the beta-D-Glcp residue (GlcI) is substituted by phosphocholine at O-6 and the distal heptose residue (HepIII) by PEtn at O-3, respectively. In a subpopulation of glycoforms O-2 of HepIII was substituted by beta-D-Galp-(1-->4)-beta-D-Glcp-(1--> or beta-D-Glcp-(1-->. Considerable heterogeneity of the LPS was due to the extent of substitution by O-acetyl groups (Ac) and ester-linked glycine of the core oligosaccharide. The location for glycine was found to be at Kdo. Prominent acetylation sites were found to be at GlcI, HepIII, and the proximal heptose (HepI) residue of the triheptosyl moiety. Moreover, GlcI was acetylated at O-3 and/or O-4 and HepI was acetylated at O-2 as evidenced by capillary electrophoresis ESI-MSn in combination with NMR analyses. This is the first study to show that an acetyl group can substitute HepI of the inner-core region of H. influenzae LPS. PMID:16199021

  20. Lactose metabolism in Streptococcus lactis: phosphorylation of galactose and glucose moieties in vivo.

    PubMed Central

    Thompson, J

    1979-01-01

    Starved cells of Streptococcus lactis ML3 grown previously on lactose, galactose, or maltose were devoid of adenosine 5'-triphosphate contained only three glycolytic intermediates: 3-phosphoglycerate, 2-phosphoglycerate, and phosphoenolpyruvate (PEP). The three metabolites (total concentration, ca 40 mM) served as the intracellular PEP potential for sugar transport via PEP-dependent phosphotransferase systems. When accumulation of [14C]lactose by iodoacetate-inhibited starved cells was abolished within 1 s of commencement of transport, a phosphorylated disaccharide was identified by autoradiography. The compound was isolated by ion-exchange (borate) chromatography, and enzymatic analysis showed that the derivative was 6-phosphoryl-O-beta-D-galactopyranosyl (1 leads to 4')-alpha-D-glucopyranose (lactose 6-phosphate). After maximum lactose uptake (ca. 15 mM in 15 s) the cells were collected by membrane filtration and extracted with trichloroacetic acid. Neither free nor phosphorylated lactose was detected in cell extracts, but enzymatic analysis revealed high levels of galactose 6-phosphate and glucose 6-phosphate. The starved organisms rapidly accumulated glucose, 2-deoxy-D-glucose, methyl-beta-D-thiogalactopyranoside, and o-nitrophenyl-beta-D-galactopyranoside in phosphorylated form to intracellular concentrations of 32, 32, 42, and 38.5 mM, respectively. In contrast, maximum accumulation of lactose (ca. 15 mM) was only 40 to 50% that of the monosaccharides. From the stoichiometry of PEP-dependent lactose transport and the results of enzymatic analysis, it was concluded that (i) ca. 60% of the PEP potential was utilized via the lactose phosphotransferase system for phosphorylation of the galactosyl moiety of the disaccharide, and (ii) the residual potential (ca. 40%) was consumed during phosphorylation of the glucose moiety. Images PMID:118155

  1. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    SciTech Connect

    Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

    2015-04-17

    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression.

  2. 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

  3. Role of Carnitine Acetyltransferases in Acetyl Coenzyme A Metabolism in Aspergillus nidulans ▿

    PubMed Central

    Hynes, Michael J.; Murray, Sandra L.; Andrianopoulos, Alex; Davis, Meryl A.

    2011-01-01

    The flow of carbon metabolites between cellular compartments is an essential feature of fungal metabolism. During growth on ethanol, acetate, or fatty acids, acetyl units must enter the mitochondrion for metabolism via the tricarboxylic acid cycle, and acetyl coenzyme A (acetyl-CoA) in the cytoplasm is essential for the biosynthetic reactions and for protein acetylation. Acetyl-CoA is produced in the cytoplasm by acetyl-CoA synthetase during growth on acetate and ethanol while β-oxidation of fatty acids generates acetyl-CoA in peroxisomes. The acetyl-carnitine shuttle in which acetyl-CoA is reversibly converted to acetyl-carnitine by carnitine acetyltransferase (CAT) enzymes is important for intracellular transport of acetyl units. In the filamentous ascomycete Aspergillus nidulans, a cytoplasmic CAT, encoded by facC, is essential for growth on sources of cytoplasmic acetyl-CoA while a second CAT, encoded by the acuJ gene, is essential for growth on fatty acids as well as acetate. We have shown that AcuJ contains an N-terminal mitochondrial targeting sequence and a C-terminal peroxisomal targeting sequence (PTS) and is localized to both peroxisomes and mitochondria, independent of the carbon source. Mislocalization of AcuJ to the cytoplasm does not result in loss of growth on acetate but prevents growth on fatty acids. Therefore, while mitochondrial AcuJ is essential for the transfer of acetyl units to mitochondria, peroxisomal localization is required only for transfer from peroxisomes to mitochondria. Peroxisomal AcuJ was not required for the import of acetyl-CoA into peroxisomes for conversion to malate by malate synthase (MLS), and export of acetyl-CoA from peroxisomes to the cytoplasm was found to be independent of FacC when MLS was mislocalized to the cytoplasm. PMID:21296915

  4. Histone H3 acetylation in the postmortem Parkinson's disease primary motor cortex.

    PubMed

    Gebremedhin, Kibrom G; Rademacher, David J

    2016-08-01

    Although the role of epigenetics in Parkinson's disease (PD) has not been extensively studied, α-synuclein, the main component of Lewy bodies, decreased histone H3 acetylation. Here, we determined if there were histone acetylation changes in the primary motor cortex which, according to the Braak model, is one of the last brain regions affected in PD. Net histone H3 acetylation, histone H3 lysine 9 (H3K9), histone H3 lysine 14 (H3K14), histone H3 lysine 18 (H3K18), and histone H3 lysine 23 (H3K23) acetylation was assessed in the primary motor cortex of those affected and unaffected by PD. There was net increase in histone H3 acetylation due to increased H3K14 and H3K18 acetylation. There was a decrease in H3K9 acetylation. No between-groups difference was detected in H3K23 acetylation. Relationships between Unified Lewy Body Staging scores and histone H3 acetylation and substantia nigra depigmentation scores and histone H3 acetylation were observed. No relationships were detected between postmortem interval and histone H3 acetylation and expired age and histone H3 acetylation. These correlational data support the notion that the histone H3 acetylation changes observed here are not due to the postmortem interval or aging. Instead, they are due to PD and/or factors that covary with PD. The data suggest enhanced gene transcription in the primary motor cortex of the PD brain due to increase H3K14 and H3K18 acetylation. This effect is partially offset by a decreased H3K9 acetylation, which might repress gene transcription. PMID:27241718

  5. Acetyl Eburicoic Acid from Laetiporus sulphureus var. miniatus Suppresses Inflammation in Murine Macrophage RAW 264.7 Cells

    PubMed Central

    Saba, Evelyn; Son, Youngmin; Jeon, Bo Ra; Kim, Seong-Eun; Lee, In-Kyoung

    2015-01-01

    The basidiomycete Laetiporus sulphureus var. miniatus belongs to the Aphyllophorales, Polyporaceae, and grows on the needleleaf tree. The fruiting bodies of Laetiporus species are known to produce N-methylated tyramine derivatives, polysaccharides, and various lanostane triterpenoids. As part of our ongoing effort to discover biologically active compounds from wood-rotting fungi, an anti-inflammatory triterpene, LSM-H7, has been isolated from the fruiting body of L. sulphureus var. miniatus and identified as acetyl eburicoic acid. LSM-H7 dose-dependently inhibited the NO production in RAW 264.7 cells without any cytotoxicity at the tested concentrations. Furthermore it suppressed the production of proinflammatory cytokines, mainly inducible nitric oxide synthase, cyclooxygenase-2, interleukin (IL)-1β, IL-6 and tumor necrosis factor α, when compared with glyceraldehyde 3-phosphate dehydrogenase. These data suggest that LSM-H7 is a crucial component for the anti-inflammatory activity of L. sulphureus var. miniatus. PMID:26190920

  6. Astrocyte Reactivity Following Blast Exposure Involves Aberrant Histone Acetylation

    PubMed Central

    Bailey, Zachary S.; Grinter, Michael B.; VandeVord, Pamela J.

    2016-01-01

    Blast induced neurotrauma (BINT) is a prevalent injury within military and civilian populations. The injury is characterized by persistent inflammation at the cellular level which manifests as a multitude of cognitive and functional impairments. Epigenetic regulation of transcription offers an important control mechanism for gene expression and cellular function which may underlie chronic inflammation and result in neurodegeneration. We hypothesize that altered histone acetylation patterns may be involved in blast induced inflammation and the chronic activation of glial cells. This study aimed to elucidate changes to histone acetylation occurring following injury and the roles these changes may have within the pathology. Sprague Dawley rats were subjected to either a 10 or 17 psi blast overpressure within an Advanced Blast Simulator (ABS). Sham animals underwent the same procedures without blast exposure. Memory impairments were measured using the Novel Object Recognition (NOR) test at 2 and 7 days post-injury. Tissues were collected at 7 days for Western blot and immunohistochemistry (IHC) analysis. Sham animals showed intact memory at each time point. The novel object discrimination decreased significantly between two and 7 days for each injury group (p < 0.05). This is indicative of the onset of memory impairment. Western blot analysis showed glial fibrillary acidic protein (GFAP), a known marker of activated astrocytes, was elevated in the prefrontal cortex (PFC) following blast exposure for both injury groups. Analysis of histone protein extract showed no changes in the level of any total histone proteins within the PFC. However, acetylation levels of histone H2b, H3, and H4 were decreased in both groups (p < 0.05). Co-localization immunofluorescence was used to further investigate any potential correlation between decreased histone acetylation and astrocyte activation. These experiments showed a similar decrease in H3 acetylation in astrocytes exposed to a 17

  7. Activity of influenza C virus O-acetylesterase with O-acetyl-containing compounds.

    PubMed

    Garcia-Sastre, A; Villar, E; Manuguerra, J C; Hannoun, C; Cabezas, J A

    1991-01-15

    Influenza C virus (strain C/Johannesburg/1/66) was grown, harvested, purified and used as source for the enzyme O-acetylesterase (N-acyl-O-acetylneuraminate O-acetylhydrolase; EC 3.1.1.53). This activity was studied and characterized with regard to some new substrates. The pH optimum of the enzyme is around 7.6, its stability at different pH values shows a result similar to that of the pH optimum, and its activity is well maintained in the pH range from 7.0 to 8.5 (all these tests were performed with 4-nitrophenyl acetate as substrate). Remarkable differences were found in the values of both Km and Vmax, with the synthetic substrates 4-nitrophenyl acetate, 2-nitrophenyl acetate, 4-methylumbelliferyl acetate, 1-naphthyl acetate and fluorescein diacetate. The use of 4-nitrophenyl acetate, 4-methylumbelliferyl acetate or 1-naphthyl acetate as substrate seems to be convenient for routine work, but it is better to carry out the measurements in parallel with those on bovine submandibular gland mucin (the latter is a natural and commercially available substrate). It was found that 4-acetoxybenzoic acid, as well as the methyl ester of 2-acetoxybenzoic acid, but not 2-acetoxybenzoic acid itself, are cleaved by this enzyme. Triacetin, di-O-acetyladenosine, tri-O-acetyladenosine, and di-O-acetyl-N-acetyladenosine phosphate, hitherto unreported as substrates for this viral esterase, are hydrolysed at different rates by this enzyme. We conclude that the O-acetylesterase from influenza C virus has a broad specificity towards both synthetic and natural non-sialic acid-containing substrates. Zn2+, Mn2+ and Pb2+ (as their chloride salts), N-acetylneuraminic acid, 4-methyl-umbelliferone and 2-acetoxybenzoic acid (acetylsalicylic acid) did not act as inhibitors. PMID:1991039

  8. Phosphorylation of epigenetic "readers, writers and erasers": Implications for developmental reprogramming and the epigenetic basis for health and disease.

    PubMed

    Treviño, Lindsey S; Wang, Quan; Walker, Cheryl L

    2015-07-01

    Epigenetic reprogramming that occurs during critical periods of development can increase the susceptibility to many diseases in adulthood. Programming of the epigenome during development occurs via the activity of a variety of epigenetic modifiers, including "readers, writers and erasers" of histone methyl marks. Posttranslational modification of these programmers can alter their activity, resulting in global or gene-specific changes in histone methylation and gene transcription. This review summarizes what is currently known about phosphorylation of histone methyltransferases ("writers"), demethylases ("erasers") and effector proteins ("readers) that program the epigenome, and the impact of this posttranslational modification on their activity. Understanding how the activity of these epigenetic programmers is perturbed by environmental exposures via changes in phosphorylation is key to understanding mechanisms of developmental reprogramming and the epigenetic basis of health and disease. PMID:25841987

  9. Phosphorylation of Tip60 by GSK-3 determines the induction of PUMA and apoptosis by p53

    PubMed Central

    Charvet, Céline; Wissler, Manuela; Brauns-Schubert, Prisca; Wang, Shang-Jui; Tang, Yi; Sigloch, Florian C.; Mellert, Hestia; Brandenburg, Martin; Lindner, Silke E.; Breit, Bernhard; Green, Douglas R.; McMahon, Steven B.; Borner, Christoph; Gu, Wei; Maurer, Ulrich

    2011-01-01

    Summary Activation of p53 by DNA damage results in either cell cycle arrest, allowing DNA repair and cell survival, or induction of apoptosis. As these opposite outcomes are both mediated by p53 stabilization, additional mechanisms to determine this decision must exist. Here we show that glycogen synthase kinase-3 (GSK-3) is required for the p53-mediated induction of the pro-apoptotic BH3 only-protein PUMA, an essential mediator of p53-induced apoptosis. Inhibition of GSK-3 protected from cell death induced by DNA damage and promoted increased long-term cell survival. We demonstrate that GSK-3 phosphorylates serine 86 of the p53-acetyltransferase Tip60. A Tip60S86A mutant was less active to induce p53 K120 acetylation, Histone 4 acetylation and expression of PUMA. Our data suggest that GSK-3 mediated Tip60S86-phosphorylation provides a link between PI3K signaling and the choice for or against apoptosis induction by p53. PMID:21658600

  10. Tyrosine phosphorylation of RACK1 triggers cardiomyocyte hypertrophy by regulating the interaction between p300 and GATA4.

    PubMed

    Suzuki, Hidetoshi; Katanasaka, Yasufumi; Sunagawa, Yoichi; Miyazaki, Yusuke; Funamoto, Masafumi; Wada, Hiromichi; Hasegawa, Koji; Morimoto, Tatsuya

    2016-09-01

    The zinc finger protein GATA4 is a transcription factor involved in cardiomyocyte hypertrophy. It forms a functional complex with the intrinsic histone acetyltransferase (HAT) p300. The HAT activity of p300 is required for the acetylation and transcriptional activity of GATA4, as well as for cardiomyocyte hypertrophy and the development of heart failure. In the present study, we have identified Receptor for Activated Protein Kinase C1 (RACK1) as a novel GATA4-binding protein using tandem affinity purification and mass spectrometry analyses. We found that exogenous RACK1 repressed phenylephrine (PE)-induced hypertrophic responses, such as myofibrillar organization, increased cell size, and hypertrophy-associated gene transcription, in cultured cardiomyocytes. RACK1 physically interacted with GATA4 and the overexpression of RACK1 reduced PE-induced formation of the p300/GATA4 complex and the acetylation and DNA binding activity of GATA4. In response to hypertrophic stimulation in cultured cardiomyocytes and in the hearts of hypertensive heart disease model rats, the tyrosine phosphorylation of RACK1 was increased, and the binding between GATA4 and RACK1 was reduced. In addition, the tyrosine phosphorylation of RACK1 was required for the disruption of the RACK1/GATA4 complex and for the formation of the p300/GATA4 complex. These findings demonstrate that RACK1 is involved in p300/GATA4-dependent hypertrophic responses in cardiomyocytes and is a promising therapeutic target for heart failure. PMID:27208796

  11. Activity of the response regulator CiaR in mutants of Streptococcus pneumoniae R6 altered in acetyl phosphate production

    PubMed Central

    Marx, Patrick; Meiers, Marina; Brückner, Reinhold

    2015-01-01

    The two-component regulatory system (TCS) CiaRH of Streptococcus pneumoniae is implicated in competence, ß-lactam resistance, maintenance of cell integrity, bacteriocin production, host colonization, and virulence. Depending on the growth conditions, CiaR can be highly active in the absence of its cognate kinase CiaH, although phosphorylation of CiaR is required for DNA binding and gene regulation. To test the possibility that acetyl phosphate (AcP) could be the alternative phosphodonor, genes involved in pyruvate metabolism were disrupted to alter cellular levels of acetyl phosphate. Inactivating the genes of pyruvate oxidase SpxB, phosphotransacetylase Pta, and acetate kinase AckA, resulted in very low AcP levels and in strongly reduced CiaR-mediated gene expression in CiaH-deficient strains. Therefore, alternative phosphorylation of CiaR appears to proceed via AcP. The AcP effect on CiaR is not detected in strains with CiaH. Attempts to obtain elevated AcP by preventing its degradation by acetate kinase AckA, were not successful in CiaH-deficient strains with a functional SpxB, the most important enzyme for AcP production in S. pneumoniae. The ciaH-spxB-ackA mutant producing intermediate amounts of AcP could be constructed and showed a promoter activation, which was much higher than expected. Since activation was dependent on AcP, it can apparently be used more efficiently for CiaR phosphorylation in the absence of AckA. Therefore, high AcP levels in the absence of CiaH and AckA may cause extreme overexpression of the CiaR regulon leading to synthetic lethality. AckA is also involved in a regulatory response, which is mediated by CiaH. Addition of acetate to the growth medium switch CiaH from kinase to phosphatase. This switch is lost in the absence of AckA indicating metabolism of acetate is required, which starts with the production of AcP by AckA. Therefore, AckA plays a special regulatory role in the control of the CiaRH TCS. PMID:25642214

  12. DNA methylation in fibrosis.

    PubMed

    Dowson, Christopher; O'Reilly, Steven

    2016-09-01

    Fibrosis is characterised by an exuberant wound healing response and the major cell type responsible is the myofibroblast. The myofibroblast is typified by excessive ECM production and contractile activity and is demarcated by alpha-smooth muscle actin expression. What has recently come to light is that the activation of the fibroblast to myofibroblast may be under epigenetic control, specifically methylation. Methylation of DNA is a conserved mechanism to precisely regulate gene expression in a specific context. Hypermethylation leads to gene repression and hypomethylation results in gene induction. Methylation abnormalities have recently been uncovered in fibrosis, both organ specific and widespread fibrosis. The fact that these methylation changes are rapid and reversible lends themselves amenable to therapeutic intervention. This review considers the role of methylation in fibrosis and the activation of the myofibroblasts and how this could be targeted for fibrosis. Fibrosis is of course currently intractable to therapeutics and is a leading cause of morbidity and mortality and is an urgent unmet clinical need. PMID:27346523

  13. Early Developmental and Evolutionary Origins of Gene Body DNA Methylation Patterns in Mammalian Placentas.

    PubMed

    Schroeder, Diane I; Jayashankar, Kartika; Douglas, Kory C; Thirkill, Twanda L; York, Daniel; Dickinson, Pete J; Williams, Lawrence E; Samollow, Paul B; Ross, Pablo J; Bannasch, Danika L; Douglas, Gordon C; LaSalle, Janine M

    2015-08-01

    Over the last 20-80 million years the mammalian placenta has taken on a variety of morphologies through both divergent and convergent evolution. Recently we have shown that the human placenta genome has a unique epigenetic pattern of large partially methylated domains (PMDs) and highly methylated domains (HMDs) with gene body DNA methylation positively correlating with level of gene expression. In order to determine the evolutionary conservation of DNA methylation patterns and transcriptional regulatory programs in the placenta, we performed a genome-wide methylome (MethylC-seq) analysis of human, rhesus macaque, squirrel monkey, mouse, dog, horse, and cow placentas as well as opossum extraembryonic membrane. We found that, similar to human placenta, mammalian placentas and opossum extraembryonic membrane have globally lower levels of methylation compared to somatic tissues. Higher relative gene body methylation was the conserved feature across all mammalian placentas, despite differences in PMD/HMDs and absolute methylation levels. Specifically, higher methylation over the bodies of genes involved in mitosis, vesicle-mediated transport, protein phosphorylation, and chromatin modification was observed compared with the rest of the genome. As in human placenta, higher methylation is associated with higher gene expression and is predictive of genic location across species. Analysis of DNA methylation in oocytes and preimplantation embryos shows a conserved pattern of gene body methylation similar to the placenta. Intriguingly, mouse and cow oocytes and mouse early embryos have PMD/HMDs but their placentas do not, suggesting that PMD/HMDs are a feature of early preimplantation methylation patterns that become lost during placental development in some species and following implantation of the embryo. PMID:26241857

  14. Early Developmental and Evolutionary Origins of Gene Body DNA Methylation Patterns in Mammalian Placentas

    PubMed Central

    Schroeder, Diane I.; Jayashankar, Kartika; Douglas, Kory C.; Thirkill, Twanda L.; York, Daniel; Dickinson, Pete J.; Williams, Lawrence E.; Samollow, Paul B.; Ross, Pablo J.; Bannasch, Danika L.; Douglas, Gordon C.; LaSalle, Janine M.

    2015-01-01

    Over the last 20-80 million years the mammalian placenta has taken on a variety of morphologies through both divergent and convergent evolution. Recently we have shown that the human placenta genome has a unique epigenetic pattern of large partially methylated domains (PMDs) and highly methylated domains (HMDs) with gene body DNA methylation positively correlating with level of gene expression. In order to determine the evolutionary conservation of DNA methylation patterns and transcriptional regulatory programs in the placenta, we performed a genome-wide methylome (MethylC-seq) analysis of human, rhesus macaque, squirrel monkey, mouse, dog, horse, and cow placentas as well as opossum extraembryonic membrane. We found that, similar to human placenta, mammalian placentas and opossum extraembryonic membrane have globally lower levels of methylation compared to somatic tissues. Higher relative gene body methylation was the conserved feature across all mammalian placentas, despite differences in PMD/HMDs and absolute methylation levels. Specifically, higher methylation over the bodies of genes involved in mitosis, vesicle-mediated transport, protein phosphorylation, and chromatin modification was observed compared with the rest of the genome. As in human placenta, higher methylation is associated with higher gene expression and is predictive of genic location across species. Analysis of DNA methylation in oocytes and preimplantation embryos shows a conserved pattern of gene body methylation similar to the placenta. Intriguingly, mouse and cow oocytes and mouse early embryos have PMD/HMDs but their placentas do not, suggesting that PMD/HMDs are a feature of early preimplantation methylation patterns that become lost during placental development in some species and following implantation of the embryo. PMID:26241857

  15. MeCP2 phosphorylation limits psychostimulant-induced behavioral and neuronal plasticity.

    PubMed

    Deng, Jie V; Wan, Yehong; Wang, Xiaoting; Cohen, Sonia; Wetsel, William C; Greenberg, Michael E; Kenny, Paul J; Calakos, Nicole; West, Anne E

    2014-03-26

    The methyl-DNA binding protein MeCP2 is emerging as an important regulator of drug reinforcement processes. Psychostimulants induce phosphorylation of MeCP2 at Ser421; however, the functional significance of this posttranslational modification for addictive-like behaviors was unknown. Here we show that MeCP2 Ser421Ala knock-in mice display both a reduced threshold for the induction of locomotor sensitization by investigator-administered amphetamine and enhanced behavioral sensitivity to the reinforcing properties of self-administered cocaine. These behavioral differences were accompanied in the knock-in mice by changes in medium spiny neuron intrinsic excitability and nucleus accumbens gene expression typically observed in association with repeated exposure to these drugs. These data show that phosphorylation of MeCP2 at Ser421 functions to limit the circuit plasticities in the nucleus accumbens that underlie addictive-like behaviors. PMID:24671997

  16. MeCP2 Phosphorylation Limits Psychostimulant-Induced Behavioral and Neuronal Plasticity

    PubMed Central

    Deng, Jie V.; Wan, Yehong; Wang, Xiaoting; Cohen, Sonia; Wetsel, William C.; Greenberg, Michael E.; Kenny, Paul J.; Calakos, Nicole

    2014-01-01

    The methyl-DNA binding protein MeCP2 is emerging as an important regulator of drug reinforcement processes. Psychostimulants induce phosphorylation of MeCP2 at Ser421; however, the functional significance of this posttranslational modification for addictive-like behaviors was unknown. Here we show that MeCP2 Ser421Ala knock-in mice display both a reduced threshold for the induction of locomotor sensitization by investigator-administered amphetamine and enhanced behavioral sensitivity to the reinforcing properties of self-administered cocaine. These behavioral differences were accompanied in the knock-in mice by changes in medium spiny neuron intrinsic excitability and nucleus accumbens gene expression typically observed in association with repeated exposure to these drugs. These data show that phosphorylation of MeCP2 at Ser421 functions to limit the circuit plasticities in the nucleus accumbens that underlie addictive-like behaviors. PMID:24671997

  17. Multiple Mass Isotopomer Tracing of Acetyl-CoA Metabolism in Langendorff-perfused Rat Hearts

    PubMed Central

    Li, Qingling; Deng, Shuang; Ibarra, Rafael A.; Anderson, Vernon E.; Brunengraber, Henri; Zhang, Guo-Fang

    2015-01-01

    We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (≈citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [13C2,2H3]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [13C6]glucose or [1,2-13C2]palmitate) or/and M1 acetyl-CoA (e.g. [1-13C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA. PMID:25645937

  18. Herpesvirus Genome Recognition Induced Acetylation of Nuclear IFI16 Is Essential for Its Cytoplasmic Translocation, Inflammasome and IFN-β Responses.

    PubMed

    Ansari, Mairaj Ahmed; Dutta, Sujoy; Veettil, Mohanan Valiya; Dutta, Dipanjan; Iqbal, Jawed; Kumar, Binod; Roy, Arunava; Chikoti, Leela; Singh, Vivek Vikram; Chandran, Bala

    2015-07-01

    The IL-1β and type I interferon-β (IFN-β) molecules are important inflammatory cytokines elicited by the eukaryotic host as innate immune responses against invading pathogens and danger signals. Recently, a predominantly nuclear gamma-interferon-inducible protein 16 (IFI16) involved in transcriptional regulation has emerged as an innate DNA sensor which induced IL-1β and IFN-β production through inflammasome and STING activation, respectively. Herpesvirus (KSHV, EBV, and HSV-1) episomal dsDNA genome recognition by IFI16 leads to IFI16-ASC-procaspase-1 inflammasome association, cytoplasmic translocation and IL-1β production. Independent of ASC, HSV-1 genome recognition results in IFI16 interaction with STING in the cytoplasm to induce interferon-β production. However, the mechanisms of IFI16-inflammasome formation, cytoplasmic redistribution and STING activation are not known. Our studies here demonstrate that recognition of herpesvirus genomes in the nucleus by IFI16 leads into its interaction with histone acetyltransferase p300 and IFI16 acetylation resulting in IFI16-ASC interaction, inflammasome assembly, increased interaction with Ran-GTPase, cytoplasmic redistribution, caspase-1 activation, IL-1β production, and interaction with STING which results in IRF-3 phosphorylation, nuclear pIRF-3 localization and interferon-β production. ASC and STING knockdowns did not affect IFI16 acetylation indicating that this modification is upstream of inflammasome-assembly and STING-activation. Vaccinia virus replicating in the cytoplasm did not induce nuclear IFI16 acetylation and cytoplasmic translocation. IFI16 physically associates with KSHV and HSV-1 genomes as revealed by proximity ligation microscopy and chromatin-immunoprecipitation studies which is not hampered by the inhibition of acetylation, thus suggesting that acetylation of IFI16 is not required for its innate sensing of nuclear viral genomes. Collectively, these studies identify the increased nuclear

  19. Herpesvirus Genome Recognition Induced Acetylation of Nuclear IFI16 Is Essential for Its Cytoplasmic Translocation, Inflammasome and IFN-β Responses

    PubMed Central

    Ansari, Mairaj Ahmed; Dutta, Sujoy; Veettil, Mohanan Valiya; Dutta, Dipanjan; Iqbal, Jawed; Kumar, Binod; Roy, Arunava; Chikoti, Leela; Singh, Vivek Vikram; Chandran, Bala

    2015-01-01

    The IL-1β and type I interferon-β (IFN-β) molecules are important inflammatory cytokines elicited by the eukaryotic host as innate immune responses against invading pathogens and danger signals. Recently, a predominantly nuclear gamma-interferon-inducible protein 16 (IFI16) involved in transcriptional regulation has emerged as an innate DNA sensor which induced IL-1β and IFN-β production through inflammasome and STING activation, respectively. Herpesvirus (KSHV, EBV, and HSV-1) episomal dsDNA genome recognition by IFI16 leads to IFI16-ASC-procaspase-1 inflammasome association, cytoplasmic translocation and IL-1β production. Independent of ASC, HSV-1 genome recognition results in IFI16 interaction with STING in the cytoplasm to induce interferon-β production. However, the mechanisms of IFI16-inflammasome formation, cytoplasmic redistribution and STING activation are not known. Our studies here demonstrate that recognition of herpesvirus genomes in the nucleus by IFI16 leads into its interaction with histone acetyltransferase p300 and IFI16 acetylation resulting in IFI16-ASC interaction, inflammasome assembly, increased interaction with Ran-GTPase, cytoplasmic redistribution, caspase-1 activation, IL-1β production, and interaction with STING which results in IRF-3 phosphorylation, nuclear pIRF-3 localization and interferon-β production. ASC and STING knockdowns did not affect IFI16 acetylation indicating that this modification is upstream of inflammasome-assembly and STING-activation. Vaccinia virus replicating in the cytoplasm did not induce nuclear IFI16 acetylation and cytoplasmic translocation. IFI16 physically associates with KSHV and HSV-1 genomes as revealed by proximity ligation microscopy and chromatin-immunoprecipitation studies which is not hampered by the inhibition of acetylation, thus suggesting that acetylation of IFI16 is not required for its innate sensing of nuclear viral genomes. Collectively, these studies identify the increased nuclear

  20. [Spectrophotometric evaluation of N-acetyl-beta-glucosaminidase in urine].

    PubMed

    Potere, C; Di Cosmo, C; Riario-Sforza, G; Di Silverio, F; Albertazzi, A; Cappelli, P

    1982-01-01

    A spectrophotometric method for the assay of N-Acetyl-beta-Glucosaminidase activity in human undiluted urines is described. The application of this method is recommended for its sensitivity (2,6 X 10(-4)M) and its rapid performance, because it represents a good alternative to current methods and essentially to the fluorimetric technique with which it has a significant statistical correlation. Estimates of normal individuals aged between 1-70 years are reported. PMID:7168631

  1. Selective recognition of acetylated histones by bromodomains in transcriptional co-activators

    PubMed Central

    Hassan, Ahmed H.; Awad, Salma; Al-Natour, Zeina; Othman, Samah; Mustafa, Farah; Rizvi, Tahir A.

    2006-01-01

    Bromodomains are present in many chromatin-associated proteins such as the SWI/SNF and RSC chromatin remodelling and the SAGA HAT (histone acetyltransferase) complexes, and can bind to acetylated lysine residues in the N-terminal tails of the histones. Lysine acetylation is a histone modification that forms a stable epigenetic mark on chromatin for bromodomain-containing proteins to dock and in turn regulate gene expression. In order to better understand how bromodomains read the ‘histone code’ and interact with acetylated histones, we have tested the interactions of several bromodomains within transcriptional co-activators with differentially acetylated histone tail peptides and HAT-acetylated histones. Using GST (glutathione S-transferase) pull-down assays, we show specificity of binding of some bromodomains to differentially acetylated H3 and H4 peptides as well as HAT-acetylated histones. Our results reveal that the Swi2/Snf2 bromodomain interacts with various acetylated H3 and H4 peptides, whereas the Gcn5 bromodomain interacts only with acetylated H3 peptides and tetra-acetylated H4 peptides. Additionally we show that the Spt7 bromodomain interacts with acetylated H3 peptides weakly, but not with acetylated H4 peptides. Some bromodomains such as the Bdf1-2 do not interact with most of the acetylated peptides tested. Results of the peptide experiments are confirmed with tests of interactions between these bromodomains and HAT-acetylated histones. Furthermore, we demonstrate that the Swi2/Snf2 bromodomain is important for the binding and the remodelling activity of the SWI/SNF complex on hyperacetylated nucleosomes. The selective recognition of the bromodomains observed in the present study accounts for the broad effects of bromodomain-containing proteins observed on binding to histones. PMID:17049045

  2. Mechanism of action of clostridial glycine reductase: Isolation and characterization of a covalent acetyl enzyme intermediate

    SciTech Connect

    Arkowitz, R.A.; Abeles, R.H. )

    1991-04-23

    Clostridial glycine reductase consists of proteins A, B, and C and catalyzes the reaction glycine + P{sub i} + 2e{sup {minus}} {yields} acetyl phosphate + NH{sub 4}{sup +}. Evidence was previously obtained that is consistent with the involvement of an acyl enzyme intermediate in this reaction. The authors now demonstrate that protein C catalyzes exchange of ({sup 32}P)P{sub i} into acetyl phosphate, providing additional support for an acetyl enzyme intermediate on protein C. Furthermore, they have isolated acetyl protein C and shown that it is qualitatively, catalytically competent. Acetyl protein C can be obtained through the forward reaction from protein C and Se-(carboxymethyl)selenocysteine-protein A, which is generated by the reaction of glycine with proteins A and B. Acetyl protein C can also be generated through the reverse reaction by the addition of acetyl phosphate to protein C. Both procedures lead to the same acetyl enzyme. The acetyl enzyme reacts with P{sub i} to give acetyl phosphate. When ({sup 14}C)acetyl protein C is denaturated with TCA and redissolved with urea, radioactivity remained associated with the protein. Treatment with KBH{sub 4} removes all the radioactivity associated with protein C, resulting in the formation of ({sup 14}C)ethanol. They conclude that a thiol group on protein C is acetylated. Proteins A and C together catalyze the exchange of tritium atoms from ({sup 3}H)H{sub 2}O into acetyl phosphate. This exchange reaction supports the proposal that an enol of the acetyl enzyme is an intermediate in the reaction sequence.

  3. N-Acetyl-4-aminophenol (paracetamol), N-acetyl-2-aminophenol and acetanilide in urine samples from the general population, individuals exposed to aniline and paracetamol users.

    PubMed

    Dierkes, Georg; Weiss, Tobias; Modick, Hendrik; Käfferlein, Heiko Udo; Brüning, Thomas; Koch, Holger M

    2014-01-01

    Epidemiological studies suggest associations between the use of N-acetyl-4-aminophenol (paracetamol) during pregnancy and increased risks of reproductive disorders in the male offspring. Previously we have reported a ubiquitous urinary excretion of N-acetyl-4-aminophenol in the general population. Possible sources are (1) direct intake of paracetamol through medication, (2) paracetamol residues in the food chain and (3) environmental exposure to aniline or related substances that are metabolized into N-acetyl-4-aminophenol. In order to elucidate the origins of the excretion of N-acetyl-4-aminophenol in urine and to contribute to the understanding of paracetamol and aniline metabolism in humans we developed a rapid, turbulent-flow HPLC-MS/MS method with isotope dilution for the simultaneous quantification of N-acetyl-4-aminophenol and two other aniline related metabolites, N-acetyl-2-aminophenol and acetanilide. We applied this method to three sets of urine samples: (1) individuals with no known exposure to aniline and also no recent paracetamol medication; (2) individuals after occupational exposure to aniline but no paracetamol medication and (3) paracetamol users. We confirmed the omnipresent excretion of N-acetyl-4-aminophenol. Additionally we revealed an omnipresent excretion of N-acetyl-2-aminophenol. In contrast, acetanilide was only found after occupational exposure to aniline, not in the general population or after paracetamol use. The results lead to four preliminary conclusions: (1) other sources than aniline seem to be responsible for the major part of urinary N-acetyl-4-aminophenol in the general population; (2) acetanilide is a metabolite of aniline in man and a valuable biomarker for aniline in occupational settings; (3) aniline baseline levels in the general population measured after chemical hydrolysis do not seem to originate from acetanilide and hence not from a direct exposure to aniline itself and (4) N-acetyl-2-aminophenol does not seem to be

  4. Acetylation modification regulates GRP78 secretion in colon cancer cells

    PubMed Central

    Li, Zongwei; Zhuang, Ming; Zhang, Lichao; Zheng, Xingnan; Yang, Peng; Li, Zhuoyu

    2016-01-01

    High glucose-regulated protein 78 (GRP78) expression contributes to the acquisition of a wide range of phenotypic cancer hallmarks, and the pleiotropic oncogenic functions of GRP78 may result from its diverse subcellular distribution. Interestingly, GRP78 has been reported to be secreted from solid tumour cells, participating in cell-cell communication in the tumour microenvironment. However, the mechanism underlying this secretion remains elusive. Here, we report that GRP78 is secreted from colon cancer cells via exosomes. Histone deacetylase (HDAC) inhibitors blocked GRP78 release by inducing its aggregation in the ER. Mechanistically, HDAC inhibitor treatment suppressed HDAC6 activity and led to increased GRP78 acetylation; acetylated GRP78 then bound to VPS34, a class III phosphoinositide-3 kinase, consequently preventing the sorting of GRP78 into multivesicular bodies (MVBs). Of note, we found that mimicking GRP78 acetylation by substituting the lysine at residue 633, one of the deacetylated sites of HDAC6, with a glutamine resulted in decreased GRP78 secretion and impaired tumour cell growth in vitro. Our study thus reveals a hitherto-unknown mechanism of GRP78 secretion and may also provide implications for the therapeutic use of HDAC inhibitors. PMID:27460191

  5. Acetylation modification regulates GRP78 secretion in colon cancer cells.

    PubMed

    Li, Zongwei; Zhuang, Ming; Zhang, Lichao; Zheng, Xingnan; Yang, Peng; Li, Zhuoyu

    2016-01-01

    High glucose-regulated protein 78 (GRP78) expression contributes to the acquisition of a wide range of phenotypic cancer hallmarks, and the pleiotropic oncogenic functions of GRP78 may result from its diverse subcellular distribution. Interestingly, GRP78 has been reported to be secreted from solid tumour cells, participating in cell-cell communication in the tumour microenvironment. However, the mechanism underlying this secretion remains elusive. Here, we report that GRP78 is secreted from colon cancer cells via exosomes. Histone deacetylase (HDAC) inhibitors blocked GRP78 release by inducing its aggregation in the ER. Mechanistically, HDAC inhibitor treatment suppressed HDAC6 activity and led to increased GRP78 acetylation; acetylated GRP78 then bound to VPS34, a class III phosphoinositide-3 kinase, consequently preventing the sorting of GRP78 into multivesicular bodies (MVBs). Of note, we found that mimicking GRP78 acetylation by substituting the lysine at residue 633, one of the deacetylated sites of HDAC6, with a glutamine resulted in decreased GRP78 secretion and impaired tumour cell growth in vitro. Our study thus reveals a hitherto-unknown mechanism of GRP78 secretion and may also provide implications for the therapeutic use of HDAC inhibitors. PMID:27460191

  6. Selective cleavage enhanced by acetylating the side chain of lysine.

    PubMed

    Fu, Leixiaomeng; Chen, Tingting; Xue, Gaiqing; Zu, Lily; Fang, Weihai

    2013-01-01

    Selective cleavage is of great interest in mass spectrometry studies as it can help sequence identification by promoting simple fragmentation pattern of peptides and proteins. In this work, the collision-induced dissociation of peptides containing internal lysine and acetylated lysine residues were studied. The experimental and computational results revealed that multiple fragmentation pathways coexisted when the lysine residue was two amino acid residues away from N-terminal of the peptide. After acetylation of the lysine side-chain, b(n)+ ions were the most abundant primary fragment products and the Lys(Ac)-Gly amide bond became the dominant cleavage site via an oxazolone pathway. Acetylating the side-chain of lysine promoted the selective cleavage of Lys-Xxx amide bond and generated much more information of the peptide backbone sequence. The results re-evaluate the selective cleavage due to the lysine basic side-chain and provide information for studying the post-translational modification of proteins and other bio-molecules containing Lys residues. PMID:23303756

  7. Chromatin decondensed by acetylation shows an elevated radiation response

    SciTech Connect

    Nackerdien, Z.; Michie, J.; Boehm, L.

    1989-02-01

    V-79 Chinese hamster lung fibroblasts exposed to 5 mM n-sodium butyrate were irradiated with 60Co gamma rays and cell survival was determined by the cell colony assay. In a separate set of experiments the acetylated chromatin obtained from these cells was irradiated and the change of molecular weight of the DNA was evaluated by alkaline sucrose density centrifugation. At a survival level of 10(-2) to 10(-4) cells exposed to butyrate were found to be 1.3-1.4 times more radiosensitive than control cells. Exposure of isolated chromatin to 100 Gy of 60Co gamma irradiation generated 0.9 +/- 0.03 single-strand breaks (ssb) per 10 Gy per 10(8) Da and 2.0 +/- 0.3 ssb/10 Gy/10(8) Da for control and acetylated chromatin, respectively. The elevated radiation sensitivity of chromatin relaxed by acetylation is in good agreement with previous results on chromatin expanded by histone H1 depletion. Packing and accessibility of DNA in chromatin appear to be major factors which influence the radiation sensitivity. The intrinsic radiation sensitivity of chromatin in various packing states is discussed in light of the variation of radiation sensitivity of whole cells in the cell cycle which incorporates repair.

  8. Carbon isotope fractionation and the acetyl-CoA pathway

    NASA Astrophysics Data System (ADS)

    Blaser, Martin; Conrad, Ralf

    2010-05-01

    Homoacetogenic bacteria can catalyze the reductive synthesis of acetate from CO2 via the acetyl-CoA pathway. Besides this unifying property homoacetogenic bacteria constitute a metabolically and phylogenetically diverse bacteriological group. Therefore their environmental role is difficult to address. It has been recognized that in methanogenic environments homoacetogenic bacteria contribute to the degradation of organic matter. The natural abundance of 13C may be used to understand the functional impact of homoacetogenic bacteria in the soil environment. To distinguish the acetyl-CoA pathway from other dominant processes, the isotopic composition of acetate and CO2 can be determined and the fractionation factors of the individual processes may be used to discriminate between the dominant pathways. To characterize the fractionation factor associated with the acetyl-CoA pathway the phylogenetic and metabolic diversity needs to be considered. Therefore the fractionation factor of substrate utilization and product formation of different homoacetogens (Acetobacterium woodii, Sporomusa ovata, Thermoanaerobacter kivui, Morella thermoautotrophica) has been studied under pure culture conditions in two defined minimal medium with H2/CO2 as sole source of carbon and energy. It became obvious that the cultivation conditions have a major impact on the obtained fractionation factors.

  9. Phosphorylation Modulates Catalytic Activity of Mycobacterial Sirtuins

    PubMed Central

    Yadav, Ghanshyam S.; Ravala, Sandeep K.; Malhotra, Neha; Chakraborti, Pradip K.

    2016-01-01

    Sirtuins are NAD+-dependent deacetylases involved in the regulation of diverse cellular processes and are conserved throughout phylogeny. Here we report about in vitro transphosphorylation of the only NAD+-dependent deacetylase (mDAC) present in the genome of Mycobacterium tuberculosis by eukaryotic-type Ser/Thr kinases, particularly PknA. The phosphorylated mDAC displayed decreased deacetylase activity compared to its unphosphorylated counterpart. Mass-spectrometric study identified seven phosphosites in mDAC; however, mutational analysis highlighted major contribution of Thr-214 for phosphorylation of the protein. In concordance to this observation, variants of mDAC substituting Thr-214 with either Ala (phospho-ablated) or Glu (phosphomimic) exhibited significantly reduced deacetylase activity suggesting phosphorylation mediated control of enzymatic activity. To assess the role of phosphorylation towards functionality of mDAC, we opted for a sirtuin knock-out strain of Escherichia coli (Δdac), where interference of endogenous mycobacterial kinases could be excluded. The Δdac strain in nutrient deprived acetate medium exhibited compromised growth and complementation with mDAC reversed this phenotype. The phospho-ablated or phosphomimic variant, on the other hand, was unable to restore the functionality of mDAC indicating the role of phosphorylation per se in the process. We further over-expressed mDAC or mDAC-T214A as His-tagged protein in M. smegmatis, where endogenous eukaryotic-type Ser/Thr kinases are present. Anti-phosphothreonine antibody recognized both mDAC and mDAC-T214A proteins in western blotting. However, the extent of phosphorylation as adjudged by scanning the band intensity, was significantly low in the mutant protein (mDAC-T214A) compared to that of the wild-type (mDAC). Furthermore, expression of PknA in the mDAC complemented Δdac strain was able to phosphorylate M. tuberculosis sirtuin. The growth profile of this culture in acetate medium was

  10. Phosphorylated tau and the neurodegenerative foldopathies.

    PubMed

    Kosik, Kenneth S; Shimura, Hideki

    2005-01-01

    Many studies have implicated phosphorylated tau in the Alzheimer disease process. However, the cellular fate of phosphorylated tau has only recently been described. Recent work has shown that tau phosphorylation at substrate sites for the kinases Cdk5 and GSK3-beta can trigger the binding of tau to the chaperones Hsc70 and Hsp27. The binding of phosphorylated tau to Hsc70 implied that the complex may be a substrate for the E3 ligase CHIP and this possibility was experimentally verified. The presence of this system in cells suggests that phosphorylated tau may hold toxic dangers for cell viability, and the response of the cell is to harness a variety of protective mechanisms. These include binding to chaperones, which may prevent more toxic conformations of the protein, ubiquitination which will direct the protein to the proteasome, segregation of tau aggregates from the cellular machinery, and recruitment of Hsp27 which will confer anti-apoptotic properties to the cell. PMID:15615647

  11. Extensive phosphorylation of AMPA receptors in neurons.

    PubMed

    Diering, Graham H; Heo, Seok; Hussain, Natasha K; Liu, Bian; Huganir, Richard L

    2016-08-16

    Regulation of AMPA receptor (AMPAR) function is a fundamental mechanism controlling synaptic strength during long-term potentiation/depression and homeostatic scaling. AMPAR function and membrane trafficking is controlled by protein-protein interactions, as well as by posttranslational modifications. Phosphorylation of the GluA1 AMPAR subunit at S845 and S831 play especially important roles during synaptic plasticity. Recent controversy has emerged regarding the extent to which GluA1 phosphorylation may contribute to synaptic plasticity. Here we used a variety of methods to measure the population of phosphorylated GluA1-containing AMPARs in cultured primary neurons and mouse forebrain. Phosphorylated GluA1 represents large fractions from 12% to 50% of the total population under basal and stimulated conditions in vitro and in vivo. Furthermore, a large fraction of synapses are positive for phospho-GluA1-containing AMPARs. Our results support the large body of research indicating a prominent role of GluA1 phosphorylation in synaptic plasticity. PMID:27482106

  12. Protein phosphorylation systems in postmortem human brain

    SciTech Connect

    Walaas, S.I.; Perdahl-Wallace, E.; Winblad, B.; Greengard, P. )

    1989-01-01

    Protein phosphorylation systems regulated by cyclic adenosine 3',5'-monophosphate (cyclic AMP), or calcium in conjunction with calmodulin or phospholipid/diacylglycerol, have been studied by phosphorylation in vitro of particulate and soluble fractions from human postmortem brain samples. One-dimensional or two-dimensional gel electrophoretic protein separations were used for analysis. Protein phosphorylation catalyzed by cyclic AMP-dependent protein kinase was found to be highly active in both particulate and soluble preparations throughout the human CNS, with groups of both widely distributed and region-specific substrates being observed in different brain nuclei. Dopamine-innervated parts of the basal ganglia and cerebral cortex contained the phosphoproteins previously observed in rodent basal ganglia. In contrast, calcium/phospholipid-dependent and calcium/calmodulin-dependent protein phosphorylation systems were less prominent in human postmortem brain than in rodent brain, and only a few widely distributed substrates for these protein kinases were found. Protein staining indicated that postmortem proteolysis, particularly of high-molecular-mass proteins, was prominent in deeply located, subcortical regions in the human brain. Our results indicate that it is feasible to use human postmortem brain samples, when obtained under carefully controlled conditions, for qualitative studies on brain protein phosphorylation. Such studies should be of value in studies on human neurological and/or psychiatric disorders.

  13. Sugar phosphorylation activity in ruminal acetogens.

    PubMed

    Jiang, W; Pinder, R S; Patterson, J A; Ricke, S C

    2012-01-01

    Acetogenic bacteria Acetitomaculum ruminis, Acetobacterium woodii, and Eubacterium limosum were compared for phosphoenolpyruvate (PEP) and ATP-dependent phosphorylation of glucose and 2-deoxy-glucose. Rate of phosphorylation activity was measured in toluene-treated acetogenic cells using PEP and ATP and radiolabled glucose or 2-deoxy glucose. Eubacterium limosum, most likely has a glucose phosphotransferase system (PTS). In contrast, A. ruminis, and A. woodii had PEP-dependent glucose phosphorylation rates very similar to control rates, suggesting the lack of PTS activity. These results were confirmed by PEP dependent 2-deoxyglucose phosphorylation data. The rates of ATP-dependent glucose phosphorylation were higher than PEP-dependent glucose dependent in all organisms surveyed. Only E. limosum appeared to have PTS. The presence of PTS in E. limosum could explain why it is not capable of utilizing sugars and H(2)/CO(2) simultaneously and why acetogenesis is not as prominant in the rumen because of the availability of carbohydrates as alternative energy substrates. PMID:22423990

  14. CpGIMethPred: computational model for predicting methylation status of CpG islands in human genome

    PubMed Central

    2013-01-01

    DNA methylation is an inheritable chemical modification of cytosine, and represents one of the most important epigenetic events. Computational prediction of the DNA methylation status can be employed to speed up the genome-wide methylation profiling, and to identify the key features that are correlated with various methylation patterns. Here, we develop CpGIMethPred, the support vector machine-based models to predict the methylation status of the CpG islands in the human genome under normal conditions. The features for prediction include those that have been previously demonstrated effective (CpG island specific attributes, DNA sequence composition patterns, DNA structure patterns, distribution patterns of conserved transcription factor binding sites and conserved elements, and histone methylation status) as well as those that have not been extensively explored but are likely to contribute additional information from a biological point of view (nucleosome positioning propensities, gene functions, and histone acetylation status). Statistical tests are performed to identify the features that are significantly correlated with the methylation status of the CpG islands, and principal component analysis is then performed to decorrelate the selected features. Data from the Human Epigenome Project (HEP) are used to train, validate and test the predictive models. Specifically, the models are trained and validated by using the DNA methylation data obtained in the CD4 lymphocytes, and are then tested for generalizability using the DNA methylation data obtained in the other 11 normal tissues and cell types. Our experiments have shown that (1) an eight-dimensional feature space that is selected via the principal component analysis and that combines all categories of information is effective for predicting the CpG island methylation status, (2) by incorporating the information regarding the nucleosome positioning, gene functions, and histone acetylation, the models can achieve

  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. In vivo differentiation of N-acetyl aspartyl glutamate from N-acetyl aspartate at 3 Tesla.

    PubMed

    Edden, Richard A E; Pomper, Martin G; Barker, Peter B

    2007-06-01

    A method is described that allows the in vivo differentiation of N-acetyl aspartate (NAA) from N-acetyl aspartyl glutamate (NAAG) by in vivo MR spectroscopy (MRS) at 3 Tesla (3T). The method, which is based on MEGA-point-resolved spectroscopy (PRESS) editing, selectively targets the aspartyl spin system of one species while deliberately removing the other species from the spectrum. This allows quantitative measurements of NAA and NAAG without the need for fitting of unresolved peaks. White matter concentrations of NAA (6.7 +/- 0.3 mM) and NAAG (2.2 +/- 0.3 mM) were measured in 10 healthy volunteers to demonstrate the method. PMID:17534922

  17. Phosphorylation state-dependent interaction between AKAP7δ/γ and phospholamban increases phospholamban phosphorylation.

    PubMed

    Rigatti, Marc; Le, Andrew V; Gerber, Claire; Moraru, Ion I; Dodge-Kafka, Kimberly L

    2015-09-01

    Changes in heart rate and contractility in response to sympathetic stimulation occur via activation of cAMP dependent protein kinase A (PKA), leading to phosphorylation of numerous substrates that alter Ca(2+) cycling. Phosphorylation of these substrates is coordinated by A-kinase anchoring proteins (AKAPs), which recruit PKA to specific substrates [1]. Phosphorylation of the PKA substrate phospholamban (PLB) is a critical determinant of Ca(2+) re-entry into the sarcoplasmic reticulum and is coordinated by AKAP7δ/γ [2,3]. Here, we further these findings by showing that phosphorylation of PLB requires interaction with AKAP7δ/γ and that this interaction occurs only when PLB is unphosphorylated. Additionally, we find that two mutants of PLB (R9C and Δ14), which are associated with dilated cardiomyopathy in humans, prevent association with AKAP7δ/γ and display reduced phosphorylation in vitro. This finding implicates the AKAP7δ/γ-PLB interaction in the pathology of the disease phenotype. Further exploration of the AKAP7δ/γ-PLB association demonstrated a phosphorylation state-dependence of the interaction. Computational modeling revealed that this mode of interaction allows for small amounts of AKAP and PKA (100-200nM) to regulate the phosphorylation of large quantities of PLB (50μM). Our results confirm that AKAP7γ/δ binding to PLB is important for phosphorylation of PLB, and describe a novel phosphorylation state-dependent binding mechanism that explains how phosphorylation of highly abundant PKA substrates can be regulated by AKAPs present at ~100-200 fold lower concentrations. PMID:26027516

  18. Metabolic production of methylated selenium species requires adequate methylation status

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Obesity negatively impacts methylation status and markers of methylation status vary according to selenium status in supplemented subjects. We have proposed that disruptions in methylation capacity induced by obesity compromise demonstrable anti-cancer effects of Se supplementation. In order to addr...

  19. Leucine-684: A conserved residue of an AMP-acetyl CoA synthetase (AceCS) from Leishmania donovani is involved in substrate recognition, catalysis and acetylation.

    PubMed

    Soumya, Neelagiri; Tandan, Hitendra; Damre, Mangesh V; Gangwal, Rahul P; Sangamwar, Abhay T; Singh, Sushma

    2016-04-15

    AMP-acetyl CoA synthetase (AMP-AceCS) is a key enzyme which catalyzes the activation of acetate to acetyl CoA, an important intermediate at the cross roads of various anabolic and catabolic pathways. Multiple sequence alignment of Leishmania donovani AceCS with other organisms revealed the presence of a highly conserved leucine residue at 684 position which is known to be crucial for acetylation by protein acetyl transferases in other organisms. In an attempt to understand the role of leucine residue at 684 position in L. donovani acetyl CoA synthetase (LdAceCS), it was mutated to proline (P) by site directed mutagenesis. Kinetic analysis of the L684P-LdAceCS mutant revealed approximately two fold increased binding affinity with acetate, whereas fivefold decreased affinity was observed with ATP. There was insignificant change in secondary structure as revealed by CD however, two fold decreased fluorescence intensity was observed at an emission maxima of 340nm. Interestingly, L684P mutation abolished the acetylation of the mutant enzyme indicating the importance of L684 in acetylation of the enzyme. Changes in biochemical parameters of the mutant protein were validated by homology modeling of the wild type and mutant LdAceCS enzyme using Salmonella enterica AceCS crystal structure as template. Our data provides evidence for the role of leucine 684 residue in substrate recognition, catalysis and acetylation of the AceCS enzyme. PMID:26794803

  20. Phosphorylated silk fibroin matrix for methotrexate release.

    PubMed

    Volkov, Vadim; Sárria, Marisa P; Gomes, Andreia C; Cavaco-Paulo, Artur

    2015-01-01

    Silk-based matrix was produced for delivery of a model anticancer drug, methotrexate (MTX). The calculation of net charge of silk fibroin and MTX was performed to better understand the electrostatic interactions during matrix formation upon casting. Silk fibroin films were cast at pH 7.2 and pH 3.5. Protein kinase A was used to prepare phosphorylated silk fibroin. The phosphorylation content of matrix was controlled by mixing at specific ratios the phosphorylated and unphosphorylated solutions. In vitro release profiling data suggest that the observed interactions are mainly structural and not electrostatical. The release of MTX is facilitated by use of proteolytic enzymes and higher pHs. The elevated β-sheet content and crystallinity of the acidified-cast fibroin solution seem not to favor drug retention. All the acquired data underline the prevalence of structural interactions over electrostatical interactions between methotrexate and silk fibroin. PMID:25435334

  1. Phosphorylation of RACK1 in plants

    SciTech Connect

    Chen, Jay -Gui

    2015-08-31

    Receptor for Activated C Kinase 1 (RACK1) is a versatile scaffold protein that interacts with a large, diverse group of proteins to regulate various signaling cascades. RACK1 has been shown to regulate hormonal signaling, stress responses and multiple processes of growth and development in plants. However, little is known about the molecular mechanism underlying these regulations. Recently, it has been demonstrated that Arabidopsis RACK1 is phosphorylated by an atypical serine/threonine protein kinase, WITH NO LYSINE 8 (WNK8). Furthermore, RACK1 phosphorylation by WNK8 negatively regulates RACK1 function by influencing its protein stability. In conclusion, these findings promote a new regulatory system in which the action of RACK1 is controlled by phosphorylation and subsequent protein degradation.

  2. Phosphorylation of RACK1 in plants

    DOE PAGESBeta

    Chen, Jay -Gui

    2015-08-31

    Receptor for Activated C Kinase 1 (RACK1) is a versatile scaffold protein that interacts with a large, diverse group of proteins to regulate various signaling cascades. RACK1 has been shown to regulate hormonal signaling, stress responses and multiple processes of growth and development in plants. However, little is known about the molecular mechanism underlying these regulations. Recently, it has been demonstrated that Arabidopsis RACK1 is phosphorylated by an atypical serine/threonine protein kinase, WITH NO LYSINE 8 (WNK8). Furthermore, RACK1 phosphorylation by WNK8 negatively regulates RACK1 function by influencing its protein stability. In conclusion, these findings promote a new regulatory systemmore » in which the action of RACK1 is controlled by phosphorylation and subsequent protein degradation.« less

  3. DNA Methylation in Osteoarthritis

    PubMed Central

    den Hollander, Wouter; Meulenbelt, Ingrid

    2015-01-01

    Osteoarthritis (OA) is a prevalent disease of articular joints and primarily characterized by degradation and calcification of articular cartilage. Presently, no effective treatment other than pain relief exists and patients ultimately need to undergo replacement surgery of the affected joint. During disease progression articular chondrocytes, the single cell type present in articular cartilage, show altered transcriptional profiles and undergo phenotypic changes that resemble the terminal differentiation route apparent in growth plate chondrocytes. Hence, given its prominent function in both regulating gene expression and maintaining cellular phenotypes, DNA methylation of CpG dinucleotides is intensively studied in the context of OA. An increasing number of studies have been published that employed a targeted approach on genes known to play a role in OA pathophysiology. As of such, it has become clear that OA responsive DNA methylation changes seem to mediate disease associated aberrant gene expression. Furthermore, established OA susceptibility alleles such as GDF5 and DIO2 appear to confer OA risk via DNA methylation and respective pathophysiological expression changes. In more recent years, genome wide profiling of DNA methylation in OA affected articular cartilage has emerged as a powerful tool to address the epigenetic changes in their entirety, which has resulted in the identification of putative patient subgroups as well as generic OA associated pathways. PMID:27019616

  4. DNA Methylation in Osteoarthritis.

    PubMed

    den Hollander, Wouter; Meulenbelt, Ingrid

    2015-12-01

    Osteoarthritis (OA) is a prevalent disease of articular joints and primarily characterized by degradation and calcification of articular cartilage. Presently, no effective treatment other than pain relief exists and patients ultimately need to undergo replacement surgery of the affected joint. During disease progression articular chondrocytes, the single cell type present in articular cartilage, show altered transcriptional profiles and undergo phenotypic changes that resemble the terminal differentiation route apparent in growth plate chondrocytes. Hence, given its prominent function in both regulating gene expression and maintaining cellular phenotypes, DNA methylation of CpG dinucleotides is intensively studied in the context of OA. An increasing number of studies have been published that employed a targeted approach on genes known to play a role in OA pathophysiology. As of such, it has become clear that OA responsive DNA methylation changes seem to mediate disease associated aberrant gene expression. Furthermore, established OA susceptibility alleles such as GDF5 and DIO2 appear to confer OA risk via DNA methylation and respective pathophysiological expression changes. In more recent years, genome wide profiling of DNA methylation in OA affected articular cartilage has emerged as a powerful tool to address the epigenetic changes in their entirety, which has resulted in the identification of putative patient subgroups as well as generic OA associated pathways. PMID:27019616

  5. Nutrients and DNA Methylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Epigenetics is a new mechanism responsible for development, aging, and disease process such as cancer development. One major epigenetic phenomenon is DNA methylation, which attributes to gene expression and integrity. Deepening the knowledge on one-carbon metabolism is very important to understandin...

  6. Methyl Bromide Poisoning

    PubMed Central

    Rathus, E. M.; Landy, P. J.

    1961-01-01

    Seven cases of methyl bromide poisoning which occurred amongst workers engaged on a fumigation project are described. The methods adopted for investigation of the environmental situation are discussed and the measurement of blood bromide levels on random samples of workers is suggested as an index of the effectiveness of equipment and working methods. PMID:13739738

  7. Thiophanate-methyl

    Integrated Risk Information System (IRIS)

    Thiophanate - methyl ; CASRN 23564 - 05 - 8 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 Noncarcino

  8. Pirimiphos-methyl

    Integrated Risk Information System (IRIS)

    Pirimiphos - methyl ; CASRN 29232 - 93 - 7 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 Noncarcinog

  9. Methyl isobutyl ketone (MIBK)

    Integrated Risk Information System (IRIS)

    Methyl Isobutyl Ketone ( MIBK ) ; CASRN 108 - 10 - 1 ; 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

  10. Haloxyfop-methyl

    Integrated Risk Information System (IRIS)

    Haloxyfop - methyl ; CASRN 69806 - 40 - 2 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 Noncarcinoge

  11. Methyl ethyl ketone (MEK)

    Integrated Risk Information System (IRIS)

    Methyl ethyl ketone ( MEK ) ( CASRN 78 - 93 - 3 ) 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 Nonc

  12. Chloromethyl methyl ether (CMME)

    Integrated Risk Information System (IRIS)

    Chloromethyl methyl ether ( CMME ) ; CASRN 107 - 30 - 2 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 fo

  13. Kenaf methyl esters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Additional or alternative feedstocks are one of the major areas of interest regarding biodiesel. In this paper, for the first time, the fuel properties of kenaf (Hibiscus cannabinus L.) seed oil methyl esters are comprehensively reported. This biodiesel is also relatively unique by containing small ...

  14. Kapok oil methyl esters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The increased need for biodiesel feedstocks has caused various vegetable oils to be examined for this purpose. In the present work, the methyl esters of kapok (Ceiba pentandra) oil were prepared. The essential fuel properties were comprehensively determined and evaluated in comparison to specificati...

  15. Src kinase regulation by phosphorylation and dephosphorylation

    SciTech Connect

    Roskoski, Robert . E-mail: biocrr@lsuhsc.edu

    2005-05-27

    Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTP{alpha}, PTP{epsilon}, and PTP{lambda}. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.

  16. 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

  17. Protein phosphorylation is involved in bacterial chemotaxis.

    PubMed Central

    Hess, J F; Oosawa, K; Matsumura, P; Simon, M I

    1987-01-01

    The nature of the biochemical signal that is involved in the excitation response in bacterial chemotaxis is not known. However, ATP is required for chemotaxis. We have purified all of the proteins involved in signal transduction and show that the product of the cheA gene is rapidly autophosphorylated, while some mutant CheA proteins cannot be phosphorylated. The presence of stoichiometric levels of two other purified components in the chemotaxis system, the CheY and CheZ proteins, induces dephosphorylation. We suggest that the phosphorylation of CheA by ATP plays a central role in signal transduction in chemotaxis. Images PMID:3313398

  18. Sgf29 binds histone H3K4me2/3 and is required for SAGA complex recruitment and histone H3 acetylation

    SciTech Connect

    Bian, Chuanbing; Xu, Chao; Ruan, Jianbin; Lee, Kenneth K.; Burke, Tara L.; Tempel, Wolfram; Barsyte, Dalia; Li, Jing; Wu, Minhao; Zhou, Bo O.; Fleharty, Brian E.; Paulson, Ariel; Allali-Hassani, Abdellah; Zhou, Jin-Qiu; Mer, Georges; Grant, Patrick A.; Workman, Jerry L.; Zang, Jianye; Min, Jinrong

    2011-09-28

    The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.

  19. Lysine deacetylase inhibition attenuates hypertension and is accompanied by acetylation of mineralocorticoid receptor instead of histone acetylation in spontaneously hypertensive rats.

    PubMed

    Seok, Young Mi; Lee, Hae Ahm; Park, Kwon Moo; Hwangbo, Mi-Hyang; Kim, In Kyeom

    2016-07-01

    Inhibition of lysine deacetylase (KDAC) attenuated development of hypertension in spontaneously hypertensive rats (SHRs). We hypothesized that KDAC inhibition attenuates hypertension and is accompanied by acetylation of mineralocorticoid receptors (MR) instead of histone acetylation in SHRs. Valproate (VPA, 0.71 % wt/vol), an inhibitor of class I KDACs, was administered in drinking water to 7-week-old SHRs and Wistar Kyoto rats for 11 weeks. MR acetylation was determined by immunoprecipitation with anti-MR antibody followed by western blot with anti-acetyl-lysine antibody. Expression levels of acetylated histone H3, KDACs, MR target genes, or MR corepressors in the kidney cortex were measured by using western blot analysis or real-time PCR. Recruitment of MR and RNA polymerase II (Pol II) and histone modifications on promoters of target genes were analyzed by performing a chromatin immunoprecipitation (ChIP) assay. Treatment of SHR with VPA increased MR acetylation without affecting MR expression, which attenuated development of hypertension in SHR VPA decreased expression of KDAC class I but globally increased acetylated histone H3. Although VPA treatment increased histone 3 acetylation (H3Ac) and trimethylation of the fourth lysine (H3K4me3) in the promoter regions of MR target genes, it decreased the expression of target genes as well as recruitment of MR and Pol II. These results suggest that KDAC inhibition attenuates the development of hypertension in SHRs and is accompanied by acetylation of MR that is independent of histone acetylation. PMID:27106211

  20. Acetylator genotype-dependent formation of 2-aminofluorene-hemoglobin adducts in rapid and slow acetylator Syrian hamsters congenic at the NAT2 locus.

    PubMed

    Feng, Y; Rustan, T D; Ferguson, R J; Doll, M A; Hein, D W

    1994-01-01

    Arylamine-hemoglobin adducts are a valuable dosimeter for assessing arylamine exposures and carcinogenic risk. The effects of age, sex, time-course, dose, and acetylator genotype on levels of 2-aminofluorene-hemoglobin adducts were investigated in homozygous rapid (Bio. 82.73/H-Patr) and slow (Bio. 82.73/H-Pats) acetylator hamsters congenic at the polymorphic (NAT2) acetylator locus. Following administration of a single ip dose of [3H]2-aminofluorene, peak 2-aminofluorene-hemoglobin adduct levels were achieved at 12-18 hr and retained a plateau up to 72 hr postinjection in both rapid and slow acetylator congenic hamsters. 2-Aminofluorene-hemoglobin adduct levels did not differ significantly between young (5-6 weeks) and old (32-49 weeks) hamsters or between male and female hamsters within either acetylator genotype. 2-Aminofluorene-hemoglobin adduct levels increased in a dose-dependent manner (r = 0.95, p = 0.0001) and were consistently higher in slow versus rapid acetylator congenic hamsters in studies of both time-course and dose-effect. The magnitude of the acetylator genotype-dependent difference was a function of dose; 2-aminofluorene-hemoglobin adduct levels were 1.5-fold higher in slow acetylator congenic hamsters following a 60 mg/kg 2-aminofluorene dose (p = 0.0013) but 2-fold higher following a 100 mg/kg 2-aminofluorene dose (p < 0.0001). These results show a specific and significant role for NAT2 acetylator genotype in formation of arylamine-hemoglobin adducts, which may reflect the relationship between acetylator genotype and the incidence of different cancers from arylamine exposures. PMID:8291051