Science.gov

Sample records for 14-3-3 mediates histone

  1. 14-3-3 regulates the nuclear import of class IIa histone deacetylases

    SciTech Connect

    Nishino, Tomonori G.; Miyazaki, Masaya; Hoshino, Hideto; Miwa, Yoshihiro; Horinouchi, Sueharu; Yoshida, Minoru

    2008-12-19

    Class IIa histone deacetylases (HDACs) form complexes with a class of transcriptional repressors in the nucleus. While screening for compounds that could block the association of HDAC4 with the BTB domain-containing transcriptional repressor Bach2, we discovered that phorbol 12-myristate 13-acetate (PMA) induced the cytoplasmic retention of HDAC4 mutants lacking a nuclear export signal (NES). Although PMA treatment and PKD overexpression has been proposed to facilitate the nuclear export of class IIa HDACs by creating 14-3-3 binding sites containing phosphoserines, our experiments using HDAC mutants demonstrated that PMA greatly reduces nuclear import. PMA treatment repressed the NLS activity in a manner dependent on 14-3-3 binding. These results suggest that nuclear HDAC4 is not tethered in the nucleus, but instead shuttles between the nucleus and the cytoplasm. Phosphorylation-induced 14-3-3 binding biases the balance of nucleo-cytoplasmic shuttling toward the cytoplasm by inhibiting nuclear import.

  2. Intracellular Generation of a Diterpene-Peptide Conjugate that Inhibits 14-3-3-Mediated Interactions.

    PubMed

    Parvatkar, Prakash; Kato, Nobuo; Uesugi, Motonari; Sato, Shin-Ichi; Ohkanda, Junko

    2015-12-23

    Synthetic agents that disrupt intracellular protein-protein interactions (PPIs) are highly desirable for elucidating signaling networks and developing new therapeutics. However, designing cell-penetrating large molecules equipped with the many functional groups necessary for binding to large interfaces remains challenging. Here, we describe a rational strategy for the intracellular oxime ligation-mediated generation of an amphipathic bivalent inhibitor composed of a peptide and diterpene natural product, fusicoccin, which binds 14-3-3 protein with submicromolar affinity. Our results demonstrate that co-treatment of cells with small module molecules, the aldehyde-containing fusicoccin 1 and the aminooxy-containing peptide 2, generates the corresponding conjugate 3 in cells, resulting in significant cytotoxicity. In contrast, chemically synthesized 3 is not cytotoxic, likely due to its inability to penetrate cells. Compound 3, but not 1 or 2, disrupts endogenous 14-3-3/cRaf interactions, suggesting that cell death is caused by inhibition of 14-3-3 activity. These results suggest that intracellular generation of large-sized molecules may serve as a new approach for modulating PPIs.

  3. 14-3-3 proteins play a role in the cell cycle by shielding cdt2 from ubiquitin-mediated degradation.

    PubMed

    Dar, Ashraf; Wu, David; Lee, Nicholas; Shibata, Etsuko; Dutta, Anindya

    2014-11-01

    Cdt2 is the substrate recognition adaptor of CRL4(Cdt2) E3 ubiquitin ligase complex and plays a pivotal role in the cell cycle by mediating the proteasomal degradation of Cdt1 (DNA replication licensing factor), p21 (cyclin-dependent kinase [CDK] inhibitor), and Set8 (histone methyltransferase) in S phase. Cdt2 itself is attenuated by SCF(FbxO11)-mediated proteasomal degradation. Here, we report that 14-3-3 adaptor proteins interact with Cdt2 phosphorylated at threonine 464 (T464) and shield it from polyubiquitination and consequent proteasomal degradation. Depletion of 14-3-3 proteins promotes the interaction of FbxO11 with Cdt2. Overexpressing 14-3-3 proteins shields Cdt2 that has a phospho-mimicking mutation (T464D [change of T to D at position 464]) but not Cdt2(T464A) from ubiquitination. Furthermore, the delay of the cell cycle in the G2/M phase and decrease in cell proliferation seen upon depletion of 14-3-3γ is partly due to the accumulation of the CRL4(Cdt2) substrate, Set8 methyltransferase. Therefore, the stabilization of Cdt2 is an important function of 14-3-3 proteins in cell cycle progression.

  4. 14-3-3 Proteins Play a Role in the Cell Cycle by Shielding Cdt2 from Ubiquitin-Mediated Degradation

    PubMed Central

    Dar, Ashraf; Wu, David; Lee, Nicholas; Shibata, Etsuko

    2014-01-01

    Cdt2 is the substrate recognition adaptor of CRL4Cdt2 E3 ubiquitin ligase complex and plays a pivotal role in the cell cycle by mediating the proteasomal degradation of Cdt1 (DNA replication licensing factor), p21 (cyclin-dependent kinase [CDK] inhibitor), and Set8 (histone methyltransferase) in S phase. Cdt2 itself is attenuated by SCFFbxO11-mediated proteasomal degradation. Here, we report that 14-3-3 adaptor proteins interact with Cdt2 phosphorylated at threonine 464 (T464) and shield it from polyubiquitination and consequent proteasomal degradation. Depletion of 14-3-3 proteins promotes the interaction of FbxO11 with Cdt2. Overexpressing 14-3-3 proteins shields Cdt2 that has a phospho-mimicking mutation (T464D [change of T to D at position 464]) but not Cdt2(T464A) from ubiquitination. Furthermore, the delay of the cell cycle in the G2/M phase and decrease in cell proliferation seen upon depletion of 14-3-3γ is partly due to the accumulation of the CRL4Cdt2 substrate, Set8 methyltransferase. Therefore, the stabilization of Cdt2 is an important function of 14-3-3 proteins in cell cycle progression. PMID:25154416

  5. Induction of AID-targeting adaptor 14-3-3γ is mediated by NF-κB-dependent recruitment of CFP1 to the 5′-CpG-3′-rich 14-3-3γ promoter and is sustained by E2A

    PubMed Central

    Mai, Thach; Pone, Egest J.; Li, Guideng; Lam, Tonika S.; Moehlman, J’aime; Xu, Zhenming; Casali, Paolo

    2013-01-01

    Class switch DNA recombination (CSR) crucially diversifies antibody biological effectors functions. 14-3-3γ specifically binds to the 5′-AGCT-3′ repeats in the IgH locus switch (S) regions. By directly interacting with the C-terminal region of activation-induced cytidine deaminase (AID), 14-3-3γ targets this enzyme to S regions to mediate CSR. Here, we showed that 14-3-3γ was expressed in germinal center B cells in vivo and induced in B cells by T-dependent and T-independent primary CSR-inducing stimuli in vitro in humans and mice. Induction of 14-3-3γ was rapid, peaking within 3 h of stimulation by lipopolysaccharides (LPS), and sustained over the course of AID and CSR induction. It was dependent on recruitment of NF-κB to the 14-3-3γ gene promoter. The NF-κB recruitment enhanced the occupancy of the CpG island within the 14-3-3γ promoter by CFP1, a component of the COMPASS histone methyltransferase complex, and promoter-specific enrichment of histone 3 lysine 4 trimethylation (H3K4me3), which is indicative of open chromatin state and marks transcription-competent promoters. NF-κB also potentiated the binding of B cell lineage-specific factor E2A to an E-box motif located immediately downstream of the two closely-spaced transcription start sites (TSSs) for sustained 14-3-3γ expression and CSR induction. Thus, 14-3-3γ induction in CSR is enabled by the CFP1-mediated H3K4me3 enrichment in the promoter, dependent on NF-κB and sustained by E2A. PMID:23851690

  6. Induction of activation-induced cytidine deaminase-targeting adaptor 14-3-3γ is mediated by NF-κB-dependent recruitment of CFP1 to the 5'-CpG-3'-rich 14-3-3γ promoter and is sustained by E2A.

    PubMed

    Mai, Thach; Pone, Egest J; Li, Guideng; Lam, Tonika S; Moehlman, J'aime; Xu, Zhenming; Casali, Paolo

    2013-08-15

    Class switch DNA recombination (CSR) crucially diversifies Ab biologic effector functions. 14-3-3γ specifically binds to the 5'-AGCT-3' repeats in the IgH locus switch (S) regions. By interacting directly with the C-terminal region of activation-induced cytidine deaminase (AID), 14-3-3γ targets this enzyme to S regions to mediate CSR. In this study, we showed that 14-3-3γ was expressed in germinal center B cells in vivo and induced in B cells by T-dependent and T-independent primary CSR-inducing stimuli in vitro in humans and mice. Induction of 14-3-3γ was rapid, peaking within 3 h of stimulation by LPSs, and sustained over the course of AID and CSR induction. It was dependent on recruitment of NF-κB to the 14-3-3γ gene promoter. The NF-κB recruitment enhanced the occupancy of the CpG island within the 14-3-3γ promoter by CFP1, a component of the COMPASS histone methyltransferase complex, and promoter-specific enrichment of histone 3 lysine 4 trimethylation (H3K4me3), which is indicative of open chromatin state and marks transcription-competent promoters. NF-κB also potentiated the binding of B cell lineage-specific factor E2A to an E-box motif located immediately downstream of the two closely-spaced transcription start sites for sustained 14-3-3γ expression and CSR induction. Thus, 14-3-3γ induction in CSR is enabled by the CFP1-mediated H3K4me3 enrichment in the promoter, dependent on NF-κB and sustained by E2A.

  7. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells.

    PubMed

    Li, Tong; Paudel, Hemant K

    2016-01-01

    Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer's disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445-6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ. PMID:27548710

  8. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells

    PubMed Central

    Li, Tong; Paudel, Hemant K.

    2016-01-01

    Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer’s disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445–6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ. PMID:27548710

  9. Melatonin synthesis: 14-3-3-dependent activation and inhibition of arylalkylamine N-acetyltransferase mediated by phosphoserine-205.

    PubMed

    Ganguly, Surajit; Weller, Joan L; Ho, Anthony; Chemineau, Philippe; Malpaux, Benoit; Klein, David C

    2005-01-25

    The nocturnal increase in circulating melatonin in vertebrates is regulated by the activity of arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the melatonin pathway (serotonin --> N-acetylserotonin --> melatonin). Large changes in activity are linked to cyclic AMP-dependent protein kinase-mediated phosphorylation of AANAT T31. Phosphorylation of T31 promotes binding of AANAT to the dimeric 14-3-3 protein, which activates AANAT by increasing arylalkylamine affinity. In the current study, a putative second AANAT cyclic AMP-dependent protein kinase phosphorylation site, S205, was found to be approximately 55% phosphorylated at night, when T31 is approximately 40% phosphorylated. These findings indicate that ovine AANAT is dual-phosphorylated. Moreover, light exposure at night decreases T31 and S205 phosphorylation, consistent with a regulatory role of both sites. AANAT peptides containing either T31 or S205 associate with 14-3-3zeta in a phosphorylation-dependent manner; binding through phosphorylated (p)T31 is stronger than that through pS205, consistent with the location of only pT31 in a mode I binding motif, one of two recognized high-affinity 14-3-3-binding motifs AANAT protein binds to 14-3-3zeta through pT31 or pS205. Two-site binding lowers the Km for arylalkylamine substrate to approximately 30 microM. In contrast, single-site pS205 binding increases the Km to approximately 1,200 microM. Accordingly, the switch from dual to single pS205 binding of AANAT to 14-3-3 changes the Km for substrates by approximately 40-fold. pS205 seems to be part of a previously unrecognized 14-3-3-binding motif-pS/pT (X1-2)-COOH, referred to here as mode III.

  10. Histone Deacetylase 6 (HDAC6) Promotes the Pro-survival Activity of 14-3-3ζ via Deacetylation of Lysines within the 14-3-3ζ Binding Pocket*

    PubMed Central

    Mortenson, Jeffrey B.; Heppler, Lisa N.; Banks, Courtney J.; Weerasekara, Vajira K.; Whited, Matthew D.; Piccolo, Stephen R.; Johnson, William E.; Thompson, J. Will; Andersen, Joshua L.

    2015-01-01

    The phospho-binding protein 14-3-3ζ acts as a signaling hub controlling a network of interacting partners and oncogenic pathways. We show here that lysines within the 14-3-3ζ binding pocket and protein-protein interface can be modified by acetylation. The positive charge on two of these lysines, Lys49 and Lys120, is critical for coordinating 14-3-3ζ-phosphoprotein interactions. Through screening, we identified HDAC6 as the Lys49/Lys120 deacetylase. Inhibition of HDAC6 blocks 14-3-3ζ interactions with two well described interacting partners, Bad and AS160, which triggers their dephosphorylation at Ser112 and Thr642, respectively. Expression of an acetylation-refractory K49R/K120R mutant of 14-3-3ζ rescues both the HDAC6 inhibitor-induced loss of interaction and Ser112/Thr642 phosphorylation. Furthermore, expression of the K49R/K120R mutant of 14-3-3ζ inhibits the cytotoxicity of HDAC6 inhibition. These data demonstrate a novel role for HDAC6 in controlling 14-3-3ζ binding activity. PMID:25770209

  11. Suppression of 14-3-3γ-mediated surface expression of ANO1 inhibits cancer progression of glioblastoma cells

    PubMed Central

    Lee, Young-Sun; Lee, Jae Kwang; Bae, Yeonju; Lee, Bok-Soon; Kim, Eunju; Cho, Chang-Hoon; Ryoo, Kanghyun; Yoo, Jiyun; Kim, Chul-Ho; Yi, Gwan-Su; Lee, Seok-Geun; Lee, C. Justin; Kang, Sang Soo; Hwang, Eun Mi; Park, Jae-Yong

    2016-01-01

    Anoctamin-1 (ANO1) acts as a Ca2+-activated Cl− channel in various normal tissues, and its expression is increased in several different types of cancer. Therefore, understanding the regulation of ANO1 surface expression is important for determining its physiological and pathophysiological functions. However, the trafficking mechanism of ANO1 remains elusive. Here, we report that segment a (N-terminal 116 amino acids) of ANO1 is crucial for its surface expression, and we identified 14-3-3γ as a binding partner for anterograde trafficking using yeast two-hybrid screening. The surface expression of ANO1 was enhanced by 14-3-3γ, and the Thr9 residue of ANO1 was critical for its interaction with 14-3-3γ. Gene silencing of 14-3-3γ and/or ANO1 demonstrated that suppression of ANO1 surface expression inhibited migration and invasion of glioblastoma cells. These findings provide novel therapeutic implications for glioblastomas, which are associated with poor prognosis. PMID:27212225

  12. 14-3-3 proteins regulate a cell-intrinsic switch from sonic hedgehog-mediated commissural axon attraction to repulsion after midline crossing.

    PubMed

    Yam, Patricia T; Kent, Christopher B; Morin, Steves; Farmer, W Todd; Alchini, Ricardo; Lepelletier, Léa; Colman, David R; Tessier-Lavigne, Marc; Fournier, Alyson E; Charron, Frédéric

    2012-11-21

    Axons must switch responsiveness to guidance cues during development for correct pathfinding. Sonic Hedgehog (Shh) attracts spinal cord commissural axons ventrally toward the floorplate. We show that after crossing the floorplate, commissural axons switch their response to Shh from attraction to repulsion, so that they are repelled anteriorly by a posterior-high/anterior-low Shh gradient along the longitudinal axis. This switch is recapitulated in vitro with dissociated commissural neurons as they age, indicating that the switch is intrinsic and time dependent. 14-3-3 protein inhibition converted Shh-mediated repulsion of aged dissociated neurons to attraction and prevented the correct anterior turn of postcrossing commissural axons in vivo, an effect mediated through PKA. Conversely, overexpression of 14-3-3 proteins was sufficient to drive the switch from Shh-mediated attraction to repulsion both in vitro and in vivo. Therefore, we identify a 14-3-3 protein-dependent mechanism for a cell-intrinsic temporal switch in the polarity of axon turning responses.

  13. Tomato 14-3-3 protein TFT7 interacts with a MAP kinase kinase to regulate immunity-associated programmed cell death mediated by diverse disease resistance proteins.

    PubMed

    Oh, Chang-Sik; Martin, Gregory B

    2011-04-22

    Programmed cell death (PCD) associated with immunity is triggered when a plant disease resistance (R) protein recognizes a corresponding pathogen virulence protein. In tomato, detection by the host Pto kinase of the Pseudomonas syringae proteins AvrPto or AvrPtoB causes localized PCD. Previously, we reported that both MAPKKKα (mitogen-activated protein kinase kinase kinase) and the tomato 14-3-3 protein 7 (TFT7) positively regulate Pto-mediated PCD in tomato and Nicotiana benthamiana. In addition, in contrast to MAPKKKα, TFT7 is required for PCD mediated by four other R proteins. Here we investigate why TFT7 is required for PCD induced by diverse R proteins in plants. We discovered that a MAPKK, SlMKK2, which acts downstream of SlMAPKKKα, also interacts with TFT7 in plant cells. Gene silencing experiments revealed that the orthologous genes of both SlMKK2 and TFT7 in N. benthamiana are required for PCD mediated by the same set of R proteins. SlMKK2 and its orthologs contain a 14-3-3 binding site in their N terminus, and Thr(33) in this site is required for interaction with TFT7 in vivo. Like the structurally similar human 14-3-3ε protein, TFT7 forms a homodimer in vivo. Because TFT7 interacts with both SlMAPKKKα and SlMKK2 and also forms a homodimer, we propose that TFT7 may coordinately recruit these client proteins for efficient signal transfer, leading to PCD induction. PMID:21378171

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

    PubMed Central

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

    2013-01-01

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

  15. Environmental responses mediated by histone variants.

    PubMed

    Talbert, Paul B; Henikoff, Steven

    2014-11-01

    Fluctuations in the ambient environment can trigger chromatin disruptions, involving replacement of nucleosomes or exchange of their histone subunits. Unlike canonical histones, which are available only during S-phase, replication-independent histone variants are present throughout the cell cycle and are adapted for chromatin repair. The H2A.Z variant mediates responses to environmental perturbations including fluctuations in temperature and seasonal variation. Phosphorylation of histone H2A.X rapidly marks double-strand DNA breaks for chromatin repair, which is mediated by both H2A and H3 histone variants. Other histones are used as weapons in conflicts between parasites and their hosts, which suggests broad involvement of histone variants in environmental responses beyond chromatin repair. PMID:25150594

  16. Epstein-Barr virus Rta-mediated transactivation of p21 and 14-3-3σ arrests cells at the G1/S transition by reducing cyclin E/CDK2 activity.

    PubMed

    Huang, Sheng-Yen; Hsieh, Min-Jie; Chen, Chu-Ying; Chen, Yen-Ju; Chen, Jen-Yang; Chen, Mei-Ru; Tsai, Ching-Hwa; Lin, Su-Fang; Hsu, Tsuey-Ying

    2012-01-01

    Many herpesviral immediate-early proteins promote their robust lytic phase replications by hijacking the cell cycle machinery. Previously, lytic replication of Epstein-Barr virus (EBV) was found to be concurrent with host cell cycle arrest. In this study, we showed that ectopic expression of EBV immediate-early protein Rta in HEp-2 cells resulted in increased G1/S population, hypophosphorylation of pRb and decreased incorporation of 5-bromo-2'-deoxyuridine. In addition, EBV Rta transcriptionally upregulates the expressions of p21 and 14-3-3σ in HEp-2 cells, 293 cells and nasopharyngeal carcinoma TW01 cells. Although p21 and 14-3-3σ are known targets for p53, Rta-mediated p21 and 14-3-3σ transactivation can be detected in the absence of p53. In addition, results from luciferase reporter assays indicated that direct binding of Rta to either promoter sequences is not required for activation. On the other hand, a special class of Sp1-responsive elements was involved in Rta-mediated transcriptional activation on both promoters. Finally, Rta-induced p21 expression diminished the activity of CDK2/cyclin E complex, and, Rta-induced 14-3-3σ expression sequestered CDK1 and CDK2 in the cytoplasm. Based on these results, we hypothesize that through the disruption of CDK1 and CDK2 activities, EBV Rta might contribute to cell cycle arrest in EBV-infected epithelial cells during viral reactivation. PMID:21918011

  17. Royal Jelly-Mediated Prolongevity and Stress Resistance in Caenorhabditis elegans Is Possibly Modulated by the Interplays of DAF-16, SIR-2.1, HCF-1, and 14-3-3 Proteins.

    PubMed

    Wang, Xiaoxia; Cook, Lauren F; Grasso, Lindsay M; Cao, Min; Dong, Yuqing

    2015-07-01

    Recent studies suggest that royal jelly (RJ) and its related substances may have antiaging properties. However, the molecular mechanisms underlying the beneficial effects remain elusive. We report that the effects of RJ and enzyme-treated RJ (eRJ) on life span and health span in Caenorhabditis elegans (C elegans) are modulated by the sophisticated interplays of DAF-16, SIR-2.1, HCF-1, and 14-3-3 proteins. Dietary supplementation with RJ or eRJ increased C. elegans life span in a dose-dependent manner. The RJ and eRJ consumption increased the tolerance of C elegans to oxidative stress, ultraviolet irradiation, and heat shock stress. Our genetic analyses showed that RJ/eRJ-mediated life-span extension requires insulin/IGF-1 signaling and the activities of DAF-16, SIR-2.1, HCF-1, and FTT-2, a 14-3-3 protein. Earlier studies reported that DAF-16/FOXO, SIR-2.1/SIRT1, FTT-2, and HCF-1 have extensive interplays in worms and mammals. Our present findings suggest that RJ/eRJ-mediated promotion of longevity and stress resistance in C elegans is dependent on these conserved interplays. From an evolutionary point of view, this study not only provides new insights into the molecular mechanisms of RJ's action on health span promotion in C elegans, but also has imperative implications in using RJ/eRJ as nutraceuticals to delay aging and age-related disorders.

  18. Histone chaperone-mediated nucleosome assembly process.

    PubMed

    Fan, Hsiu-Fang; Liu, Zi-Ning; Chow, Sih-Yao; Lu, Yi-Han; Li, Hsin

    2015-01-01

    A huge amount of information is stored in genomic DNA and this stored information resides inside the nucleus with the aid of chromosomal condensation factors. It has been reported that the repeat nucleosome core particle (NCP) consists of 147-bp of DNA and two copies of H2A, H2B, H3 and H4. Regulation of chromosomal structure is important to many processes inside the cell. In vivo, a group of histone chaperones facilitate and regulate nucleosome assembly. How NCPs are constructed with the aid of histone chaperones remains unclear. In this study, the histone chaperone-mediated nucleosome assembly process was investigated using single-molecule tethered particle motion (TPM) experiments. It was found that Asf1 is able to exert more influence than Nap1 and poly glutamate acid (PGA) on the nucleosome formation process, which highlights Asf1's specific role in tetrasome formation. Thermodynamic parameters supported a model whereby energetically favored nucleosomal complexes compete with non-nucleosomal complexes. In addition, our kinetic findings propose the model that histone chaperones mediate nucleosome assembly along a path that leads to enthalpy-favored products with free histones as reaction substrates. PMID:25611318

  19. Histone Chaperone-Mediated Nucleosome Assembly Process

    PubMed Central

    Fan, Hsiu-Fang; Liu, Zi-Ning; Chow, Sih-Yao; Lu, Yi-Han; Li, Hsin

    2015-01-01

    A huge amount of information is stored in genomic DNA and this stored information resides inside the nucleus with the aid of chromosomal condensation factors. It has been reported that the repeat nucleosome core particle (NCP) consists of 147-bp of DNA and two copies of H2A, H2B, H3 and H4. Regulation of chromosomal structure is important to many processes inside the cell. In vivo, a group of histone chaperones facilitate and regulate nucleosome assembly. How NCPs are constructed with the aid of histone chaperones remains unclear. In this study, the histone chaperone-mediated nucleosome assembly process was investigated using single-molecule tethered particle motion (TPM) experiments. It was found that Asf1 is able to exert more influence than Nap1 and poly glutamate acid (PGA) on the nucleosome formation process, which highlights Asf1’s specific role in tetrasome formation. Thermodynamic parameters supported a model whereby energetically favored nucleosomal complexes compete with non-nucleosomal complexes. In addition, our kinetic findings propose the model that histone chaperones mediate nucleosome assembly along a path that leads to enthalpy-favored products with free histones as reaction substrates. PMID:25611318

  20. Histone chaperone-mediated nucleosome assembly process.

    PubMed

    Fan, Hsiu-Fang; Liu, Zi-Ning; Chow, Sih-Yao; Lu, Yi-Han; Li, Hsin

    2015-01-01

    A huge amount of information is stored in genomic DNA and this stored information resides inside the nucleus with the aid of chromosomal condensation factors. It has been reported that the repeat nucleosome core particle (NCP) consists of 147-bp of DNA and two copies of H2A, H2B, H3 and H4. Regulation of chromosomal structure is important to many processes inside the cell. In vivo, a group of histone chaperones facilitate and regulate nucleosome assembly. How NCPs are constructed with the aid of histone chaperones remains unclear. In this study, the histone chaperone-mediated nucleosome assembly process was investigated using single-molecule tethered particle motion (TPM) experiments. It was found that Asf1 is able to exert more influence than Nap1 and poly glutamate acid (PGA) on the nucleosome formation process, which highlights Asf1's specific role in tetrasome formation. Thermodynamic parameters supported a model whereby energetically favored nucleosomal complexes compete with non-nucleosomal complexes. In addition, our kinetic findings propose the model that histone chaperones mediate nucleosome assembly along a path that leads to enthalpy-favored products with free histones as reaction substrates.

  1. 14-3-3-Pred: improved methods to predict 14-3-3-binding phosphopeptides

    PubMed Central

    Madeira, Fábio; Tinti, Michele; Murugesan, Gavuthami; Berrett, Emily; Stafford, Margaret; Toth, Rachel; Cole, Christian; MacKintosh, Carol; Barton, Geoffrey J.

    2015-01-01

    Motivation: The 14-3-3 family of phosphoprotein-binding proteins regulates many cellular processes by docking onto pairs of phosphorylated Ser and Thr residues in a constellation of intracellular targets. Therefore, there is a pressing need to develop new prediction methods that use an updated set of 14-3-3-binding motifs for the identification of new 14-3-3 targets and to prioritize the downstream analysis of >2000 potential interactors identified in high-throughput experiments. Results: Here, a comprehensive set of 14-3-3-binding targets from the literature was used to develop 14-3-3-binding phosphosite predictors. Position-specific scoring matrix, support vector machines (SVM) and artificial neural network (ANN) classification methods were trained to discriminate experimentally determined 14-3-3-binding motifs from non-binding phosphopeptides. ANN, position-specific scoring matrix and SVM methods showed best performance for a motif window spanning from −6 to +4 around the binding phosphosite, achieving Matthews correlation coefficient of up to 0.60. Blind prediction showed that all three methods outperform two popular 14-3-3-binding site predictors, Scansite and ELM. The new methods were used for prediction of 14-3-3-binding phosphosites in the human proteome. Experimental analysis of high-scoring predictions in the FAM122A and FAM122B proteins confirms the predictions and suggests the new 14-3-3-predictors will be generally useful. Availability and implementation: A standalone prediction web server is available at http://www.compbio.dundee.ac.uk/1433pred. Human candidate 14-3-3-binding phosphosites were integrated in ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome database. Contact: cmackintosh@dundee.ac.uk or gjbarton@dundee.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25735772

  2. 14-3-3 Proteins in Guard Cell Signaling.

    PubMed

    Cotelle, Valérie; Leonhardt, Nathalie

    2015-01-01

    Guard cells are specialized cells located at the leaf surface delimiting pores which control gas exchanges between the plant and the atmosphere. To optimize the CO2 uptake necessary for photosynthesis while minimizing water loss, guard cells integrate environmental signals to adjust stomatal aperture. The size of the stomatal pore is regulated by movements of the guard cells driven by variations in their volume and turgor. As guard cells perceive and transduce a wide array of environmental cues, they provide an ideal system to elucidate early events of plant signaling. Reversible protein phosphorylation events are known to play a crucial role in the regulation of stomatal movements. However, in some cases, phosphorylation alone is not sufficient to achieve complete protein regulation, but is necessary to mediate the binding of interactors that modulate protein function. Among the phosphopeptide-binding proteins, the 14-3-3 proteins are the best characterized in plants. The 14-3-3s are found as multiple isoforms in eukaryotes and have been shown to be involved in the regulation of stomatal movements. In this review, we describe the current knowledge about 14-3-3 roles in the regulation of their binding partners in guard cells: receptors, ion pumps, channels, protein kinases, and some of their substrates. Regulation of these targets by 14-3-3 proteins is discussed and related to their function in guard cells during stomatal movements in response to abiotic or biotic stresses.

  3. Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

    PubMed

    Teichmann, Aline; Vargas, Daiani M; Monteiro, Karina M; Meneghetti, Bruna V; Dutra, Cristine S; Paredes, Rodolfo; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique B

    2015-04-01

    The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization. PMID:25748451

  4. Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

    PubMed

    Teichmann, Aline; Vargas, Daiani M; Monteiro, Karina M; Meneghetti, Bruna V; Dutra, Cristine S; Paredes, Rodolfo; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique B

    2015-04-01

    The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization.

  5. 14-3-3 proteins regulate Tctp–Rheb interaction for organ growth in Drosophila

    PubMed Central

    Le, Thao Phuong; Vuong, Linh Thuong; Kim, Ah-Ram; Hsu, Ya-Chieh; Choi, Kwang-Wook

    2016-01-01

    14-3-3 family proteins regulate multiple signalling pathways. Understanding biological functions of 14-3-3 proteins has been limited by the functional redundancy of conserved isotypes. Here we provide evidence that 14-3-3 proteins regulate two interacting components of Tor signalling in Drosophila, translationally controlled tumour protein (Tctp) and Rheb GTPase. Single knockdown of 14-3-3ɛ or 14-3-3ζ isoform does not show obvious defects in organ development but causes synergistic genetic interaction with Tctp and Rheb to impair tissue growth. 14-3-3 proteins physically interact with Tctp and Rheb. Knockdown of both 14-3-3 isoforms abolishes the binding between Tctp and Rheb, disrupting organ development. Depletion of 14-3-3s also reduces the level of phosphorylated S6 kinase, phosphorylated Thor/4E-BP and cyclin E (CycE). Growth defects from knockdown of 14-3-3 and Tctp are suppressed by CycE overexpression. This study suggests a novel mechanism of Tor regulation mediated by 14-3-3 interaction with Tctp and Rheb. PMID:27151460

  6. Dual binding of 14-3-3 protein regulates Arabidopsis nitrate reductase activity.

    PubMed

    Chi, Jen-Chih; Roeper, Juliane; Schwarz, Guenter; Fischer-Schrader, Katrin

    2015-03-01

    14-3-3 proteins represent a family of ubiquitous eukaryotic proteins involved in numerous signal transduction processes and metabolic pathways. One important 14-3-3 target in higher plants is nitrate reductase (NR), whose activity is regulated by different physiological conditions. Intra-molecular electron transfer in NR is inhibited following 14-3-3 binding to a conserved phospho-serine motif located in hinge 1, a surface exposed loop between the catalytic molybdenum and central heme domain. Here we describe a novel 14-3-3 binding site within the NR N-terminus, an acidic motif conserved in NRs of higher plants, which significantly contributes to 14-3-3-mediated inhibition of NR. Deletion or mutation of the N-terminal acidic motif resulted in a significant loss of 14-3-3 mediated inhibition of Ser534 phosphorylated NR-Mo-heme (residues 1-625), a previously established model of NR regulation. Co-sedimentation and crosslinking studies with NR peptides comprising each of the two binding motifs demonstrated direct binding of either peptide to 14-3-3. Surface plasmon resonance spectroscopy disclosed high-affinity binding of 14-3-3ω to the well-known phospho-hinge site and low-affinity binding to the N-terminal acidic motif. A binding groove-deficient 14-3-3ω variant retained interaction to the acidic motif, but lost binding to the phospho-hinge motif. To our knowledge, NR is the first enzyme that harbors two independent 14-3-3 binding sites with different affinities, which both need to be occupied by 14-3-3ω to confer full inhibition of NR activity under physiological conditions. PMID:25578809

  7. Scaffold functions of 14-3-3 adaptors in B cell immunoglobulin class switch DNA recombination.

    PubMed

    Lam, Tonika; Thomas, Lisa M; White, Clayton A; Li, Guideng; Pone, Egest J; Xu, Zhenming; Casali, Paolo

    2013-01-01

    Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID) expression and AID targeting to switch (S) regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5'-AGCT-3' repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S-S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3β, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit) and PKA-RIα (regulatory inhibitory subunit) and uracil DNA glycosylase (Ung). 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180-198) or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR). 14-3-3 adaptors colocalized with AID and replication protein A (RPA) in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr), an accessory protein of human immunodeficiency virus type-1 (HIV-1), which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR.

  8. 14-3-3 proteins in plant-pathogen interactions.

    PubMed

    Lozano-Durán, Rosa; Robatzek, Silke

    2015-05-01

    14-3-3 proteins define a eukaryotic-specific protein family with a general role in signal transduction. Primarily, 14-3-3 proteins act as phosphosensors, binding phosphorylated client proteins and modulating their functions. Since phosphorylation regulates a plethora of different physiological responses in plants, 14-3-3 proteins play roles in multiple signaling pathways, including those controlling metabolism, hormone signaling, cell division, and responses to abiotic and biotic stimuli. Increasing evidence supports a prominent role of 14-3-3 proteins in regulating plant immunity against pathogens at various levels. In this review, potential links between 14-3-3 function and the regulation of plant-pathogen interactions are discussed, with a special focus on the regulation of 14-3-3 proteins in response to pathogen perception, interactions between 14-3-3 proteins and defense-related proteins, and 14-3-3 proteins as targets of pathogen effectors.

  9. Phosphorylation and Interaction with the 14-3-3 Protein of the Plasma Membrane H+-ATPase are Involved in the Regulation of Magnesium-Mediated Increases in Aluminum-Induced Citrate Exudation in Broad Bean (Vicia faba. L).

    PubMed

    Chen, Qi; Kan, Qi; Wang, Ping; Yu, Wenqian; Yu, Yuzhen; Zhao, Yan; Yu, Yongxiong; Li, Kunzhi; Chen, Limei

    2015-06-01

    Several studies have shown that external application of micromolar magnesium (Mg) can increase the resistance of legumes to aluminum (Al) stress by enhancing Al-induced citrate exudation. However, the exact mechanism underlying this regulation remains unknown. In this study, the physiological and molecular mechanisms by which Mg enhances Al-induced citrate exudation to alleviate Al toxicity were investigated in broad bean. Micromolar concentrations of Mg that alleviated Al toxicity paralleled the stimulation of Al-induced citrate exudation and increased the activity of the plasma membrane (PM) H(+)-ATPase. Northern blot analysis shows that a putative MATE-like gene (multidrug and toxic compound extrusion) was induced after treatment with Al for 4, 8 and 12 h, whereas the mRNA abundance of the MATE-like gene showed no significant difference between Al plus Mg and Al-only treatments during the entire treatment period. Real-time reverse transcription-PCR (RT-PCR) and Western blot analyses suggest that the transcription and translation of the PM H(+)-ATPase were induced by Al but not by Mg. In contrast, immunoprecipitation suggests that Mg enhanced the phosphorylation levels of VHA2 and its interaction with the vf14-3-3b protein under Al stress. Taken together, our results suggest that micromolar concentrations of Mg can alleviate the Al rhizotoxicity by increasing PM H(+)-ATPase activity and Al-induced citrate exudation in YD roots. This enhancement is likely to be attributable to Al-induced increases in the expression of the MATE-like gene and vha2 and Mg-induced changes in the phosphorylation levels of VHA2, thus changing its interaction with the vf14-3-3b protein.

  10. 14-3-3 proteins: a historic overview.

    PubMed

    Aitken, Alastair

    2006-06-01

    This chapter includes a historic overview of 14-3-3 proteins with an emphasis on the differences between potentially cancer-relevant isoforms on the genomic, protein and functional level. The focus will therefore be on mammalian 14-3-3s although many important developments in the field have involved Drosophila 14-3-3 proteins for example and the cross-fertilisation from parallel studies on plant 14-3-3 should not be underestimated. In the major part of this review I will attempt to focus on some novel data and aspects of 14-3-3 structure and function, in particular regulation of 14-3-3 isoforms by oncogene-related protein kinase phosphorylation and aspects of 14-3-3 research with which newcomers to the field may be less familiar.

  11. 14-3-3ζ coordinates adipogenesis of visceral fat

    PubMed Central

    Lim, Gareth E.; Albrecht, Tobias; Piske, Micah; Sarai, Karnjit; Lee, Jason T. C; Ramshaw, Hayley S.; Sinha, Sunita; Guthridge, Mark A.; Acker-Palmer, Amparo; Lopez, Angel F.; Clee, Susanne M.; Nislow, Corey; Johnson, James D.

    2015-01-01

    The proteins that coordinate complex adipogenic transcriptional networks are poorly understood. 14-3-3ζ is a molecular adaptor protein that regulates insulin signalling and transcription factor networks. Here we report that 14-3-3ζ-knockout mice are strikingly lean from birth with specific reductions in visceral fat depots. Conversely, transgenic 14-3-3ζ overexpression potentiates obesity, without exacerbating metabolic complications. Only the 14-3-3ζ isoform is essential for adipogenesis based on isoform-specific RNAi. Mechanistic studies show that 14-3-3ζ depletion promotes autophagy-dependent degradation of C/EBP-δ, preventing induction of the master adipogenic factors, Pparγ and C/EBP-α. Transcriptomic data indicate that 14-3-3ζ acts upstream of hedgehog signalling-dependent upregulation of Cdkn1b/p27Kip1. Indeed, concomitant knockdown of p27Kip1 or Gli3 rescues the early block in adipogenesis induced by 14-3-3ζ knockdown in vitro. Adipocyte precursors in 14-3-3ζKO embryos also appear to have greater Gli3 and p27Kip1 abundance. Together, our in vivo and in vitro findings demonstrate that 14-3-3ζ is a critical upstream driver of adipogenesis. PMID:26220403

  12. 14-3-3 proteins and plant development.

    PubMed

    Fulgosi, Hrvoje; Soll, Jürgen; de Faria Maraschin, Simone; Korthout, Henrie A A J; Wang, Mei; Testerink, Christa

    2002-12-01

    The 14-3-3 proteins are a family of ubiquitous regulatory molecules which have been found in virtually every eukaryotic organism and tissue. Discovered 34 years ago, 14-3-3 proteins have first been studied in mammalian nervous tissues, but in the past decade their indispensable role in various plant regulatory and metabolic pathways has been increasingly established. We now know that 14-3-3 members regulate fundamental processes of nitrogen assimilation and carbon assimilation, play an auxiliary role in regulation of starch synthesis, ATP production, peroxide detoxification, and participate in modulation of several other important biochemical pathways. Plant development and seed germination appear also to be under control of factors whose interaction with 14-3-3 molecules is crucial for their activation. Located within the nucleus, 14-3-3 isoforms are constituents of transcription factor complexes and interact with components of abscisic acid (ABA)-induced gene expression machinery. In addition, in animal cells they participate in nucleo-cytoplasmic trafficking and molecular sequestration. Cytoplasmic 14-3-3 members form a guidance complex with chloroplast destined preproteins and facilitate their import into these photosynthetic organelles. Recently, several 14-3-3s have been identified within chloroplasts where they could be involved in targeting and insertion of thylakoid proteins. The identification of 14-3-3 isoform specificity, and in particular the elucidation of the signal transduction mechanisms connecting 14-3-3 members with physiological responses, are central and developing topics of current research in this field.

  13. Decreased expression of 14-3-3 in Paracoccidioides brasiliensis confirms its involvement in fungal pathogenesis.

    PubMed

    Marcos, Caroline Maria; Silva, Julhiany de Fátima ds; Oliveira, Haroldo Cesar de; Assato, Patrícia Akemi; Singulani, Junya de Lacorte; Lopez, Angela Maria; Tamayo, Diana Patricia; Hernandez-Ruiz, Orville; McEwen, Juan G; Mendes-Giannini, Maria José Soares; Fusco-Almeida, Ana Marisa

    2016-01-01

    The interaction between the fungal pathogen Paracoccidioides brasiliensis and host cells is usually mediated by specific binding events between adhesins on the fungal surface and receptors on the host extracellular matrix or cell surface. One molecule implicated in the P. brasiliensis-host interaction is the 14-3-3 protein. The 14-3-3 protein belongs to a family of conserved regulatory molecules that are expressed in all eukaryotic cells and are involved in diverse cellular functions. Here, we investigated the relevance of the 14-3-3 protein to the virulence of P. brasiliensis. Using antisense RNA technology and Agrobacterium tumefaciens-mediated transformation, we generated a 14-3-3-silenced strain (expression reduced by ˜55%). This strain allowed us to investigate the interaction between 14-3-3 and the host and to correlate the functions of P. brasiliensis 14-3-3 with cellular features, such as morphological characteristics and virulence, that are important for pathogenesis. PMID:26646480

  14. Molecular characterization of a novel 14-3-3 protein gene (Hb14-3-3c) from Hevea brasiliensis.

    PubMed

    Yang, Zi-Ping; Li, Hui-Liang; Guo, Dong; Tian, Wei-Min; Peng, Shi-Qing

    2012-04-01

    The cDNA encoding a 14-3-3 protein, designated as Hb14-3-3c, was isolated from Hevea brasiliensis. Hb14-3-3c was 1,269 bp long containing a 795 bp open reading frame encoding a putative protein of 264 amino acids, flanked by a 146 bp 5'UTR and a 328 bp 3' UTR. The predicted molecular mass of Hb14-3-3c is 29.67 kDa, with an isoelectric point of 4.52 and the deduced protein showed high similarity to the 14-3-3 protein from other plant species. Expression analysis revealed more significant accumulation of Hb14-3-3c transcripts in latex than in leaves, buds and flowers. The transcription of Hb14-3-3c in latex was induced by jasmonate and ethephon. Overproduction of recombinant Hb14-3-3c protein gave the Escherichia coli cells more tolerance on Co(2+), Cu(2+) and Zn(2+). Through yeast two-hybrid screening, 11 interaction partners of the Hb14-3-3c, which are involved in rubber biosynthesis, stress-related responses, defence etc., were identified in rubber tree latex. Taking these data together, it is proposed that the Hb14-3-3c may participate in regulation of rubber biosynthesis. Thus, the results of this study provide novel insights into the 14-3-3 signaling related to rubber biosynthesis, stress-related responses in rubber tree. PMID:21947841

  15. The pro-inflammatory cytokine 14-3-3ε is a ligand of CD13 in cartilage

    PubMed Central

    Nefla, Meriam; Sudre, Laure; Denat, Guillaume; Priam, Sabrina; Andre-Leroux, Gwenaëlle; Berenbaum, Francis; Jacques, Claire

    2015-01-01

    ABSTRACT Osteoarthritis is a whole-joint disease characterized by the progressive destruction of articular cartilage involving abnormal communication between subchondral bone and cartilage. Our team previously identified 14-3-3ε protein as a subchondral bone soluble mediator altering cartilage homeostasis. The aim of this study was to investigate the involvement of CD13 (also known as aminopeptidase N, APN) in the chondrocyte response to 14-3-3ε. After identifying CD13 in chondrocytes, we knocked down CD13 with small interfering RNA (siRNA) and blocking antibodies in articular chondrocytes. 14-3-3ε-induced MMP-3 and MMP-13 was significantly reduced with CD13 knockdown, which suggests that it has a crucial role in 14-3-3ε signal transduction. Aminopeptidase N activity was identified in chondrocytes, but the activity was unchanged after stimulation with 14-3-3ε. Direct interaction between CD13 and 14-3-3ε was then demonstrated by surface plasmon resonance. Using labeled 14-3-3ε, we also found that 14-3-3ε binds to the surface of chondrocytes in a manner that is dependent on CD13. Taken together, these results suggest that 14-3-3ε might directly bind to CD13, which transmits its signal in chondrocytes to induce a catabolic phenotype similar to that observed in osteoarthritis. The 14-3-3ε–CD13 interaction could be a new therapeutic target in osteoarthritis. PMID:26208633

  16. 14-3-3 proteins as potential therapeutic targets

    PubMed Central

    Zhao, Jing; Meyerkord, Cheryl L.; Du, Yuhong; Khuri, Fadlo R.; Fu, Haian

    2011-01-01

    The 14-3-3 family of phosphoserine/phosphothreonine-binding proteins dynamically regulates the activity of client proteins in various signaling pathways that control diverse physiological and pathological processes. In response to environmental cues, 14-3-3 proteins orchestrate the highly regulated flow of signals through complex networks of molecular interactions to achieve well-controlled physiological outputs, such as cell proliferation or differentiation. Accumulating evidence now supports the concept that either an abnormal state of 14-3-3 protein expression, or dysregulation of 14-3-3/client protein interactions, contributes to the development of a large number of human diseases. In particular, clinical investigations in the field of oncology have demonstrated a correlation between upregulated 14-3-3 levels and poor survival of cancer patients. These studies highlight the rapid emergence of 14-3-3 proteins as a novel class of molecular target for potential therapeutic intervention. The current status of 14-3-3 modulator discovery is discussed. PMID:21983031

  17. A tale of tails: How histone tails mediate chromatin compaction in different salt and linker histone environments

    PubMed Central

    Arya, Gaurav; Schlick, Tamar

    2009-01-01

    To elucidate the role of the histone tails in chromatin compaction and in higher-order folding of chromatin under physiological conditions, we extend a mesoscale model of chromatin [Arya, Zhang, and Schlick, Biophys. J. 91, 133 (2006); Arya and Schlick, Proc. Natl. Acad. Sci. USA 103, 16236 (2006)] to account for divalent cations (Mg2+) and linker histones. Configurations of 24-nucleosome oligonucleosomes in different salt environments and in the presence and absence of linker histones are sampled by a mixture of local and global Monte Carlo methods. Analyses of the resulting ensembles reveals a dynamic synergism between the histone tails, linker histones, and physiological ions in forming compact higher-order structures of chromatin. In the presence of monovalent salt alone, oligonucleosomes remain relatively unfolded and the histone tails do not mediate many internucleosomal interactions. Upon the addition of linker histones and divalent cations, the oligonucleosomes undergo a significant compaction triggered by: a dramatic increase in the internucleosomal interactions mediated by the histone tails; formation of a rigid linker DNA “stem” around the linker histones' C-terminal domains; and reducion in the electrostatic repulsion between linker DNAs via sharp bending in some linker DNAs caused by the divalent cations. Among all histone tails, the H4 tails mediate the most internucleosomal interactions, consistent with experimental observations, followed by the H3, H2A, and H2B tails in decreasing order. Apart from mediating internucleosomal interactions, the H3 tails also contribute to chromatin compaction by attaching to the entering and exiting linker DNA to screen electrotatic repulsion among the linker DNAs. This tendency of the H3 tails to attach to linker DNA, however, decreases significantly upon the addition of linker histones due to competition effects. The H2A and H2B tails do not mediate significant internucleosomal interactions but are important for

  18. Increased 14-3-3 phosphorylation observed in Parkinson's disease reduces neuroprotective potential of 14-3-3 proteins.

    PubMed

    Slone, Sunny Rae; Lavalley, Nicholas; McFerrin, Michael; Wang, Bing; Yacoubian, Talene Alene

    2015-07-01

    14-3-3 proteins are key regulators of cell survival. We have previously demonstrated that 14-3-3 levels are decreased in an alpha-synuclein (αsyn) mouse model of Parkinson's disease (PD), and that overexpression of certain 14-3-3 isoforms is protective in several PD models. Here we examine whether changes in 14-3-3 phosphorylation may contribute to the neurodegenerative process in PD. We examine three key 14-3-3 phosphorylation sites that normally regulate 14-3-3 function, including serine 58 (S58), serine 184 (S184), and serine/threonine 232 (S/T232), in several models of PD and in human PD brain. We observed that an increase in S232 phosphorylation is observed in rotenone-treated neuroblastoma cells, in cells overexpressing αsyn, and in human PD brains. Alterations in S58 phosphorylation were less consistent in these models, and we did not observe any phosphorylation changes at S184. Phosphorylation at S232 induced by rotenone is reduced by casein kinase inhibitors, and is not dependent on αsyn. Mutation of the S232 site affected 14-3-3θ's neuroprotective effects against rotenone and 1-methyl-4-phenylpyridinium (MPP(+)), with the S232D mutant lacking any protective effect compared to wildtype or S232A 14-3-3θ. The S232D mutant partially reduced the ability of 14-3-3θ to inhibit Bax activation in response to rotenone. Based on these findings, we propose that phosphorylation of 14-3-3s at serine 232 contributes to the neurodegenerative process in PD. PMID:25862939

  19. Calcium-mediated histone modifications regulate alternative splicing in cardiomyocytes

    PubMed Central

    Sharma, Alok; Nguyen, Hieu; Geng, Cuiyu; Hinman, Melissa N.; Luo, Guangbin; Lou, Hua

    2014-01-01

    In cardiomyocytes, calcium is known to control gene expression at the level of transcription, whereas its role in regulating alternative splicing has not been explored. Here we report that, in mouse primary or embryonic stem cell-derived cardiomyocytes, increased calcium levels induce robust and reversible skipping of several alternative exons from endogenously expressed genes. Interestingly, we demonstrate a calcium-mediated splicing regulatory mechanism that depends on changes of histone modifications. Specifically, the regulation occurs through changes in calcium-responsive kinase activities that lead to alterations in histone modifications and subsequent changes in the transcriptional elongation rate and exon skipping. We demonstrate that increased intracellular calcium levels lead to histone hyperacetylation along the body of the genes containing calcium-responsive alternative exons by disrupting the histone deacetylase-to-histone acetyltransferase balance in the nucleus. Consequently, the RNA polymerase II elongation rate increases significantly on those genes, resulting in skipping of the alternative exons. These studies reveal a mechanism by which calcium-level changes in cardiomyocytes impact on the output of gene expression through altering alternative pre-mRNA splicing patterns. PMID:25368158

  20. 14-3-3 Proteins are Regulators of Autophagy

    PubMed Central

    Pozuelo-Rubio, Mercedes

    2012-01-01

    14-3-3 proteins are implicated in the regulation of proteins involved in a variety of signaling pathways. 14-3-3-dependent protein regulation occurs through phosphorylation-dependent binding that results, in many cases, in the release of survival signals in cells. Autophagy is a cell digestion process that contributes to overcoming nutrient deprivation and is initiated under stress conditions. However, whether autophagy is a cell survival or cell death mechanism remains under discussion and may depend on context. Nevertheless, autophagy is a cellular process that determines cell fate and is tightly regulated by different signaling pathways, some of which, for example MAPK, PI3K and mTOR, are tightly regulated by 14-3-3 proteins. It is therefore important to understand the role of 14-3-3 protein in modulating the autophagic process. Within this context, direct binding of 14-3-3 to mTOR regulatory proteins, such as TSC2 and PRAS40, connects 14-3-3 with autophagy regulatory processes. In addition, 14-3-3 binding to human vacuolar protein sorting 34 (hVps34), a class III phosphatidylinositol-3-kinase (PI3KC3), indicates the involvement of 14-3-3 proteins in regulating autophagosome formation. hVps34 is involved in vesicle trafficking processes such as autophagy, and its activation is needed for initiation of autophagy. Chromatography and overlay techniques suggest that hVps34 directly interacts with 14-3-3 proteins under physiological conditions, thereby maintaining hVps34 in an inactive state. In contrast, nutrient starvation promotes dissociation of the 14-3-3–hVps34 complex, thereby enhancing hVps34 lipid kinase activity. Thus, 14-3-3 proteins are regulators of autophagy through regulating key components of the autophagic machinery. This review summarizes the role of 14-3-3 protein in the control of target proteins involved in regulating the master switches of autophagy. PMID:24710529

  1. 14-3-3 zeta down-regulates p53 in mammary epithelial cells and confers luminal filling.

    PubMed

    Danes, Christopher G; Wyszomierski, Shannon L; Lu, Jing; Neal, Christopher L; Yang, Wentao; Yu, Dihua

    2008-03-15

    Recent progress in diagnostic tools allows many breast cancers to be detected at an early preinvasive stage. Thus, a better understanding of the molecular basis of early breast cancer progression is essential. Previously, we discovered that 14-3-3 zeta is overexpressed in >40% of advanced breast cancers, and this overexpression predicts poor patient survival. Here, we examined at what stage of breast disease 14-3-3 zeta overexpression occurs, and we found that increased expression of 14-3-3 zeta begins at atypical ductal hyperplasia, an early stage of breast disease. To determine whether 14-3-3 zeta overexpression is a decisive early event in breast cancer, we overexpressed 14-3-3 zeta in MCF10A cells and examined its effect in a three-dimensional culture model. We discovered that 14-3-3 zeta overexpression severely disrupted the acini architecture resulting in luminal filling. Proper lumen formation is a result of anoikis, apoptosis due to detachment from the basement membrane. We found that 14-3-3 zeta overexpression conferred resistance to anoikis. Additionally, 14-3-3 zeta overexpression in MCF10A cells and in mammary epithelial cells (MEC) from 14-3-3 zeta transgenic mice reduced expression of p53, which is known to mediate anoikis. Mechanistically, 14-3-3 zeta induced hyperactivation of the phosphoinositide 3-kinase/Akt pathway which led to phosphorylation and translocation of the MDM2 E3 ligase resulting in increased p53 degradation. Ectopic expression of p53 restored luminal apoptosis in 14-3-3 zeta-overexpressing MCF10A acini in three-dimensional cultures. These data suggest that 14-3-3 zeta overexpression is a critical event in early breast disease, and down-regulation of p53 is one of the mechanisms by which 14-3-3 zeta alters MEC acini structure and increases the risk of breast cancer.

  2. Discovery and structural characterization of a small molecule 14-3-3 protein-protein interaction inhibitor

    PubMed Central

    Zhao, Jing; Du, Yuhong; Horton, John R.; Upadhyay, Anup K.; Lou, Bin; Bai, Yan; Zhang, Xing; Du, Lupei; Li, Minyong; Wang, Binghe; Zhang, Lixin; Barbieri, Joseph T.; Khuri, Fadlo R.; Cheng, Xiaodong; Fu, Haian

    2011-01-01

    The 14-3-3 family of phosphoserine/threonine-recognition proteins engage multiple nodes in signaling networks that control diverse physiological and pathophysiological functions and have emerged as promising therapeutic targets for such diseases as cancer and neurodegenerative disorders. Thus, small molecule modulators of 14-3-3 are much needed agents for chemical biology investigations and therapeutic development. To analyze 14-3-3 function and modulate its activity, we conducted a chemical screen and identified 4-[(2Z)-2-[4-formyl-6-methyl-5-oxo-3-(phosphonatooxymethyl)pyridin-2-ylidene]hydrazinyl]benzoate as a 14-3-3 inhibitor, which we termed FOBISIN (FOurteen-three-three BInding Small molecule INhibitor) 101. FOBISIN101 effectively blocked the binding of 14-3-3 with Raf-1 and proline-rich AKT substrate, 40 kDa and neutralized the ability of 14-3-3 to activate exoenzyme S ADP-ribosyltransferase. To provide a mechanistic basis for 14-3-3 inhibition, the crystal structure of 14-3-3ζ in complex with FOBISIN101 was solved. Unexpectedly, the double bond linking the pyridoxal-phosphate and benzoate moieties was reduced by X-rays to create a covalent linkage of the pyridoxal-phosphate moiety to lysine 120 in the binding groove of 14-3-3, leading to persistent 14-3-3 inactivation. We suggest that FOBISIN101-like molecules could be developed as an entirely unique class of 14-3-3 inhibitors, which may serve as radiation-triggered therapeutic agents for the treatment of 14-3-3-mediated diseases, such as cancer. PMID:21908710

  3. Histone deacetylase-mediated morphological transition in Candida albicans.

    PubMed

    Kim, Jueun; Lee, Ji-Eun; Lee, Jung-Shin

    2015-12-01

    Candida albicans is the most common opportunistic fungal pathogen, which switches its morphology from single-cell yeast to filament through the various signaling pathways responding to diverse environmental cues. Various transcriptional factors such as Nrg1, Efg1, Brg1, Ssn6, and Tup1 are the key components of these signaling pathways. Since C. albicans can regulate its transcriptional gene expressions using common eukaryotic regulatory systems, its morphological transition by these signaling pathways could be linked to the epigenetic regulation by chromatin structure modifiers. Histone proteins, which are critical components of eukaryotic chromatin structure, can regulate the eukaryotic chromatin structure through their own modifications such as acetylation, methylation, phosphorylation and ubiquitylation. Recent studies revealed that various histone modifications, especially histone acetylation and deacetylation, participate in morphological transition of C. albicans collaborating with well-known transcription factors in the signaling pathways. Here, we review recent studies about chromatin-mediated morphological transition of C. albicans focusing on the interaction between transcription factors in the signaling pathways and histone deacetylases.

  4. Cofilin regulator 14-3-3zeta is an evolutionarily conserved protein required for phagocytosis and microbial resistance.

    PubMed

    Ulvila, Johanna; Vanha-aho, Leena-Maija; Kleino, Anni; Vähä-Mäkilä, Mari; Vuoksio, Milka; Eskelinen, Sinikka; Hultmark, Dan; Kocks, Christine; Hallman, Mikko; Parikka, Mataleena; Rämet, Mika

    2011-05-01

    Phagocytosis is an ancient cellular process that plays an important role in host defense. In Drosophila melanogaster phagocytic, macrophage-like hemocytes recognize and ingest microbes. We performed an RNAi-based in vitro screen in the Drosophila hemocyte cell line S2 and identified Abi, cpa, cofilin regulator 14-3-3ζ, tlk, CG2765, and CG15609 as mediators of bacterial phagocytosis. Of these identified genes, 14-3-3ζ had an evolutionarily conserved role in phagocytosis: bacterial phagocytosis was compromised when 14-3-3ζ was targeted with RNAi in primary Drosophila hemocytes and when the orthologous genes Ywhab and Ywhaz were silenced in zebrafish and mouse RAW 264.7 cells, respectively. In Drosophila and zebrafish infection models, 14-3-3ζ was required for resistance against Staphylococcus aureus. We conclude that 14-3-3ζ is essential for phagocytosis and microbial resistance in insects and vertebrates. PMID:21208897

  5. 14-3-3 proteins: Macro-regulators with great potential for improving abiotic stress tolerance in plants.

    PubMed

    Liu, Qing; Zhang, Shaohong; Liu, Bin

    2016-08-12

    14-3-3 proteins (14-3-3s) are highly conserved regulatory proteins that are uniquely eukaryotic, and deeply involved in protein-protein interactions that mediate diverse signaling pathways. In plants, 14-3-3s have been validated to regulate many biological processes, such as metabolism, light and hormone signaling, cell-cycle control and protein trafficking. Recent years we have also witnessed an increasing number of reports describing the functions of 14-3-3s in plant stress responses through interactions with key proteins in both biotic and abiotic stresses. In this review, we highlight the advances that have been made in investigating the roles of 14-3-3s in plant abiotic stress tolerance. These advances provide a framework for our understanding of how signals are integrated to perceive and respond to the abiotic stresses in plants. PMID:27233603

  6. Expression of 14-3-3 transcript isoforms in response to ethanol exposure and their regulation by miRNAs.

    PubMed

    Mathew, Divya Elizabeth; Larsen, Kaitlyn; Janeczek, Paulina; Lewohl, Joanne M

    2016-09-01

    The 14-3-3 proteins are a family of highly conserved molecular chaperones involved in the regulation of a number of key cellular functions including metabolism, stress response, protein trafficking, cell-cycle control, signal transduction, transcription, apoptosis and neurotransmission. 14-3-3 proteins have also been implicated in the pathophysiology of neurodegenerative disorders including Alzheimer disease and Parkinson disease. Recent studies have also shown that 14-3-3s are differentially expressed in the frontal cortex of human alcoholics suggesting a potential role in the pathophysiology of alcohol use disorders. Here we measured the expression of 14-3-3 transcripts in HEK293T cells in response to chronic ethanol treatment. Five of the seven transcripts (14-3-3β, 14-3-3γ, 14-3-3ζ, 14-3-3ε and 14-3-3θ) were significantly down-regulated following chronic exposure to ethanol for a five day period with these changes persisting even after withdrawal from ethanol treatment. One transcript, 14-3-3σ, was significantly up-regulated following chronic ethanol exposure and 14-3-3η showed no differences in expression in the same treatment model. The pattern of expression changes is similar to those seen in the frontal cortex of human alcoholics. To investigate the role of miRNAs in mediating the expression changes we measured the expression of the 14-3-3 transcripts following transfection with miR-203, miR-144 and miR-7 mimics. Although these miRNAs had predicted target sites in the 3'untranslated region of each 14-3-3 isoform, only miR-203 resulted in a down-regulation of 14-3-3θ transcript. In addition, the expression of 14-3-3γ was upregulated following transfection with miR-7 and miR-144 mimics. MiRNA regulation of these isoforms following alcohol exposure may lead to alterations in neurotransmission, the balance between cell survival and cell death, as well as changing the rewarding effects of alcohol. PMID:27370936

  7. Eimeria tenella: 14-3-3 protein interacts with telomerase.

    PubMed

    Zhao, Na; Gong, Pengtao; Cheng, Baiqi; Li, Jianhua; Yang, Zhengtao; Li, He; Yang, Ju; Zhang, Guocai; Zhang, Xichen

    2014-10-01

    Telomerase, consisting of telomerase RNA and telomerase reverse transcriptase (TERT), is responsible for the maintenance of the end of linear chromosomes. TERT, as the catalytic subunit of telomerase, plays a critical role in telomerase activity. Researches indicate TERT-associated proteins participate in the regulation of telomerase assembly, posttranslational modification, localization, and enzymatic function. Here, the telomerase RNA-binding domain of Eimeria tenella TERT (EtTRBD) was cloned into pGBKT7 and performed as the bait. α-Galactosidase assay showed that the bait plasmid did not activate Gal4 reporter gene. Further, we isolated an EtTRBD-associated protein, 14-3-3, by yeast two-hybrid screening using the constructed bait plasmid. To confirm the interaction, EtTRBD and 14-3-3 were expressed by prokaryotic and eukaryotic expression systems. Pull-down assays by purified proteins demonstrated a direct bind between EtTRBD and 14-3-3. Co-immunoprecipitation techniques successfully validated that 14-3-3 interacted with EtTRBD in 293T cells. The protein-protein interaction provides a starting point for more in-depth studies on telomerase and telomere regulation in E. tenella.

  8. 14-3-3 in Thoracic Aortic Aneurysms

    PubMed Central

    Chakravarti, Ritu; Gupta, Karishma; Swain, Mamuni; Willard, Belinda; Scholtz, Jaclyn; Svensson, Lars G.; Roselli, Eric E.; Pettersson, Gosta; Johnston, Douglas R.; Soltesz, Edward G.; Yamashita, Michifumi; Stuehr, Dennis; Daly, Thomas M.; Hoffman, Gary S.

    2015-01-01

    Objective Large vessel vasculitides (LVV) are a group of autoimmune diseases characterized by injury to and anatomic modifications of large vessels, including the aorta and its branch vessels. Disease etiology is unknown. This study was undertaken to identify antigen targets within affected vessel walls in aortic root, ascending aorta, and aortic arch surgical specimens from patients with LVV, including giant cell arteritis, Takayasu arteritis, and isolated focal aortitis. Methods Thoracic aortic aneurysm specimens and autologous blood were acquired from consenting patients who underwent aorta reconstruction procedures. Aorta proteins were extracted from both patients with LVV and age-, race-, and sex-matched disease controls with noninflammatory aneurysms. A total of 108 serum samples from patients with LVV, matched controls, and controls with antinuclear antibodies, different forms of vasculitis, or sepsis were tested. Results Evaluation of 108 serum samples and 22 aortic tissue specimens showed that 78% of patients with LVV produced antibodies to 14-3-3 proteins in the aortic wall (93.7% specificity), whereas controls were less likely to do so (6.7% produced antibodies). LVV patient sera contained autoantibody sufficient to immunoprecipitate 14-3-3 protein(s) from aortic lysates. Three of 7 isoforms of 14-3-3 were found to be up-regulated in aorta specimens from patients with LVV, and 2 isoforms (ε and ζ) were found to be antigenic in LVV. Conclusion This is the first study to use sterile, snap-frozen thoracic aorta biopsy specimens to identify autoantigens in LVV. Our findings indicate that 78% of patients with LVV have antibody reactivity to 14-3-3 protein(s). The precise role of these antibodies and 14-3-3 proteins in LVV pathogenesis deserves further study. PMID:25917817

  9. Histone H4 Lys 20 monomethylation by histone methylase SET8 mediates Wnt target gene activation.

    PubMed

    Li, Zhenfei; Nie, Fen; Wang, Sheng; Li, Lin

    2011-02-22

    Histone methylation has an important role in transcriptional regulation. However, unlike H3K4 and H3K9 methylation, the role of H4K20 monomethylation (H4K20me-1) in transcriptional regulation remains unclear. Here, we show that Wnt3a specifically stimulates H4K20 monomethylation at the T cell factor (TCF)-binding element through the histone methylase SET8. Additionally, SET8 is crucial for activation of the Wnt reporter gene and target genes in both mammalian cells and zebrafish. Furthermore, SET8 interacts with lymphoid enhancing factor-1 (LEF1)/TCF4 directly, and this interaction is regulated by Wnt3a. Therefore, we conclude that SET8 is a Wnt signaling mediator and is recruited by LEF1/TCF4 to regulate the transcription of Wnt-activated genes, possibly through H4K20 monomethylation at the target gene promoters. Our findings also indicate that H4K20me-1 is a marker for gene transcription activation, at least in canonical Wnt signaling. PMID:21282610

  10. Circulating Extracellular Histones Are Clinically Relevant Mediators of Multiple Organ Injury.

    PubMed

    Kawai, Chihiro; Kotani, Hirokazu; Miyao, Masashi; Ishida, Tokiko; Jemail, Leila; Abiru, Hitoshi; Tamaki, Keiji

    2016-04-01

    Extracellular histones are a damage-associated molecular pattern (DAMP) involved in the pathogenesis of various diseases. The mechanisms of histone-mediated injury in certain organs have been extensively studied, but an understanding of the pathophysiological role of histone-mediated injury in multiple organ injury remains elusive. To elucidate this role, we systemically subjected C57BL/6 mice to various doses of histones and performed a chronological evaluation of the morphological and functional changes in the lungs, liver, and kidneys. Notably, histone administration ultimately led to death after a dose-dependent aggravation of multiple organ injury. In chronological studies, pulmonary and hepatic injuries occurred within 15 minutes, whereas renal injuries presented at a later phase, suggesting that susceptibility to extracellular histones varies among organs. Histones bound to pulmonary and hepatic endothelial cells immediately after administration, leading to endothelial damage, which could be ameliorated by pretreatment with heparin. Furthermore, release of another DAMP, high-mobility group protein box 1, followed the histone-induced tissue damage, and an antibody against the molecule ameliorated hepatic and renal failure in a late phase. These findings indicate that extracellular histones induce multiple organ injury in two progressive stages-direct injury to endothelial cells and the subsequent release of other DAMPs-and that combination therapies against extracellular histones and high-mobility group protein box 1 may be a promising strategy for treating multiple organ injury.

  11. Overexpression of several Arabidopsis histone genes increases agrobacterium-mediated transformation and transgene expression in plants.

    PubMed

    Tenea, Gabriela N; Spantzel, Joerg; Lee, Lan-Ying; Zhu, Yanmin; Lin, Kui; Johnson, Susan J; Gelvin, Stanton B

    2009-10-01

    The Arabidopsis thaliana histone H2A-1 is important for Agrobacterium tumefaciens-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, results in decreased T-DNA integration into the genome of Arabidopsis roots, whereas overexpression of HTA1 increases transformation frequency. To understand the mechanism by which HTA1 enhances transformation, we investigated the effects of overexpression of numerous Arabidopsis histones on transformation and transgene expression. Transgenic Arabidopsis containing cDNAs encoding histone H2A (HTA), histone H4 (HFO), and histone H3-11 (HTR11) displayed increased transformation susceptibility, whereas histone H2B (HTB) and most histone H3 (HTR) cDNAs did not increase transformation. A parallel increase in transient gene expression was observed when histone HTA, HFO, or HTR11 overexpression constructs were cotransfected with double- or single-stranded forms of a gusA gene into tobacco (Nicotiana tabacum) protoplasts. However, these cDNAs did not increase expression of a previously integrated transgene. We identified the N-terminal 39 amino acids of H2A-1 as sufficient to increase transient transgene expression in plants. After transfection, transgene DNA accumulates more rapidly in the presence of HTA1 than with a control construction. Our results suggest that certain histones enhance transgene expression, protect incoming transgene DNA during the initial stages of transformation, and subsequently increase the efficiency of Agrobacterium-mediated transformation.

  12. Up-regulated 14-3-3β and 14-3-3ζ proteins in prefrontal cortex of subjects with schizophrenia: effect of psychotropic treatment.

    PubMed

    Rivero, Guadalupe; Gabilondo, Ane M; García-Sevilla, Jesús A; La Harpe, Romano; Morentín, Benito; Meana, J Javier

    2015-02-01

    14-3-3 is a family of conserved regulatory proteins that bind to a multitude of functionally diverse signalling proteins. Various genetic studies and gene expression and proteomic analyses have involved 14-3-3 proteins in schizophrenia (SZ). On the other hand, studies about the status of these proteins in major depressive disorder (MD) are still missing. Immunoreactivity values of cytosolic 14-3-3β and 14-3-3ζ proteins were evaluated by Western blot in prefrontal cortex (PFC) of subjects with schizophrenia (SZ; n=22), subjects with major depressive disorder (MD; n=21) and age-, gender- and postmortem delay-matched control subjects (n=52). The modulation of 14-3-3β and 14-3-3ζ proteins by psychotropic medication was also assessed. The analysis of both proteins in SZ subjects with respect to matched control subjects showed increased 14-3-3β (Δ=33±10%, p<0.05) and 14-3-3ζ (Δ=29±6%, p<0.05) immunoreactivity in antipsychotic-free but not in antipsychotic-treated SZ subjects. Immunoreactivity values of 14-3-3β and 14-3-3ζ were not altered in MD subjects. These results show the specific up-regulation of 14-3-3β and 14-3-3ζ proteins in PFC of SZ subjects and suggest a possible down-regulation of both proteins by antipsychotic treatment.

  13. Up-regulated 14-3-3β and 14-3-3ζ proteins in prefrontal cortex of subjects with schizophrenia: effect of psychotropic treatment.

    PubMed

    Rivero, Guadalupe; Gabilondo, Ane M; García-Sevilla, Jesús A; La Harpe, Romano; Morentín, Benito; Meana, J Javier

    2015-02-01

    14-3-3 is a family of conserved regulatory proteins that bind to a multitude of functionally diverse signalling proteins. Various genetic studies and gene expression and proteomic analyses have involved 14-3-3 proteins in schizophrenia (SZ). On the other hand, studies about the status of these proteins in major depressive disorder (MD) are still missing. Immunoreactivity values of cytosolic 14-3-3β and 14-3-3ζ proteins were evaluated by Western blot in prefrontal cortex (PFC) of subjects with schizophrenia (SZ; n=22), subjects with major depressive disorder (MD; n=21) and age-, gender- and postmortem delay-matched control subjects (n=52). The modulation of 14-3-3β and 14-3-3ζ proteins by psychotropic medication was also assessed. The analysis of both proteins in SZ subjects with respect to matched control subjects showed increased 14-3-3β (Δ=33±10%, p<0.05) and 14-3-3ζ (Δ=29±6%, p<0.05) immunoreactivity in antipsychotic-free but not in antipsychotic-treated SZ subjects. Immunoreactivity values of 14-3-3β and 14-3-3ζ were not altered in MD subjects. These results show the specific up-regulation of 14-3-3β and 14-3-3ζ proteins in PFC of SZ subjects and suggest a possible down-regulation of both proteins by antipsychotic treatment. PMID:25549848

  14. Cancer Chemoprotection Through Nutrient-mediated Histone Modifications

    PubMed Central

    Gao, Yifeng; Tollefsbol, Trygve O.

    2016-01-01

    Epigenetics, the study of heritable changes in gene expression without modifying the nucleotide sequence, is among the most important topics in medicinal chemistry and cancer chemoprotection. Among those changes, DNA methylation and histone modification have been shown to be associated with various types of cancers in a number of ways, many of which are regulated by dietary components that are mostly found in plants. Although, mechanisms of nutrient components affecting histone acetylation/deacetylation in cancer are widely studied, how those natural compounds affect cancer through other histone modifications, such as methylation, phosphorylation and ubiquitylation, is rarely reviewed. Thus, this review article discusses impacts recently studied on histone acetylation as well as other histone modifications by dietary components, such as genistein, resveratrol, curcumin, epigallocatechin-3-gallate (EGCG), 3,3′-diindolylmethane (DIM), diallyl disulfide, garcinol, procyanidin B3, quercetin, sulforaphane and other isothiocyanates, in various types of cancer. PMID:25891109

  15. Dexamethasone downregulated the expression of CSF 14-3-3β protein in mice with eosinophilic meningitis caused by Angiostrongylus cantonensis infection.

    PubMed

    Tsai, Hung-Chin; Lee, Bi-Yao; Yen, Chuan-Min; Wann, Shue-Ren; Lee, Susan Shin-Jung; Chen, Yao-Shen; Tai, Ming-Hong

    2014-03-01

    Angiostrongylus cantonensis is the main causative agent of human eosinophilic meningitis in Southeast Asia and the Pacific Islands. A previous study demonstrated that the 14-3-3β protein is a neuropathological marker in monitoring neuronal damage in meningitis. Steroids are commonly used in patients with eosinophilic meningitis caused by A. cantonensis infection. However, the mechanism by which steroids act in eosinophilic meningitis is unknown. We hypothesized that the beneficial effect of steroids on eosinophilic meningitis is partially mediated by the down-regulation of 14-3-3β protein expression in the cerebrospinal fluid (CSF). In this animal study, we determined the dynamic changes of 14-3-3β protein in mice with eosinophilic meningitis. The 14-3-3β protein in serum and CSF was increased in week 2 and 3 after infections. Dexamethasone administration significantly decreased the amounts of CSF 14-3-3β protein. By developing an in-house ELISA to measure 14-3-3β protein, it was found that the amounts of 14-3-3β protein in the CSF and serum increased over a three-week period after infection. There was a remarkable reduction of 14-3-3β protein in the CSF after 2 weeks of dexamethasone treatment. In conclusion, the administration of corticosteroids in mice with eosinophilic meningitis decreased the expression of 14-3-3β protein in the CSF.

  16. Functional responses and molecular mechanisms involved in histone-mediated platelet activation.

    PubMed

    Carestia, A; Rivadeneyra, L; Romaniuk, M A; Fondevila, C; Negrotto, S; Schattner, M

    2013-11-01

    Histones are highly alkaline proteins found in cell nuclei and they can be released by either dying or inflammatory cells. The recent observations that histones are major components of neutrophil extracellular traps and promote platelet aggregation and platelet-dependent thrombin generation have shown that these proteins are potent prothrombotic molecules. Because the mechanism(s) of platelet activation by histones are not completely understood, we explored the ability of individual recombinant human histones H1, H2A, H2B, H3 and H4 to induce platelet activation as well as the possible molecular mechanisms involved. All histones were substrates for platelet adhesion and spreading and triggered fibrinogen binding, aggregation, von Willebrand factor release, P-selectin and phosphatidylserine (PS) exposure and the formation of platelet-leukocyte aggregates; however, H4 was the most potent. Histone-mediated fibrinogen binding, P-selectin and PS exposure and the formation of mixed aggregates were potentiated by thrombin. Histones induced the activation of ERK, Akt, p38 and NFκB. Accordingly, histone-induced platelet activation was significantly impaired by pretreatment of platelets with inhibitors of ERK (U 0126), PI3K/Akt (Ly 294002), p38 (SB 203580) and NFκB (BAY 11-7082 and Ro 106-9920). Preincubation of platelets with either aspirin or dexamethasone markedly decreased fibrinogen binding and the adhesion mediated by histones without affecting P-selectin exposure. Functional platelet responses induced by H3 and H4, but not H1, H2A and H2B, were partially mediated through interaction with Toll-like receptors -2 and -4. Our data identify histones as important triggers of haemostatic and proinflammatory platelet responses, and only haemostatic responses are partially inhibited by anti-inflammatory drugs. PMID:23965842

  17. Functional responses and molecular mechanisms involved in histone-mediated platelet activation.

    PubMed

    Carestia, A; Rivadeneyra, L; Romaniuk, M A; Fondevila, C; Negrotto, S; Schattner, M

    2013-11-01

    Histones are highly alkaline proteins found in cell nuclei and they can be released by either dying or inflammatory cells. The recent observations that histones are major components of neutrophil extracellular traps and promote platelet aggregation and platelet-dependent thrombin generation have shown that these proteins are potent prothrombotic molecules. Because the mechanism(s) of platelet activation by histones are not completely understood, we explored the ability of individual recombinant human histones H1, H2A, H2B, H3 and H4 to induce platelet activation as well as the possible molecular mechanisms involved. All histones were substrates for platelet adhesion and spreading and triggered fibrinogen binding, aggregation, von Willebrand factor release, P-selectin and phosphatidylserine (PS) exposure and the formation of platelet-leukocyte aggregates; however, H4 was the most potent. Histone-mediated fibrinogen binding, P-selectin and PS exposure and the formation of mixed aggregates were potentiated by thrombin. Histones induced the activation of ERK, Akt, p38 and NFκB. Accordingly, histone-induced platelet activation was significantly impaired by pretreatment of platelets with inhibitors of ERK (U 0126), PI3K/Akt (Ly 294002), p38 (SB 203580) and NFκB (BAY 11-7082 and Ro 106-9920). Preincubation of platelets with either aspirin or dexamethasone markedly decreased fibrinogen binding and the adhesion mediated by histones without affecting P-selectin exposure. Functional platelet responses induced by H3 and H4, but not H1, H2A and H2B, were partially mediated through interaction with Toll-like receptors -2 and -4. Our data identify histones as important triggers of haemostatic and proinflammatory platelet responses, and only haemostatic responses are partially inhibited by anti-inflammatory drugs.

  18. Modulation of GluK2a subunit-containing kainate receptors by 14-3-3 proteins.

    PubMed

    Sun, Changcheng; Qiao, Haifa; Zhou, Qin; Wang, Yan; Wu, Yuying; Zhou, Yi; Li, Yong

    2013-08-23

    Kainate receptors (KARs) are one of the ionotropic glutamate receptors that mediate excitatory postsynaptic currents (EPSCs) with characteristically slow kinetics. Although mechanisms for the slow kinetics of KAR-EPSCs are not totally understood, recent evidence has implicated a regulatory role of KAR-associated proteins. Here, we report that decay kinetics of GluK2a-containing receptors is modulated by closely associated 14-3-3 proteins. 14-3-3 binding requires PKC-dependent phosphorylation of serine residues localized in the carboxyl tail of the GluK2a subunit. In transfected cells, 14-3-3 binding to GluK2a slows desensitization kinetics of both homomeric GluK2a and heteromeric GluK2a/GluK5 receptors. Moreover, KAR-EPSCs at mossy fiber-CA3 synapses decay significantly faster in the 14-3-3 functional knock-out mice. Collectively, these results demonstrate that 14-3-3 proteins are an important regulator of GluK2a-containing KARs and may contribute to the slow decay kinetics of native KAR-EPSCs. PMID:23861400

  19. 14-3-3 isoforms are induced by aldosterone and participate in its regulation of epithelial sodium channels.

    PubMed

    Liang, Xiubin; Peters, Kathryn W; Butterworth, Michael B; Frizzell, Raymond A

    2006-06-16

    Aldosterone increases sodium absorption across renal collecting duct cells primarily by increasing the apical membrane expression of ENaC, the sodium entry channel. Nedd4-2, a ubiquitin-protein isopeptide ligase, tags ENaC with ubiquitin for internalization and degradation, but when it is phosphorylated by the aldosterone-induced kinase, SGK1, Nedd4-2 is inhibited and apical ENaC density and sodium absorption increase. We evaluated the hypothesis that 14-3-3 proteins participate in the aldosterone-mediated regulation of ENaC by associating with phosphorylated Nedd4-2. Mouse cortical collecting duct (mCCD) epithelia cultured on filters expressed several 14-3-3 isoforms; this study focused on an isoform whose expression was induced 3-fold by aldosterone, 14-3-3beta. In polarized mCCD epithelia, aldosterone elicited significant, time-dependent increases in the expression of alpha-ENaC, SGK1, phospho-Nedd4-2, and 14-3-3beta without altering total Nedd4-2. Aldosterone decreased the interaction of alpha-ENaC with Nedd4-2, and with similar kinetics increased the association of 14-3-3beta with phospho-Nedd4-2. Short interfering RNA-induced knockdown of 14-3-3beta blunted the aldosterone-induced increase in alpha-ENaC expression, returned alpha-ENaC-Nedd4-2 binding toward prealdosterone levels, and blocked the aldosterone-stimulated increase in transepithelial sodium transport. Incubation of cell extracts with a selective phospho-Nedd4-2 antibody blocked the aldosterone-induced association of 14-3-3beta with Nedd4-2, implicating SGK1 phosphorylation at Ser-328 as the primary site of 14-3-3beta binding. Our studies show that aldosterone increases the expression of 14-3-3beta, which interacts with phospho-Nedd4-2 to block its interaction with ENaC, thus enhancing sodium absorption by increasing apical membrane ENaC density. PMID:16613846

  20. An obligatory heterodimer of 14-3-3beta and 14-3-3epsilon is required for aldosterone regulation of the epithelial sodium channel.

    PubMed

    Liang, Xiubin; Butterworth, Michael B; Peters, Kathryn W; Walker, William H; Frizzell, Raymond A

    2008-10-10

    Increased distal nephron sodium absorption in response to aldosterone involves Nedd4-2 phosphorylation, which blocks its ability to ubiquitylate ENaC and increases apical membrane channel density by reducing its endocytosis. Our prior work (Liang, X., Peters, K. W., Butterworth, M. B., and Frizzell, R. A. (2006) J. Biol. Chem. 281, 16323-16332) showed that aldosterone selectively increased 14-3-3 protein isoform expression and that the association of 14-3-3beta with phospho-Nedd4-2 was required for sodium transport stimulation. The knockdown of 14-3-3beta alone nearly eliminated the response to aldosterone, despite the expression of other 14-3-3 isoforms in cortical collecting duct (CCD) cells. To further examine this marked effect of 14-3-3beta knockdown, we evaluated the hypothesis that phospho-Nedd4-2 binding prefers a heterodimer composed of two different 14-3-3 isoforms. We tested this concept in polarized CCD cells using RNA interference and assays of sodium transport and of the interaction of Nedd4-2 with 14-3-3epsilon, a second aldosterone-induced isoform. As observed previously for 14-3-3beta knockdown, small interfering RNA-induced reduction of 14-3-3epsilon markedly attenuated aldosterone-stimulated ENaC expression and sodium transport and increased the interaction of Nedd4-2 with ENaC toward prealdosterone levels. After aldosterone induction, 14-3-3beta and 14-3-3epsilon were quantitatively co-immunoprecipitated from CCD cell lysates, and the association of both isoforms with Nedd4-2 increased. Finally, the knockdown of either 14-3-3beta or 14-3-3epsilon reduced the association of Nedd4-2 with the other isoform. We conclude that the two aldosterone-induced 14-3-3 isoforms, beta and epsilon, interact with phospho-Nedd4-2 as an obligatory heterodimer, blocking its interaction with ENaC and thereby increasing apical ENaC density and sodium transport. PMID:18687683

  1. Identification of a functional splice variant of 14-3-3E1 in rainbow trout

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 14-3-3 proteins are a family of regulatory proteins involved in diverse cellular processes. The presence of 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 isoforms suggest functional specificity of the isoforms. In this study, we report the identification and charact...

  2. Genetic variations of 14-3-3E1 isoform in rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The highly conserved family of 14-3-3 proteins functions in the regulation of a wide variety of cellular processes. The presence of 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 isoforms suggest functional specificity of the isoforms. Several studies have observed diffe...

  3. The role of 14-3-3{beta} in transcriptional activation of estrogen receptor {alpha} and its involvement in proliferation of breast cancer cells

    SciTech Connect

    Kim, Yoonseo; Kim, Hyungjin; Jang, Sung-Wuk; Ko, Jesang

    2011-10-14

    Highlights: {yields} 14-3-3{beta} interacts with ER{alpha} and the interaction is Akt-dependent. {yields} 14-3-3{beta} regulates the transcriptional activity of ER{alpha} in a ligand-dependent manner. {yields} 14-3-3{beta} increases expressions of ER{alpha} target genes. {yields} 14-3-3{beta} increases breast cancer cell proliferation. -- Abstract: The estrogen receptor (ER) functions as a transcription factor that mediates the effects of estrogen. ER{alpha}, which plays a crucial role in the development and progression of breast cancer, is activated by estrogen binding, leading to receptor phosphorylation, dimerization, and recruitment of co-activators and chaperons to the estrogen-bound receptor complex. The 14-3-3 proteins bind to target proteins via phosphorylation and influence many cellular events by altering their subcellular localization or acting as a chaperone. However, regulation of ER{alpha} expression and transactivation by the 14-3-3 proteins has not been reported. We demonstrate that 14-3-3{beta} functions as a positive regulator of ER{alpha} through a direct protein-protein interaction in an estrogen-dependent manner. Ectopic expression of 14-3-3{beta} stimulated ER{alpha}-mediated transcriptional activity in MCF-7 breast cancer cells. Enhanced ER{alpha} transcriptional activity due to 14-3-3{beta} increased the expressions of the endogenous ER{alpha} target genes, leading to proliferation of breast cancer cells. We suggest that 14-3-3{beta} has oncogenic potential in breast cancer via binding to ER{alpha} and activation of the transcriptional activity of ER{alpha}.

  4. Higher order Arabidopsis 14-3-3 mutants show 14-3-3 involvement in primary root growth both under control and abiotic stress conditions

    PubMed Central

    van Kleeff, P. J. M.; Jaspert, N.; Li, K. W.; Rauch, S.; Oecking, C.; de Boer, A. H.

    2014-01-01

    Arabidopsis 14-3-3 proteins are a family of conserved proteins that interact with numerous partner proteins in a phospho-specific manner, and can affect the target proteins in a number of ways; e.g. modification of enzymatic activity. We isolated T-DNA insertion lines in six 14-3-3 genes within the non-epsilon group that phylogenetically group in three closely related gene pairs. In total, 6 single, 3 double, 12 triple, and 3 quadruple mutants were generated. The mutants were phenotyped for primary root growth on control plates: single and double mutants were indistinguishable from WT, whereas six triples and all quadruples showed a shorter primary root. In addition, length of the first epidermal cell with a visible root hair bulge (LEH) was used to determine primary root elongation on medium containing mannitol and 1-aminocyclopropane-1-carboxylic acid (ACC). This analysis showed clear differences depending on the stress and 14-3-3 gene combinations. Next to the phenotypic growth analyses, a 14-3-3 pull-down assay on roots treated with and without mannitol showed that mannitol stress strongly affects the 14-3-3 interactome. In conclusion, we show gene specificity and functional redundancy among 14-3-3 proteins in primary root elongation under control and under abiotic stress conditions and changes in the 14-3-3 interactome during the onset of stress adaptation. PMID:25189593

  5. Involvement of 14-3-3 Proteins in Regulating Tumor Progression of Hepatocellular Carcinoma.

    PubMed

    Wu, Yi-Ju; Jan, Yee-Jee; Ko, Bor-Sheng; Liang, Shu-Man; Liou, Jun-Yang

    2015-01-01

    There are seven mammalian isoforms of the 14-3-3 protein, which regulate multiple cellular functions via interactions with phosphorylated partners. Increased expression of 14-3-3 proteins contributes to tumor progression of various malignancies. Several isoforms of 14-3-3 are overexpressed and associate with higher metastatic risks and poorer survival rates of hepatocellular carcinoma (HCC). 14-3-3β and 14-3-3ζ regulate HCC cell proliferation, tumor growth and chemosensitivity via modulating mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) and p38 signal pathways. Moreover, 14-3-3ε suppresses E-cadherin and induces focal adhesion kinase (FAK) expression, thereby enhancing epithelial-mesenchymal transition (EMT) and HCC cell migration. 14-3-3ζ forms complexes with αB-crystallin, which induces EMT and is the cause of sorafenib resistance in HCC. Finally, a recent study has indicated that 14-3-3σ induces heat shock protein 70 (HSP70) expression, which increases HCC cell migration. These results suggest that selective 14-3-3 isoforms contribute to cell proliferation, EMT and cell migration of HCC by regulating distinct targets and signal pathways. Targeting 14-3-3 proteins together with specific downstream effectors therefore has potential to be therapeutic and prognostic factors of HCC. In this article, we will overview 14-3-3's regulation of its downstream factors and contributions to HCC EMT, cell migration and proliferation. PMID:26083935

  6. 14-3-3σ confers cisplatin resistance in esophageal squamous cell carcinoma cells via regulating DNA repair molecules.

    PubMed

    Lai, Kenneth K Y; Chan, Kin Tak; Choi, Mei Yuk; Wang, Hector K; Fung, Eva Y M; Lam, Ho Yu; Tan, Winnie; Tung, Lai Nar; Tong, Daniel K H; Sun, Raymond W Y; Lee, Nikki P; Law, Simon

    2016-02-01

    Esophageal squamous cell carcinoma (ESCC) is the predominant type of esophageal cancer in Asia. Cisplatin is commonly used in chemoradiation for unresectable ESCC patients. However, the treatment efficacy is diminished in patients with established cisplatin resistance. To understand the mechanism leading to the development of cisplatin resistance in ESCC, we compared the proteomes from a cisplatin-resistant HKESC-2R cell line with its parental-sensitive counterpart HKESC-2 to identify key molecule involved in this process. Mass spectrometry analysis detected 14-3-3σ as the most abundant molecule expressed exclusively in HKESC-2R cells, while western blot result further validated it to be highly expressed in HKESC-2R cells when compared to HKESC-2 cells. Ectopic expression of 14-3-3σ increased cisplatin resistance in HKESC-2 cells, while its suppression sensitized SLMT-1 cells to cisplatin. Among the molecules involved in drug detoxification, drug transportation, and DNA repair, the examined DNA repair molecules HMGB1 and XPA were found to be highly expressed in HKESC-2R cells with high 14-3-3σ expression. Subsequent manipulation of 14-3-3σ by both overexpression and knockdown approaches concurrently altered the expression of HMGB1 and XPA. 14-3-3σ, HMGB1, and XPA were preferentially expressed in cisplatin-resistant SLMT-1 cells when compared to those more sensitive to cisplatin. In ESCC patients with poor response to cisplatin-based chemoradiation, their pre-treatment tumors expressed higher expression of HMGB1 than those with response to such treatment. In summary, our results demonstrate that 14-3-3σ induces cisplatin resistance in ESCC cells and that 14-3-3σ-mediated cisplatin resistance involves DNA repair molecules HMGB1 and XPA. Results from this study provide evidences for further work in researching the potential use of 14-3-3σ and DNA repair molecules HMGB1 and XPA as biomarkers and therapeutic targets for ESCC.

  7. 14-3-3 phosphoprotein interaction networks - does isoform diversity present functional interaction specification?

    PubMed

    Paul, Anna-Lisa; Denison, Fiona C; Schultz, Eric R; Zupanska, Agata K; Ferl, Robert J

    2012-01-01

    The 14-3-3 proteins have emerged as major phosphoprotein interaction proteins and thereby constitute a key node in the Arabidopsis Interactome Map, a node through which a large number of important signals pass. Throughout their history of discovery and description, the 14-3-3s have been described as protein families and there has been some evidence that the different 14-3-3 family members within any organism might carry isoform-specific functions. However, there has also been evidence for redundancy of 14-3-3 function, suggesting that the perceived 14-3-3 diversity may be the accumulation of neutral mutations over evolutionary time and as some 14-3-3 genes develop tissue or organ-specific expression. This situation has led to a currently unresolved question - does 14-3-3 isoform sequence diversity indicate functional diversity at the biochemical or cellular level? We discuss here some of the key observations on both sides of the resulting debate, and present a set of contrastable observations to address the theory functional diversity does exist among 14-3-3 isoforms. The resulting model suggests strongly that there are indeed functional specificities in the 14-3-3s of Arabidopsis. The model further suggests that 14-3-3 diversity and specificity should enter into the discussion of 14-3-3 roles in signal transduction and be directly approached in 14-3-3 experimentation. It is hoped that future studies involving 14-3-3s will continue to address specificity in experimental design and analysis. PMID:22934100

  8. IFNγ-induced suppression of β-catenin signaling: evidence for roles of Akt and 14.3.3ζ

    PubMed Central

    Nava, Porfirio; Kamekura, Ryuta; Quirós, Miguel; Medina-Contreras, Oscar; Hamilton, Ross W.; Kolegraff, Keli N.; Koch, Stefan; Candelario, Aurora; Romo-Parra, Hector; Laur, Oskar; Hilgarth, Roland S.; Denning, Timothy L.; Parkos, Charles A.; Nusrat, Asma

    2014-01-01

    The proinflammatory cytokine interferon γ (IFNγ ) influences intestinal epithelial cell (IEC) homeostasis in a biphasic manner by acutely stimulating proliferation that is followed by sustained inhibition of proliferation despite continued mucosal injury. β-Catenin activation has been classically associated with increased IEC proliferation. However, we observed that IFNγ inhibits IEC proliferation despite sustained activation of Akt/β-catenin signaling. Here we show that inhibition of Akt/β-catenin–mediated cell proliferation by IFNγ is associated with the formation of a protein complex containing phosphorylated β-catenin 552 (pβ-cat552) and 14.3.3ζ. Akt1 served as a bimodal switch that promotes or inhibits β-catenin transactivation in response to IFNγ stimulation. IFNγ initially promotes β-catenin transactivation through Akt-dependent C-terminal phosphorylation of β-catenin to promote its association with 14.3.3ζ. Augmented β-catenin transactivation leads to increased Akt1 protein levels, and active Akt1 accumulates in the nucleus, where it phosphorylates 14.3.3ζ to translocate 14.3.3ζ/β-catenin from the nucleus, thereby inhibiting β-catenin transactivation and IEC proliferation. These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation. PMID:25079689

  9. The role of the 14-3-3 protein family in health, disease, and drug development.

    PubMed

    Aghazadeh, Yasaman; Papadopoulos, Vassilios

    2016-02-01

    14-3-3 proteins regulate intracellular signaling pathways, such as signal transduction, protein trafficking, cell cycle, and apoptosis. In addition to the ubiquitous roles of 14-3-3 isoforms, unique tissue-specific functions are also described for each isoform. Owing to their role in regulating cell cycle, protein trafficking, and steroidogenesis, 14-3-3 proteins are prevalent in human diseases, such as cancer, neurodegeneration, and reproductive disorders, and, therefore, serve as valuable drug targets. In this review, we summarize the role of 14-3-3 proteins in normal and disease states, with a focus on 14-3-3γ and ɛ. We also discuss drug compounds targeting 14-3-3 proteins and their potential therapeutic uses. PMID:26456530

  10. A Negative Regulatory Mechanism Involving 14-3-3ζ Limits Signaling Downstream of ROCK to Regulate Tissue Stiffness in Epidermal Homeostasis.

    PubMed

    Kular, Jasreen; Scheer, Kaitlin G; Pyne, Natasha T; Allam, Amr H; Pollard, Anthony N; Magenau, Astrid; Wright, Rebecca L; Kolesnikoff, Natasha; Moretti, Paul A; Wullkopf, Lena; Stomski, Frank C; Cowin, Allison J; Woodcock, Joanna M; Grimbaldeston, Michele A; Pitson, Stuart M; Timpson, Paul; Ramshaw, Hayley S; Lopez, Angel F; Samuel, Michael S

    2015-12-21

    ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities.

  11. A Negative Regulatory Mechanism Involving 14-3-3ζ Limits Signaling Downstream of ROCK to Regulate Tissue Stiffness in Epidermal Homeostasis.

    PubMed

    Kular, Jasreen; Scheer, Kaitlin G; Pyne, Natasha T; Allam, Amr H; Pollard, Anthony N; Magenau, Astrid; Wright, Rebecca L; Kolesnikoff, Natasha; Moretti, Paul A; Wullkopf, Lena; Stomski, Frank C; Cowin, Allison J; Woodcock, Joanna M; Grimbaldeston, Michele A; Pitson, Stuart M; Timpson, Paul; Ramshaw, Hayley S; Lopez, Angel F; Samuel, Michael S

    2015-12-21

    ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities. PMID:26702834

  12. Interactions of c-Raf-1 with phosphatidylserine and 14-3-3.

    PubMed

    McPherson, R A; Harding, A; Roy, S; Lane, A; Hancock, J F

    1999-07-01

    Activation of Raf-1 occurs at the plasma membrane. We recently showed that 14-3-3 must be complexed with Raf-1 for efficient recruitment to the plasma membrane and activation by Ras, but that 14-3-3 is completely displaced from Raf-1 following plasma membrane binding. We show here that the Raf-1 zinc finger is not absolutely required for 14-3-3 binding but is required to stabilize the interaction between Raf-1 and 14-3-3. Incubation of Raf-1 with phosphatidylserine, an inner plasma membrane phospholipid, results in removal of 14-3-3 and an increase in Raf-1 kinase activity, whereas removal of 14-3-3 from Raf-1 using specific phosphopeptides substantially reduces Raf-1 basal kinase activity. Displacement of 14-3-3 from activated Raf-1 by phosphopeptides has no effect on kinase activity if Raf-1 is first removed from solution, but completely eradicates kinase activity of soluble activated Raf-1. These results suggest a mechanism for the removal of 14-3-3 from Raf-1 at the plasma membrane and show that removal of 14-3-3 from Raf-1 has markedly different effects depending on experimental conditions.

  13. Dynamic imaging of interaction between protein 14-3-3 and Bid in living cells

    NASA Astrophysics Data System (ADS)

    Chen, Tongsheng; Xing, Da; Wang, Jinjun

    2006-02-01

    The 14-3-3 proteins are known to sequester certain pro-apoptotic members of this family. BH3- interacting domain death agonist (Bid) may contribute to tumor necrosis factor α(TNF-α)-induced neuronal death, although regulation by 14-3-3 has not been reported. In this study we examined whether 14-3-3 proteins interact with Bid/tBid during TNF-α-induced cell death. The TNF-αtriggered Bid cleavage and tBid translocated to mitochondria. Human lung adenocarcinoma cells were co-transfected with both CFP-Bid and 14-3-3-YFP plasmids, and the dynamical interaction between the Bid/tBid and 14-3-3 were performed on laser confocal fluorescence microscope in single living cell during TNF-α-induced cell apoptosis. The Bid distribute equally only in the cytoplasm of healthy cells, and the 14-3-3 protein distribute not only in the cytoplasm but also in the nucleus of healthy cells. Our data showed that the tBid aggregate, but the 14-3-3 protein does not aggregate as the tBid, and the 14-3-3 protein separate from the aggregated tBid, implying that the 14-3-3 proteins do not interact with the aggregated tBid after TNF-αtreatment.

  14. Mediator, TATA-binding Protein, and RNA Polymerase II Contribute to Low Histone Occupancy at Active Gene Promoters in Yeast*

    PubMed Central

    Ansari, Suraiya A.; Paul, Emily; Sommer, Sebastian; Lieleg, Corinna; He, Qiye; Daly, Alexandre Z.; Rode, Kara A.; Barber, Wesley T.; Ellis, Laura C.; LaPorta, Erika; Orzechowski, Amanda M.; Taylor, Emily; Reeb, Tanner; Wong, Jason; Korber, Philipp; Morse, Randall H.

    2014-01-01

    Transcription by RNA polymerase II (Pol II) in eukaryotes requires the Mediator complex, and often involves chromatin remodeling and histone eviction at active promoters. Here we address the role of Mediator in recruitment of the Swi/Snf chromatin remodeling complex and its role, along with components of the preinitiation complex (PIC), in histone eviction at inducible and constitutively active promoters in the budding yeast Saccharomyces cerevisiae. We show that recruitment of the Swi/Snf chromatin remodeling complex to the induced CHA1 promoter, as well as its association with several constitutively active promoters, depends on the Mediator complex but is independent of Mediator at the induced MET2 and MET6 genes. Although transcriptional activation and histone eviction at CHA1 depends on Swi/Snf, Swi/Snf recruitment is not sufficient for histone eviction at the induced CHA1 promoter. Loss of Swi/Snf activity does not affect histone occupancy of several constitutively active promoters; in contrast, higher histone occupancy is seen at these promoters in Mediator and PIC component mutants. We propose that an initial activator-dependent, nucleosome remodeling step allows PIC components to outcompete histones for occupancy of promoter sequences. We also observe reduced promoter association of Mediator and TATA-binding protein in a Pol II (rpb1-1) mutant, indicating mutually cooperative binding of these components of the transcription machinery and indicating that it is the PIC as a whole whose binding results in stable histone eviction. PMID:24727477

  15. 14-3-3 Protein Masks the DNA Binding Interface of Forkhead Transcription Factor FOXO4*

    PubMed Central

    Silhan, Jan; Vacha, Petr; Strnadova, Pavla; Vecer, Jaroslav; Herman, Petr; Sulc, Miroslav; Teisinger, Jan; Obsilova, Veronika; Obsil, Tomas

    2009-01-01

    The role of 14-3-3 proteins in the regulation of FOXO forkhead transcription factors is at least 2-fold. First, the 14-3-3 binding inhibits the interaction between the FOXO and the target DNA. Second, the 14-3-3 proteins prevent nuclear reimport of FOXO factors by masking their nuclear localization signal. The exact mechanisms of these processes are still unclear, mainly due to the lack of structural data. In this work, we used fluorescence spectroscopy to investigate the mechanism of the 14-3-3 protein-dependent inhibition of FOXO4 DNA-binding properties. Time-resolved fluorescence measurements revealed that the 14-3-3 binding affects fluorescence properties of 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid moiety attached at four sites within the forkhead domain of FOXO4 that represent important parts of the DNA binding interface. Observed changes in 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid fluorescence strongly suggest physical contacts between the 14-3-3 protein and labeled parts of the FOXO4 DNA binding interface. The 14-3-3 protein binding, however, does not cause any dramatic conformational change of FOXO4 as documented by the results of tryptophan fluorescence experiments. To build a realistic model of the FOXO4·14-3-3 complex, we measured six distances between 14-3-3 and FOXO4 using Förster resonance energy transfer time-resolved fluorescence experiments. The model of the complex suggests that the forkhead domain of FOXO4 is docked within the central channel of the 14-3-3 protein dimer, consistent with our hypothesis that 14-3-3 masks the DNA binding interface of FOXO4. PMID:19416966

  16. ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome

    PubMed Central

    Tinti, Michele; Madeira, Fábio; Murugesan, Gavuthami; Hoxhaj, Gerta; Toth, Rachel; MacKintosh, Carol

    2014-01-01

    The dimeric 14-3-3 proteins dock onto pairs of phosphorylated Ser and Thr residues on hundreds of proteins, and thereby regulate many events in mammalian cells. To facilitate global analyses of these interactions, we developed a web resource named ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome, which integrates multiple data sets on 14-3-3-binding phosphoproteins. ANIA also pinpoints candidate 14-3-3-binding phosphosites using predictor algorithms, assisted by our recent discovery that the human 14-3-3-interactome is highly enriched in 2R-ohnologues. 2R-ohnologues are proteins in families of two to four, generated by two rounds of whole genome duplication at the origin of the vertebrate animals. ANIA identifies candidate ‘lynchpins’, which are 14-3-3-binding phosphosites that are conserved across members of a given 2R-ohnologue protein family. Other features of ANIA include a link to the catalogue of somatic mutations in cancer database to find cancer polymorphisms that map to 14-3-3-binding phosphosites, which would be expected to interfere with 14-3-3 interactions. We used ANIA to map known and candidate 14-3-3-binding enzymes within the 2R-ohnologue complement of the human kinome. Our projections indicate that 14-3-3s dock onto many more human kinases than has been realized. Guided by ANIA, PAK4, 6 and 7 (p21-activated kinases 4, 6 and 7) were experimentally validated as a 2R-ohnologue family of 14-3-3-binding phosphoproteins. PAK4 binding to 14-3-3 is stimulated by phorbol ester, and involves the ‘lynchpin’ site phosphoSer99 and a major contribution from Ser181. In contrast, PAK6 and PAK7 display strong phorbol ester-independent binding to 14-3-3, with Ser113 critical for the interaction with PAK6. These data point to differential 14-3-3 regulation of PAKs in control of cell morphology. Database URL: https://ania-1433.lifesci.dundee.ac.uk/prediction/webserver/index.py PMID:24501395

  17. ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome.

    PubMed

    Tinti, Michele; Madeira, Fábio; Murugesan, Gavuthami; Hoxhaj, Gerta; Toth, Rachel; Mackintosh, Carol

    2014-01-01

    The dimeric 14-3-3 proteins dock onto pairs of phosphorylated Ser and Thr residues on hundreds of proteins, and thereby regulate many events in mammalian cells. To facilitate global analyses of these interactions, we developed a web resource named ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome, which integrates multiple data sets on 14-3-3-binding phosphoproteins. ANIA also pinpoints candidate 14-3-3-binding phosphosites using predictor algorithms, assisted by our recent discovery that the human 14-3-3-interactome is highly enriched in 2R-ohnologues. 2R-ohnologues are proteins in families of two to four, generated by two rounds of whole genome duplication at the origin of the vertebrate animals. ANIA identifies candidate 'lynchpins', which are 14-3-3-binding phosphosites that are conserved across members of a given 2R-ohnologue protein family. Other features of ANIA include a link to the catalogue of somatic mutations in cancer database to find cancer polymorphisms that map to 14-3-3-binding phosphosites, which would be expected to interfere with 14-3-3 interactions. We used ANIA to map known and candidate 14-3-3-binding enzymes within the 2R-ohnologue complement of the human kinome. Our projections indicate that 14-3-3s dock onto many more human kinases than has been realized. Guided by ANIA, PAK4, 6 and 7 (p21-activated kinases 4, 6 and 7) were experimentally validated as a 2R-ohnologue family of 14-3-3-binding phosphoproteins. PAK4 binding to 14-3-3 is stimulated by phorbol ester, and involves the 'lynchpin' site phosphoSer99 and a major contribution from Ser181. In contrast, PAK6 and PAK7 display strong phorbol ester-independent binding to 14-3-3, with Ser113 critical for the interaction with PAK6. These data point to differential 14-3-3 regulation of PAKs in control of cell morphology. Database URL: https://ania-1433.lifesci.dundee.ac.uk/prediction/webserver/index.py.

  18. Association of 14-3-3 Proteins to β1-Adrenergic Receptors Modulates Kv11.1 K+ Channel Activity in Recombinant Systems

    PubMed Central

    Tutor, Antonio S.; Delpón, Eva; Caballero, Ricardo; Gómez, Ricardo; Núñez, Lucía; Vaquero, Miguel; Tamargo, Juan; Penela, Petronila

    2006-01-01

    We identify a new mechanism for the β1-adrenergic receptor (β1AR)-mediated regulation of human ether-a-go-go–related gene (HERG) potassium channel (Kv11.1). We find that the previously reported modulatory interaction between Kv11.1 channels and 14-3-3ε proteins is competed by wild type β1AR by means of a novel interaction between this receptor and 14-3-3ε. The association between β1AR and 14-3-3ε is increased by agonist stimulation in both transfected cells and heart tissue and requires cAMP-dependent protein kinase (PKA) activity. The β1AR/14-3-3ε association is direct, since it can be recapitulated using purified 14-3-3ε and β1AR fusion proteins and is abolished in cells expressing β1AR phosphorylation–deficient mutants. Biochemical and electrophysiological studies of the effects of isoproterenol on Kv11.1 currents recorded using the whole-cell patch clamp demonstrated that β1AR phosphorylation–deficient mutants do not recruit 14-3-3ε away from Kv11.1 and display a markedly altered agonist-mediated modulation of Kv11.1 currents compared with wild-type β1AR, increasing instead of inhibiting current amplitudes. Interestingly, such differential modulation is not observed in the presence of 14-3-3 inhibitors. Our results suggest that the dynamic association of 14-3-3 proteins to both β1AR and Kv11.1 channels is involved in the adrenergic modulation of this critical regulator of cardiac repolarization and refractoriness. PMID:16914520

  19. 14-3-3s are Potential Biomarkers for HIV-related Neurodegeneration

    PubMed Central

    Morales, Diana; Skoulakis, Efthimios M.; Acevedo, Summer F.

    2013-01-01

    Over the last decade, it has become evident that 14-3-3 proteins are essential for primary cell functions. These proteins are abundant throughout the body, including the central nervous system (CNS) and interact with other proteins in both cell cycle and apoptotic pathways. Examination of cerebral spinal fluid (CSF) in humans, suggest that 14-3-3s including 14-3-3ε (YWHAE), are upregulated in several neurological diseases and loss or duplication of the YWHAE gene leads to Miller-Dieker Syndrome (MDS). The goal of this review is to examine the utility of 14-3-3s as a marker of Human Immune deficiency virus (HIV)-dependent neurodegeneration, and also as a tool to track disease progression. To that end we describe mechanisms implicating 14-3-3s in neurological diseases and summarize evidence of its interactions with HIV accessory and co-receptor proteins. PMID:22811265

  20. Identification of a redox-modulatory interaction between selenoprotein W and 14-3-3 protein.

    PubMed

    Jeon, Yeong Ha; Ko, Kwan Young; Lee, Jea Hwang; Park, Ki Jun; Jang, Jun Ki; Kim, Ick Young

    2016-01-01

    Selenoprotein W (SelW) contains a selenocysteine (Sec, U) in a conserved CXXU motif corresponding to the CXXC redox motif of thioredoxin, suggesting a putative redox function of SelW. We have previously reported that the binding of 14-3-3 protein to its target proteins, including CDC25B, Rictor and TAZ, is inhibited by the interaction of 14-3-3 protein with SelW. However, the binding mechanism is unclear. In this study, we sought to determine the binding site of SelW to understand the regulatory mechanism of the interaction between SelW and 14-3-3 and its biological effects. Phosphorylated Ser(pS) or Thr(pT) residues in RSXpSXP or RXXXp(S/T)XP motifs are well-known common 14-3-3-binding sites, but Thr41, Ser59, and T69 of SelW, which are computationally predicted to serve are phosphorylation sites, were neither phosphorylation sites nor sites involved in the interaction. A mutant SelW in which Sec13 is changed to Ser (U13S) was unable to interact with 14-3-3 protein and thus did not inhibit the interaction of 14-3-3 to other target proteins. However, other Cys mutants of SelW(C10S, C33S and C37S) normally interacted with 14-3-3 protein. The interaction of SelW to 14-3-3 protein was enhanced by diamide or H2O2 and decreased by dithiothreitol (DTT). Taken together, these findings demonstrate that the Sec of SelW is involved in its interaction with 14-3-3 protein and that this interaction is increased under oxidative stress conditions. Thus, SelW may have a regulatory function in redox cell signaling by interacting with 14-3-3 protein. PMID:26474786

  1. Interaction network of the 14-3-3 protein in the ancient protozoan parasite Giardia duodenalis.

    PubMed

    Lalle, Marco; Camerini, Serena; Cecchetti, Serena; Sayadi, Ahmed; Crescenzi, Marco; Pozio, Edoardo

    2012-05-01

    14-3-3s are phosphoserine/phosphotreonine binding proteins that play pivotal roles as regulators of multiple cellular processes in eukaryotes. The flagellated protozoan parasite Giardia duodenalis, the causing agent of giardiasis, is a valuable simplified eukaryotic model. A single 14-3-3 isoform (g14-3-3) is expressed in Giardia, and it is directly involved in the differentiation of the parasite into cyst. To define the overall functions of g14-3-3, the protein interactome has been investigated. A transgenic G. duodenalis strain was engineered to express a FLAG-tagged g14-3-3 under its own promoter. Affinity chromatography coupled with tandem mass spectrometry analysis have been used to purify and identify FLAG-g14-3-3-associated proteins from trophozoites and encysting parasites. A total of 314 putative g14-3-3 interaction partners were identified, including proteins involved in several pathways. Some interactions seemed to be peculiar of one specific stage, while others were shared among the different stages. Furthermore, the interaction of g14-3-3 with the giardial homologue of the CDC7 protein kinase (gCDC7) was characterized, leading to the identification of a multiprotein complex containing not only g14-3-3 and gCDC7 but also a newly identified and highly divergent homologue of DBF4, the putative regulatory subunit of gCDC7. The relevance of g14-3-3 interactions in G. duodenalis biology was discussed.

  2. Hyperglycemia decreases expression of 14-3-3 proteins in an animal model of stroke.

    PubMed

    Jeon, Seong-Jun; Sung, Jin-Hee; Koh, Phil-Ok

    2016-07-28

    Diabetes is a severe metabolic disorder and a major risk factor for stroke. Stroke severity is worse in patients with diabetes compared to the non-diabetic population. The 14-3-3 proteins are a family of conserved acidic proteins that are ubiquitously expressed in cells and tissues. These proteins are involved in many cellular processes including metabolic pathways, signal transduction, protein trafficking, protein synthesis, and cell cycle control. This study investigated 14-3-3 proteins expression in the cerebral cortex of animals with diabetes, cerebral ischemic injury and a combination of both diabetes and cerebral ischemic injury. Diabetes was induced by intraperitoneal injection of streptozotocin (40mg/kg) in adult male rats. After 4 weeks of treatment, middle cerebral artery occlusion (MCAO) was performed for the induction of focal cerebral ischemia and cerebral cortex tissue was collected 24h after MCAO. We confirmed that diabetes increases infarct volume following MCAO compared to non-diabetic animals. In diabetic animals with MCAO injury, reduction of 14-3-3 β/α, 14-3-3 ζ/δ, 14-3-3 γ, and 14-3-3 ε isoforms was detected. The expression of these proteins was significantly decreased in diabetic animals with MCAO injury compared to diabetic-only and MCAO-only animals. Moreover, Western blot analysis ascertained the decreased expression of 14-3-3 family proteins in diabetic animals with MCAO injury, including β/α, ζ/δ, γ, ε, τ, and η isoforms. These results show the changes of 14-3-3 proteins expression in streptozotocin-induced diabetic animals with MCAO injury. Thus, these findings suggest that decreases in 14-3-3 proteins might be involved in the regulation of 14-3-3 proteins under the presence of diabetes following MCAO. PMID:27177727

  3. Histone deacetylase 3 (HDAC 3) as emerging drug target in NF-κB-mediated inflammation

    PubMed Central

    Leus, Niek G.J.; Zwinderman, Martijn R.H.; Dekker, Frank J.

    2016-01-01

    Activation of inflammatory gene expression is regulated, among other factors, by post-translational modifications of histone proteins. The most investigated type of histone modifications are lysine acetylations. Histone deacetylases (HDACs) remove acetylations from lysines, thereby influencing (inflammatory) gene expression. Intriguingly, apart from histones, HDACs also target non-histone proteins. The nuclear factor κB (NF-κB) pathway is an important regulator in the expression of numerous inflammatory genes, and acetylation plays a crucial role in regulating its responses. Several studies have shed more light on the role of HDAC 1-3 in inflammation with a particular pro-inflammatory role for HDAC 3. Nevertheless, the HDAC-NF-κB interactions in inflammatory signalling have not been fully understood. An important challenge in targeting the regulatory role of HDACs in the NF-κB pathway is the development of highly potent small molecules that selectively target HDAC iso-enzymes. This review focuses on the role of HDAC 3 in (NF-κB-mediated) inflammation and NF-κB lysine acetylation. In addition, we address the application of frequently used small molecule HDAC inhibitors as an approach to attenuate inflammatory responses, and their potential as novel therapeutics. Finally, recent progress and future directions in medicinal chemistry efforts aimed at HDAC 3-selective inhibitors are discussed. PMID:27371876

  4. Hypothalamic leptin action is mediated by histone deacetylase 5.

    PubMed

    Kabra, Dhiraj G; Pfuhlmann, Katrin; García-Cáceres, Cristina; Schriever, Sonja C; Casquero García, Veronica; Kebede, Adam Fiseha; Fuente-Martin, Esther; Trivedi, Chitrang; Heppner, Kristy; Uhlenhaut, N Henriette; Legutko, Beata; Kabra, Uma D; Gao, Yuanqing; Yi, Chun-Xia; Quarta, Carmelo; Clemmensen, Christoffer; Finan, Brian; Müller, Timo D; Meyer, Carola W; Paez-Pereda, Marcelo; Stemmer, Kerstin; Woods, Stephen C; Perez-Tilve, Diego; Schneider, Robert; Olson, Eric N; Tschöp, Matthias H; Pfluger, Paul T

    2016-01-01

    Hypothalamic leptin signalling has a key role in food intake and energy-balance control and is often impaired in obese individuals. Here we identify histone deacetylase 5 (HDAC5) as a regulator of leptin signalling and organismal energy balance. Global HDAC5 KO mice have increased food intake and greater diet-induced obesity when fed high-fat diet. Pharmacological and genetic inhibition of HDAC5 activity in the mediobasal hypothalamus increases food intake and modulates pathways implicated in leptin signalling. We show HDAC5 directly regulates STAT3 localization and transcriptional activity via reciprocal STAT3 deacetylation at Lys685 and phosphorylation at Tyr705. In vivo, leptin sensitivity is substantially impaired in HDAC5 loss-of-function mice. Hypothalamic HDAC5 overexpression improves leptin action and partially protects against HFD-induced leptin resistance and obesity. Overall, our data suggest that hypothalamic HDAC5 activity is a regulator of leptin signalling that adapts food intake and body weight to our dietary environment. PMID:26923837

  5. Hypothalamic leptin action is mediated by histone deacetylase 5

    PubMed Central

    Kabra, Dhiraj G.; Pfuhlmann, Katrin; García-Cáceres, Cristina; Schriever, Sonja C.; Casquero García, Veronica; Kebede, Adam Fiseha; Fuente-Martin, Esther; Trivedi, Chitrang; Heppner, Kristy; Uhlenhaut, N. Henriette; Legutko, Beata; Kabra, Uma D.; Gao, Yuanqing; Yi, Chun-Xia; Quarta, Carmelo; Clemmensen, Christoffer; Finan, Brian; Müller, Timo D.; Meyer, Carola W.; Paez-Pereda, Marcelo; Stemmer, Kerstin; Woods, Stephen C.; Perez-Tilve, Diego; Schneider, Robert; Olson, Eric N.; Tschöp, Matthias H.; Pfluger, Paul T.

    2016-01-01

    Hypothalamic leptin signalling has a key role in food intake and energy-balance control and is often impaired in obese individuals. Here we identify histone deacetylase 5 (HDAC5) as a regulator of leptin signalling and organismal energy balance. Global HDAC5 KO mice have increased food intake and greater diet-induced obesity when fed high-fat diet. Pharmacological and genetic inhibition of HDAC5 activity in the mediobasal hypothalamus increases food intake and modulates pathways implicated in leptin signalling. We show HDAC5 directly regulates STAT3 localization and transcriptional activity via reciprocal STAT3 deacetylation at Lys685 and phosphorylation at Tyr705. In vivo, leptin sensitivity is substantially impaired in HDAC5 loss-of-function mice. Hypothalamic HDAC5 overexpression improves leptin action and partially protects against HFD-induced leptin resistance and obesity. Overall, our data suggest that hypothalamic HDAC5 activity is a regulator of leptin signalling that adapts food intake and body weight to our dietary environment. PMID:26923837

  6. 14-3-3β protein expression in eosinophilic meningitis caused by Angiostrongylus cantonensis infection

    PubMed Central

    2014-01-01

    Background Angiostrongylus cantonensis is a parasite endemic in the Southeast Asian and Pacific regions. Humans are incidentally infected either by eating uncooked intermediate hosts or by consuming vegetables containing the living third-stage larvae. The 14-3-3β protein is a cerebrospinal fluid (CSF) marker of neuronal damage during the development of Creutzfeldt-Jakob disease. In addition, increased 14-3-3β protein is also found in CSF from patients with a variety of neurological disorders. The goal of this study is to determine the roles of serum/CSF14-3-3β protein in patients with eosinophilic meningitis. Methods In a cohort study among nine Thai laborers with eosinophilic meningitis due to eating raw snails (Pomacea canaliculata), we examined the CSF weekly while patients were still hospitalized and followed up the serum for 6 months. The levels of 14-3-3β protein in CSF were analyzed by western blot and an in-house 14-3-3β enzyme-linked immunosorbent assay (ELISA) measurement was established and tested in an animal model of eosinophilic meningitis. Results The elevated 14-3-3β level was detected in the CSF from eight out of nine (81%) patients After 2 weeks of treatment, all patients showed a declined level or cleared of 14-3-3β protein in the CSF. By developing an in-house ELISA for measurement of 14-3-3β protein, it was found that the serum 14-3-3β level was significantly increased in patients during initial visit. . This finding was consistent to the animal experiment result in which there was severe blood brain barrier damage three weeks after infection and increased 14-3-3β protein expression in the CSF and serum by western blot and in house ELISA. After treatment, the serum 14-3-3β level in meningitis patients was rapidly returned to normal threshold. There was a correlation between initial CSF 14-3-3β level with severity of headache (r = 0.692, p = 0.039), CSF pleocytosis (r = 0.807, p = 0.009) and eosinophilia (r = 0

  7. 14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression

    PubMed Central

    Mukhopadhyay, Amitabha; Sehgal, Lalit; Bose, Arunabha; Gulvady, Anushree; Senapati, Parijat; Thorat, Rahul; Basu, Srikanta; Bhatt, Khyati; Hosing, Amol S.; Balyan, Renu; Borde, Lalit; Kundu, Tapas K.; Dalal, Sorab N.

    2016-01-01

    More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering. PMID:27253419

  8. The cell cycle regulator 14-3-3σ opposes and reverses cancer metabolic reprogramming.

    PubMed

    Phan, Liem; Chou, Ping-Chieh; Velazquez-Torres, Guermarie; Samudio, Ismael; Parreno, Kenneth; Huang, Yaling; Tseng, Chieh; Vu, Thuy; Gully, Chris; Su, Chun-Hui; Wang, Edward; Chen, Jian; Choi, Hyun-Ho; Fuentes-Mattei, Enrique; Shin, Ji-Hyun; Shiang, Christine; Grabiner, Brian; Blonska, Marzenna; Skerl, Stephen; Shao, Yiping; Cody, Dianna; Delacerda, Jorge; Kingsley, Charles; Webb, Douglas; Carlock, Colin; Zhou, Zhongguo; Hsieh, Yun-Chih; Lee, Jaehyuk; Elliott, Andrew; Ramirez, Marc; Bankson, Jim; Hazle, John; Wang, Yongxing; Li, Lei; Weng, Shaofan; Rizk, Nibal; Wen, Yu Ye; Lin, Xin; Wang, Hua; Wang, Huamin; Zhang, Aijun; Xia, Xuefeng; Wu, Yun; Habra, Mouhammed; Yang, Wei; Pusztai, Lajos; Yeung, Sai-Ching; Lee, Mong-Hong

    2015-01-01

    Extensive reprogramming of cellular energy metabolism is a hallmark of cancer. Despite its importance, the molecular mechanism controlling this tumour metabolic shift remains not fully understood. Here we show that 14-3-3σ regulates cancer metabolic reprogramming and protects cells from tumorigenic transformation. 14-3-3σ opposes tumour-promoting metabolic programmes by enhancing c-Myc poly-ubiquitination and subsequent degradation. 14-3-3σ demonstrates the suppressive impact on cancer glycolysis, glutaminolysis, mitochondrial biogenesis and other major metabolic processes of tumours. Importantly, 14-3-3σ expression levels predict overall and recurrence-free survival rates, tumour glucose uptake and metabolic gene expression in breast cancer patients. Thus, these results highlight that 14-3-3σ is an important regulator of tumour metabolism, and loss of 14-3-3σ expression is critical for cancer metabolic reprogramming. We anticipate that pharmacologically elevating the function of 14-3-3σ in tumours could be a promising direction for targeted anticancer metabolism therapy development in future. PMID:26179207

  9. Clinical implication of 14-3-3 epsilon expression in gastric cancer

    PubMed Central

    Leal, Mariana Ferreira; Calcagno, Danielle Queiroz; Demachki, Sâmia; Assumpção, Paulo Pimentel; Chammas, Roger; Burbano, Rommel Rodríguez; Smith, Marília de Arruda Cardoso

    2012-01-01

    AIM: To evaluate for the first time the protein and mRNA expression of 14-3-3ε in gastric carcinogenesis. METHODS: 14-3-3ε protein expression was determined by western blotting, and mRNA expression was examined by real-time quantitative RT-PCR in gastric tumors and their matched non-neoplastic gastric tissue samples. RESULTS: Authors observed a significant reduction of 14-3-3ε protein expression in gastric cancer (GC) samples compared to their matched non-neoplastic tissue. Reduced levels of 14-3-3ε were also associated with diffuse-type GC and early-onset of this pathology. Our data suggest that reduced 14-3-3ε may have a role in gastric carcinogenesis process. CONCLUSION: Our results reveal that the reduced 14-3-3ε expression in GC and investigation of 14-3-3ε interaction partners may help to elucidate the carcinogenesis process. PMID:22509086

  10. The cell cycle regulator 14-3-3σ opposes and reverses cancer metabolic reprogramming

    PubMed Central

    Phan, Liem; Chou, Ping-Chieh; Velazquez-Torres, Guermarie; Samudio, Ismael; Parreno, Kenneth; Huang, Yaling; Tseng, Chieh; Vu, Thuy; Gully, Chris; Su, Chun-Hui; Wang, Edward; Chen, Jian; Choi, Hyun-Ho; Fuentes-Mattei, Enrique; Shin, Ji-Hyun; Shiang, Christine; Grabiner, Brian; Blonska, Marzenna; Skerl, Stephen; Shao, Yiping; Cody, Dianna; Delacerda, Jorge; Kingsley, Charles; Webb, Douglas; Carlock, Colin; Zhou, Zhongguo; Hsieh, Yun-Chih; Lee, Jaehyuk; Elliott, Andrew; Ramirez, Marc; Bankson, Jim; Hazle, John; Wang, Yongxing; Li, Lei; Weng, Shaofan; Rizk, Nibal; Wen, Yu Ye; Lin, Xin; Wang, Hua; Wang, Huamin; Zhang, Aijun; Xia, Xuefeng; Wu, Yun; Habra, Mouhammed; Yang, Wei; Pusztai, Lajos; Yeung, Sai-Ching; Lee, Mong-Hong

    2015-01-01

    Summary Extensive reprogramming of cellular energy metabolism is a hallmark of cancer. Despite its importance, the molecular mechanism controlling this tumour metabolic shift remains not fully understood. Here we show that 14-3-3σ regulates cancer metabolic reprogramming and protects cells from tumourigenic transformation. 14-3-3σ opposes tumour-promoting metabolic programs by enhancing c-Myc poly-ubiquitination and subsequent degradation. 14-3-3σ demonstrates the suppressive impact on cancer glycolysis, glutaminolysis, mitochondrial biogenesis and other major metabolic processes of tumours. Importantly, 14-3-3σ expression levels predict overall and recurrence-free survival rates, tumour glucose uptake and metabolic gene expression in breast cancer patients. Thus, these results highlight that 14-3-3σ is an important regulator of tumour metabolism, and loss of 14-3-3σ expression is critical for cancer metabolic reprogramming. We anticipate that pharmacologically elevating the function of 14-3-3σ in tumours could be a promising direction for targeted anti-cancer metabolism therapy development in future. PMID:26179207

  11. Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana.

    PubMed

    Chang, Ing-Feng; Curran, Amy; Woolsey, Rebekah; Quilici, David; Cushman, John C; Mittler, Ron; Harmon, Alice; Harper, Jeffrey F

    2009-06-01

    In eukaryotes, 14-3-3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a "tandem affinity purification" tag and expressed in transgenic plants. Purified complexes were analyzed by tandem MS. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 12 14-3-3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (i) Ion transport, such as a K(+) channel (GORK), a Cl(-) channel (CLCg), Ca(2+) channels belonging to the glutamate receptor family (1.2, 2.1, 2.9, 3.4, 3.7); (ii) hormone signaling, such as ACC synthase (isoforms ACS-6, -7 and -8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (iii) transcription, such as 7 WRKY family transcription factors; (iv) metabolism, such as phosphoenol pyruvate carboxylase; and (v) lipid signaling, such as phospholipase D (beta and gamma). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300.

  12. Dual role for 14-3-3 proteins and ABF transcription factors in gibberellic acid and abscisic acid signalling in barley (Hordeum vulgare) aleurone cells.

    PubMed

    Schoonheim, Peter J; Costa Pereira, Daniel D A; De Boer, Albertus H

    2009-05-01

    The balance of gibberellins [gibberellic acid (GA)] and abscisic acid (ABA) is a determining factor during transition of embryogenesis and seed germination. Recently, we showed that 14-3-3 proteins are important in ABA signalling in barley aleurone cells. Using 14-3-3 RNAi constructs in the barley aleurone transient expression system, we demonstrate here that silencing of each 14-3-3 isoform suppresses GA induction of the alpha-amylase gene. 14-3-3 Proteins interact with ABA-responsive element (ABRE) binding factors HvABF1, 2 and 3, and here we show that these transcription factors also interact with the ABA-responsive kinase PKABA1, a kinase that mediates cross-talk between the GA and ABA pathway. ABF1 and ABF2 have a function in both signalling pathways as: (1) ectopic expression of wild-type ABF1 and mutant ABF2, lacking the 14-3-3 interaction domain, transactivates the ABA inducible HVA1 gene; and (2) GA induction of the alpha-amylase gene is repressed by ectopic expression of wild-type ABF1 and 2. Mutant ABF1 and 2 were still effective repressors of GA signalling. In summary, our data provide evidence that 14-3-3 proteins and members of the ABF transcription factor family have a regulatory function in the GA pathway and suggest that PKABA1 and ABF transcription factors are cross-talk intermediates in ABA and GA signalling.

  13. Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis.

    PubMed

    Liang, Xiubin; Da Paula, Ana Carina; Bozóky, Zoltán; Zhang, Hui; Bertrand, Carol A; Peters, Kathryn W; Forman-Kay, Julie D; Frizzell, Raymond A

    2012-03-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)-regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. The 14-3-3β, γ, and ε isoforms were expressed in airway cells and interacted with CFTR in coimmunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3β and ε binding to immature and mature CFTR (bands B and C), and 14-3-3 overexpression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3β increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3β knockdown decreased CFTR B and C bands (70 and 55%) and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3β interacted with the CFTR regulatory region, and by nuclear magnetic resonance analysis, this interaction occurred at known PKA phosphorylated sites. In coimmunoprecipitation assays, forskolin stimulated the CFTR/14-3-3β interaction while reducing CFTR's interaction with coat protein complex 1 (COP1). Thus 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the endoplasmic reticulum. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion.

  14. 14-3-3 protein is a regulator of the mitochondrial and chloroplast ATP synthase

    PubMed Central

    Bunney, Tom D.; van Walraven, Hendrika S.; de Boer, Albertus H.

    2001-01-01

    Mitochondrial and chloroplast ATP synthases are key enzymes in plant metabolism, providing cells with ATP, the universal energy currency. ATP synthases use a transmembrane electrochemical proton gradient to drive synthesis of ATP. The enzyme complexes function as miniature rotary engines, ensuring energy coupling with very high efficiency. Although our understanding of the structure and functioning of the synthase has made enormous progress in recent years, our understanding of regulatory mechanisms is still rather preliminary. Here we report a role for 14-3-3 proteins in the regulation of ATP synthases. These 14-3-3 proteins are highly conserved phosphoserine/phosphothreonine-binding proteins that regulate a wide range of enzymes in plants, animals, and yeast. Recently, the presence of 14-3-3 proteins in chloroplasts was illustrated, and we show here that plant mitochondria harbor 14-3-3s within the inner mitochondrial-membrane compartment. There, the 14-3-3 proteins were found to be associated with the ATP synthases, in a phosphorylation-dependent manner, through direct interaction with the F1 β-subunit. The activity of the ATP synthases in both organelles is drastically reduced by recombinant 14-3-3. The rapid reduction in chloroplast ATPase activity during dark adaptation was prevented by a phosphopeptide containing the 14-3-3 interaction motif, demonstrating a role for endogenous 14-3-3 in the down-regulation of the CFoF1 activity. We conclude that regulation of the ATP synthases by 14-3-3 represents a mechanism for plant adaptation to environmental changes such as light/dark transitions, anoxia in roots, and fluctuations in nutrient supply. PMID:11274449

  15. Overexpression of 14-3-3z promotes tau phosphorylation at Ser262 and accelerates proteosomal degradation of synaptophysin in rat primary hippocampal neurons.

    PubMed

    Qureshi, Hamid Y; Han, Dong; MacDonald, Ryen; Paudel, Hemant K

    2013-01-01

    b-Amyloid peptide accumulation, tau hyperphosphorylation, and synapse loss are characteristic neuropathological symptoms of Alzheimer's disease (AD). Tau hyperphosphorylation is suggested to inhibit the association of tau with microtubules, making microtubules unstable and causing neurodegeneration. The mechanism of tau phosphorylation in AD brain, therefore, is of considerable significance. Although PHF-tau is phosphorylated at over 40 Ser/Thr sites, Ser(262) phosphorylation was shown to mediate b-amyloid neurotoxicity and formation of toxic tau lesions in the brain. In vitro, PKA is one of the kinases that phosphorylates tau at Ser(262), but the mechanism by which it phosphorylates tau in AD brain is not very clear. 14-3-3z is associated with neurofibrillary tangles and is upregulated in AD brain. In this study, we show that 14-3-3z promotes tau phosphorylation at Ser(262) by PKA in differentiating neurons. When overexpressed in rat hippocampal primary neurons, 14-3-3z causes an increase in Ser(262) phosphorylation, a decrease in the amount of microtubule-bound tau, a reduction in the amount of polymerized microtubules, as well as microtubule instability. More importantly, the level of pre-synaptic protein synaptophysin was significantly reduced. Downregulation of synaptophysin in 14-3-3z overexpressing neurons was mitigated by inhibiting the proteosome, indicating that 14-3-3z promotes proteosomal degradation of synaptophysin. When 14-3-3z overexpressing neurons were treated with the microtubule stabilizing drug taxol, tau Ser(262) phosphorylation decreased and synaptophysin level was restored. Our data demonstrate that overexpression of 14-3-3z accelerates proteosomal turnover of synaptophysin by promoting the destabilization of microtubules. Synaptophysin is involved in synapse formation and neurotransmitter release. Our results suggest that 14-3-3z may cause synaptic pathology by reducing synaptophysin levels in the brains of patients suffering from AD. PMID

  16. 14-3-3σ regulates keratinocyte proliferation and differentiation by modulating Yap1 cellular localization

    PubMed Central

    Sambandam, Sumitha A.T.; Kasetti, Ramesh Babu; Xue, Lei; Dean, Douglas C.; Lu, Qingxian; Li, Qiutang

    2015-01-01

    The homozygous repeated epilation (Er/Er) mouse mutant of the gene encoding 14-3-3σ displays an epidermal phenotype characterized by hyperproliferative keratinocytes and undifferentiated epidermis. Heterozygous Er/+ mice develop spontaneous skin tumors and are highly sensitive to tumor-promoting DMBA/TPA induction. The molecular mechanisms underlying 14-3-3σ regulation of epidermal proliferation, differentiation, and tumor formation have not been well elucidated. In the present study, we found that Er/Er keratinocytes failed to sequester Yap1 in the cytoplasm, leading to its nuclear localization during epidermal development in vivo and under differentiation-inducing culture conditions in vitro. In addition, enhanced Yap1 nuclear localization was also evident in DMBA/TPA-induced tumors from Er/+ skin. Furthermore, shRNA knockdown of Yap1 expression in Er/Er keratinocytes inhibited their proliferation, suggesting that YAP1 functions as a downstream effector of 14-3-3σ controlling epidermal proliferation. We then demonstrated that keratinocytes express all seven 14-3-3 protein isoforms, some of which form heterodimers with 14-3-3σ, either full-length WT or the mutant form found in Er/Er mice. However Er 14-3-3σ does not interact with Yap1, as demonstrated by co-immunoprecipitation. We conclude that Er 14-3-3σ disrupts the interaction between 14-3-3 and Yap1, thus fails to block Yap1 nuclear transcriptional function, causing continued progenitor expansion and inhibition of differentiation in Er/Er epidermis. PMID:25668240

  17. Alternations of 14-3-3 θ and β protein levels in brain during experimental sepsis.

    PubMed

    Memos, Nikolaos; Kataki, Agapi; Chatziganni, Emmy; Nikolopoulou, Marilena; Skoulakis, Euthimios; Consoulas, Christos; Zografos, George; Konstadoulakis, Manousos

    2011-09-01

    The 14-3-3 family members play a crucial role in the determination of cell fate, exerting their antiapoptotic activity through directly interfering with the critical function of the mitochondrial core proapoptotic machinery. Dimerization of 14-3-3 is vital for the interaction with many of its client proteins and is regulated by phosphorylation. In a previous study, we observed time-dependent neuronal apoptosis during sepsis. Therefore, in the present study, we sought to evaluate the expression of 14-3-3 θ and β isoforms in septic brain and their association with apoptosis. Sepsis was induced by a CLP model in Wistar rats that were sacrificed at predefined time points. Flow cytometric analysis showed a sepsis-induced, time-dependent alteration of 14-3-3 θ and β isoforms in both Neun(+) and GFAP(+) cells. 14-3-3 θ was linearly correlated with apoptosis, and stratified analysis for alive and apoptotic neuronal cells demonstrated a gradual down-regulation of θ isoform in alive neurons and astrocytes. The phospho-P38 (pP38) MAP kinase levels were altered in a time-dependent manner during sepsis, presenting a peak at 6 hr post-CLP. A significant correlation between the two isoforms of 14-3-3 was observed in septic rats, with the θ isoform predominant at all time points. The hippocampus, Purkinje cells, and glia-like cells showed intense immunohistochemical reactivity for 14-3-3 θ isoform, whereas the choroid plexus showed constantly increased β isoform expression. Our results showed that sepsis alters the expression of both 14-3-3 θ and β isoforms in a time-, cell-, and topography-dependent manner. PMID:21618583

  18. Structural Modulation of Phosducin by Phosphorylation and 14-3-3 Protein Binding

    PubMed Central

    Rezabkova, Lenka; Kacirova, Miroslava; Sulc, Miroslav; Herman, Petr; Vecer, Jaroslav; Stepanek, Miroslav; Obsilova, Veronika; Obsil, Tomas

    2012-01-01

    Phosducin (Pdc), a highly conserved phosphoprotein, plays an important role in the regulation of G protein signaling, transcriptional control, and modulation of blood pressure. Pdc is negatively regulated by phosphorylation followed by binding to the 14-3-3 protein, whose role is still unclear. To gain insight into the role of 14-3-3 in the regulation of Pdc function, we studied structural changes of Pdc induced by phosphorylation and 14-3-3 protein binding using time-resolved fluorescence spectroscopy. Our data show that the phosphorylation of the N-terminal domain of Pdc at Ser-54 and Ser-73 affects the structure of the whole Pdc molecule. Complex formation with 14-3-3 reduces the flexibility of both the N- and C-terminal domains of phosphorylated Pdc, as determined by time-resolved tryptophan and dansyl fluorescence. Therefore, our data suggest that phosphorylated Pdc undergoes a conformational change when binding to 14-3-3. These changes involve the Gtβγ binding surface within the N-terminal domain of Pdc, and thus could explain the inhibitory effect of 14-3-3 on Pdc function. PMID:23199924

  19. 14-3-3 fusion oncogenes in high-grade endometrial stromal sarcoma

    PubMed Central

    Lee, Cheng-Han; Ou, Wen-Bin; Mariño-Enriquez, Adrian; Zhu, Meijun; Mayeda, Mark; Wang, Yuexiang; Guo, Xiangqian; Brunner, Alayne L.; Amant, Frédéric; French, Christopher A.; West, Robert B.; McAlpine, Jessica N.; Gilks, C. Blake; Yaffe, Michael B.; Prentice, Leah M.; McPherson, Andrew; Jones, Steven J. M.; Marra, Marco A.; Shah, Sohrab P.; van de Rijn, Matt; Huntsman, David G.; Dal Cin, Paola; Debiec-Rychter, Maria; Nucci, Marisa R.; Fletcher, Jonathan A.

    2012-01-01

    14-3-3 proteins are ubiquitously expressed regulators of various cellular functions, including proliferation, metabolism, and differentiation, and altered 14-3-3 expression is associated with development and progression of cancer. We report a transforming 14-3-3 oncoprotein, which we identified through conventional cytogenetics and whole-transcriptome sequencing analysis as a highly recurrent genetic mechanism in a clinically aggressive form of uterine sarcoma: high-grade endometrial stromal sarcoma (ESS). The 14-3-3 oncoprotein results from a t(10;17) genomic rearrangement, leading to fusion between 14-3-3ε (YWHAE) and either of two nearly identical FAM22 family members (FAM22A or FAM22B). Expression of YWHAE–FAM22 fusion oncoproteins was demonstrated by immunoblot in t(10;17)-bearing frozen tumor and cell line samples. YWHAE–FAM22 fusion gene knockdowns were performed with shRNAs and siRNAs targeting various FAM22A exons in an t(10;17)-bearing ESS cell line (ESS1): Fusion protein expression was inhibited, with corresponding reduction in cell growth and migration. YWHAE–FAM22 maintains a structurally and functionally intact 14-3-3ε (YWHAE) protein-binding domain, which is directed to the nucleus by a FAM22 nuclear localization sequence. In contrast to classic ESS, harboring JAZF1 genetic fusions, YWHAE–FAM22 ESS display high-grade histologic features, a distinct gene-expression profile, and a more aggressive clinical course. Fluorescence in situ hybridization analysis demonstrated absolute specificity of YWHAE–FAM22A/B genetic rearrangement for high-grade ESS, with no fusions detected in other uterine and nonuterine mesenchymal tumors (55 tumor types, n = 827). These discoveries reveal diagnostically and therapeutically relevant models for characterizing aberrant 14-3-3 oncogenic functions. PMID:22223660

  20. Visualization and Biochemical Analyses of the Emerging Mammalian 14-3-3-Phosphoproteome*

    PubMed Central

    Johnson, Catherine; Tinti, Michele; Wood, Nicola T.; Campbell, David G.; Toth, Rachel; Dubois, Fanny; Geraghty, Kathryn M.; Wong, Barry H. C.; Brown, Laura J.; Tyler, Jennifer; Gernez, Aurélie; Chen, Shuai; Synowsky, Silvia; MacKintosh, Carol

    2011-01-01

    Hundreds of candidate 14-3-3-binding (phospho)proteins have been reported in publications that describe one interaction at a time, as well as high-throughput 14-3-3-affinity and mass spectrometry-based studies. Here, we transcribed these data into a common format, deposited the collated data from low-throughput studies in MINT (http://mint.bio.uniroma2.it/mint), and compared the low- and high-throughput data in VisANT graphs that are easy to analyze and extend. Exploring the graphs prompted questions about technical and biological specificity, which were addressed experimentally, resulting in identification of phosphorylated 14-3-3-binding sites in the mitochondrial import sequence of the iron-sulfur cluster assembly enzyme (ISCU), cytoplasmic domains of the mitochondrial fission factor (MFF), and endoplasmic reticulum-tethered receptor expression-enhancing protein 4 (REEP4), RNA regulator SMAUG2, and cytoskeletal regulatory proteins, namely debrin-like protein (DBNL) and kinesin light chain (KLC) isoforms. Therefore, 14-3-3s undergo physiological interactions with proteins that are destined for diverse subcellular locations. Graphing and validating interactions underpins efforts to use 14-3-3-phosphoproteomics to identify mechanisms and biomarkers for signaling pathways in health and disease. PMID:21725060

  1. Suppression of death-associated protein kinase 2 by interaction with 14-3-3 proteins.

    PubMed

    Yuasa, Keizo; Ota, Reina; Matsuda, Shinya; Isshiki, Kinuka; Inoue, Masahiro; Tsuji, Akihiko

    2015-08-14

    Death-associated protein kinase 2 (DAPK2), a Ca(2+)/calmodulin-regulated serine/threonine kinase, induces apoptosis. However, the signaling mechanisms involved in this process are unknown. Using a proteomic approach, we identified 14-3-3 proteins as novel DAPK2-interacting proteins. The 14-3-3 family has the ability to bind to phosphorylated proteins via recognition of three conserved amino acid motifs (mode 1-3 motifs), and DAPK2 contains the mode 3 motif ((pS/pT)X1-2-COOH). The interaction of 14-3-3 proteins with DAPK2 was dependent on the phosphorylation of Thr(369), and effectively suppressed DAPK2 kinase activity and DAPK2-induced apoptosis. Furthermore, we revealed that the 14-3-3 binding site Thr(369) of DAPK2 was phosphorylated by the survival kinase Akt. Our findings suggest that DAPK2-induced apoptosis is negatively regulated by Akt and 14-3-3 proteins.

  2. Functional identification of a novel 14-3-3 epsilon splicing variant suggests dimerization is not necessary for 14-3-3 epsilon to inhibit UV-induced apoptosis

    SciTech Connect

    Han, Dingding; Ye, Guangming; Liu, Tingting; Chen, Cong; Yang, Xianmei; Wan, Bo; Pan, Yuanwang; Yu, Long

    2010-05-28

    14-3-3 proteins function as a dimer and have been identified to involve in diverse signaling pathways. Here we reported the identification of a novel splicing variant of human 14-3-3 epsilon (14-3-3 epsilon sv), which is derived from a novel exon 1' insertion. The insertion contains a stop codon and leads to a truncated splicing variant of 14-3-3 epsilon. The splicing variant is translated from the exon 2 and results in the deletion of an N-terminal {alpha}-helix which is crucial for the dimerization. Therefore, the 14-3-3 epsilon sv could not form a dimer with 14-3-3 zeta. However, after UV irradiation 14-3-3 epsilon sv could also support cell survival, suggesting monomer of 14-3-3 epsilon is sufficient to protect cell from apoptosis.

  3. Histone density is maintained during transcription mediated by the chromatin remodeler RSC and histone chaperone NAP1 in vitro.

    PubMed

    Kuryan, Benjamin G; Kim, Jessica; Tran, Nancy Nga H; Lombardo, Sarah R; Venkatesh, Swaminathan; Workman, Jerry L; Carey, Michael

    2012-02-01

    ATPases and histone chaperones facilitate RNA polymerase II (pol II) elongation on chromatin. In vivo, the coordinated action of these enzymes is necessary to permit pol II passage through a nucleosome while restoring histone density afterward. We have developed a biochemical system recapitulating this basic process. Transcription through a nucleosome in vitro requires the ATPase remodels structure of chromatin (RSC) and the histone chaperone nucleosome assembly protein 1 (NAP1). In the presence of NAP1, RSC generates a hexasome. Despite the propensity of RSC to evict histones, NAP1 reprograms the reaction such that the hexasome is retained on the template during multiple rounds of transcription. This work has implications toward understanding the mechanism of pol II elongation on chromatin. PMID:22308335

  4. Histone density is maintained during transcription mediated by the chromatin remodeler RSC and histone chaperone NAP1 in vitro.

    PubMed

    Kuryan, Benjamin G; Kim, Jessica; Tran, Nancy Nga H; Lombardo, Sarah R; Venkatesh, Swaminathan; Workman, Jerry L; Carey, Michael

    2012-02-01

    ATPases and histone chaperones facilitate RNA polymerase II (pol II) elongation on chromatin. In vivo, the coordinated action of these enzymes is necessary to permit pol II passage through a nucleosome while restoring histone density afterward. We have developed a biochemical system recapitulating this basic process. Transcription through a nucleosome in vitro requires the ATPase remodels structure of chromatin (RSC) and the histone chaperone nucleosome assembly protein 1 (NAP1). In the presence of NAP1, RSC generates a hexasome. Despite the propensity of RSC to evict histones, NAP1 reprograms the reaction such that the hexasome is retained on the template during multiple rounds of transcription. This work has implications toward understanding the mechanism of pol II elongation on chromatin.

  5. Impaired Binding of 14-3-3 to C-RAF in Noonan Syndrome Suggests New Approaches in Diseases with Increased Ras Signaling▿

    PubMed Central

    Molzan, Manuela; Schumacher, Benjamin; Ottmann, Corinna; Baljuls, Angela; Polzien, Lisa; Weyand, Michael; Thiel, Philipp; Rose, Rolf; Rose, Micheline; Kuhenne, Philipp; Kaiser, Markus; Rapp, Ulf R.; Kuhlmann, Jürgen; Ottmann, Christian

    2010-01-01

    The Ras-RAF-mitogen-activated protein kinase (Ras-RAF-MAPK) pathway is overactive in many cancers and in some developmental disorders. In one of those disorders, namely, Noonan syndrome, nine activating C-RAF mutations cluster around Ser259, a regulatory site for inhibition by 14-3-3 proteins. We show that these mutations impair binding of 14-3-3 proteins to C-RAF and alter its subcellular localization by promoting Ras-mediated plasma membrane recruitment of C-RAF. By presenting biophysical binding data, the 14-3-3/C-RAFpS259 crystal structure, and cellular analyses, we indicate a mechanistic link between a well-described human developmental disorder and the impairment of a 14-3-3/target protein interaction. As a broader implication of these findings, modulating the C-RAFSer259/14-3-3 protein-protein interaction with a stabilizing small molecule may yield a novel potential approach for treatment of diseases resulting from an overactive Ras-RAF-MAPK pathway. PMID:20679480

  6. Histone Modifications Are Associated with Δ9-Tetrahydrocannabinol-mediated Alterations in Antigen-specific T Cell Responses*

    PubMed Central

    Yang, Xiaoming; Hegde, Venkatesh L.; Rao, Roshni; Zhang, Jiajia; Nagarkatti, Prakash S.; Nagarkatti, Mitzi

    2014-01-01

    Marijuana is one of the most abused drugs due to its psychotropic effects. Interestingly, it is also used for medicinal purposes. The main psychotropic component in marijuana, Δ9-tetrahydrocannabinol (THC), has also been shown to mediate potent anti-inflammatory properties. Whether the immunomodulatory activity of THC is mediated by epigenetic regulation has not been investigated previously. In this study, we employed ChIP-Seq technology to examine the in vivo effect of THC on global histone methylation in lymph node cells of mice immunized with a superantigen, staphylococcal enterotoxin B. We compared genome-wide histone H3 Lys-4, Lys-27, Lys-9, and Lys-36 trimethylation and histone H3 Lys-9 acetylation patterns in such cells exposed to THC or vehicle. Our results showed that THC treatment leads to the association of active histone modification signals to Th2 cytokine genes and suppressive modification signals to Th1 cytokine genes, indicating that such a mechanism may play a critical role in the THC-mediated switch from Th1 to Th2. At the global level, a significant portion of histone methylation and acetylation regions were altered by THC. However, the overall distribution of these histone methylation signals among the genomic features was not altered significantly by THC, suggesting that THC activates the expression of a subset of genes while suppressing the expression of another subset of genes through histone modification. Functional classification of these histone marker-associated genes showed that these differentially associated genes were involved in various cellular functions, from cell cycle regulation to metabolism, suggesting that THC had a pleiotropic effect on gene expression in immune cells. Altogether, the current study demonstrates for the first time that THC may modulate immune response through epigenetic regulation involving histone modifications. PMID:24841204

  7. 14-3-3, an integrator of cell mechanics and cytokinesis.

    PubMed

    Robinson, Douglas N

    2010-11-01

    One of the goals of understanding cytokinesis is to uncover the molecular regulation of the cellular mechanical properties that drive cell shape change. Such regulatory pathways are likely to be used at multiple stages of a cell's life, but are highly featured during cell division. Recently, we demonstrated that 14-3-3 (encoded by a single gene in the social amoeba Dictyostelium discoideum) serves to integrate key cytoskeletal components-microtubules, Rac and myosin II-to control cell mechanics and cytokinesis. As 14-3-3 proteins are frequently altered in a variety of human tumors, we extend these observations to suggest possible additional roles for how 14-3-3 proteins may contribute to tumorigenesis. PMID:21686271

  8. Diagnosing Sporadic Creutzfeldt-Jakob Disease: Accuracy of CSF 14-3-3 Protein Test of the Spinal Fluid

    MedlinePlus

    ... JAKOB DISEASE: ACCURACY OF THE 14-3-3 PROTEIN TEST OF THE SPINAL FLUID This information sheet ... help you understand how the 14-3-3 protein test helps in diagnosing sporadic Creutzfeldt-Jakob disease ( ...

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

    PubMed

    Kuo, Yin-Ming; Andrews, Andrew J

    2013-01-01

    Lysine acetyltransferases (KATs) play a unique role in regulating gene transcription as well as maintaining the epigenetic state of the cell. KATs such as Gcn5 and p300/CBP can modify multiple residues on a single histone; however, order and specificity of acetylation can be altered by factors such as histone chaperones, subunit proteins or external stimulus. While the importance of acetylation is well documented, it has been difficult to quantitatively measure the specificity and selectivity of acetylation at different residues within a histone. In this paper, we demonstrate a label-free quantitative high throughput mass spectrometry-based assay capable of quantitatively monitoring all known acetylation sites of H3 simultaneously. Using this assay, we are able to analyze the steady-state enzyme kinetics of Gcn5, an evolutionarily conserved KAT. In doing so, we measured Gcn5-mediated acetylation at six residues (K14>K9 ≈ K23> K18> K27 ≈ K36) and the catalytic efficiency (k(cat)/K(m)) for K9, K14, K18, and K23 as well as the nonenzymatic acetylation rate. We observed selectivity differences of up to -4 kcal/mol between K14 and K18, the highest and lowest measurable k(cat)/K(m). These data provide a first look at quantitating the specificity and selectivity of multiple lysines on a single substrate (H3) by Gcn5. PMID:23437046

  10. Deletion of host histone acetyltransferases and deacetylases strongly affects Agrobacterium-mediated transformation of Saccharomyces cerevisiae.

    PubMed

    Soltani, Jalal; van Heusden, Gerard Paul H; Hooykaas, Paul J J

    2009-09-01

    Agrobacterium tumefaciens is a plant pathogen that genetically transforms plant cells by transferring a part of its Ti-plasmid, the T-strand, to the host cell. Under laboratory conditions, it can also transform cells from many different nonplant organisms, including the yeast Saccharomyces cerevisiae. Collections of S. cerevisiae strains have been developed with systematic deletion of all coding sequences. Here, we used these collections to identify genes involved in the Agrobacterium-mediated transformation (AMT) of S. cerevisiae. We found that deletion of genes (GCN5, NGG1, YAF9 and EAF7) encoding subunits of the SAGA, SLIK, ADA and NuA4 histone acetyltransferase complexes highly increased the efficiency of AMT, while deletion of genes (HDA2, HDA3 and HST4) encoding subunits of histone deacetylase complexes decreased AMT. These effects are specific for AMT as the efficiency of chemical (lithium acetate) transformation was not or only slightly affected by these deletions. Our data are consistent with a positive role of host histone deacetylation in AMT.

  11. dLKR/SDH regulates hormone-mediated histone arginine methylation and transcription of cell death genes.

    PubMed

    Cakouros, Dimitrios; Mills, Kathryn; Denton, Donna; Paterson, Alicia; Daish, Tasman; Kumar, Sharad

    2008-08-11

    The sequential modifications of histones form the basis of the histone code that translates into either gene activation or repression. Nuclear receptors recruit a cohort of histone-modifying enzymes in response to ligand binding and regulate proliferation, differentiation, and cell death. In Drosophila melanogaster, the steroid hormone ecdysone binds its heterodimeric receptor ecdysone receptor/ultraspiracle to spatiotemporally regulate the transcription of several genes. In this study, we identify a novel cofactor, Drosophila lysine ketoglutarate reductase (dLKR)/saccharopine dehydrogenase (SDH), that is involved in ecdysone-mediated transcription. dLKR/SDH binds histones H3 and H4 and suppresses ecdysone-mediated transcription of cell death genes by inhibiting histone H3R17me2 mediated by the Drosophila arginine methyl transferase CARMER. Our data suggest that the dynamic recruitment of dLKR/SDH to ecdysone-regulated gene promoters controls the timing of hormone-induced gene expression. In the absence of dLKR/SDH, histone methylation occurs prematurely, resulting in enhanced gene activation. Consistent with these observations, the loss of dLKR/SDH in Drosophila enhances hormone-regulated gene expression, affecting the developmental timing of gene activation. PMID:18695041

  12. Regulation of the Regulators: Post-Translational Modifications, Subcellular, and Spatiotemporal Distribution of Plant 14-3-3 Proteins

    PubMed Central

    Wilson, Rashaun S.; Swatek, Kirby N.; Thelen, Jay J.

    2016-01-01

    14-3-3 proteins bind to and modulate the activity of phosphorylated proteins that regulate a variety of metabolic processes in eukaryotes. Multiple 14-3-3 isoforms are expressed in most organisms and display redundancy in both sequence and function. Plants contain the largest number of 14-3-3 isoforms. For example, Arabidopsis thaliana contains thirteen 14-3-3 genes, each of which is expressed. Interest in the plant 14-3-3 field has swelled over the past decade, largely due to the vast number of possibilities for 14-3-3 metabolic regulation. As the field progresses, it is essential to understand these proteins' activities at both the spatiotemporal and subcellular levels. This review summarizes current knowledge of 14-3-3 proteins in plants, including 14-3-3 interactions, regulatory functions, isoform specificity, and post-translational modifications. We begin with a historical overview and structural analysis of 14-3-3 proteins, which describes the basic principles of 14-3-3 function, and then discuss interactions and regulatory effects of plant 14-3-3 proteins in specific tissues and subcellular compartments. We conclude with a summary of 14-3-3 phosphorylation and current knowledge of the functional effects of this modification in plants. PMID:27242818

  13. Circulating histones are major mediators of systemic inflammation and cellular injury in patients with acute liver failure

    PubMed Central

    Wen, Zongmei; Lei, Zhen; Yao, Lu; Jiang, Ping; Gu, Tao; Ren, Feng; Liu, Yan; Gou, Chunyan; Li, Xiuhui; Wen, Tao

    2016-01-01

    Acute liver failure (ALF) is a life-threatening systemic disorder. Here we investigated the impact of circulating histones, recently identified inflammatory mediators, on systemic inflammation and liver injury in murine models and patients with ALF. We analyzed histone levels in blood samples from 62 patients with ALF, 60 patients with chronic liver disease, and 30 healthy volunteers. We incubated patients' sera with human L02 hepatocytes and monocytic U937 cells to assess cellular damage and cytokine production. d-galactosamine plus lipopolysaccharide (GalN/LPS), concanavalin A (ConA), and acetaminophen (APAP) were given to C57BL/6N mice to induce liver injury, respectively, and the pathogenic role of circulating histones was studied. Besides, the protective effect of nonanticoagulant heparin, which can bind histones, was evaluated with in vivo and ex vivo investigations. We observed that circulating histones were significantly increased in patients with ALF, and correlated with disease severity and mortality. Significant systemic inflammation was also pronounced in ALF patients, which were associated with histone levels. ALF patients' sera induced significant L02 cell death and stimulated U937 cells to produce cytokines, which were abrogated by nonanticoagulant heparin. Furthermore, circulating histones were all released remarkably in GalN/LPS, ConA, and APAP-treated mice, and associated with high levels of inflammatory cytokines. Heparin reduced systemic inflammation and liver damage in mice, suggesting that it could interfere with histone-associated liver injury. Collectively, these findings demonstrate that circulating histones are critical mediators of systemic inflammation and cellular damage in ALF, which may be potentially translatable for clinical use. PMID:27685635

  14. Identification of 14-3-3 Family in Common Bean and Their Response to Abiotic Stress.

    PubMed

    Li, Ruihua; Jiang, Xiaotong; Jin, Donghao; Dhaubhadel, Sangeeta; Bian, Shaomin; Li, Xuyan

    2015-01-01

    14-3-3s are a class of conserved regulatory proteins ubiquitously found in eukaryotes, which play important roles in a variety of cellular processes including response to diverse stresses. Although much has been learned about 14-3-3s in several plant species, it remains unknown in common bean. In this study, 9 common bean 14-3-3s (PvGF14s) were identified by exhaustive data mining against the publicly available common bean genomic database. A phylogenetic analysis revealed that each predicted PvGF14 was clustered with two GmSGF14 paralogs from soybean. Both epsilon-like and non-epsilon classes of PvGF14s were found in common bean, and the PvGF14s belonging to each class exhibited similar gene structure. Among 9 PvGF14s, only 8 are transcribed in common bean. Expression patterns of PvGF14s varied depending on tissue type, developmental stage and exposure of plants to stress. A protein-protein interaction study revealed that PvGF14a forms dimer with itself and with other PvGF14 isoforms. This study provides a first comprehensive look at common bean 14-3-3 proteins, a family of proteins with diverse functions in many cellular processes, especially in response to stresses.

  15. Polycations Globally Enhance Binding of 14-3-3 omega to Target Proteins in Spinach Leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The binding of 14-3-3' to phosphorylated NR (pNR) is stimulated by cations such as Mg2+ or spermine, and decreased by 5'-AMP. In order to determine whether binding to other cellular proteins is affected similarly, Far-Western overlays of extracts prepared from light- or dark-treated spinach (Spinac...

  16. Identification of 14-3-3 Family in Common Bean and Their Response to Abiotic Stress

    PubMed Central

    Dhaubhadel, Sangeeta; Bian, Shaomin; Li, Xuyan

    2015-01-01

    14-3-3s are a class of conserved regulatory proteins ubiquitously found in eukaryotes, which play important roles in a variety of cellular processes including response to diverse stresses. Although much has been learned about 14-3-3s in several plant species, it remains unknown in common bean. In this study, 9 common bean 14-3-3s (PvGF14s) were identified by exhaustive data mining against the publicly available common bean genomic database. A phylogenetic analysis revealed that each predicted PvGF14 was clustered with two GmSGF14 paralogs from soybean. Both epsilon-like and non-epsilon classes of PvGF14s were found in common bean, and the PvGF14s belonging to each class exhibited similar gene structure. Among 9 PvGF14s, only 8 are transcribed in common bean. Expression patterns of PvGF14s varied depending on tissue type, developmental stage and exposure of plants to stress. A protein-protein interaction study revealed that PvGF14a forms dimer with itself and with other PvGF14 isoforms. This study provides a first comprehensive look at common bean 14-3-3 proteins, a family of proteins with diverse functions in many cellular processes, especially in response to stresses. PMID:26599110

  17. Identification of 14-3-3β Gene as a Novel miR-152 Target Using a Proteome-based Approach*

    PubMed Central

    Jasinski-Bergner, Simon; Stehle, Franziska; Gonschorek, Evamaria; Kalich, Jana; Schulz, Kristin; Huettelmaier, Stefan; Braun, Juliane; Seliger, Barbara

    2014-01-01

    Recent studies demonstrated that miR-152 overexpression down-regulates the nonclassical human leukocyte antigen (HLA) class I molecule HLA-G in human tumors thereby contributing to their immune surveillance. Using two-dimensional gel electrophoresis followed by MALDI-TOF mass spectrometry, the protein expression profile of HLA-G+, miR-152low cells, and their miR-152-overexpressing (miRhigh) counterparts was compared leading to the identification of 24 differentially expressed proteins. These were categorized according to their function and localization demonstrating for most of them an important role in the initiation and progression of tumors. The novel miR-152 target 14-3-3 protein β/α/YWHAB (14-3-3β) is down-regulated upon miR-152 overexpression, although its overexpression was often found in tumors of distinct origin. The miR-152-mediated reduction of the 14-3-3β expression was accompanied by an up-regulation of BAX protein expression resulting in a pro-apoptotic phenotype. In contrast, the reconstitution of 14-3-3β expression in miR-152high cells increased the expression of the anti-apoptotic BCL2 gene, enhances the proliferative activity in the presence of the cytostatic drug paclitaxel, and causes resistance to apoptosis induced by this drug. By correlating clinical microarray data with the patients' outcome, a link between 14-3-3β and HLA-G expression was found, which could be associated with poor prognosis and overall survival of patients with tumors. Because miR-152 controls both the expression of 14-3-3β and HLA-G, it exerts a dual role in tumor cells by both altering the immunogenicity and the tumorigenicity. PMID:25228695

  18. Regulation of presynaptic anchoring of the scaffold protein Bassoon by phosphorylation-dependent interaction with 14-3-3 adaptor proteins.

    PubMed

    Schröder, Markus S; Stellmacher, Anne; Romorini, Stefano; Marini, Claudia; Montenegro-Venegas, Carolina; Altrock, Wilko D; Gundelfinger, Eckart D; Fejtova, Anna

    2013-01-01

    The proper organization of the presynaptic cytomatrix at the active zone is essential for reliable neurotransmitter release from neurons. Despite of the virtual stability of this tightly interconnected proteinaceous network it becomes increasingly clear that regulated dynamic changes of its composition play an important role in the processes of synaptic plasticity. Bassoon, a core component of the presynaptic cytomatrix, is a key player in structural organization and functional regulation of presynaptic release sites. It is one of the most highly phosphorylated synaptic proteins. Nevertheless, to date our knowledge about functions mediated by any one of the identified phosphorylation sites of Bassoon is sparse. In this study, we have identified an interaction of Bassoon with the small adaptor protein 14-3-3, which depends on phosphorylation of the 14-3-3 binding motif of Bassoon. In vitro phosphorylation assays indicate that phosphorylation of the critical Ser-2845 residue of Bassoon can be mediated by a member of the 90-kDa ribosomal S6 protein kinase family. Elimination of Ser-2845 from the 14-3-3 binding motif results in a significant decrease of Bassoon's molecular exchange rates at synapses of living rat neurons. We propose that the phosphorylation-induced 14-3-3 binding to Bassoon modulates its anchoring to the presynaptic cytomatrix. This regulation mechanism might participate in molecular and structural presynaptic remodeling during synaptic plasticity.

  19. Protective effect of the long pentraxin PTX3 against histone-mediated endothelial cell cytotoxicity in sepsis.

    PubMed

    Daigo, Kenji; Nakakido, Makoto; Ohashi, Riuko; Fukuda, Rie; Matsubara, Koichi; Minami, Takashi; Yamaguchi, Naotaka; Inoue, Kenji; Jiang, Shuying; Naito, Makoto; Tsumoto, Kouhei; Hamakubo, Takao

    2014-01-01

    Pentraxin 3 (PTX3), a member of the long pentraxin subfamily within the family of pentraxins, is a soluble pattern recognition molecule that functions in the innate immune system. Innate immunity affords the infected host protection against sepsis, a potentially life-threatening inflammatory response to infection. Extracellular histones are considered to be the main cause of septic death because of their cytotoxic effect on endothelial cells, which makes them a potential therapeutic target. We found that PTX3 interacted with histones to form coaggregates, which depended on polyvalent interactions and disorder in the secondary structure of PTX3. PTX3 exerted a protective effect, both in vitro and in vivo, against histone-mediated cytotoxicity toward endothelial cells. Additionally, the intraperitoneal administration of PTX3 reduced mortality in mouse models of sepsis. The amino-terminal domain of PTX3, which was required for coaggregation with histones, was sufficient to protect against cytotoxicity. Our results suggest that the host-protective effects of PTX3 in sepsis are a result of its coaggregation with histones rather than its ability to mediate pattern recognition. This long pentraxin-specific effect provides a potential basis for the treatment of sepsis directed at protecting cells from the toxic effects of extracellular histones.

  20. Down-regulation of 14-3-3β exerts anti-cancer effects through inducing ER stress in human glioma U87 cells: Involvement of CHOP–Wnt pathway

    SciTech Connect

    Cao, Lei; Lei, Hui; Chang, Ming-Ze; Liu, Zhi-Qin; Bie, Xiao-Hua

    2015-07-10

    We previously identified 14-3-3β as a tumor-specific isoform of 14-3-3 protein in astrocytoma, but its functional role in glioma cells and underlying mechanisms are poorly understood. In the present study, we investigated the effects of 14-3-3β inhibition in human glioma U87 cells using specific targeted small interfering RNA (siRNA). The results showed that 14-3-3β is highly expressed in U87 cells but not in normal astrocyte SVGp12 cells. Knockdown of 14-3-3β by Si-14-3-3β transfection significantly decreased the cell viability but increased the LDH release in a time-dependent fashion in U87 cells, and these effects were accompanied with G0/G1 cell cycle arrest and apoptosis. In addition, 14-3-3β knockdown induced ER stress in U87 cells, as evidenced by ER calcium release, increased expression of XBP1S mRNA and induction of ER related pro-apoptotic factors. Down-regulation of 14-3-3β significantly decreased the nuclear localization of β-catenin and inhibited Topflash activity, which was shown to be reversely correlated with CHOP. Furthermore, Si-CHOP and sFRP were used to inhibit CHOP and Wnt, respectively. The results showed that the anti-cancer effects of 14-3-3β knockdown in U87 cells were mediated by increased expression of CHOP and followed inhibition of Wnt/β-catenin pathway. In summary, the remarkable efficiency of 14-3-3β knockdown to induce apoptotic cell death in U87 cells may find therapeutic application for the treatment of glioma patients. - Highlights: • Knockdown of 14-3-3β leads to cytotoxicity in human glioma U87 cells. • Knockdown of 14-3-3β induces cell cycle arrest and apoptosis in U87 cells. • Knockdown of 14-3-3β results in ER stress in U87 cells. • Knockdown of 14-3-3β inhibits Wnt/β-catenin pathway via CHOP activation.

  1. 14-3-3ζ Interacts with Stat3 and Regulates Its Constitutive Activation in Multiple Myeloma Cells

    PubMed Central

    Li, Wenliang; Xiong, Qian; Yang, Mingkun; Zheng, Peng; Li, Chongyang; Pei, Jianfeng; Ge, Feng

    2012-01-01

    The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner and function as adapter or scaffold proteins in signal transduction pathways. One family member, 14-3-3ζ, is believed to function in cell signaling, cycle control, and apoptotic death. A systematic proteomic analysis done in our laboratory has identified signal transducers and activators of transcription 3 (Stat3) as a novel 14-3-3ζ interacting protein. Following our initial finding, in this study, we provide evidence that 14-3-3ζ interacts physically with Stat3. We further demonstrate that phosphorylation of Stat3 at Ser727 is vital for 14-3-3ζ interaction and mutation of Ser727 to Alanine abolished 14-3-3ζ/Stat3 association. Inhibition of 14-3-3ζ protein expression in U266 cells inhibited Stat3 Ser727 phosphorylation and nuclear translocation, and decreased both Stat3 DNA binding and transcriptional activity. Moreover, 14-3-3ζ is involved in the regulation of protein kinase C (PKC) activity and 14-3-3ζ binding to Stat3 protects Ser727 dephosphorylation from protein phosphatase 2A (PP2A). Taken together, our findings support the model that multiple signaling events impinge on Stat3 and that 14-3-3ζ serves as an essential coordinator for different pathways to regulate Stat3 activation and function in MM cells. PMID:22279540

  2. Molecular tweezers modulate 14-3-3 protein-protein interactions.

    PubMed

    Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

    2013-03-01

    Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins--a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)--in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions. PMID:23422566

  3. Histone methyltransferase Ash1L mediates activity-dependent repression of neurexin-1α.

    PubMed

    Zhu, Τao; Liang, Chen; Li, Dongdong; Tian, Miaomiao; Liu, Sanxiong; Gao, Guanjun; Guan, Ji-Song

    2016-05-27

    Activity-dependent transcription is critical for the regulation of long-term synaptic plasticity and plastic rewiring in the brain. Here, we report that the transcription of neurexin1α (nrxn1α), a presynaptic adhesion molecule for synaptic formation, is regulated by transient neuronal activation. We showed that 10 minutes of firing at 50 Hz in neurons repressed the expression of nrxn1α for 24 hours in a primary cortical neuron culture through a transcriptional repression mechanism. By performing a screening assay using a synthetic zinc finger protein (ZFP) to pull down the proteins enriched near the nrxn1α promoter region in vivo, we identified that Ash1L, a histone methyltransferase, is enriched in the nrxn1α promoter. Neuronal activity triggered binding of Ash1L to the promoter and enriched the histone marker H3K36me2 at the nrxn1α promoter region. Knockout of Ash1L in mice completely abolished the activity-dependent repression of nrxn1α. Taken together, our results reveal that a novel process of activity-dependent transcriptional repression exists in neurons and that Ash1L mediates the long-term repression of nrxn1α, thus implicating an important role for epigenetic modification in brain functioning.

  4. Histone methyltransferase Ash1L mediates activity-dependent repression of neurexin-1α

    PubMed Central

    Zhu, Τao; Liang, Chen; Li, Dongdong; Tian, Miaomiao; Liu, Sanxiong; Gao, Guanjun; Guan, Ji-Song

    2016-01-01

    Activity-dependent transcription is critical for the regulation of long-term synaptic plasticity and plastic rewiring in the brain. Here, we report that the transcription of neurexin1α (nrxn1α), a presynaptic adhesion molecule for synaptic formation, is regulated by transient neuronal activation. We showed that 10 minutes of firing at 50 Hz in neurons repressed the expression of nrxn1α for 24 hours in a primary cortical neuron culture through a transcriptional repression mechanism. By performing a screening assay using a synthetic zinc finger protein (ZFP) to pull down the proteins enriched near the nrxn1α promoter region in vivo, we identified that Ash1L, a histone methyltransferase, is enriched in the nrxn1α promoter. Neuronal activity triggered binding of Ash1L to the promoter and enriched the histone marker H3K36me2 at the nrxn1α promoter region. Knockout of Ash1L in mice completely abolished the activity-dependent repression of nrxn1α. Taken together, our results reveal that a novel process of activity-dependent transcriptional repression exists in neurons and that Ash1L mediates the long-term repression of nrxn1α, thus implicating an important role for epigenetic modification in brain functioning. PMID:27229316

  5. Butyrate mediates decrease of histone acetylation centered on transcription start sites and down-regulation of associated genes

    PubMed Central

    Rada-Iglesias, Alvaro; Enroth, Stefan; Ameur, Adam; Koch, Christoph M.; Clelland, Gayle K.; Respuela-Alonso, Patricia; Wilcox, Sarah; Dovey, Oliver M.; Ellis, Peter D.; Langford, Cordelia F.; Dunham, Ian; Komorowski, Jan; Wadelius, Claes

    2007-01-01

    Butyrate is a histone deacetylase inhibitor (HDACi) with anti-neoplastic properties, which theoretically reactivates epigenetically silenced genes by increasing global histone acetylation. However, recent studies indicate that a similar number or even more genes are down-regulated than up-regulated by this drug. We treated hepatocarcinoma HepG2 cells with butyrate and characterized the levels of acetylation at DNA-bound histones H3 and H4 by ChIP-chip along the ENCODE regions. In contrast to the global increases of histone acetylation, many genomic regions close to transcription start sites were deacetylated after butyrate exposure. In order to validate these findings, we found that both butyrate and trichostatin A treatment resulted in histone deacetylation at selected regions, while nucleosome loss or changes in histone H3 lysine 4 trimethylation (H3K4me3) did not occur in such locations. Furthermore, similar histone deacetylation events were observed when colon adenocarcinoma HT-29 cells were treated with butyrate. In addition, genes with deacetylated promoters were down-regulated by butyrate, and this was mediated at the transcriptional level by affecting RNA polymerase II (POLR2A) initiation/elongation. Finally, the global increase in acetylated histones was preferentially localized to the nuclear periphery, indicating that it might not be associated to euchromatin. Our results are significant for the evaluation of HDACi as anti-tumourogenic drugs, suggesting that previous models of action might need to be revised, and provides an explanation for the frequently observed repression of many genes during HDACi treatment. PMID:17567991

  6. T cell tolerance induced by histone deacetylase inhibitor is mediated by P21cip1.

    PubMed

    Gilbert, Kathleen M; Boger, Susan R; Price, Peter; Fifer, E Kim

    2005-01-01

    MEB [n-butyrate 2-(4-morpholinyl) ethyl butyrate hydrochloride], a histone deacetylase inhibitor and G1 blocker, has been shown to induce unresponsiveness in antigen-activated Th1 cells. MEB was tested for here for its ability to inactivate naive alloantigen-specific T cells from DBA/2 and C57BL/10 mice. Since T cells from these two strains of mice have been shown to differ in their cell cycle regulation, it we hoped that this comparison would provide information concerning the role of cycle regulatory proteins in mediating MEB-induced T cell unresponsiveness. MEB inhibited proliferation in a one-way mixed lymphocyte reaction (MLR) in which spleen cells from DBA/2 mice (H-2d) or C57BL/10 mice (H-2b) were stimulated with spleen cells from C57BL/10 or DBA/2 mice, respectively. C57BL/10 responder T cells isolated from the MEB-treated primary MLR remained unresponsive to alloantigen following restimulation in a secondary MLR that did not contain MEB. T cells from DBA/2 mice were less sensitive to MEB-induced unresponsiveness and required a longer exposure or pretreatment with IL-2 to become tolerant. In all cases responsiveness to MEB-induced tolerance in the alloantigen-stimulated T cells corresponded with the levels of the cyclin-dependent kinase inhibitor p21cip1. Additional experiments showed that T cells from p21cip1-deficient mice, unlike T cells from p21cip1 wild-type littermates, were resistant to MEB-induced tolerance. These results underscore the role of p21cip1 in mediating T cell tolerance induced by the histone deacetylase inhibitor MEB.

  7. Dynamic interaction between 14-3-3zeta and bax during TNF-α-induced apoptosis in living cells

    NASA Astrophysics Data System (ADS)

    Gao, Xuejuan; Xing, Da; Chen, Tongsheng

    2006-09-01

    Bax, a proapoptotic member of the Bcl-2 family, localizes largely in the cytoplasm but redistributes to mitochondria and undergoes oligomerization to induce the release of apoptogenic factors such as cytochrome c in response to apoptotic stimuli. Cytoplasmic protein 14-3-3zeta binds to Bax and, upon apoptotic stimulation, releases Bax by a caspase-independent mechanism. However, the direct interaction of the cytoplasmic 14-3-3zeta and Bax in living cells has not been observed. In present study, to monitor the dynamic interaction between 14-3-3zeta and Bax in living cells in real time during apoptosis induced by tumor necrosis factor (TNF-α), DsRed-14-3-3zeta plasmid is constructed. By cotransfecting DsRed- 14-3-3zeta and GFP-Bax plasmids into human lung adenocarcinoma cells (ASTC-a-1), we observe the dynamic interaction between Bax and 14-3-3zeta using fluorescence resonance energy transfer (FRET) technique on laser scanning confocal microscope. The results show that 14-3-3zeta remains in the cytoplasm but GFP-Bax translocates to mitochondria completely after TNF-α stimulation. These results reveal that 14-3-3zeta binds directly to Bax in healthy cells, and that 14-3-3zeta negatively regulates Bax translocation to mitochondria during TNF-α-induced apoptosis.

  8. Histone H2A-mediated transient cytokine gene delivery induces efficient antitumor responses in murine neuroblastoma.

    PubMed

    Balicki, D; Reisfeld, R A; Pertl, U; Beutler, E; Lode, H N

    2000-10-10

    A major goal of cancer immunotherapy is the induction of a cell-mediated antitumor response in poorly immunogenic malignancies. We tested the hypothesis that this can be achieved by cytokine gene therapy with a novel histone H2A-based transient transfection procedure. This was tested by using cytokine genes encoding for IL-2 and a single chain IL-12 (scIL-12) fusion protein in a recently developed murine neuroblastoma model. Here, we demonstrate that cytokine gene transfer of IL-2 and scIL-12 with histone H2A results in the induction of an antitumor immune response that is superior in some respects to gene transfer with Superfect, a commercially available activated dendrimer commonly used to effect transfection with plasmids. Three lines of evidence support this contention. First, histone H2A-mediated transfection of IL-2 induces a natural killer cell-induced rejection of primary tumors in contrast to Superfect, which produces only a partial reduction in primary tumor growth. Second, the induction of a T cell-mediated protective tumor immunity following gene transfer of scIL-12 is more efficient with the histone H2A-mediated gene transfer because rejection of a lethal wild-type tumor cell challenge is accompanied by the greatest degree of MHC class I-restricted tumor cell killing in vitro. Third, histone H2A-mediated scIL-12 gene therapy induces the greatest release of mIFN-gamma from splenocytes of vaccinated animals in contrast to Superfect and other controls.

  9. Interaction of Ubinuclein-1, a nuclear and adhesion junction protein, with the 14-3-3 epsilon protein in epithelial cells: implication of the PKA pathway.

    PubMed

    Conti, Audrey; Sueur, Charlotte; Lupo, Julien; Brazzolotto, Xavier; Burmeister, Wim P; Manet, Evelyne; Gruffat, Henri; Morand, Patrice; Boyer, Véronique

    2013-03-01

    Ubinuclein-1 is a NACos (Nuclear and Adhesion junction Complex components) protein which shuttles between the nucleus and tight junctions, but its function in the latter is not understood. Here, by co-immunoprecipitation and confocal analysis, we show that Ubinuclein-1 interacts with the 14-3-3ɛ protein both in HT29 colon cells, and AGS gastric cells. This interaction is mediated by an Ubinuclein-1 phosphoserine motif. We show that the arginine residues (R56, R60 and R132) which form the 14-3-3ɛ ligand binding site are responsible for the binding of 14-3-3ɛ to phosphorylated Ubinuclein-1. Furthermore, we demonstrate that in vitro Ubinuclein-1 can be directly phosphorylated by cAMP-dependent protein kinase A. This in vitro phosphorylation allows binding of wildtype 14-3-3ɛ. Moreover, treatment of the cells with inhibitors of the cAMP-dependent protein kinase, KT5720 or H89, modifies the subcellular localization of Ubinuclein-1. Indeed, KT5720 and H89 greatly increase the staining of Ubinuclein-1 at the tight junctions in AGS gastric cells. In the presence of the kinase inhibitor KT5720, the amount of Ubinuclein-1 in the NP40 insoluble fraction is increased, together with actin. Moreover, treatment of the cells with KT5720 or H89 induces the concentration of Ubinuclein-1 at tricellular intersections of MDCK cells. Taken together, our findings demonstrate novel cell signaling trafficking by Ubinuclein-1 via association with 14-3-3ɛ following Ubinuclein-1 phosphorylation by the cAMP-dependent protein kinase-A.

  10. Transcriptional Regulation of YWHAZ, the Gene Encoding 14-3-3ζ

    PubMed Central

    Kasinski, Andrea; Dong, Xueyuan; Khuri, Fadlo R.; Boss, Jeremy; Fu, Haian

    2014-01-01

    Aberrant expression of oncogenic 14-3-3 proteins is correlated with poor survival of cancer patients. While the underlying mechanism of the abnormal expression in tumors remains elusive for the six oncogenic 14-3-3 isoforms; the potential involvement of a transcriptional component has been suggested. Unfortunately, little experimental data has been reported to support this hypothesis. In this study we describe the genetic structure of YWHAZ, the gene encoding 14-3-3ζ, including the identification of previously unreported transcript variants. In total, five transcript variants were revealed and their expressions confirmed in a panel of cell lines. Expressed sequence tag (EST) database mining and in vitro rapid-amplification of cDNA ends (RACE) confirmed that one variant, 1c, represents >80% of the expressed transcripts, which is also the most efficiently translated. An analysis of the proximal promoter of this variant revealed a functional Cyclic-AMP Response Element (CRE). Factors that bound to the CRE element were recognized through fractionation and subsequent EMSAs. This analysis identified CREB and ATF-1 as the trans-interacting factors. Cell-based assays confirm that ATF-1, and to a lesser extent CREB, bind the endogenous YWHAZ promoter especially under TNF-α stimulating conditions. In support of a role of ATF-1 in the regulation of YWHAZ, silencing of ATF-1 resulted in a marked reduction in two of the five YWHAZ transcripts. These data suggest a novel mechanism for the transcriptional regulation of a major pro-survival gene, YWHAZ, by ATF-1. PMID:24690670

  11. Differential interaction and aggregation of 3-repeat and 4-repeat tau isoforms with 14-3-3{zeta} protein

    SciTech Connect

    Sadik, Golam; Tanaka, Toshihisa; Kato, Kiyoko; Yanagi, Kentaro; Kudo, Takashi; Takeda, Masatoshi

    2009-05-22

    Tau isoforms, 3-repeat (3R) and 4-repeat tau (4R), are differentially involved in neuronal development and in several tauopathies. 14-3-3 protein binds to tau and 14-3-3/tau association has been found both in the development and in tauopathies. To understand the role of 14-3-3 in the differential regulation of tau isoforms, we have performed studies on the interaction and aggregation of 3R-tau and 4R-tau, either phosphorylated or unphosphorylated, with 14-3-3{zeta}. We show by surface plasmon resonance studies that the interaction between unphosphorylated 3R-tau and 14-3-3{zeta} is {approx}3-folds higher than that between unphosphorylated 4R-tau and 14-3-3{zeta}. Phosphorylation of tau by protein kinase A (PKA) increases the affinity of both 3R- and 4R-tau for 14-3-3{zeta} to a similar level. An in vitro aggregation assay employing both transmission electron microscopy and fluorescence spectroscopy revealed the aggregation of unphosphorylated 4R-tau to be significantly higher than that of unphosphorylated 3R-tau following the induction of 14-3-3{zeta}. The filaments formed from 3R- and 4R-tau were almost similar in morphology. In contrast, the aggregation of both 3R- and 4R-tau was reduced to a similar low level after phosphorylation with PKA. Taken together, these results suggest that 14-3-3{zeta} exhibits a similar role for tau isoforms after PKA-phosphorylation, but a differential role for unphosphorylated tau. The significant aggregation of 4R-tau by 14-3-3{zeta} suggests that 14-3-3 may act as an inducer in the generation of 4R-tau-predominant neurofibrillary tangles in tauopathies.

  12. GATA2 regulates GATA1 expression through LSD1-mediated histone modification

    PubMed Central

    Guo, Yidi; Fu, Xueqi; Huo, Bo; Wang, Yongsen; Sun, Jing; Meng, Lingyuan; Hao, Tian; Zhao, Zhizhuang Joe; Hu, Xin

    2016-01-01

    The dynamic and reversed expression of GATA1 and GATA2 are essential for proper erythroid differentiation. Our previous work demonstrates that LSD1, a histone H3K4 demethylase, represses GATA2 expression at late stage of erythroid differentiation. K562 and MEL cells were used and cultured in Roswell Park Memorial Institute-1640 medium (RPMI) and Dulbecco’s modified Eagle’s medium (DMEM), respectively. Western blot assay was used to examine the GATA1, GATA2, TAL1, HDAC1, HDAC2, CoREST and β-actin protein. The immunoprecipitation assay and GST pull-down assay were employed to detect the precipitated protein complexes and investigate the interaction between the proteins. The small interfering RNA (siRNA) and nonspecific control siRNA were synthesized to silence the target genes. Double fluorescence immunostaining was used to observe the association of LSD1 with GATA2 in K562 cells. The results indicated that knockdown of LSD1 in K562 cell causes increased H3K4 di-methylation at GATA1 locus and activates GATA1 expression, demonstrating that LSD1 represses GATA1 expression through LSD1-mediated histone demethylation. Upon induced erythroid differentiation of K562 cells, the interaction between GATA2 and LSD1 is decreased, consistent with a de-repression of GATA1 expression. Meanwhile, the interaction between TAL1 and LSD1 is increased, which forms a complex that efficiently suppresses GATA2 expression. In conclusion, these observations reveal an elegant mechanism to modulate GATA1 and GATA2 expression during erythroid differentiation. While LSD1 mainly forms complex with GATA2 to repress GATA1 expression in hematopoietic progenitor cells, it mostly forms complex with TAL1 to repress GATA2 expression in differentiated cells. PMID:27347333

  13. Binding of 14-3-3 reader proteins to phosphorylated DNMT1 facilitates aberrant DNA methylation and gene expression

    PubMed Central

    Estève, Pierre-Olivier; Zhang, Guoqiang; Ponnaluri, V.K. Chaithanya; Deepti, Kanneganti; Chin, Hang Gyeong; Dai, Nan; Sagum, Cari; Black, Karynne; Corrêa, Ivan R.; Bedford, Mark T.; Cheng, Xiaodong; Pradhan, Sriharsa

    2016-01-01

    Mammalian DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for maintenance methylation. Phosphorylation of Ser143 (pSer143) stabilizes DNMT1 during DNA replication. Here, we show 14-3-3 is a reader protein of DNMT1pSer143. In mammalian cells 14-3-3 colocalizes and binds DNMT1pSer143 post-DNA replication. The level of DNMT1pSer143 increased with overexpression of 14-3-3 and decreased by its depletion. Binding of 14-3-3 proteins with DNMT1pSer143 resulted in inhibition of DNA methylation activity in vitro. In addition, overexpression of 14-3-3 in NIH3T3 cells led to decrease in DNMT1 specific activity resulting in hypomethylation of the genome that was rescued by transfection of DNMT1. Genes representing cell migration, mobility, proliferation and focal adhesion pathway were hypomethylated and overexpressed. Furthermore, overexpression of 14-3-3 also resulted in enhanced cell invasion. Analysis of TCGA breast cancer patient data showed significant correlation for DNA hypomethylation and reduced patient survival with increased 14-3-3 expressions. Therefore, we suggest that 14-3-3 is a crucial reader of DNMT1pSer143 that regulates DNA methylation and altered gene expression that contributes to cell invasion. PMID:26553800

  14. Comparative analysis of the 14-3-3 gene and its expression in Echinococcus granulosus and Echinococcus multilocularis metacestodes.

    PubMed

    Siles-Lucas, M; Nunes, C P; Zaha, A

    2001-03-01

    It was suggested that the unlimited proliferative capacity of the Echinococcus multilocularis metacestode may be related to overproduction of the 14-3-3 protein. As is known, the proliferative capacities of E. granulosus and E. multilocularis metacestodes are very different. By comparing the expression levels of the 14-3-3 gene between in vitro-obtained E. granulosus and E. multilocularis metacestodes, we were able to provide experimental evidence of the potential relation between 14-3-3 over-expression and tumour-like growth in E. multilocularis metacestodes. RT-PCR and Northern blot experiments indicated that 14-3-3 expression level is about 4-fold higher in the E. multilocularis metacestode. This differential expression was confirmed both by immunoblotting and immunocytochemistry experiments, which allowed detection of the protein in the cyst wall from E. multilocularis but not in the cyst wall from E. granulosus. The alignment of the Echinococcus 14-3-3 cDNA sequence with known 14-3-3 isoforms from other organisms, grouped the parasite sequence into the tumour growth-related isoforms. The known relation between over-expression of some 14-3-3 isoforms and tumour-related processes, together with the present results, suggest that the Echinococcus 14-3-3 protein could be one of the molecules responsible for the differences between E. granulosus and E. multilocularis metacestode growth behaviour.

  15. Site-specific regulatory interaction between spinach leaf sucrose-phosphate synthase and 14-3-3 proteins

    NASA Technical Reports Server (NTRS)

    Toroser, D.; Athwal, G. S.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    We report an Mg2+-dependent interaction between spinach leaf sucrose-phosphate synthase (SPS) and endogenous 14-3-3 proteins, as evidenced by co-elution during gel filtration and co-immunoprecipitation. The content of 14-3-3s associated with an SPS immunoprecipitate was inversely related to activity, and was specifically reduced when tissue was pretreated with 5-aminoimidazole-4-carboxamide riboside, suggesting metabolite control in vivo. A synthetic phosphopeptide based on Ser-229 was shown by surface plasmon resonance to bind a recombinant plant 14-3-3, and addition of the phosphorylated SPS-229 peptide was found to stimulate the SPS activity of an SPS:14-3-3 complex. Taken together, the results suggest a regulatory interaction of 14-3-3 proteins with Ser-229 of SPS.

  16. Validation of 14-3-3 Protein as a Marker in Sporadic Creutzfeldt-Jakob Disease Diagnostic.

    PubMed

    Schmitz, Matthias; Ebert, Elisabeth; Stoeck, Katharina; Karch, André; Collins, Steven; Calero, Miguel; Sklaviadis, Theodor; Laplanche, Jean-Louis; Golanska, Ewa; Baldeiras, Ines; Satoh, Katsuya; Sanchez-Valle, Raquel; Ladogana, Anna; Skinningsrud, Anders; Hammarin, Anna-Lena; Mitrova, Eva; Llorens, Franc; Kim, Yong Sun; Green, Alison; Zerr, Inga

    2016-05-01

    At present, the testing of 14-3-3 protein in cerebrospinal fluid (CSF) is a standard biomarker test in suspected sporadic Creutzfeldt-Jakob disease (sCJD) diagnosis. Increasing 14-3-3 test referrals in CJD reference laboratories in the last years have led to an urgent need to improve established 14-3-3 test methods. The main result of our study was the validation of a commercially available 14-3-3 ELISA next to the commonly used Western blot method as a high-throughput screening test. Hereby, 14-3-3 protein expression was quantitatively analyzed in CSF of 231 sCJD and 2035 control patients. We obtained excellent sensitivity/specificity values of 88 and 96% that are comparable to the established Western blot method. Since standard protocols and preanalytical sample handling have become more important in routine diagnostic, we investigated in a further step the reproducibility and stability of 14-3-3 as a biomarker for human prion diseases. Ring trial data from 2009 to 2013 revealed an increase of Fleiss' kappa from 0.51 to 0.68 indicating an improving reliability of 14-3-3 protein detection. The stability of 14-3-3 protein under short-term and long-term storage conditions at various temperatures and after repeated freezing/thawing cycles was confirmed. Contamination of CSF samples with blood appears likely to be an important factor at a concentration of more than 2500 erythrocytes/μL. Hemolysis of erythrocytes with significant release of 14-3-3 protein started after 2 days at room temperature. We first define clear standards for the sample handling, short- and long-term storage of CSF samples as well as the handling of blood- contaminated samples which may result in artificially elevated CSF levels of 14-3-3.

  17. Histone H2B Monoubiquitination Mediated by HISTONE MONOUBIQUITINATION1 and HISTONE MONOUBIQUITINATION2 Is Involved in Anther Development by Regulating Tapetum Degradation-Related Genes in Rice1[OPEN

    PubMed Central

    Cao, Hong; Li, Xiaoying; Wang, Zhi; Ding, Meng; Sun, Yongzhen; Dong, Fengqin; Chen, Fengying; Liu, Li’an; Doughty, James; Li, Yong; Liu, Yong-Xiu

    2015-01-01

    Histone H2B monoubiquitination (H2Bub1) is an important regulatory mechanism in eukaryotic gene transcription and is essential for normal plant development. However, the function of H2Bub1 in reproductive development remains elusive. Here, we report rice (Oryza sativa) HISTONE MONOUBIQUITINATION1 (OsHUB1) and OsHUB2, the homologs of Arabidopsis (Arabidopsis thaliana) HUB1 and HUB2 proteins, which function as E3 ligases in H2Bub1, are involved in late anther development in rice. oshub mutants exhibit abnormal tapetum development and aborted pollen in postmeiotic anthers. Knockout of OsHUB1 or OsHUB2 results in the loss of H2Bub1 and a reduction in the levels of dimethylated lysine-4 on histone 3 (H3K4me2). Anther transcriptome analysis revealed that several key tapetum degradation-related genes including OsC4, rice Cysteine Protease1 (OsCP1), and Undeveloped Tapetum1 (UDT1) were down-regulated in the mutants. Further, chromatin immunoprecipitation assays demonstrate that H2Bub1 directly targets OsC4, OsCP1, and UDT1 genes, and enrichment of H2Bub1 and H3K4me2 in the targets is consistent to some degree. Our studies suggest that histone H2B monoubiquitination, mediated by OsHUB1 and OsHUB2, is an important epigenetic modification that in concert with H3K4me2, modulates transcriptional regulation of anther development in rice. PMID:26143250

  18. Cotton (Gossypium hirsutum) 14-3-3 proteins participate in regulation of fibre initiation and elongation by modulating brassinosteroid signalling.

    PubMed

    Zhou, Ying; Zhang, Ze-Ting; Li, Mo; Wei, Xin-Zheng; Li, Xiao-Jie; Li, Bing-Ying; Li, Xue-Bao

    2015-02-01

    Cotton (Gossypium hirsutum) fibre is an important natural raw material for textile industry in the world. Understanding the molecular mechanism of fibre development is important for the development of future cotton varieties with superior fibre quality. In this study, overexpression of Gh14-3-3L in cotton promoted fibre elongation, leading to an increase in mature fibre length. In contrast, suppression of expression of Gh14-3-3L, Gh14-3-3e and Gh14-3-3h in cotton slowed down fibre initiation and elongation. As a result, the mature fibres of the Gh14-3-3 RNAi transgenic plants were significantly shorter than those of wild type. This 'short fibre' phenotype of the 14-3-3 RNAi cotton could be partially rescued by application of 2,4-epibrassinolide (BL). Expression levels of the BR-related and fibre-related genes were altered in the Gh14-3-3 transgenic fibres. Furthermore, we identified Gh14-3-3 interacting proteins (including GhBZR1) in cotton. Site mutation assay revealed that Ser163 in GhBZR1 and Lys51/56/53 in Gh14-3-3L/e/h were required for Gh14-3-3-GhBZR1 interaction. Nuclear localization of GhBZR1 protein was induced by BR, and phosphorylation of GhBZR1 by GhBIN2 kinase was helpful for its binding to Gh14-3-3 proteins. Additionally, 14-3-3-regulated GhBZR1 protein may directly bind to GhXTH1 and GhEXP promoters to regulate gene expression for responding rapid fibre elongation. These results suggested that Gh14-3-3 proteins may be involved in regulating fibre initiation and elongation through their interacting with GhBZR1 to modulate BR signalling. Thus, our study provides the candidate intrinsic genes for improving fibre yield and quality by genetic manipulation.

  19. Molecular Characterization of the 14-3-3 Gene Family in Brachypodium distachyon L. Reveals High Evolutionary Conservation and Diverse Responses to Abiotic Stresses.

    PubMed

    Cao, Hui; Xu, Yuxing; Yuan, Linlin; Bian, Yanwei; Wang, Lihui; Zhen, Shoumin; Hu, Yingkao; Yan, Yueming

    2016-01-01

    The 14-3-3 gene family identified in all eukaryotic organisms is involved in a wide range of biological processes, particularly in resistance to various abiotic stresses. Here, we performed the first comprehensive study on the molecular characterization, phylogenetics, and responses to various abiotic stresses of the 14-3-3 gene family in Brachypodium distachyon L. A total of seven 14-3-3 genes from B. distachyon and 120 from five main lineages among 12 species were identified, which were divided into five well-conserved subfamilies. The molecular structure analysis showed that the plant 14-3-3 gene family is highly evolutionarily conserved, although certain divergence had occurred in different subfamilies. The duplication event investigation revealed that segmental duplication seemed to be the predominant form by which the 14-3-3 gene family had expanded. Moreover, seven critical amino acids were detected, which may contribute to functional divergence. Expression profiling analysis showed that BdGF14 genes were abundantly expressed in the roots, but showed low expression in the meristems. All seven BdGF14 genes showed significant expression changes under various abiotic stresses, including heavy metal, phytohormone, osmotic, and temperature stresses, which might play important roles in responses to multiple abiotic stresses mainly through participating in ABA-dependent signaling and reactive oxygen species-mediated MAPK cascade signaling pathways. In particular, BdGF14 genes generally showed upregulated expression in response to multiple stresses of high temperature, heavy metal, abscisic acid (ABA), and salicylic acid (SA), but downregulated expression under H2O2, NaCl, and polyethylene glycol (PEG) stresses. Meanwhile, dynamic transcriptional expression analysis of BdGF14 genes under longer treatments with heavy metals (Cd(2+), Cr(3+), Cu(2+), and Zn(2+)) and phytohormone (ABA) and recovery revealed two main expression trends in both roots and leaves: up

  20. Molecular Characterization of the 14-3-3 Gene Family in Brachypodium distachyon L. Reveals High Evolutionary Conservation and Diverse Responses to Abiotic Stresses

    PubMed Central

    Cao, Hui; Xu, Yuxing; Yuan, Linlin; Bian, Yanwei; Wang, Lihui; Zhen, Shoumin; Hu, Yingkao; Yan, Yueming

    2016-01-01

    The 14-3-3 gene family identified in all eukaryotic organisms is involved in a wide range of biological processes, particularly in resistance to various abiotic stresses. Here, we performed the first comprehensive study on the molecular characterization, phylogenetics, and responses to various abiotic stresses of the 14-3-3 gene family in Brachypodium distachyon L. A total of seven 14-3-3 genes from B. distachyon and 120 from five main lineages among 12 species were identified, which were divided into five well-conserved subfamilies. The molecular structure analysis showed that the plant 14-3-3 gene family is highly evolutionarily conserved, although certain divergence had occurred in different subfamilies. The duplication event investigation revealed that segmental duplication seemed to be the predominant form by which the 14-3-3 gene family had expanded. Moreover, seven critical amino acids were detected, which may contribute to functional divergence. Expression profiling analysis showed that BdGF14 genes were abundantly expressed in the roots, but showed low expression in the meristems. All seven BdGF14 genes showed significant expression changes under various abiotic stresses, including heavy metal, phytohormone, osmotic, and temperature stresses, which might play important roles in responses to multiple abiotic stresses mainly through participating in ABA-dependent signaling and reactive oxygen species-mediated MAPK cascade signaling pathways. In particular, BdGF14 genes generally showed upregulated expression in response to multiple stresses of high temperature, heavy metal, abscisic acid (ABA), and salicylic acid (SA), but downregulated expression under H2O2, NaCl, and polyethylene glycol (PEG) stresses. Meanwhile, dynamic transcriptional expression analysis of BdGF14 genes under longer treatments with heavy metals (Cd2+, Cr3+, Cu2+, and Zn2+) and phytohormone (ABA) and recovery revealed two main expression trends in both roots and leaves: up-down and up

  1. Phosphorylation-related modification at the dimer interface of 14-3-3ω dramatically alters monomer interaction dynamics.

    PubMed

    Denison, Fiona C; Gökirmak, Tufan; Ferl, Robert J

    2014-01-01

    14-3-3 proteins are generally believed to function as dimers in a broad range of eukaryotic signaling pathways. The consequences of altering dimer stability are not fully understood. Phosphorylation at Ser58 in the dimer interface of mammalian 14-3-3 isoforms has been reported to destabilise dimers. An equivalent residue, Ser62, is present across most Arabidopsis isoforms but the effects of phosphorylation have not been studied in plants. Here, we assessed the effects of phosphorylation at the dimer interface of Arabidopsis 14-3-3ω. Protein kinase A phosphorylated 14-3-3ω at Ser62 and also at a previously unreported residue, Ser67, resulting in a monomer-sized band on native-PAGE. Phosphorylation at Ser62 alone, or with additional Ser67 phosphorylation, was investigated using phosphomimetic versions of 14-3-3ω. In electrophoretic and chromatographic analyses, these mutants showed mobilities intermediate between dimers and monomers. Mobility was increased by detergents, by reducing protein concentration, or by increasing pH or temperature. Urea gradient gels showed complex structural transitions associated with alterations of dimer stability, including a previously unreported 14-3-3 aggregation phenomenon. Overall, our analyses showed that dimer interface modifications such as phosphorylation reduce dimer stability, dramatically affecting the monomer-dimer equilibrium and denaturation trajectory. These findings may have dramatic implications for 14-3-3 structure and function in vivo.

  2. 14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function

    PubMed Central

    Schoenwaelder, Simone M.; Darbousset, Roxane; Cranmer, Susan L.; Ramshaw, Hayley S.; Orive, Stephanie L.; Sturgeon, Sharelle; Yuan, Yuping; Yao, Yu; Krycer, James R.; Woodcock, Joanna; Maclean, Jessica; Pitson, Stuart; Zheng, Zhaohua; Henstridge, Darren C.; van der Wal, Dianne; Gardiner, Elizabeth E.; Berndt, Michael C.; Andrews, Robert K.; James, David E.; Lopez, Angel F.; Jackson, Shaun P.

    2016-01-01

    The 14-3-3 family of adaptor proteins regulate diverse cellular functions including cell proliferation, metabolism, adhesion and apoptosis. Platelets express numerous 14-3-3 isoforms, including 14-3-3ζ, which has previously been implicated in regulating GPIbα function. Here we show an important role for 14-3-3ζ in regulating arterial thrombosis. Interestingly, this thrombosis defect is not related to alterations in von Willebrand factor (VWF)–GPIb adhesive function or platelet activation, but instead associated with reduced platelet phosphatidylserine (PS) exposure and procoagulant function. Decreased PS exposure in 14-3-3ζ-deficient platelets is associated with more sustained levels of metabolic ATP and increased mitochondrial respiratory reserve, independent of alterations in cytosolic calcium flux. Reduced platelet PS exposure in 14-3-3ζ-deficient mice does not increase bleeding risk, but results in decreased thrombin generation and protection from pulmonary embolism, leading to prolonged survival. Our studies define an important role for 14-3-3ζ in regulating platelet bioenergetics, leading to decreased platelet PS exposure and procoagulant function. PMID:27670677

  3. Evolution of signal multiplexing by 14-3-3-binding 2R-ohnologue protein families in the vertebrates

    PubMed Central

    Tinti, Michele; Johnson, Catherine; Toth, Rachel; Ferrier, David E. K.; MacKintosh, Carol

    2012-01-01

    14-3-3 proteins regulate cellular responses to stimuli by docking onto pairs of phosphorylated residues on target proteins. The present study shows that the human 14-3-3-binding phosphoproteome is highly enriched in 2R-ohnologues, which are proteins in families of two to four members that were generated by two rounds of whole genome duplication at the origin of the vertebrates. We identify 2R-ohnologue families whose members share a ‘lynchpin’, defined as a 14-3-3-binding phosphosite that is conserved across members of a given family, and aligns with a Ser/Thr residue in pro-orthologues from the invertebrate chordates. For example, the human receptor expression enhancing protein (REEP) 1–4 family has the commonest type of lynchpin motif in current datasets, with a phosphorylatable serine in the –2 position relative to the 14-3-3-binding phosphosite. In contrast, the second 14-3-3-binding sites of REEPs 1–4 differ and are phosphorylated by different kinases, and hence the REEPs display different affinities for 14-3-3 dimers. We suggest a conceptual model for intracellular regulation involving protein families whose evolution into signal multiplexing systems was facilitated by 14-3-3 dimer binding to lynchpins, which gave freedom for other regulatory sites to evolve. While increased signalling complexity was needed for vertebrate life, these systems also generate vulnerability to genetic disorders. PMID:22870394

  4. Sporadic Creutzfeldt-Jakob disease diagnostic accuracy is improved by a new CSF ELISA 14-3-3γ assay.

    PubMed

    Leitão, M J; Baldeiras, I; Almeida, M R; Ribeiro, M H; Santos, A C; Ribeiro, M; Tomás, J; Rocha, S; Santana, I; Oliveira, C R

    2016-05-13

    Protein 14-3-3 is a reliable marker of rapid neuronal damage, specifically increased in cerebrospinal fluid (CSF) of sporadic Creutzfeldt-Jakob disease (sCJD) patients. Its detection is usually performed by Western Blot (WB), prone to methodological issues. Our aim was to evaluate the diagnostic performance of a recently developed quantitative enzyme-linked immunosorbent (ELISA) assay for 14-3-3γ, in comparison with WB and other neurodegeneration markers. CSF samples from 145 patients with suspicion of prion disease, later classified as definite sCJD (n=72) or Non-prion diseases (Non-CJD; n=73) comprised our population. 14-3-3 protein was determined by WB and ELISA. Total Tau (t-Tau) and phosphorylated Tau (p-Tau) were also evaluated. Apolipoprotein E gene (ApoE) and prionic protein gene (PRNP) genotyping was assessed. ELISA 14-3-3γ levels were significantly increased in sCJD compared to Non-CJD patients (p<0.001), showing very good accuracy (AUC=0.982; sensitivity=97%; specificity=94%), and matching WB results in 81% of all cases. It strongly correlated with t-Tau and p-Tau (p<0.0001), showing slightly higher specificity (14-3-3 WB - 63%; Tau - 90%; p-Tau/t-Tau ratio - 88%). From WB inconclusive results (n=44), ELISA 14-3-3γ correctly classified 41 patients. Additionally, logistic regression analysis selected ELISA 14-3-3γ as the best single predictive marker for sCJD (overall accuracy=93%). ApoE and PRNP genotypes did not influence ELISA 14-3-3γ levels. Despite specificity for 14-3-3γ isoform, ELISA results not only match WB evaluation but also help discrimination of inconclusive results. Our results therefore reinforce this assay as a single screening test, allowing higher sample throughput and unequivocal results. PMID:26940479

  5. Identification of 14-3-3 Proteins Phosphopeptide-Binding Specificity Using an Affinity-Based Computational Approach.

    PubMed

    Li, Zhao; Tang, Jijun; Guo, Fei

    2016-01-01

    The 14-3-3 proteins are a highly conserved family of homodimeric and heterodimeric molecules, expressed in all eukaryotic cells. In human cells, this family consists of seven distinct but highly homologous 14-3-3 isoforms. 14-3-3σ is the only isoform directly linked to cancer in epithelial cells, which is regulated by major tumor suppressor genes. For each 14-3-3 isoform, we have 1,000 peptide motifs with experimental binding affinity values. In this paper, we present a novel method for identifying peptide motifs binding to 14-3-3σ isoform. First, we propose a sampling criteria to build a predictor for each new peptide sequence. Then, we select nine physicochemical properties of amino acids to describe each peptide motif. We also use auto-cross covariance to extract correlative properties of amino acids in any two positions. Finally, we consider elastic net to predict affinity values of peptide motifs, based on ridge regression and least absolute shrinkage and selection operator (LASSO). Our method tests on the 1,000 known peptide motifs binding to seven 14-3-3 isoforms. On the 14-3-3σ isoform, our method has overall pearson-product-moment correlation coefficient (PCC) and root mean squared error (RMSE) values of 0.84 and 252.31 for N-terminal sublibrary, and 0.77 and 269.13 for C-terminal sublibrary. We predict affinity values of 16,000 peptide sequences and relative binding ability across six permutated positions similar with experimental values. We identify phosphopeptides that preferentially bind to 14-3-3σ over other isoforms. Several positions on peptide motifs are in the same amino acid category with experimental substrate specificity of phosphopeptides binding to 14-3-3σ. Our method is fast and reliable and is a general computational method that can be used in peptide-protein binding identification in proteomics research. PMID:26828594

  6. Transcriptional increase and misexpression of 14-3-3 epsilon in sea urchin embryos exposed to UV-B

    PubMed Central

    Russo, Roberta; Zito, Francesca; Costa, Caterina; Bonaventura, Rosa

    2010-01-01

    Members of the 14-3-3 protein family are involved in many important cellular events, including stress response, survival and apoptosis. Genes of the 14-3-3 family are conserved from plants to humans, and some members are responsive to UV radiation. Here, we report the isolation of the complete cDNA encoding the 14-3-3 epsilon isoform from Paracentrotus lividus sea urchin embryos, referred to as Pl14-3-3ε, and the phylogenetic relationship with other homologues described in different phyla. Pl14-3-3ε mRNA levels were measured by QPCR during development and found to increase from the mesenchyme blastula to the prism stage. In response to UV-B (312 nm) exposure, early stage embryos collected 2 h later showed a 2.3-fold (at 400 J/m2) and a 2.7-fold (at 800 J/m2) increase in Pl14-3-3ε transcript levels compared with controls. The spatial expression of Pl14-3-3ε mRNA, detected by whole mount in situ hybridization in both control and UV-B exposed embryos, harvested at late developmental stages, showed transcripts to be located in the archenteron of gastrula stage and widely distributed in all germ layers, respectively. The Pl14-3-3ε mRNA delocalization parallels the failure in archenteron elongation observed morphologically, as well as the lack of specific endoderm markers, investigated by indirect immuno-fluorescence on whole mount embryos. Results confirm the involvement of 14-3-3ε in the stress response elicited by UV-B and demonstrate, for the first time, its contribution at the transcriptional level in the sea urchin embryo. PMID:20607471

  7. G9a-mediated histone methylation regulates cadmium-induced male fertility damage in pubertal mice.

    PubMed

    Li, Min; Liu, Chuan; Yang, Lingling; Zhang, Lei; Chen, Chunhai; He, Mindi; Lu, Yonghui; Feng, Wei; Pi, Huifeng; Zhang, Yanwen; Zhong, Min; Yu, Zhengping; Zhou, Zhou

    2016-06-11

    Increasing evidence suggests that cadmium (Cd) is associated with male fertility damage. However, the effects of histone modification on Cd-induced male fertility damage remain obscure. This study aims to evaluate the roles of histone methylation mediated by euchromatin histone methyltransferase (EHMT2/G9a) in regulating Cd-induced male fertility damage. We exposed 4-week-old male C57BL/6J mice to Cd by intraperitoneal injection at 2mg/kg for 1, 3 and 5days. Our data showed that Cd exposure decreased the numbers of impregnated females and litter sizes, which was concomitant with sperm count reduction, histological changes in the cauda epididymal ducts and seminiferous epithelium, and testicular cell apoptosis as evaluated by terminal dUTP nick-end labeling (TUNEL) assay and immunoblotting with increased levels of cleaved caspase 3, PARP and Bax and a decreased level of Bcl-2. Cd-induced male fertility damage was accompanied by enhanced G9a activity followed by increased histone H3 lysine 9 monomethylation (H3K9me1) and dimethylation (H3K9me2) in testes. Furthermore, inhibition of G9a by BIX-01294 normalized H3K9me1 and H3K9me2 to basal levels and prevented Cd-induced male fertility damage and testicular cell apoptosis. Our present study revealed that G9a-mediated histone methylation plays a critical role in Cd-induced male fertility damage and testicular cell apoptosis. PMID:27060504

  8. PIWI homologs mediate Histone H4 mRNA localization to planarian chromatoid bodies

    PubMed Central

    Rouhana, Labib; Weiss, Jennifer A.; King, Ryan S.; Newmark, Phillip A.

    2014-01-01

    The well-known regenerative abilities of planarian flatworms are attributed to a population of adult stem cells called neoblasts that proliferate and differentiate to produce all cell types. A characteristic feature of neoblasts is the presence of large cytoplasmic ribonucleoprotein granules named chromatoid bodies, the function of which has remained largely elusive. This study shows that histone mRNAs are a common component of chromatoid bodies. Our experiments also demonstrate that accumulation of histone mRNAs, which is typically restricted to the S phase of eukaryotic cells, is extended during the cell cycle of neoblasts. The planarian PIWI homologs SMEDWI-1 and SMEDWI-3 are required for proper localization of germinal histone H4 (gH4) mRNA to chromatoid bodies. The association between histone mRNA and chromatoid body components extends beyond gH4 mRNA, since transcripts of other core histone genes were also found in these structures. Additionally, piRNAs corresponding to loci of every core histone type have been identified. Altogether, this work provides evidence that links PIWI proteins and chromatoid bodies to histone mRNA regulation in planarian stem cells. The molecular similarities between neoblasts and undifferentiated cells of other organisms raise the possibility that PIWI proteins might also regulate histone mRNAs in stem cells and germ cells of other metazoans. PMID:24903754

  9. The paradox of histone deacetylase inhibitor-mediated modulation of cellular responses to radiation.

    PubMed

    Karagiannis, Tom C; El-Osta, Assam

    2006-02-01

    Given the widespread use of radiotherapy in cancer, there has been a longstanding interest in the development of chemical compounds that can modify cellular responses to ionizing radiation. Additionally, recent terrorism threats suggesting attacks with 'dirty bombs' containing combinations of radioactive isotopes with conventional explosives, has increased the interest in compounds that can protect from radiation injury. Histone deacetylase inhibitors represent a new class of compounds that can modulate the effects of radiation. Research with histone deacetylase inhibitors has largely focussed on the consequences of their ability to alter gene transcription via histone acetylation and on their properties as anti-cancer agents. They have been shown to cause cell cycle and growth arrest, differentiation and in certain cases apoptosis in cell cultures and in vivo. In addition to their intrinsic anti-cancer properties, numerous studies have demonstrated that histone deacetylase inhibitors can modulate cellular responses to other toxicity-inducing modalities including ionizing radiation. The consensus is that histone deacetylase inhibitors markedly enhance the sensitivity of cells to radiation by altering numerous molecular pathways. Intriguingly, a report has also shown that histone deacetylase inhibitors can reduce radiation induced acute and late skin damage using a well-established animal model of cutaneous radiation syndrome. Hence, there is an emerging interest in potential use of histone deacetylase inhibitors as radiation sensitizers or protectors. This review focuses on the different mechanisms by which histone deacetylase inhibitors modify cellular responses to ionizing radiation. PMID:16418577

  10. Genome-Wide Identification, Classification, and Expression Analysis of 14-3-3 Gene Family in Populus

    PubMed Central

    Tian, Fengxia; Wang, Tan; Xie, Yuli; Zhang, Jin; Hu, Jianjun

    2015-01-01

    Background In plants, 14-3-3 proteins are encoded by a large multigene family and are involved in signaling pathways to regulate plant development and protection from stress. Although twelve Populus 14-3-3s were identified based on the Populus trichocarpa genome V1.1 in a previous study, no systematic analysis including genome organization, gene structure, duplication relationship, evolutionary analysis and expression compendium has been conducted in Populus based on the latest P. trichocarpa genome V3.0. Principal Findings Here, a comprehensive analysis of Populus 14-3-3 family is presented. Two new 14-3-3 genes were identified based on the latest P. trichocarpa genome. In P. trichocarpa, fourteen 14-3-3 genes were grouped into ε and non-ε group. Exon-intron organizations of Populus 14-3-3s are highly conserved within the same group. Genomic organization analysis indicated that purifying selection plays a pivotal role in the retention and maintenance of Populus 14-3-3 family. Protein conformational analysis indicated that Populus 14-3-3 consists of a bundle of nine α-helices (α1-α9); the first four are essential for formation of the dimer, while α3, α5, α7, and α9 form a conserved peptide-binding groove. In addition, α1, α3, α5, α7, and α9 were evolving at a lower rate, while α2, α4, and α6 were evolving at a relatively faster rate. Microarray analyses showed that most Populus 14-3-3s are differentially expressed across tissues and upon exposure to various stresses. Conclusions The gene structures and their coding protein structures of Populus 14-3-3s are highly conserved among group members, suggesting that members of the same group might also have conserved functions. Microarray and qRT-PCR analyses showed that most Populus 14-3-3s were differentially expressed in various tissues and were induced by various stresses. Our investigation provided a better understanding of the complexity of the 14-3-3 gene family in poplars. PMID:25867623

  11. Potential role for PADI-mediated histone citrullination in preimplantation development

    PubMed Central

    2012-01-01

    Background The peptidylarginine deiminases (PADIs) convert positively charged arginine residues to neutrally charged citrulline on protein substrates in a process that is known as citrullination or deimination. Previous reports have documented roles for histone citrullination in chromatin remodeling and gene regulation in several tissue types, however, a potential role for histone citrullination in chromatin-based activities during early embryogenesis has not been investigated. Results In the present study, we tested by laser scanning confocal indirect immunofluorescence microscopy whether specific arginine residues on the histone H3 and H4 N-terminal tails (H4R3, H3R2 + 8 + 17, and H3R26) were citrullinated in mouse oocytes and preimplantation embryos. Results showed that all of the tested residues were deiminated with each site showing a unique localization pattern during early development. Given these findings, we next tested whether inhibition of PADI activity using the PADI-specific inhibitor, Cl-amidine, may affect embryonic development. We found that treatment of pronuclear stage zygotes with Cl-amidine reduces both histone H3 and H4 tail citrullination and also potently blocks early cleavage divisions in vitro. Additionally, we found that the Cl-amidine treatment reduces acetylation at histone H3K9, H3K18, and H4K5 while having no apparent effect on the repressive histone H3K9 dimethylation modification. Lastly, we found that treatment of zygotes with trichostatin A (TSA) to induce hyperacetylation also resulted in an increase in histone citrullination at H3R2 + 8 + 17. Conclusions Given the observed effects of Cl-amidine on embryonic development and the well documented correlation between histone acetylation and transcriptional activation, our findings suggest that histone citrullination may play an important role in facilitating gene expression in early embryos by creating a chromatin environment that is permissive for histone acetylation

  12. 14-3-3 Proteins SGF14c and SGF14l Play Critical Roles during Soybean Nodulation1[W][OA

    PubMed Central

    Radwan, Osman; Wu, Xia; Govindarajulu, Manjula; Libault, Marc; Neece, David J.; Oh, Man-Ho; Berg, R. Howard; Stacey, Gary; Taylor, Christopher G.; Huber, Steven C.; Clough, Steven J.

    2012-01-01

    The soybean (Glycine max) genome contains 18 members of the 14-3-3 protein family, but little is known about their association with specific phenotypes. Here, we report that the Glyma0529080 Soybean G-box Factor 14-3-3c (SGF14c) and Glyma08g12220 (SGF14l) genes, encoding 14-3-3 proteins, appear to play essential roles in soybean nodulation. Quantitative reverse transcription-polymerase chain reaction and western-immunoblot analyses showed that SGF14c mRNA and protein levels were specifically increased in abundance in nodulated soybean roots 10, 12, 16, and 20 d after inoculation with Bradyrhizobium japonicum. To investigate the role of SGF14c during soybean nodulation, RNA interference was employed to silence SGF14c expression in soybean roots using Agrobacterium rhizogenes-mediated root transformation. Due to the paleopolyploid nature of soybean, designing a specific RNA interference sequence that exclusively targeted SGF14c was not possible. Therefore, two highly similar paralogs (SGF14c and SGF14l) that have been shown to function as dimers were silenced. Transcriptomic and proteomic analyses showed that mRNA and protein levels were significantly reduced in the SGF14c/SGF14l-silenced roots, and these roots exhibited reduced numbers of mature nodules. In addition, SGF14c/SGF14l-silenced roots contained large numbers of arrested nodule primordia following B. japonicum inoculation. Transmission electron microscopy further revealed that the host cytoplasm and membranes, except the symbiosome membrane, were severely degraded in the failed nodules. Altogether, transcriptomic, proteomic, and cytological data suggest a critical role of one or both of these 14-3-3 proteins in early development stages of soybean nodules. PMID:23060368

  13. Increased Histone Deacetylase Activity Involved in the Suppressed Invasion of Cancer Cells Survived from ALA-Mediated Photodynamic Treatment

    PubMed Central

    Li, Pei-Tzu; Tsai, Yi-Jane; Lee, Ming-Jen; Chen, Chin-Tin

    2015-01-01

    Previously, we have found that cancer cells survived from 5-Aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) have abnormal mitochondrial function and suppressed cellular invasiveness. Here we report that both the mRNA expression level and enzymatic activity of histone deacetylase (HDAC) were elevated in the PDT-derived variants with dysfunctional mitochondria. The activated HDAC deacetylated histone H3 and further resulted in the reduced migration and invasion, which correlated with the reduced expression of the invasion-related genes, matrix metalloproteinase 9 (MMP9), paternally expressed gene 1 (PEG1), and miR-355, the intronic miRNA. Using chromatin immunoprecipitation, we further demonstrate the reduced amount of acetylated histone H3 on the promoter regions of MMP9 and PEG1, supporting the down-regulation of these two genes in PDT-derived variants. These results indicate that HDAC activation induced by mitochondrial dysfunction could modulate the cellular invasiveness and its related gene expression. This argument was further verified in the 51-10 cybrid cells with the 4977 bp mtDNA deletion and A375 ρ0 cells with depleted mitochondria. These results indicate that mitochondrial dysfunction might suppress tumor invasion through modulating histone acetylation. PMID:26473836

  14. Histone deacetylase 4 possesses intrinsic nuclear import and export signals.

    PubMed

    Wang, A H; Yang, X J

    2001-09-01

    Nucleocytoplasmic trafficking of histone deacetylase 4 (HDAC4) plays an important role in regulating its function, and binding of 14-3-3 proteins is necessary for its cytoplasmic retention. Here, we report the identification of nuclear import and export sequences of HDAC4. While its N-terminal 118 residues modulate the nuclear localization, residues 244 to 279 constitute an authentic, strong nuclear localization signal. Mutational analysis of this signal revealed that three arginine-lysine clusters are necessary for its nuclear import activity. As for nuclear export, leucine-rich sequences located in the middle part of HDAC4 do not function as nuclear export signals. By contrast, a hydrophobic motif (MXXLXVXV) located at the C-terminal end serves as a nuclear export signal that is necessary for cytoplasmic retention of HDAC4. This motif is required for CRM1-mediated nuclear export of HDAC4. Furthermore, binding of 14-3-3 proteins promotes cytoplasmic localization of HDAC4 by both inhibiting its nuclear import and stimulating its nuclear export. Unlike wild-type HDAC4, a point mutant with abrogated MEF2-binding ability remains cytoplasmic upon exogenous expression of MEF2C, supporting the notion that direct MEF2 binding targets HDAC4 to the nucleus. Therefore, HDAC4 possesses intrinsic nuclear import and export signals for its dynamic nucleocytoplasmic shuttling, and association with 14-3-3 and MEF2 proteins affects such shuttling and thus directs HDAC4 to the cytoplasm and the nucleus, respectively.

  15. CRL4(WDR23)-Mediated SLBP Ubiquitylation Ensures Histone Supply during DNA Replication.

    PubMed

    Brodersen, Mia M L; Lampert, Fabienne; Barnes, Christopher A; Soste, Martin; Piwko, Wojciech; Peter, Matthias

    2016-05-19

    To maintain genome integrity and epigenetic information, mammalian cells must carefully coordinate the supply and deposition of histones during DNA replication. Here we report that the CUL4 E3 ubiquitin ligase complex CRL4(WDR23) directly regulates the stem-loop binding protein (SLBP), which orchestrates the life cycle of histone transcripts including their stability, maturation, and translation. Lack of CRL4(WDR23) activity is characterized by depletion of histones resulting in inhibited DNA replication and a severe slowdown of growth in human cells. Detailed analysis revealed that CRL4(WDR23) is required for efficient histone mRNA 3' end processing to produce mature histone mRNAs for translation. CRL4(WDR23) binds and ubiquitylates SLBP in vitro and in vivo, and this modification activates SLBP function in histone mRNA 3' end processing without affecting its protein levels. Together, these results establish a mechanism by which CUL4 regulates DNA replication and possible additional chromatin transactions by controlling the concerted expression of core histones.

  16. Dual phosphorylation of Btk by Akt/protein kinase b provides docking for 14-3-3ζ, regulates shuttling, and attenuates both tonic and induced signaling in B cells.

    PubMed

    Mohammad, Dara K; Nore, Beston F; Hussain, Alamdar; Gustafsson, Manuela O; Mohamed, Abdalla J; Smith, C I Edvard

    2013-08-01

    Bruton's tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in the Btk gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation.

  17. 14-3-3-dependent inhibition of the deubiquitinating activity of UBPY and its cancellation in the M phase

    SciTech Connect

    Mizuno, Emi; Kitamura, Naomi; Komada, Masayuki

    2007-10-01

    The deubiquitinating enzyme UBPY, also known as USP8, regulates cargo sorting and membrane traffic at early endosomes. Here we demonstrate the regulatory mechanism of the UBPY catalytic activity. We identified 14-3-3 {epsilon}, {gamma}, and {zeta} as UBPY-binding proteins using co-immunoprecipitation followed by mass spectrometric analysis. The 14-3-3 binding of UBPY was inhibited by mutating the consensus 14-3-3-binding motif RSYS{sup 680}SP, by phosphatase treatment, and by competition with the Ser{sup 680}-phosphorylated RSYS{sup 680}SP peptide. Metabolic labeling with [{sup 32}P]orthophosphate and immunoblotting using antibody against the phosphorylated 14-3-3-binding motif showed that Ser{sup 680} is a major phosphorylation site in UBPY. These results indicated that 14-3-3s bind to the region surrounding Ser{sup 680} in a phosphorylation-dependent manner. The mutation at Ser{sup 680} led to enhanced ubiquitin isopeptidase activity of UBPY toward poly-ubiquitin chains and a cellular substrate, epidermal growth factor receptor, in vitro and in vivo. Moreover, addition of 14-3-3{epsilon} inhibited the UBPY activity in vitro. Finally, UBPY was dephosphorylated at Ser{sup 680} and dissociated from 14-3-3s in the M phase, resulting in enhanced activity of UBPY during cell division. We conclude that UBPY is catalytically inhibited in a phosphorylation-dependent manner by 14-3-3s during the interphase, and this regulation is cancelled in the M phase.

  18. Changes in histone acetylation as potential mediators of pupal diapause in the flesh fly, Sarcophaga bullata.

    PubMed

    Reynolds, J A; Bautista-Jimenez, Robin; Denlinger, D L

    2016-09-01

    The growing appreciation that epigenetic processes are integral to the responses of many organisms to changes in the environment suggests a possible role for epigenetics in coordination of insect diapause. The results we present suggest that histone modification may be one type of epigenetic process that contributes to regulation of pupal diapause in the flesh fly, Sarcophaga bullata. Reduction in total histone H3 acetylation in diapausing pupae, shifts in mRNA expression profiles of genes encoding histone acetyltransferase (HAT) and histone deacetylase (HDAC) in pre-diapause, diapause and post-diapause flies compared to their nondiapause counterparts, and alterations in HDAC enzyme activity during and post-diapause lend support to the hypothesis that this specific type of histone modification is involved in regulating diapause programming, maintenance, and termination. Transcription of genes encoding HDAC1, HDAC3, HDAC6, and Sirtuin2 were all upregulated in photosensitive first instar larvae programmed to enter pupal diapause, suggesting that histone deacetylation may be linked to the early decision to enter diapause. A 50% reduction in transcription of hdac3 and a corresponding 30% reduction in HDAC activity during diapause suggest that removal of acetyl groups from histones primarily occurs prior to diapause entry and that further histone deacetylation is not necessary to maintain diapause. Transcription of the HDAC genes was quickly elevated when diapause was terminated, followed by an increase in enzyme activity after a short delay. A maternal effect operating in these flies prevents pupal diapause in progeny whose mothers experienced pupal diapause, even if the progeny are reared in strong diapause-inducing short-day conditions. Such nondiapausing pupae had HDAC transcription profiles nearly identical to the profiles seen in nondiapausing pupae generated under a long-day photoperiod. Together, these results provide consistent evidence for histone acetylation

  19. Changes in histone acetylation as potential mediators of pupal diapause in the flesh fly, Sarcophaga bullata.

    PubMed

    Reynolds, J A; Bautista-Jimenez, Robin; Denlinger, D L

    2016-09-01

    The growing appreciation that epigenetic processes are integral to the responses of many organisms to changes in the environment suggests a possible role for epigenetics in coordination of insect diapause. The results we present suggest that histone modification may be one type of epigenetic process that contributes to regulation of pupal diapause in the flesh fly, Sarcophaga bullata. Reduction in total histone H3 acetylation in diapausing pupae, shifts in mRNA expression profiles of genes encoding histone acetyltransferase (HAT) and histone deacetylase (HDAC) in pre-diapause, diapause and post-diapause flies compared to their nondiapause counterparts, and alterations in HDAC enzyme activity during and post-diapause lend support to the hypothesis that this specific type of histone modification is involved in regulating diapause programming, maintenance, and termination. Transcription of genes encoding HDAC1, HDAC3, HDAC6, and Sirtuin2 were all upregulated in photosensitive first instar larvae programmed to enter pupal diapause, suggesting that histone deacetylation may be linked to the early decision to enter diapause. A 50% reduction in transcription of hdac3 and a corresponding 30% reduction in HDAC activity during diapause suggest that removal of acetyl groups from histones primarily occurs prior to diapause entry and that further histone deacetylation is not necessary to maintain diapause. Transcription of the HDAC genes was quickly elevated when diapause was terminated, followed by an increase in enzyme activity after a short delay. A maternal effect operating in these flies prevents pupal diapause in progeny whose mothers experienced pupal diapause, even if the progeny are reared in strong diapause-inducing short-day conditions. Such nondiapausing pupae had HDAC transcription profiles nearly identical to the profiles seen in nondiapausing pupae generated under a long-day photoperiod. Together, these results provide consistent evidence for histone acetylation

  20. Akt Phosphorylates Connexin43 on Ser373, a “Mode-1” Binding Site for 14-3-3

    PubMed Central

    PARK, DARREN J.; WALLICK, CHRISTOPHER J.; MARTYN, KENDRA D.; LAU, ALAN F.; JIN, CHENGSHI; WARN-CRAMER, BONNIE J.

    2009-01-01

    Connexin43 (Cx43) is a membrane-spanning protein that forms channels that bridge the gap between adjacent cells and this allows for the intercellular exchange of information. Cx43 is regulated by phosphorylation and by interacting proteins. “Mode-1” interaction with 14-3-3 requires phosphorylation of Ser373 on Cx43 (Park et al. 2006). Akt phosphorylates and targets a number of proteins to interactions with 14-3-3. Here we demonstrate that Akt phosphorylates Cx43 on Ser373 and Ser369; antibodies recognizing Akt-phosphorylated sites or phospho-Ser “mode-1” 14-3-3-binding sites recognize a protein from EGF-treated cells that migrates as Cx43, and GST-14-3-3 binds to Cx43 phosphorylated endogenously in EGF-treated cells. Confocal microscopy supports the co-localization of Cx43 with Akt and with 14-3-3 at the outer edges of gap junctional plaques. These data suggest that Akt could target Cx43 to an interaction with 14-3-3 that may play a role in the forward trafficking of Cx43 multimers and/or their incorporation into existing gap junctional plaques. PMID:18163231

  1. Proteomic analysis of media from lung cancer cells reveals role of 14-3-3 proteins in cachexia

    PubMed Central

    McLean, Julie B.; Moylan, Jennifer S.; Horrell, Erin M. W.; Andrade, Francisco H.

    2015-01-01

    Aims: At the time of diagnosis, 60% of lung cancer patients present with cachexia, a severe wasting syndrome that increases morbidity and mortality. Tumors secrete multiple factors that contribute to cachectic muscle wasting, and not all of these factors have been identified. We used Orbitrap electrospray ionization mass spectrometry to identify novel cachexia-inducing candidates in media conditioned with Lewis lung carcinoma cells (LCM). Results: One-hundred and 58 proteins were confirmed in three biological replicates. Thirty-three were identified as secreted proteins, including 14-3-3 proteins, which are highly conserved adaptor proteins known to have over 200 binding partners. We confirmed the presence of extracellular 14-3-3 proteins in LCM via western blot and discovered that LCM contained less 14-3-3 content than media conditioned with C2C12 myotubes. Using a neutralizing antibody, we depleted extracellular 14-3-3 proteins in myotube culture medium, which resulted in diminished myosin content. We identified the proposed receptor for 14-3-3 proteins, CD13, in differentiated C2C12 myotubes and found that inhibiting CD13 via Bestatin also resulted in diminished myosin content. Conclusions: Our novel findings show that extracellular 14-3-3 proteins may act as previously unidentified myokines and may signal via CD13 to help maintain muscle mass. PMID:25972815

  2. 14-3-3{sigma} controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

    SciTech Connect

    Xin, Ying; Lu, Qingxian; Li, Qiutang

    2010-02-19

    14-3-3{sigma} (also called stratifin) is specifically expressed in the stratified squamous epithelium and its function was recently shown to be linked to epidermal stratification and differentiation in the skin. In this study, we investigated its role in corneal epithelium cell proliferation and differentiation. We showed that the 14-3-3{sigma} mutation in repeated epilation (Er) mutant mice results in a dominant negative truncated protein. Primary corneal epithelial cells expressing the dominant negative protein failed to undergo high calcium-induced cell cycle arrest and differentiation. We further demonstrated that blocking endogenous 14-3-3{sigma} activity in corneal epithelial cells by overexpressing dominative negative 14-3-3{sigma} led to reduced Notch activity and Notch1/2 transcription. Significantly, expression of the active Notch intracellular domain overcame the block in epithelial cell differentiation in 14-3-3{sigma} mutant-expressing corneal epithelial cells. We conclude that 14-3-3{sigma} is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

  3. 14-3-3ε and ζ Regulate Neurogenesis and Differentiation of Neuronal Progenitor Cells in the Developing Brain

    PubMed Central

    Wachi, Tomoka; Hunt, Robert F.; Baraban, Scott C.; Taya, Shinichiro; Ramshaw, Hayley; Kaibuchi, Kozo; Schwarz, Quenten P.; Lopez, Angel F.

    2014-01-01

    During brain development, neural progenitor cells proliferate and differentiate into neural precursors. These neural precursors migrate along the radial glial processes and localize at their final destination in the cortex. Numerous reports have revealed that 14-3-3 proteins are involved in many neuronal activities, although their functions in neurogenesis remain unclear. Here, using 14-3-3ε/ζ double knock-out mice, we found that 14-3-3 proteins are important for proliferation and differentiation of neural progenitor cells in the cortex, resulting in neuronal migration defects and seizures. 14-3-3 deficiency resulted in the increase of δ-catenin and the decrease of β-catenin and αN-catenin. 14-3-3 proteins regulated neuronal differentiation into neurons via direct interactions with phosphorylated δ-catenin to promote F-actin formation through a catenin/Rho GTPase/Limk1/cofilin signaling pathway. Conversely, neuronal migration defects seen in the double knock-out mice were restored by phosphomimic Ndel1 mutants, but not δ-catenin. Our findings provide new evidence that 14-3-3 proteins play important roles in neurogenesis and neuronal migration via the regulation of distinct signaling cascades. PMID:25186760

  4. Inhibition of different histone acetyltransferases (HATs) uncovers transcription-dependent and -independent acetylation-mediated mechanisms in memory formation.

    PubMed

    Merschbaecher, Katja; Hatko, Lucyna; Folz, Jennifer; Mueller, Uli

    2016-02-01

    Acetylation of histones changes the efficiency of the transcription processes and thus contributes to the formation of long-term memory (LTM). In our comparative study, we used two inhibitors to characterize the contribution of different histone acetyl transferases (HATs) to appetitive associative learning in the honeybee. For one we applied garcinol, an inhibitor of the HATs of the p300 (EP300 binding protein)/CBP (CREB-binding protein) family, and the HATs of the PCAF (p300/CBP-associated factor) family. As comparative agent we applied C646, a specific inhibitor that selectively blocks HATS of the p300/CBP family. Immunochemical analysis reveals differences in histone H3 acetylation in the honeybee brain, in response to the injection of either C646 or garcinol. Behavioral assessment reveals that the two drugs cause memory impairment of different nature when injected after associative conditioning: processes disturbed by garcinol are annihilated by the established transcription blocker actinomycin D and thus seem to require transcription processes. Actions of C646 are unaltered by actinomycin D, and thus seem to be independent of transcription. The outcome of our different approaches as summarized suggests that distinct HATs contribute to different acetylation-mediated processes in memory formation. We further deduce that the acetylation-mediated processes in memory formation comprise transcription-dependent and transcription-independent mechanisms.

  5. Histone Deacetylase Inhibition–Mediated Differentiation of RGC-5 Cells and Interaction with Survival

    PubMed Central

    Schwechter, Brandon R.; Millet, Lucia E.; Levin, Leonard A.

    2008-01-01

    PURPOSE The acetylation state of histones is modulated by histone deacetylase (HDAC) and histone acetyltransferase and is an important component in regulating gene transcription, including neuronal differentiation. The authors studied the relationship between histone acetylation and the differentiation and survival of the RGC-5 cell line and compared it with nontranscriptional-dependent differentiation with staurosporine. METHODS The retinal ganglion cell line RGC-5 was treated with trichostatin A (TSA), other HDAC inhibitors, and staurosporine; differentiation, neuritogenesis, neurotrophic factor dependence, and dependence on RNA transcription were assessed. RESULTS TSA caused significant differentiation and neuritogenesis. Differences between HDAC inhibition and staurosporine differentiation included the proportion of differentiated cells, cell viability, cell morphology, and transcriptional dependence. HDAC inhibition, but not staurosporine differentiation, resulted in RGC-5 cells that were neurotrophic factor dependent. CONCLUSIONS These results implicate two different mechanisms for RGC-5 differentiation, with a common downstream effect on neurite outgrowth but a differential effect on neurotrophic factor dependence. PMID:17525221

  6. Histone demethylase LSD1-mediated repression of GATA-2 is critical for erythroid differentiation

    PubMed Central

    Guo, Yidi; Fu, Xueqi; Jin, Yue; Sun, Jing; Liu, Ye; Huo, Bo; Li, Xiang; Hu, Xin

    2015-01-01

    Background The transcription factor GATA-2 is predominantly expressed in hematopoietic stem and progenitor cells and counteracts the erythroid-specific transcription factor GATA-1, to modulate the proliferation and differentiation of hematopoietic cells. During hematopoietic cell differentiation, GATA-2 exhibits dynamic expression patterns, which are regulated by multiple transcription factors. Methods Stable LSD1-knockdown cell lines were established by growing murine erythroleukemia (MEL) or mouse embryonic stem cells together with virus particles, in the presence of Polybrene® at 4 μg/mL, for 24–48 hours followed by puromycin selection (1 μg/mL) for 2 weeks. Real-time polymerase chain reaction (PCR)-based quantitative chromatin immunoprecipitation (ChIP) analysis was used to test whether the TAL1 transcription factor is bound to 1S promoter in the GATA-2 locus or whether LSD1 colocalizes with TAL1 at the 1S promoter. The sequential ChIP assay was utilized to confirm the role of LSD1 in the regulation of H3K4me2 at the GATA-2 locus during erythroid differentiation. Western blot analysis was employed to detect the protein expression. The alamarBlue® assay was used to examine the proliferation of the cells, and the absorbance was monitored at optical density (OD) 570 nm and OD 600 nm. Results In this study, we showed that LSD1 regulates the expression of GATA-2 during erythroid differentiation. Knockdown of LSD1 results in increased GATA-2 expression and inhibits the differentiation of MEL and embryonic stem cells. Furthermore, we demonstrated that LSD1 binds to the 1S promoter of the GATA-2 locus and suppresses GATA-2 expression, via histone demethylation. Conclusion Our data revealed that LSD1 mediates erythroid differentiation, via epigenetic modification of the GATA-2 locus. PMID:26124638

  7. CSF Tau proteins reduce misdiagnosis of sporadic Creutzfeldt-Jakob disease suspected cases with inconclusive 14-3-3 result.

    PubMed

    Leitão, M J; Baldeiras, I; Almeida, M R; Ribeiro, M H; Santos, A C; Ribeiro, M; Tomás, J; Rocha, S; Santana, I; Oliveira, C R

    2016-09-01

    Cerebrospinal fluid (CSF) 14-3-3 protein supports sporadic Creutzfeldt-Jakob (sCJD) diagnosis, but often leads to weak-positive results and lacks standardization. In this study, we explored the added diagnostic value of Total Tau (t-Tau) and phosphorylated Tau (p-Tau) in sCJD diagnosis, particularly in the cases with inconclusive 14-3-3 result. 95 definite sCJD and 287 patients without prion disease (non-CJD) were included in this study. CSF samples were collected in routine clinical diagnosis and analysed for 14-3-3 detection by Western blot (WB). CSF t-Tau and p-Tau were quantified by commercial ELISA kits and PRNP and APOE genotyping assessed by PCR-RFLP. In a regression analysis of the whole cohort, 14-3-3 protein revealed an overall accuracy of 82 % (sensitivity = 96.7 %; specificity = 75.6 %) for sCJD. Regarding 14-3-3 clear positive results, we observed no added value either of t-Tau alone or p-Tau/t-Tau ratio in the model. On the other hand, considering 14-3-3 weak-positive cases, t-Tau protein increased the overall accuracy of 14-3-3 alone from 91 to 94 % and specificity from 74 to 93 % (p < 0.05), with no sensitivity improvement. However, inclusion of p-Tau/t-Tau ratio did not significantly improve the first model (p = 0.0595). Globally, t-Tau protein allowed a further discrimination of 65 % within 14-3-3 inconclusive results. Furthermore, PRNP MV genotype showed a trend to decrease 14-3-3 sensitivity (p = 0.051), but such effect was not seen on t-Tau protein. In light of these results, we suggest that t-Tau protein assay is of significant importance as a second marker in identifying 14-3-3 false-positive results among sCJD probable cases.

  8. CSF Tau proteins reduce misdiagnosis of sporadic Creutzfeldt-Jakob disease suspected cases with inconclusive 14-3-3 result.

    PubMed

    Leitão, M J; Baldeiras, I; Almeida, M R; Ribeiro, M H; Santos, A C; Ribeiro, M; Tomás, J; Rocha, S; Santana, I; Oliveira, C R

    2016-09-01

    Cerebrospinal fluid (CSF) 14-3-3 protein supports sporadic Creutzfeldt-Jakob (sCJD) diagnosis, but often leads to weak-positive results and lacks standardization. In this study, we explored the added diagnostic value of Total Tau (t-Tau) and phosphorylated Tau (p-Tau) in sCJD diagnosis, particularly in the cases with inconclusive 14-3-3 result. 95 definite sCJD and 287 patients without prion disease (non-CJD) were included in this study. CSF samples were collected in routine clinical diagnosis and analysed for 14-3-3 detection by Western blot (WB). CSF t-Tau and p-Tau were quantified by commercial ELISA kits and PRNP and APOE genotyping assessed by PCR-RFLP. In a regression analysis of the whole cohort, 14-3-3 protein revealed an overall accuracy of 82 % (sensitivity = 96.7 %; specificity = 75.6 %) for sCJD. Regarding 14-3-3 clear positive results, we observed no added value either of t-Tau alone or p-Tau/t-Tau ratio in the model. On the other hand, considering 14-3-3 weak-positive cases, t-Tau protein increased the overall accuracy of 14-3-3 alone from 91 to 94 % and specificity from 74 to 93 % (p < 0.05), with no sensitivity improvement. However, inclusion of p-Tau/t-Tau ratio did not significantly improve the first model (p = 0.0595). Globally, t-Tau protein allowed a further discrimination of 65 % within 14-3-3 inconclusive results. Furthermore, PRNP MV genotype showed a trend to decrease 14-3-3 sensitivity (p = 0.051), but such effect was not seen on t-Tau protein. In light of these results, we suggest that t-Tau protein assay is of significant importance as a second marker in identifying 14-3-3 false-positive results among sCJD probable cases. PMID:27357003

  9. Recruitment of histone methyltransferase G9a mediates transcriptional repression of Fgf21 gene by E4BP4 protein.

    PubMed

    Tong, Xin; Zhang, Deqiang; Buelow, Katie; Guha, Anirvan; Arthurs, Blake; Brady, Hugh J M; Yin, Lei

    2013-02-22

    The liver responds to fasting-refeeding cycles by reprogramming expression of metabolic genes. Fasting potently induces one of the key hepatic hormones, fibroblast growth factor 21 (FGF21), to promote lipolysis, fatty acid oxidation, and ketogenesis, whereas refeeding suppresses its expression. We previously reported that the basic leucine zipper transcription factor E4BP4 (E4 binding protein 4) represses Fgf21 expression and disrupts its circadian oscillations in cultured hepatocytes. However, the epigenetic mechanism for E4BP4-dependent suppression of Fgf21 has not yet been addressed. Here we present evidence that histone methyltransferase G9a mediates E4BP4-dependent repression of Fgf21 during refeeding by promoting repressive histone modification. We find that Fgf21 expression is up-regulated in E4bp4 knock-out mouse liver. We demonstrate that the G9a-specific inhibitor BIX01294 abolishes suppression of the Fgf21 promoter activity by E4BP4, whereas overexpression of E4bp4 leads to increased levels of dimethylation of histone 3 lysine 9 (H3K9me2) around the Fgf21 promoter region. Furthermore, we also show that E4BP4 interacts with G9a, and knockdown of G9a blocks repression of Fgf21 promoter activity and expression in cells overexpressing E4bp4. A G9a mutant lacking catalytic activity, due to deletion of the SET domain, fails to inhibit the Fgf21 promoter activity. Importantly, acute hepatic knockdown by adenoviral shRNA targeting G9a abolishes Fgf21 repression by refeeding, concomitant with decreased levels of H3K9me2 around the Fgf21 promoter region. In summary, we show that G9a mediates E4BP4-dependent suppression of hepatic Fgf21 by enhancing histone methylation (H3K9me2) of the Fgf21 promoter. PMID:23283977

  10. The Peripheral Binding of 14-3-3γ to Membranes Involves Isoform-Specific Histidine Residues

    PubMed Central

    Ying, Ming; Halskau, Øyvind; Baumann, Anne; Rodriguez-Larrea, David; Costas, Miguel; Underhaug, Jarl; Sanchez-Ruiz, Jose M.; Martinez, Aurora

    2012-01-01

    Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states. PMID:23189152

  11. Identification and expression analysis of four 14-3-3 genes during fruit ripening in banana (Musa acuminata L. AAA group, cv. Brazilian).

    PubMed

    Li, Mei-Ying; Xu, Bi-Yu; Liu, Ju-Hua; Yang, Xiao-Liang; Zhang, Jian-Bin; Jia, Cai-Hong; Ren, Li-Cheng; Jin, Zhi-Qiang

    2012-02-01

    To investigate the regulation of 14-3-3 proteins in banana (Musa acuminata L. AAA group, cv. Brazilian) fruit postharvest ripening, four cDNAs encoding 14-3-3 proteins were isolated from banana and designated as Ma-14-3-3a, Ma-14-3-3c, Ma-14-3-3e, and Ma-14-3-3i, respectively. Amino acid sequence alignment showed that the four 14-3-3 proteins shared a highly conserved core structure and variable C-terminal as well as N-terminal regions with 14-3-3 proteins from other plant species. Phylogenetic analysis revealed that the four 14-3-3 genes belong to the non-ε groups. They were differentially and specifically expressed in various tissues. Real-time RT-PCR analysis indicated that these four genes function differentially during banana fruit postharvest ripening. Three genes, Ma-14-3-3a, Ma-14-3-3c, and Ma-14-3-3e, were significantly induced by exogenous ethylene treatment. However, gene function differed in naturally ripened fruits. Ethylene could induce Ma-14-3-3c expression during postharvest ripening, but expression patterns of Ma-14-3-3a and Ma-14-3-3e suggest that these two genes appear to be involved in regulating ethylene biosynthesis during fruit ripening. No obvious relationship emerged between Ma-14-3-3i expression in naturally ripened and 1-MCP (1-methylcyclopropene)-treated fruit groups during fruit ripening. These results indicate that the 14-3-3 proteins might be involved in various regulatory processes of banana fruit ripening. Further studies will mainly focus on revealing the detailed biological mechanisms of these four 14-3-3 genes in regulating banana fruit postharvest ripening. PMID:22009053

  12. 14-3-3 protein binds to the low molecular weight neurofilament (NFL) mRNA 3' UTR.

    PubMed

    Ge, Wei-Wen; Volkening, Kathryn; Leystra-Lantz, Cheryl; Jaffe, Howard; Strong, Michael J

    2007-01-01

    We have previously reported that altered stability of low molecular weight neurofilament (NFL) mRNA in lumbar spinal cord homogenates in amyotrophic lateral sclerosis (ALS) is associated with altered expression of trans-acting 3' UTR mRNA binding proteins. We have identified two hexanucleotide motifs as the main cis elements and, using LC/MS/MS of peptide digests of NFL 3' UTR interacting proteins from human spinal cord, observed that 14-3-3 proteins interact with these motifs. 14-3-3 beta, zeta, tau, gamma, and eta isoforms were found to be expressed in human spinal cord. Each isoform was expressed in vitro and shown to interact with NFL 3' UTR mRNA. Mutation of one or both motifs resulted in decreased 14-3-3 interaction, changes in predicted mRNA structure or alteration in stability of the mRNA. These data show a novel interaction for 14-3-3 with NFL mRNA, and suggests that 14-3-3 may play a role in regulating NFL mRNA stability.

  13. Functions of OsBZR1 and 14-3-3 proteins in brassinosteroid signaling in rice

    PubMed Central

    Bai, Ming-Yi; Zhang, Li-Ying; Gampala, Srinivas S.; Zhu, Sheng-Wei; Song, Wen-Yuan; Chong, Kang; Wang, Zhi-Yong

    2007-01-01

    Brassinosteroids (BR) are essential growth hormones found throughout the plant kingdom. BR bind to the receptor kinase BRI1 on the cell surface to activate a signal transduction pathway that regulates nuclear gene expression and plant growth. To understand the downstream BR signaling mechanism in rice, we studied the function of OsBZR1 using reverse genetic approaches and identified OsBZR1-interacting proteins. Suppressing OsBZR1 expression by RNAi resulted in dwarfism, erect leaves, reduced BR sensitivity, and altered BR-responsive gene expression in transgenic rice plants, demonstrating an essential role of OsBZR1 in BR responses in rice. Moreover, a yeast two-hybrid screen identified 14-3-3 proteins as OsBZR1-interacting proteins. Mutation of a putative 14-3-3-binding site of OsBZR1 abolished its interaction with the 14-3-3 proteins in yeast and in vivo. Such mutant OsBZR1 proteins suppressed the phenotypes of the Arabidopsis bri1–5 mutant and showed an increased nuclear distribution compared with the wild-type protein, suggesting that 14-3-3 proteins directly inhibit OsBZR1 function at least in part by reducing its nuclear localization. These results demonstrate a conserved function of OsBZR1 and an important role of 14-3-3 proteins in brassinosteroid signal transduction in rice. PMID:17699623

  14. Interactome analysis of the six cotton 14-3-3s that are preferentially expressed in fibres and involved in cell elongation

    PubMed Central

    Zhang, Ze-Ting; Zhou, Ying; Li, Yang; Shao, Su-Qiang; Li, Bing-Ying; Shi, Hai-Yan; Li, Xue-Bao

    2010-01-01

    Proteins of the 14-3-3 family regulate a divergent set of signalling pathways in all eukaryotic organisms. In this study, several cDNAs encoding 14-3-3 proteins were isolated from a cotton fibre cDNA library. The Gh14-3-3 genes share high sequence homology at the nucleotide level in the coding region and at the amino acid level. Real-time quantitative RT-PCR analysis indicated that the expression of these Gh14-3-3 genes is developmentally regulated in fibres, and reached their peak at the stage of rapid cell elongation of fibre development. Furthermore, overexpression of Gh14-3-3a, Gh14-3-3e, and Gh14-3-3L in fission yeast promoted atypical longitudinal growth of the host cells. Yeast two-hybrid analysis revealed that the interaction between cotton 14-3-3 proteins is isoform selective. Through yeast two-hybrid screening, 38 novel interaction partners of the six 14-3-3 proteins (Gh14-3-3a, Gh14-3-3e, Gh14-3-3f, Gh14-3-3g, Gh14-3-3h, and Gh14-3-3L), which are involved in plant development, metabolism, signalling transduction, and other cellular processes, were identified in cotton fibres. Taking these data together, it is proposed that the Gh14-3-3 proteins may participate in regulation of fibre cell elongation. Thus, the results of this study provide novel insights into the 14-3-3 signalling related to fibre development of cotton. PMID:20519337

  15. Histone H4 Lys 20 methyltransferase SET8 promotes androgen receptor-mediated transcription activation in prostate cancer

    SciTech Connect

    Yao, Lushuai; Li, Yanyan; Du, Fengxia; Han, Xiao; Li, Xiaohua; Niu, Yuanjie; Ren, Shancheng; Sun, Yingli

    2014-07-18

    Highlights: • Dihydrotestosterone stimulates H4K20me1 enrichment at the PSA promoter. • SET8 promotes AR-mediated transcription activation. • SET8 interacts with AR and promotes cell proliferation. - Abstract: Histone methylation status in different lysine residues has an important role in transcription regulation. The effect of H4K20 monomethylation (H4K20me1) on androgen receptor (AR)-mediated gene transcription remains unclear. Here we show that AR agonist stimulates the enrichment of H4K20me1 and SET8 at the promoter of AR target gene PSA in an AR dependent manner. Furthermore, SET8 is crucial for the transcription activation of PSA. Co-immunoprecipitation analyses demonstrate that SET8 interacts with AR. Therefore, we conclude that SET8 is involved in AR-mediated transcription activation, possibly through its interaction with AR and H4K20me1 modification.

  16. Histone methylase MLL1 and MLL3 coordinate with estrogen receptors in estrogen-mediated HOXB9 expression

    PubMed Central

    Ansari, Khairul I.; Shrestha, Bishakha; Hussain, Imran; Kasiri, Sahba; Mandal, Subhrangsu S.

    2011-01-01

    Homeobox gene HOXB9 is a critical player in development of mammary gland and sternum and in regulation of Renin which is closely linked with blood pressure control. Our studies demonstrated that HOXB9 gene is transcriptionally regulated by estrogen (E2). HOXB9 promoter contains several estrogen-response elements (ERE). Reporter assay based experiments demonstrated that HOXB9 promoter EREs are estrogen-responsive. Estrogen receptors ERα and ERβ are essential for E2-mediated transcriptional activation of HOXB9. Chromatin immuno-precipitation assay demonstrated that ERs bind to HOXB9 EREs as a function of E2. Similarly, histone methylases MLL1 and MLL3 also bind to HOXB9 EREs and play critical role in E2-mediated transcriptional activation of HOXB9. Overall, our studies demonstrated that HOXB9 is an E2-responsive gene and ERs coordinate with MLL1 and MLL3 in E2-mediated transcriptional regulation of HOXB9. PMID:21428455

  17. The histone deacetylase SIRT6 controls embryonic stem cell fate via TET-mediated production of 5-hydroxymethylcytosine.

    PubMed

    Etchegaray, Jean-Pierre; Chavez, Lukas; Huang, Yun; Ross, Kenneth N; Choi, Jiho; Martinez-Pastor, Barbara; Walsh, Ryan M; Sommer, Cesar A; Lienhard, Matthias; Gladden, Adrianne; Kugel, Sita; Silberman, Dafne M; Ramaswamy, Sridhar; Mostoslavsky, Gustavo; Hochedlinger, Konrad; Goren, Alon; Rao, Anjana; Mostoslavsky, Raul

    2015-05-01

    How embryonic stem cells (ESCs) commit to specific cell lineages and yield all cell types of a fully formed organism remains a major question. ESC differentiation is accompanied by large-scale histone and DNA modifications, but the relations between these epigenetic categories are not understood. Here we demonstrate the interplay between the histone deacetylase sirtuin 6 (SIRT6) and the ten-eleven translocation enzymes (TETs). SIRT6 targets acetylated histone H3 at Lys 9 and 56 (H3K9ac and H3K56ac), while TETs convert 5-methylcytosine into 5-hydroxymethylcytosine (5hmC). ESCs derived from Sirt6 knockout (S6KO) mice are skewed towards neuroectoderm development. This phenotype involves derepression of OCT4, SOX2 and NANOG, which causes an upregulation of TET-dependent production of 5hmC. Genome-wide analysis revealed neural genes marked with 5hmC in S6KO ESCs, thereby implicating TET enzymes in the neuroectoderm-skewed differentiation phenotype. We demonstrate that SIRT6 functions as a chromatin regulator safeguarding the balance between pluripotency and differentiation through Tet-mediated production of 5hmC.

  18. The histone acetyltransferase PsGcn5 mediates oxidative stress responses and is required for full virulence of Phytophthora sojae.

    PubMed

    Zhao, Wei; Wang, Tao; Liu, Shusen; Chen, Qingqing; Qi, Rende

    2015-10-01

    In eukaryotic organisms, histone acetyltransferase complexes are coactivators that are important for transcriptional activation by modifying chromatin. In this study, a gene (PsGcn5) from Phytophthora sojae encoding a histone acetyltransferase was identified as a homolog of one component of the histone acetyltransferase complex from yeasts to mammals. PsGcn5 was constitutively expressed in each stage tested, but had a slightly higher expression in sporulating hyphae and 3 h after infection. PsGcn5-silenced mutants were generated using polyethylene glycol-mediated protoplast stable transformation. These mutants had normal development, but compared to wild type strains they had higher sensitivity to hydrogen peroxide (H2O2) and significantly reduced virulence in soybean. Diaminobenzidine staining revealed an accumulation of H2O2 around the infection sites of PsGcn5-silenced mutants but not for wild type strains. Inhibition of the plant NADPH oxidase by diphenyleneiodonium prevented host-derived H2O2 accumulation in soybean cells and restored infectious hyphal growth of the mutants. Thus, we concluded that PsGcn5 is important for growth under conditions of oxidative stress and contributes to the full virulence of P. sojae by suppressing the host-derived reactive oxygen species.

  19. RAG1-mediated ubiquitylation of histone H3 is required for chromosomal V(D)J recombination

    PubMed Central

    Deng, Zimu; Liu, Haifeng; Liu, Xiaolong

    2015-01-01

    RAG1 and RAG2 proteins are key components in V(D)J recombination. The core region of RAG1 is capable of catalyzing the recombination reaction; however, the biological function of non-core RAG1 remains largely unknown. Here, we show that in a murine-model carrying the RAG1 ring-finger conserved cysteine residue mutation (C325Y), V(D)J recombination was abrogated at the cleavage step, and this effect was accompanied by decreased mono-ubiquitylation of histone H3. Further analyses suggest that un-ubiquitylated histone H3 restrains RAG1 to the chromatin by interacting with the N-terminal 218 amino acids of RAG1. Our data provide evidence for a model in which ubiquitylation of histone H3 mediated by the ring-finger domain of RAG1 triggers the release of RAG1, thus allowing its transition into the cleavage phase. Collectively, our findings reveal that the non-core region of RAG1 facilitates chromosomal V(D)J recombination in a ubiquitylation-dependent pathway. PMID:25572281

  20. An Analysis of CAF-1-interacting Proteins Reveals Dynamic and Direct Interactions with the KU Complex and 14-3-3 Proteins*

    PubMed Central

    Hoek, Maarten; Myers, Michael P.; Stillman, Bruce

    2011-01-01

    CAF-1 is essential in human cells for the de novo deposition of histones H3 and H4 at the DNA replication fork. Depletion of CAF-1 from various cell lines causes replication fork arrest, activation of the intra-S phase checkpoint, and global defects in chromatin structure. CAF-1 is also involved in coordinating inheritance of states of gene expression and in chromatin assembly following DNA repair. In this study, we generated cell lines expressing RNAi-resistant versions of CAF-1 and showed that the N-terminal 296 amino acids are dispensable for essential CAF-1 function in vivo. N-terminally truncated CAF-1 p150 was deficient in proliferating cell nuclear antigen (PCNA) binding, reinforcing the existence of two PCNA binding sites in human CAF-1, but the defect in PCNA binding had no effect on the recruitment of CAF-1 to chromatin after DNA damage or to resistance to DNA-damaging agents. Tandem affinity purification of CAF-1-interacting proteins under mild conditions revealed that CAF-1 was directly associated with the KU70/80 complex, part of the DNA-dependent protein kinase, and the phosphoserine/threonine-binding protein 14-3-3 ζ. CAF-1 was a substrate for DNA-dependent protein kinase, and the 14-3-3 interaction in vitro is dependent on DNA-dependent protein kinase phosphorylation. These results highlight that CAF-1 has prominent interactions with the DNA repair machinery but that the N terminus is dispensable for the role of CAF-1 in DNA replication- and repair-coupled chromatin assembly. PMID:21209461

  1. Nicotine Suppressed Fetal Adrenal StAR Expression via YY1 Mediated-Histone Deacetylation Modification Mechanism.

    PubMed

    Liu, Lian; Wang, Jian-Fei; Fan, Jie; Rao, Yi-Song; Liu, Fang; Yan, You-E; Wang, Hui

    2016-01-01

    Steroidogenic acute regulatory (StAR) protein plays a pivotal role in steroidogenesis. Previously, we have demonstrated that prenatal nicotine exposure suppressed fetal adrenal steroidogenesis via steroidogenic factor 1 deacetylation. This study further explored the potential role of the transcriptional repressor Yin Yang 1 (YY1) in nicotine-mediated StAR inhibition. Nicotine was subcutaneously administered (1.0 mg/kg) to pregnant rats twice per day and NCI-H295A cells were treated with nicotine. StAR and YY1 expression were analyzed by real-time PCR, immunohistochemistry, and Western blotting. Histone modifications and the interactions between the YY1 and StAR promoter were assessed using chromatin immunoprecipitation (ChIP). Prenatal nicotine exposure increased YY1 expression and suppressed StAR expression. ChIP assay showed that there was a decreasing trend for histone acetylation at the StAR promoter in fetal adrenal glands, whereas H3 acetyl-K14 at the YY1 promoter presented an increasing trend following nicotine exposure. Furthermore, in nicotine-treated NCI-H295A cells, nicotine enhanced YY1 expression and inhibited StAR expression. ChIP assay showed that histone acetylation decreased at the StAR promoter in NCI-H295A cells and that the interaction between the YY1 and StAR promoter increased. These data indicated that YY1-medicated histone deacetylation modification in StAR promoters might play an important role in the inhibitory effect of nicotine on StAR expression. PMID:27598153

  2. Nicotine Suppressed Fetal Adrenal StAR Expression via YY1 Mediated-Histone Deacetylation Modification Mechanism.

    PubMed

    Liu, Lian; Wang, Jian-Fei; Fan, Jie; Rao, Yi-Song; Liu, Fang; Yan, You-E; Wang, Hui

    2016-09-03

    Steroidogenic acute regulatory (StAR) protein plays a pivotal role in steroidogenesis. Previously, we have demonstrated that prenatal nicotine exposure suppressed fetal adrenal steroidogenesis via steroidogenic factor 1 deacetylation. This study further explored the potential role of the transcriptional repressor Yin Yang 1 (YY1) in nicotine-mediated StAR inhibition. Nicotine was subcutaneously administered (1.0 mg/kg) to pregnant rats twice per day and NCI-H295A cells were treated with nicotine. StAR and YY1 expression were analyzed by real-time PCR, immunohistochemistry, and Western blotting. Histone modifications and the interactions between the YY1 and StAR promoter were assessed using chromatin immunoprecipitation (ChIP). Prenatal nicotine exposure increased YY1 expression and suppressed StAR expression. ChIP assay showed that there was a decreasing trend for histone acetylation at the StAR promoter in fetal adrenal glands, whereas H3 acetyl-K14 at the YY1 promoter presented an increasing trend following nicotine exposure. Furthermore, in nicotine-treated NCI-H295A cells, nicotine enhanced YY1 expression and inhibited StAR expression. ChIP assay showed that histone acetylation decreased at the StAR promoter in NCI-H295A cells and that the interaction between the YY1 and StAR promoter increased. These data indicated that YY1-medicated histone deacetylation modification in StAR promoters might play an important role in the inhibitory effect of nicotine on StAR expression.

  3. Nicotine Suppressed Fetal Adrenal StAR Expression via YY1 Mediated-Histone Deacetylation Modification Mechanism

    PubMed Central

    Liu, Lian; Wang, Jian-Fei; Fan, Jie; Rao, Yi-Song; Liu, Fang; Yan, You-E; Wang, Hui

    2016-01-01

    Steroidogenic acute regulatory (StAR) protein plays a pivotal role in steroidogenesis. Previously, we have demonstrated that prenatal nicotine exposure suppressed fetal adrenal steroidogenesis via steroidogenic factor 1 deacetylation. This study further explored the potential role of the transcriptional repressor Yin Yang 1 (YY1) in nicotine-mediated StAR inhibition. Nicotine was subcutaneously administered (1.0 mg/kg) to pregnant rats twice per day and NCI-H295A cells were treated with nicotine. StAR and YY1 expression were analyzed by real-time PCR, immunohistochemistry, and Western blotting. Histone modifications and the interactions between the YY1 and StAR promoter were assessed using chromatin immunoprecipitation (ChIP). Prenatal nicotine exposure increased YY1 expression and suppressed StAR expression. ChIP assay showed that there was a decreasing trend for histone acetylation at the StAR promoter in fetal adrenal glands, whereas H3 acetyl-K14 at the YY1 promoter presented an increasing trend following nicotine exposure. Furthermore, in nicotine-treated NCI-H295A cells, nicotine enhanced YY1 expression and inhibited StAR expression. ChIP assay showed that histone acetylation decreased at the StAR promoter in NCI-H295A cells and that the interaction between the YY1 and StAR promoter increased. These data indicated that YY1-medicated histone deacetylation modification in StAR promoters might play an important role in the inhibitory effect of nicotine on StAR expression. PMID:27598153

  4. Histone chaperones link histone nuclear import and chromatin assembly.

    PubMed

    Keck, Kristin M; Pemberton, Lucy F

    2013-01-01

    Histone chaperones are proteins that shield histones from nonspecific interactions until they are assembled into chromatin. After their synthesis in the cytoplasm, histones are bound by different histone chaperones, subjected to a series of posttranslational modifications and imported into the nucleus. These evolutionarily conserved modifications, including acetylation and methylation, can occur in the cytoplasm, but their role in regulating import is not well understood. As part of histone import complexes, histone chaperones may serve to protect the histones during transport, or they may be using histones to promote their own nuclear localization. In addition, there is evidence that histone chaperones can play an active role in the import of histones. Histone chaperones have also been shown to regulate the localization of important chromatin modifying enzymes. This review is focused on the role histone chaperones play in the early biogenesis of histones, the distinct cytoplasmic subcomplexes in which histone chaperones have been found in both yeast and mammalian cells and the importins/karyopherins and nuclear localization signals that mediate the nuclear import of histones. We also address the role that histone chaperone localization plays in human disease. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.

  5. Drosophila 14-3-3ε has a crucial role in anti-microbial peptide secretion and innate immunity.

    PubMed

    Shandala, Tetyana; Woodcock, Joanna M; Ng, Yeap; Biggs, Lisa; Skoulakis, Efthimios M C; Brooks, Doug A; Lopez, Angel F

    2011-07-01

    The secretion of anti-microbial peptides is recognised as an essential step in innate immunity, but there is limited knowledge of the molecular mechanism controlling the release of these effectors from immune response cells. Here, we report that Drosophila 14-3-3ε mutants exhibit reduced survival when infected with either Gram-positive or Gram-negative bacteria, indicating a functional role for 14-3-3ε in innate immunity. In 14-3-3ε mutants, there was a reduced release of the anti-microbial peptide Drosomycin into the haemolymph, which correlated with an accumulation of Drosomycin-containing vesicles near the plasma membrane of cells isolated from immune response tissues. Drosomycin appeared to be delivered towards the plasma membrane in Rab4- and Rab11-positive vesicles and smaller Rab11-positive vesicles. RNAi silencing of Rab11 and Rab4 significantly blocked the anterograde delivery of Drosomycin from the perinuclear region to the plasma membrane. However, in 14-3-3ε mutants there was an accumulation of small Rab11-positive vesicles near the plasma membrane. This vesicular phenotype was similar to that observed in response to the depletion of the vesicular Syntaxin protein Syx1a. In wild-type Drosophila immune tissue, 14-3-3ε was detected adjacent to Rab11, and partially overlapping with Syx1a, on vesicles near the plasma membrane. We conclude that 14-3-3ε is required for Rab11-positive vesicle function, which in turn enables antimicrobial peptide secretion during an innate immune response.

  6. Class-Specific Evolution and Transcriptional Differentiation of 14-3-3 Family Members in Mesohexaploid Brassica rapa

    PubMed Central

    Chandna, Ruby; Augustine, Rehna; Kanchupati, Praveena; Kumar, Roshan; Kumar, Pawan; Arya, Gulab C.; Bisht, Naveen C.

    2016-01-01

    14-3-3s are highly conserved, multigene family proteins that have been implicated in modulating various biological processes. The presence of inherent polyploidy and genome complexity has limited the identification and characterization of 14-3-3 proteins from globally important Brassica crops. Through data mining of Brassica rapa, the model Brassica genome, we identified 21 members encoding 14-3-3 proteins namely, BraA.GRF14.a to BraA.GRF14.u. Phylogenetic analysis indicated that B. rapa contains both ε (epsilon) and non-ε 14-3-3 isoforms, having distinct intron-exon structural organization patterns. The non-ε isoforms showed lower divergence rate (Ks < 0.45) compared to ε protein isoforms (Ks > 0.48), suggesting class-specific divergence pattern. Synteny analysis revealed that mesohexaploid B. rapa genome has retained 1–5 orthologs of each Arabidopsis 14-3-3 gene, interspersed across its three fragmented sub-genomes. qRT-PCR analysis showed that 14 of the 21 BraA.GRF14 were expressed, wherein a higher abundance of non-ε transcripts was observed compared to the ε genes, indicating class-specific transcriptional bias. The BraA.GRF14 genes showed distinct expression pattern during plant developmental stages and in response to abiotic stress, phytohormone treatments, and nutrient deprivation conditions. Together, the distinct expression pattern and differential regulation of BraA.GRF14 genes indicated the occurrence of functional divergence of B. rapa 14-3-3 proteins during plant development and stress responses. PMID:26858736

  7. Class-Specific Evolution and Transcriptional Differentiation of 14-3-3 Family Members in Mesohexaploid Brassica rapa.

    PubMed

    Chandna, Ruby; Augustine, Rehna; Kanchupati, Praveena; Kumar, Roshan; Kumar, Pawan; Arya, Gulab C; Bisht, Naveen C

    2016-01-01

    14-3-3s are highly conserved, multigene family proteins that have been implicated in modulating various biological processes. The presence of inherent polyploidy and genome complexity has limited the identification and characterization of 14-3-3 proteins from globally important Brassica crops. Through data mining of Brassica rapa, the model Brassica genome, we identified 21 members encoding 14-3-3 proteins namely, BraA.GRF14.a to BraA.GRF14.u. Phylogenetic analysis indicated that B. rapa contains both ε (epsilon) and non-ε 14-3-3 isoforms, having distinct intron-exon structural organization patterns. The non-ε isoforms showed lower divergence rate (Ks < 0.45) compared to ε protein isoforms (Ks > 0.48), suggesting class-specific divergence pattern. Synteny analysis revealed that mesohexaploid B. rapa genome has retained 1-5 orthologs of each Arabidopsis 14-3-3 gene, interspersed across its three fragmented sub-genomes. qRT-PCR analysis showed that 14 of the 21 BraA.GRF14 were expressed, wherein a higher abundance of non-ε transcripts was observed compared to the ε genes, indicating class-specific transcriptional bias. The BraA.GRF14 genes showed distinct expression pattern during plant developmental stages and in response to abiotic stress, phytohormone treatments, and nutrient deprivation conditions. Together, the distinct expression pattern and differential regulation of BraA.GRF14 genes indicated the occurrence of functional divergence of B. rapa 14-3-3 proteins during plant development and stress responses.

  8. A 14-3-3 Family Protein from Wild Soybean (Glycine Soja) Regulates ABA Sensitivity in Arabidopsis

    PubMed Central

    Sun, Xiaoli; Sun, Mingzhe; Jia, Bowei; Chen, Chao; Qin, Zhiwei; Yang, Kejun; Shen, Yang; Meiping, Zhang; Mingyang, Cong; Zhu, Yanming

    2015-01-01

    It is widely accepted that the 14-3-3 family proteins are key regulators of multiple stress signal transduction cascades. By conducting genome-wide analysis, researchers have identified the soybean 14-3-3 family proteins; however, until now, there is still no direct genetic evidence showing the involvement of soybean 14-3-3s in ABA responses. Hence, in this study, based on the latest Glycine max genome on Phytozome v10.3, we initially analyzed the evolutionary relationship, genome organization, gene structure and duplication, and three-dimensional structure of soybean 14-3-3 family proteins systematically. Our results suggested that soybean 14-3-3 family was highly evolutionary conserved and possessed segmental duplication in evolution. Then, based on our previous functional characterization of a Glycine soja 14-3-3 protein GsGF14o in drought stress responses, we further investigated the expression characteristics of GsGF14o in detail, and demonstrated its positive roles in ABA sensitivity. Quantitative real-time PCR analyses in Glycine soja seedlings and GUS activity assays in PGsGF14O:GUS transgenic Arabidopsis showed that GsGF14o expression was moderately and rapidly induced by ABA treatment. As expected, GsGF14o overexpression in Arabidopsis augmented the ABA inhibition of seed germination and seedling growth, promoted the ABA induced stomata closure, and up-regulated the expression levels of ABA induced genes. Moreover, through yeast two hybrid analyses, we further demonstrated that GsGF14o physically interacted with the AREB/ABF transcription factors in yeast cells. Taken together, results presented in this study strongly suggested that GsGF14o played an important role in regulation of ABA sensitivity in Arabidopsis. PMID:26717241

  9. Class-Specific Evolution and Transcriptional Differentiation of 14-3-3 Family Members in Mesohexaploid Brassica rapa.

    PubMed

    Chandna, Ruby; Augustine, Rehna; Kanchupati, Praveena; Kumar, Roshan; Kumar, Pawan; Arya, Gulab C; Bisht, Naveen C

    2016-01-01

    14-3-3s are highly conserved, multigene family proteins that have been implicated in modulating various biological processes. The presence of inherent polyploidy and genome complexity has limited the identification and characterization of 14-3-3 proteins from globally important Brassica crops. Through data mining of Brassica rapa, the model Brassica genome, we identified 21 members encoding 14-3-3 proteins namely, BraA.GRF14.a to BraA.GRF14.u. Phylogenetic analysis indicated that B. rapa contains both ε (epsilon) and non-ε 14-3-3 isoforms, having distinct intron-exon structural organization patterns. The non-ε isoforms showed lower divergence rate (Ks < 0.45) compared to ε protein isoforms (Ks > 0.48), suggesting class-specific divergence pattern. Synteny analysis revealed that mesohexaploid B. rapa genome has retained 1-5 orthologs of each Arabidopsis 14-3-3 gene, interspersed across its three fragmented sub-genomes. qRT-PCR analysis showed that 14 of the 21 BraA.GRF14 were expressed, wherein a higher abundance of non-ε transcripts was observed compared to the ε genes, indicating class-specific transcriptional bias. The BraA.GRF14 genes showed distinct expression pattern during plant developmental stages and in response to abiotic stress, phytohormone treatments, and nutrient deprivation conditions. Together, the distinct expression pattern and differential regulation of BraA.GRF14 genes indicated the occurrence of functional divergence of B. rapa 14-3-3 proteins during plant development and stress responses. PMID:26858736

  10. A 14-3-3 Family Protein from Wild Soybean (Glycine Soja) Regulates ABA Sensitivity in Arabidopsis.

    PubMed

    Sun, Xiaoli; Sun, Mingzhe; Jia, Bowei; Chen, Chao; Qin, Zhiwei; Yang, Kejun; Shen, Yang; Meiping, Zhang; Mingyang, Cong; Zhu, Yanming

    2015-01-01

    It is widely accepted that the 14-3-3 family proteins are key regulators of multiple stress signal transduction cascades. By conducting genome-wide analysis, researchers have identified the soybean 14-3-3 family proteins; however, until now, there is still no direct genetic evidence showing the involvement of soybean 14-3-3s in ABA responses. Hence, in this study, based on the latest Glycine max genome on Phytozome v10.3, we initially analyzed the evolutionary relationship, genome organization, gene structure and duplication, and three-dimensional structure of soybean 14-3-3 family proteins systematically. Our results suggested that soybean 14-3-3 family was highly evolutionary conserved and possessed segmental duplication in evolution. Then, based on our previous functional characterization of a Glycine soja 14-3-3 protein GsGF14o in drought stress responses, we further investigated the expression characteristics of GsGF14o in detail, and demonstrated its positive roles in ABA sensitivity. Quantitative real-time PCR analyses in Glycine soja seedlings and GUS activity assays in PGsGF14O:GUS transgenic Arabidopsis showed that GsGF14o expression was moderately and rapidly induced by ABA treatment. As expected, GsGF14o overexpression in Arabidopsis augmented the ABA inhibition of seed germination and seedling growth, promoted the ABA induced stomata closure, and up-regulated the expression levels of ABA induced genes. Moreover, through yeast two hybrid analyses, we further demonstrated that GsGF14o physically interacted with the AREB/ABF transcription factors in yeast cells. Taken together, results presented in this study strongly suggested that GsGF14o played an important role in regulation of ABA sensitivity in Arabidopsis. PMID:26717241

  11. A 14-3-3 Family Protein from Wild Soybean (Glycine Soja) Regulates ABA Sensitivity in Arabidopsis.

    PubMed

    Sun, Xiaoli; Sun, Mingzhe; Jia, Bowei; Chen, Chao; Qin, Zhiwei; Yang, Kejun; Shen, Yang; Meiping, Zhang; Mingyang, Cong; Zhu, Yanming

    2015-01-01

    It is widely accepted that the 14-3-3 family proteins are key regulators of multiple stress signal transduction cascades. By conducting genome-wide analysis, researchers have identified the soybean 14-3-3 family proteins; however, until now, there is still no direct genetic evidence showing the involvement of soybean 14-3-3s in ABA responses. Hence, in this study, based on the latest Glycine max genome on Phytozome v10.3, we initially analyzed the evolutionary relationship, genome organization, gene structure and duplication, and three-dimensional structure of soybean 14-3-3 family proteins systematically. Our results suggested that soybean 14-3-3 family was highly evolutionary conserved and possessed segmental duplication in evolution. Then, based on our previous functional characterization of a Glycine soja 14-3-3 protein GsGF14o in drought stress responses, we further investigated the expression characteristics of GsGF14o in detail, and demonstrated its positive roles in ABA sensitivity. Quantitative real-time PCR analyses in Glycine soja seedlings and GUS activity assays in PGsGF14O:GUS transgenic Arabidopsis showed that GsGF14o expression was moderately and rapidly induced by ABA treatment. As expected, GsGF14o overexpression in Arabidopsis augmented the ABA inhibition of seed germination and seedling growth, promoted the ABA induced stomata closure, and up-regulated the expression levels of ABA induced genes. Moreover, through yeast two hybrid analyses, we further demonstrated that GsGF14o physically interacted with the AREB/ABF transcription factors in yeast cells. Taken together, results presented in this study strongly suggested that GsGF14o played an important role in regulation of ABA sensitivity in Arabidopsis.

  12. Keratin 23, a novel DPC4/Smad4 target gene which binds 14-3-3ε

    PubMed Central

    2011-01-01

    Background Inactivating mutations of SMAD4 are frequent in metastatic colorectal carcinomas. In previous analyses, we were able to show that restoration of Smad4 expression in Smad4-deficient SW480 human colon carcinoma cells was adequate to suppress tumorigenicity and invasive potential, whereas in vitro cell growth was not affected. Using this cellular model system, we searched for new Smad4 targets comparing nuclear subproteomes derived from Smad4 re-expressing and Smad4 negative SW480 cells. Methods High resolution two-dimensional (2D) gel electrophoresis was applied to identify novel Smad4 targets in the nuclear subproteome of Smad4 re-expressing SW480 cells. The identified candidate protein Keratin 23 was further characterized by tandem affinity purification. Immunoprecipitation, subfractionation and immunolocalization studies in combination with RNAi were used to validate the Keratin 23-14-3-3ε interaction. Results We identified keratins 8 and 18, heat shock proteins 60 and 70, plectin 1, as well as 14-3-3ε and γ as novel proteins present in the KRT23-interacting complex. Co-immunoprecipitation and subfractionation analyses as well as immunolocalization studies in our Smad4-SW480 model cells provided further evidence that KRT23 associates with 14-3-3ε and that Smad4 dependent KRT23 up-regulation induces a shift of the 14-3-3ε protein from a nuclear to a cytoplasmic localization. Conclusion Based on our findings we propose a new regulatory circuitry involving Smad4 dependent up-regulation of KRT23 (directly or indirectly) which in turn modulates the interaction between KRT23 and 14-3-3ε leading to a cytoplasmic sequestration of 14-3-3ε. This cytoplasmic KRT23-14-3-3 interaction may alter the functional status of the well described 14-3-3 scaffold protein, known to regulate key cellular processes, such as signal transduction, cell cycle control, and apoptosis and may thus be a previously unappreciated facet of the Smad4 tumor suppressive circuitry. PMID

  13. Posttranscriptional regulation of 14-3-3ζ by RNA-binding protein HuR modulating intestinal epithelial restitution after wounding.

    PubMed

    Hansraj, Natasha Z; Xiao, Lan; Wu, Jing; Chen, Gang; Turner, Douglas J; Wang, Jian-Ying; Rao, Jaladanki N

    2016-07-01

    The 14-3-3ζ is a member of the family of 14-3-3 proteins and participates in many aspects of cellular processes, but its regulation and involvement in gut mucosal homeostasis remain unknown. Here, we report that 14-3-3ζ expression is tightly regulated at the posttranscription level by RNA-binding protein HuR and plays an important role in early intestinal epithelial restitution after wounding. The 14-3-3ζ was highly expressed in the mucosa of gastrointestinal tract and in cultured intestinal epithelial cells (IECs). The 3' untranslated region (UTR) of the 14-3-3ζ mRNA was bound to HuR, and this association enhanced 14-3-3ζ translation without effect on its mRNA content. Conditional target deletion of HuR in IECs decreased the level of 14-3-3ζ protein in the intestinal mucosa. Silencing 14-3-3ζ by transfection with specific siRNA targeting the 14-3-3ζ mRNA suppressed intestinal epithelial restitution as indicated by a decrease in IEC migration after wounding, whereas ectopic overexpression of the wild-type 14-3-3ζ promoted cell migration. These results indicate that HuR induces 14-3-3ζ translation via interaction with its 3' UTR and that 14-3-3ζ is necessary for stimulation of IEC migration after wounding. PMID:27401462

  14. Posttranscriptional regulation of 14-3-3ζ by RNA-binding protein HuR modulating intestinal epithelial restitution after wounding.

    PubMed

    Hansraj, Natasha Z; Xiao, Lan; Wu, Jing; Chen, Gang; Turner, Douglas J; Wang, Jian-Ying; Rao, Jaladanki N

    2016-07-01

    The 14-3-3ζ is a member of the family of 14-3-3 proteins and participates in many aspects of cellular processes, but its regulation and involvement in gut mucosal homeostasis remain unknown. Here, we report that 14-3-3ζ expression is tightly regulated at the posttranscription level by RNA-binding protein HuR and plays an important role in early intestinal epithelial restitution after wounding. The 14-3-3ζ was highly expressed in the mucosa of gastrointestinal tract and in cultured intestinal epithelial cells (IECs). The 3' untranslated region (UTR) of the 14-3-3ζ mRNA was bound to HuR, and this association enhanced 14-3-3ζ translation without effect on its mRNA content. Conditional target deletion of HuR in IECs decreased the level of 14-3-3ζ protein in the intestinal mucosa. Silencing 14-3-3ζ by transfection with specific siRNA targeting the 14-3-3ζ mRNA suppressed intestinal epithelial restitution as indicated by a decrease in IEC migration after wounding, whereas ectopic overexpression of the wild-type 14-3-3ζ promoted cell migration. These results indicate that HuR induces 14-3-3ζ translation via interaction with its 3' UTR and that 14-3-3ζ is necessary for stimulation of IEC migration after wounding.

  15. Revealing the binding modes and the unbinding of 14-3-3σ proteins and inhibitors by computational methods.

    PubMed

    Hu, Guodong; Cao, Zanxia; Xu, Shicai; Wang, Wei; Wang, Jihua

    2015-01-01

    The 14-3-3σ proteins are a family of ubiquitous conserved eukaryotic regulatory molecules involved in the regulation of mitogenic signal transduction, apoptotic cell death, and cell cycle control. A lot of small-molecule inhibitors have been identified for 14-3-3 protein-protein interactions (PPIs). In this work, we carried out molecular dynamics (MD) simulations combined with molecular mechanics generalized Born surface area (MM-GBSA) method to study the binding mechanism between a 14-3-3σ protein and its eight inhibitors. The ranking order of our calculated binding free energies is in agreement with the experimental results. We found that the binding free energies are mainly from interactions between the phosphate group of the inhibitors and the hydrophilic residues. To improve the binding free energy of Rx group, we designed the inhibitor R9 with group R9 = 4-hydroxypheny. However, we also found that the binding free energy of inhibitor R9 is smaller than that of inhibitor R1. By further using the steer molecular dynamics (SMD) simulations, we identified a new hydrogen bond between the inhibitor R8 and residue Arg64 in the pulling paths. The information obtained from this study may be valuable for future rational design of novel inhibitors, and provide better structural understanding of inhibitor binding to 14-3-3σ proteins. PMID:26568041

  16. Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions.

    PubMed

    Jaumot, M; Hancock, J F

    2001-07-01

    Raf-1 activation is a complex process which involves plasma membrane recruitment, phosphorylation, protein-protein and lipid-protein interactions. We now show that PP1 and PP2A serine-threonine phosphatases also have a positive role in Ras dependent Raf-1 activation. General serine-threonine phosphatase inhibitors such sodium fluoride, or ss-glycerophosphate and sodium pyrophosphate, or specific PP1 and PP2A inhibitors including microcystin-LR, protein phosphatase 2A inhibitor I(1) or protein phosphatase inhibitor 2 all abrogate H-Ras and K-Ras dependent Raf-1 activation in vitro. A critical Raf-1 target residue for PP1 and PP2A is S259. Serine phosphatase inhibitors block the dephosphorylation of S259, which accompanies Raf-1 activation, and Ras dependent activation of mutant Raf259A is relatively resistant to serine phosphatase inhibitors. Sucrose gradient analysis demonstrates that serine phosphatase inhibition increases the total amount of 14-3-3 and Raf-1 associated with the plasma membrane and significantly alters the distribution of 14-3-3 and Raf-1 across different plasma membrane microdomains. These observations suggest that dephosphorylation of S259 is a critical early step in Ras dependent Raf-1 activation which facilitates 14-3-3 displacement. Inhibition of PP1 and PP2A therefore causes plasma membrane accumulation of Raf-1/14-3-3 complexes which cannot be activated.

  17. Suppression of Gluconeogenic Gene Expression by LSD1-Mediated Histone Demethylation

    PubMed Central

    Pan, Dongning; Mao, Chunxiao; Wang, Yong-Xu

    2013-01-01

    Aberrant gluconeogenic gene expression is associated with diabetes, glycogen storage disease, and liver cancer. However, little is known how these genes are regulated at the chromatin level. In this study, we investigated in HepG2 cells whether histone demethylation is a potential mechanism. We found that knockdown or pharmacological inhibition of histone demethylase LSD1 causes remarkable transcription activation of two gluconeogenic genes, FBP1 and G6Pase, and consequently leads to increased de novo glucose synthesis and decreased intracellular glycogen content. Mechanistically, LSD1 occupies the promoters of FBP1 and G6Pase, and modulates their H3K4 dimethylation levels. Thus, our work identifies an epigenetic pathway directly governing gluconeogenic gene expression, which might have important implications in metabolic physiology and diseases. PMID:23755305

  18. Histone turnover and chromatin accessibility: Critical mediators of neurological development, plasticity, and disease

    PubMed Central

    Wenderski, Wendy; Maze, Ian

    2016-01-01

    In postmitotic neurons, nucleosomal turnover was long considered to be a static process that is inconsequential to transcription. However, our recent studies in human and rodent brain indicate that replication-independent (RI) nucleosomal turnover, which requires the histone variant H3.3, is dynamic throughout life and is necessary for activity-dependent gene expression, synaptic connectivity, and cognition. H3.3 turnover also facilitates cellular lineage specification and plays a role in suppressing the expression of heterochromatic repetitive elements, including mutagenic transposable sequences, in mouse embryonic stem cells. In this essay, we review mechanisms and functions for RI nucleosomal turnover in brain and present the hypothesis that defects in histone dynamics may represent a common mechanism underlying neurological aging and disease. PMID:26990528

  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. Genome-Wide Identification, Phylogeny, and Expression Analyses of the 14-3-3 Family Reveal Their Involvement in the Development, Ripening, and Abiotic Stress Response in Banana

    PubMed Central

    Li, Meiying; Ren, Licheng; Xu, Biyu; Yang, Xiaoliang; Xia, Qiyu; He, Pingping; Xiao, Susheng; Guo, Anping; Hu, Wei; Jin, Zhiqiang

    2016-01-01

    Plant 14-3-3 proteins act as critical components of various cellular signaling processes and play an important role in regulating multiple physiological processes. However, less information is known about the 14-3-3 gene family in banana. In this study, 25 14-3-3 genes were identified from the banana genome. Based on the evolutionary analysis, banana 14-3-3 proteins were clustered into ε and non-ε groups. Conserved motif analysis showed that all identified banana 14-3-3 genes had the typical 14-3-3 motif. The gene structure of banana 14-3-3 genes showed distinct class-specific divergence between the ε group and the non-ε group. Most banana 14-3-3 genes showed strong transcript accumulation changes during fruit development and postharvest ripening in two banana varieties, indicating that they might be involved in regulating fruit development and ripening. Moreover, some 14-3-3 genes also showed great changes after osmotic, cold, and salt treatments in two banana varieties, suggested their potential role in regulating banana response to abiotic stress. Taken together, this systemic analysis reveals the involvement of banana 14-3-3 genes in fruit development, postharvest ripening, and response to abiotic stress and provides useful information for understanding the functions of 14-3-3 genes in banana. PMID:27713761

  1. Histone Lysine Methylation in TGF-β1 Mediated p21 Gene Expression in Rat Mesangial Cells

    PubMed Central

    Guo, Qiaoyan; Li, Xiaoxia; Han, Hongbo; Li, Chaoyuan; Liu, Shujun; Gao, Wenhui

    2016-01-01

    Transforming growth factor beta1- (TGF-β1-) induced p21-dependent mesangial cell (MC) hypertrophy plays a key role in the pathogenesis of chronic renal diseases including diabetic nephropathy (DN). Increasing evidence demonstrated the role of posttranscriptional modifications (PTMs) in the event; however, the precise regulatory mechanism of histone lysine methylation remains largely unknown. Here, we examined the roles of both histone H3 lysine 4 and lysine 9 methylations (H3K4me/H3K9me) in TGF-β1 induced p21 gene expression in rat mesangial cells (RMCs). Our results indicated that TGF-β1 upregulated the expression of p21 gene in RMCs, which was positively correlated with the increased chromatin marks associated with active genes (H3K4me1/H3K4me2/H3K4me3) and negatively correlated with the decreased levels of repressive marks (H3K9me2/H3K9me3) at p21 gene promoter. TGF-β1 also elevated the recruitment of the H3K4 methyltransferase (HMT) SET7/9 to the p21 gene promoter. SET7/9 gene silencing with small interfering RNAs (siRNAs) significantly abolished the TGF-β1 induced p21 gene expression. Taken together, these results reveal the key role of histone H3Kme in TGF-β1 mediated p21 gene expression in RMC, partly through HMT SET7/9 occupancy, suggesting H3Kme and SET7/9 may be potential renoprotective agents in managing chronic renal diseases. PMID:27247942

  2. Aurora-A mediated histone H3 phosphorylation of threonine 118 controls condensin I and cohesin occupancy in mitosis

    PubMed Central

    Wike, Candice L; Graves, Hillary K; Hawkins, Reva; Gibson, Matthew D; Ferdinand, Michelle B; Zhang, Tao; Chen, Zhihong; Hudson, Damien F; Ottesen, Jennifer J; Poirier, Michael G; Schumacher, Jill; Tyler, Jessica K

    2016-01-01

    Phosphorylation of histone H3 threonine 118 (H3 T118ph) weakens histone DNA-contacts, disrupting the nucleosome structure. We show that Aurora-A mediated H3 T118ph occurs at pericentromeres and chromosome arms during prophase and is lost upon chromosome alignment. Expression of H3 T118E or H3 T118I (a SIN mutation that bypasses the need for the ATP-dependent nucleosome remodeler SWI/SNF) leads to mitotic problems including defects in spindle attachment, delayed cytokinesis, reduced chromatin packaging, cohesion loss, cohesin and condensin I loss in human cells. In agreement, overexpression of Aurora-A leads to increased H3 T118ph levels, causing cohesion loss, and reduced levels of cohesin and condensin I on chromatin. Normal levels of H3 T118ph are important because it is required for development in fruit flies. We propose that H3 T118ph alters the chromatin structure during specific phases of mitosis to promote timely condensin I and cohesin disassociation, which is essential for effective chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.11402.001 PMID:26878753

  3. Aurora-A mediated histone H3 phosphorylation of threonine 118 controls condensin I and cohesin occupancy in mitosis.

    PubMed

    Wike, Candice L; Graves, Hillary K; Hawkins, Reva; Gibson, Matthew D; Ferdinand, Michelle B; Zhang, Tao; Chen, Zhihong; Hudson, Damien F; Ottesen, Jennifer J; Poirier, Michael G; Schumacher, Jill; Tyler, Jessica K

    2016-01-01

    Phosphorylation of histone H3 threonine 118 (H3 T118ph) weakens histone DNA-contacts, disrupting the nucleosome structure. We show that Aurora-A mediated H3 T118ph occurs at pericentromeres and chromosome arms during prophase and is lost upon chromosome alignment. Expression of H3 T118E or H3 T118I (a SIN mutation that bypasses the need for the ATP-dependent nucleosome remodeler SWI/SNF) leads to mitotic problems including defects in spindle attachment, delayed cytokinesis, reduced chromatin packaging, cohesion loss, cohesin and condensin I loss in human cells. In agreement, overexpression of Aurora-A leads to increased H3 T118ph levels, causing cohesion loss, and reduced levels of cohesin and condensin I on chromatin. Normal levels of H3 T118ph are important because it is required for development in fruit flies. We propose that H3 T118ph alters the chromatin structure during specific phases of mitosis to promote timely condensin I and cohesin disassociation, which is essential for effective chromosome segregation. PMID:26878753

  4. Arabidopsis paired amphipathic helix proteins SNL1 and SNL2 redundantly regulate primary seed dormancy via abscisic acid-ethylene antagonism mediated by histone deacetylation.

    PubMed

    Wang, Zhi; Cao, Hong; Sun, Yongzhen; Li, Xiaoying; Chen, Fengying; Carles, Annaick; Li, Yong; Ding, Meng; Zhang, Cun; Deng, Xin; Soppe, Wim J J; Liu, Yong-Xiu

    2013-01-01

    Histone (de)acetylation is a highly conserved chromatin modification that is vital for development and growth. In this study, we identified a role in seed dormancy for two members of the histone deacetylation complex in Arabidopsis thaliana, SIN3-LIKE1 (SNL1) and SNL2. The double mutant snl1 snl2 shows reduced dormancy and hypersensitivity to the histone deacetylase inhibitors trichostatin A and diallyl disulfide compared with the wild type. SNL1 interacts with HISTONE DEACETYLASE19 in vitro and in planta, and loss-of-function mutants of SNL1 and SNL2 show increased acetylation levels of histone 3 lysine 9/18 (H3K9/18) and H3K14. Moreover, SNL1 and SNL2 regulate key genes involved in the ethylene and abscisic acid (ABA) pathways by decreasing their histone acetylation levels. Taken together, we showed that SNL1 and SNL2 regulate seed dormancy by mediating the ABA-ethylene antagonism in Arabidopsis. SNL1 and SNL2 could represent a cross-link point of the ABA and ethylene pathways in the regulation of seed dormancy.

  5. The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability

    PubMed Central

    Seo, Gi Won; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Kim, Sun-Am; Lee, Yong Seok; Kim, Yu Jung; Han, Yeon Soo

    2016-01-01

    The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform. PMID:27556493

  6. 14-3-3σ Gene Loss Leads to Activation of the Epithelial to Mesenchymal Transition Due to the Stabilization of c-Jun Protein.

    PubMed

    Raychaudhuri, Kumarkrishna; Chaudhary, Neelam; Gurjar, Mansa; D'Souza, Roseline; Limzerwala, Jazeel; Maddika, Subbareddy; Dalal, Sorab N

    2016-07-29

    Loss of 14-3-3σ has been observed in multiple tumor types; however, the mechanisms by which 14-3-3σ loss leads to tumor progression are not understood. The experiments in this report demonstrate that loss of 14-3-3σ leads to a decrease in the expression of epithelial markers and an increase in the expression of mesenchymal markers, which is indicative of an induction of the epithelial to mesenchymal transition (EMT). The EMT was accompanied by an increase in migration and invasion in the 14-3-3σ(-/-) cells. 14-3-3σ(-/-) cells show increased stabilization of c-Jun, resulting in an increase in the expression of the EMT transcription factor slug. 14-3-3σ induces the ubiquitination and degradation of c-Jun in an FBW7-dependent manner. c-Jun ubiquitination is dependent on the presence of an intact nuclear export pathway as c-Jun is stabilized and localized to the nucleus in the presence of a nuclear export inhibitor. Furthermore, the absence of 14-3-3σ leads to the nuclear accumulation and stabilization of c-Jun, suggesting that 14-3-3σ regulates the subcellular localization of c-Jun. Our results have identified a novel mechanism by which 14-3-3σ maintains the epithelial phenotype by inhibiting EMT and suggest that this property of 14-3-3σ might contribute to its function as a tumor suppressor gene.

  7. 14-3-3σ Gene Loss Leads to Activation of the Epithelial to Mesenchymal Transition Due to the Stabilization of c-Jun Protein.

    PubMed

    Raychaudhuri, Kumarkrishna; Chaudhary, Neelam; Gurjar, Mansa; D'Souza, Roseline; Limzerwala, Jazeel; Maddika, Subbareddy; Dalal, Sorab N

    2016-07-29

    Loss of 14-3-3σ has been observed in multiple tumor types; however, the mechanisms by which 14-3-3σ loss leads to tumor progression are not understood. The experiments in this report demonstrate that loss of 14-3-3σ leads to a decrease in the expression of epithelial markers and an increase in the expression of mesenchymal markers, which is indicative of an induction of the epithelial to mesenchymal transition (EMT). The EMT was accompanied by an increase in migration and invasion in the 14-3-3σ(-/-) cells. 14-3-3σ(-/-) cells show increased stabilization of c-Jun, resulting in an increase in the expression of the EMT transcription factor slug. 14-3-3σ induces the ubiquitination and degradation of c-Jun in an FBW7-dependent manner. c-Jun ubiquitination is dependent on the presence of an intact nuclear export pathway as c-Jun is stabilized and localized to the nucleus in the presence of a nuclear export inhibitor. Furthermore, the absence of 14-3-3σ leads to the nuclear accumulation and stabilization of c-Jun, suggesting that 14-3-3σ regulates the subcellular localization of c-Jun. Our results have identified a novel mechanism by which 14-3-3σ maintains the epithelial phenotype by inhibiting EMT and suggest that this property of 14-3-3σ might contribute to its function as a tumor suppressor gene. PMID:27261462

  8. The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability.

    PubMed

    Seo, Gi Won; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Kim, Sun-Am; Lee, Yong Seok; Kim, Yu Jung; Han, Yeon Soo

    2016-01-01

    The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform. PMID:27556493

  9. Protein kinase B (AKT) regulates SYK activity and shuttling through 14-3-3 and importin 7.

    PubMed

    Mohammad, Dara K; Nore, Beston F; Gustafsson, Manuela O; Mohamed, Abdalla J; Smith, C I Edvard

    2016-09-01

    The Protein kinase B (AKT) regulates a plethora of intracellular signaling proteins to fine-tune signaling of multiple pathways. Here, we found that following B-cell receptor (BCR)-induced tyrosine phosphorylation of the cytoplasmic tyrosine kinase SYK and the adaptor BLNK, the AKT/PKB enzyme strongly induced BLNK (>100-fold) and SYK (>100-fold) serine/threonine phosphorylation (pS/pT). Increased phosphorylation promoted 14-3-3 binding to BLNK (37-fold) and SYK (2.5-fold) in a pS/pT-concentration dependent manner. We also demonstrated that the AKT inhibitor MK2206 reduced pS/pT of both BLNK (3-fold) and SYK (2.5-fold). Notably, the AKT phosphatase, PHLPP2 maintained the activating phosphorylation of BLNK at Y84 and increased protein stability (8.5-fold). In addition, 14-3-3 was required for the regulation SYK's interaction with BLNK and attenuated SYK binding to Importin 7 (5-fold), thereby perturbing shuttling to the nucleus. Moreover, 14-3-3 proteins also sustained tyrosine phosphorylation of SYK and BLNK. Furthermore, substitution of S295 or S297 for alanine abrogated SYK's binding to Importin 7. SYK with S295A or S297A replacements showed intense pY525/526 phosphorylation, and BLNK pY84 phosphorylation correlated with the SYK pY525/526 phosphorylation level. Conversely, the corresponding mutations to aspartic acid in SYK reduced pY525/526 phosphorylation. Collectively, these and previous results suggest that AKT and 14-3-3 proteins down-regulate the activity of several BCR-associated components, including BTK, BLNK and SYK and also inhibit SYK's interaction with Importin 7. PMID:27381982

  10. Oxidative damage of 14-3-3 zeta and gamma isoforms in Alzheimer's disease and cerebral amyloid angiopathy.

    PubMed

    Santpere, G; Puig, B; Ferrer, I

    2007-06-01

    Previous studies have shown oxidative damage resulting from amyloid Abeta exposure to cultured cells and in murine models. A target of oxidation is 14-3-3 which comprises a group of proteins involved in kinase activation and chaperone activity. The present study shows glycoxidative damage, as revealed with mono and bi-dimensional gel electrophoresis and Western blotting, followed by in-gel digestion and mass spectrometry, in the frontal cortex in Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), a neurodegenerative disease with deposition of Abeta in cerebral blood vessels and in diffuse plaques unaccompanied by intraneuronal hyper-phosphorylated tau deposition. malondialdehyde-lysine (MDA-Lys)-, but not 4-hydroxy-2-nonenal (HNE)-immunoreactive adducts, and N-carboxyethyl-lysine (CEL), but not N-carboxymethyl-lysine (CML)-products, were present in 14-3-3 involving zeta and gamma isoforms in both AD and CAA. These findings demonstrate that 14-3-3 glyco- and lipoxidation occurs in AD and CAA, probably as a direct consequence of Abeta deposition.

  11. 14-3-3 gene family in hybrid poplar and its involvement in tree defence against pathogens.

    PubMed

    Lapointe, G; Luckevich, M D; Cloutier, M; Séguin, A

    2001-06-01

    In ongoing investigations of the role of the signal transduction pathway in tree-pathogen interactions, four complete and two partial 14-3-3 cDNAs have been isolated which are members of a gene family. Comparisons of DNA sequences reveal a high degree of identity among the cDNAs, and, in some cases, higher than 75% sequence similarity with previously published sequences. Sequence analysis at the amino acid level uncovered potential phosphorylation sites, some of which were identical among the proteins, and some of which varied. Treatment of trees with chitosan, jasmonates or by wounding of leaves, caused increases in the levels of 14-3-3 mRNA transcripts. Since jasmonates and chitosan are signal transducers of defence reactions in plants, these results suggest a possible role for 14-3-3 proteins in the pathogen defence response of deciduous trees. Effects of elicitors on transcription of the pal gene were also monitored. Pal is a well-characterized, pathogen response-related gene.

  12. Histone acetyltransferase mediated regulation of FOXP3 acetylation and Treg function

    PubMed Central

    Xiao, Yan; Li, Bin; Zhou, Zhaocai; Hancock, Wayne W.; Zhang, Hongtao; Greene, Mark I.

    2010-01-01

    Regulatory T cells (Tregs) are required for the maintenance of immune homeostasis as first clearly described by Herman Waldmann’s laboratory. Dysfunction of Treg cells also leads to fatal autoimmunity in humans and mice. Conversely, the activation of different classes of Tregs operative systemically and within the cancer microenvironment can suppress host anti-tumor immune responses and promote tumor progression. Therefore, the development of new therapeutic approaches to regulate the activity of Treg cells may have considerable clinical potential. FOXP3 is the key transcriptional regulator of Treg development and function. The activity of FOXP3 is regulated by acetylation, a process catalyzed by distinct types of histone/protein acetyltransferases (HATs) that regulate the functions of many transcription factors, independently of FOXP3, as well as non-histone proteins, in addition to their effects on chromatin accessibility. Interactions between FOXP3 and these enzymes determine the suppressive function of FOXP3. Clearly, small molecules targeting these enzymes are candidates for the regulation of Treg function in vaccines and tumor therapies. PMID:20869864

  13. Transcription factor Sox4 is required for PUMA-mediated apoptosis induced by histone deacetylase inhibitor, TSA.

    PubMed

    Jang, Sang-Min; Kang, Eun-Jin; Kim, Jung-Woong; Kim, Chul-Hong; An, Joo-Hee; Choi, Kyung-Hee

    2013-08-23

    PUMA is a crucial regulator of apoptotic cell death mediated by p53-dependent and p53-independent mechanisms. In many cancer cells, PUMA expression is induced in response to DNA-damaging reagent in a p53-dependent manner. However, few studies have investigated transcription factors that lead to the induction of PUMA expression via p53-independent apoptotic signaling. In this study, we found that the transcription factor Sox4 increased PUMA expression in response to trichostatin A (TSA), a histone deacetylase inhibitor in the p53-null human lung cancer cell line H1299. Ectopic expression of Sox4 led to the induction of PUMA expression at the mRNA and protein levels, and TSA-mediated up-regulation of PUMA transcription was repressed by the knockdown of Sox4. Using luciferase assays and chromatin immunoprecipitation, we also determined that Sox4 recruits p300 on the PUMA promoter region and increases PUMA gene expression in response to TSA treatment. Taken together, these results suggest that Sox4 is required for p53-independent apoptotic cell death mediated by PUMA induction via TSA treatment.

  14. Direct interplay among histones, histone chaperones, and a chromatin boundary protein in the control of histone gene expression.

    PubMed

    Zunder, Rachel M; Rine, Jasper

    2012-11-01

    In Saccharomyces cerevisiae, the histone chaperone Rtt106 binds newly synthesized histone proteins and mediates their delivery into chromatin during transcription, replication, and silencing. Rtt106 is also recruited to histone gene regulatory regions by the HIR histone chaperone complex to ensure S-phase-specific expression. Here we showed that this Rtt106:HIR complex included Asf1 and histone proteins. Mutations in Rtt106 that reduced histone binding reduced Rtt106 enrichment at histone genes, leading to their increased transcription. Deletion of the chromatin boundary element Yta7 led to increased Rtt106:H3 binding, increased Rtt106 enrichment at histone gene regulatory regions, and decreased histone gene transcription at the HTA1-HTB1 locus. These results suggested a unique regulatory mechanism in which Rtt106 sensed the level of histone proteins to maintain the proper level of histone gene transcription. The role of these histone chaperones and Yta7 differed markedly among the histone gene loci, including the two H3-H4 histone gene pairs. Defects in silencing in rtt106 mutants could be partially accounted for by Rtt106-mediated changes in histone gene repression. These studies suggested that feedback mediated by histone chaperone complexes plays a pivotal role in regulating histone gene transcription.

  15. Histone acetyltransferase p300 promotes MKL1-mediated transactivation of catechol-O-methyltransferase gene.

    PubMed

    Liu, Zhipeng; Luo, Xuegang; Liu, Lei; Zhao, Wenwen; Guo, Shu; Guo, Yu; Wang, Nan; He, Hongpeng; Liao, Xinghua; Ma, Wenjian; Zhou, Hao; Zhang, Tongcun

    2013-12-01

    Previous studies have revealed that histone acetyltransferase p300 is recruited to the promoters of certain cardiac and smooth muscle specific genes to enhance the transactivation activity of myocardin, which is a master regulator in cardiovascular differentiation and development. Here, we found that the gene encoding catechol-O-methyltransferase (COMT), an important metabolic enzyme catalyzing the conversion of estrogen, is also a target gene of myocardin-related transcription factors (MRTFs). Megakaryoblastic leukemia 1 (MKL1, also named MRTF-A) and p300 could synergistically augment the expression of COMT gene, increase the metabolic rate of estrogen, and thus reduce the proliferation of MCF-7 breast cancer cells stimulated by estrogen. PMID:24096006

  16. PHF8 mediates histone H4 lysine 20 demethylation events involved in cell cycle progression.

    PubMed

    Liu, Wen; Tanasa, Bogdan; Tyurina, Oksana V; Zhou, Tian Yuan; Gassmann, Reto; Liu, Wei Ting; Ohgi, Kenneth A; Benner, Chris; Garcia-Bassets, Ivan; Aggarwal, Aneel K; Desai, Arshad; Dorrestein, Pieter C; Glass, Christopher K; Rosenfeld, Michael G

    2010-07-22

    While reversible histone modifications are linked to an ever-expanding range of biological functions, the demethylases for histone H4 lysine 20 and their potential regulatory roles remain unknown. Here we report that the PHD and Jumonji C (JmjC) domain-containing protein, PHF8, while using multiple substrates, including H3K9me1/2 and H3K27me2, also functions as an H4K20me1 demethylase. PHF8 is recruited to promoters by its PHD domain based on interaction with H3K4me2/3 and controls G1-S transition in conjunction with E2F1, HCF-1 (also known as HCFC1) and SET1A (also known as SETD1A), at least in part, by removing the repressive H4K20me1 mark from a subset of E2F1-regulated gene promoters. Phosphorylation-dependent PHF8 dismissal from chromatin in prophase is apparently required for the accumulation of H4K20me1 during early mitosis, which might represent a component of the condensin II loading process. Accordingly, the HEAT repeat clusters in two non-structural maintenance of chromosomes (SMC) condensin II subunits, N-CAPD3 and N-CAPG2 (also known as NCAPD3 and NCAPG2, respectively), are capable of recognizing H4K20me1, and ChIP-Seq analysis demonstrates a significant overlap of condensin II and H4K20me1 sites in mitotic HeLa cells. Thus, the identification and characterization of an H4K20me1 demethylase, PHF8, has revealed an intimate link between this enzyme and two distinct events in cell cycle progression. PMID:20622854

  17. DYRK1A phoshorylates histone H3 to differentially regulate the binding of HP1 isoforms and antagonize HP1-mediated transcriptional repression.

    PubMed

    Jang, Suk Min; Azebi, Saliha; Soubigou, Guillaume; Muchardt, Christian

    2014-06-01

    Heterochromatin protein 1 (HP1) proteins are chromatin-bound transcriptional regulators. While their chromodomain binds histone H3 methylated on lysine 9, their chromoshadow domain associates with the H3 histone fold in a region involved in chromatin remodeling. Here, we show that phosphorylation at histone H3 threonine 45 and serine 57 within this latter region differentially affects binding of the three mammalian HP1 isoforms HP1α, HP1β and HP1γ. Both phosphorylation events are dependent on the activity of the DYRK1A kinase that antagonizes HP1-mediated transcriptional repression and participates in abnormal activation of cytokine genes in Down's syndrome-associated megakaryoblastic leukemia. PMID:24820035

  18. Requirement of a novel splicing variant of human histone deacetylase 6 for TGF-{beta}1-mediated gene activation

    SciTech Connect

    Zhuang, Yan; Nguyen, Hong T.; Lasky, Joseph A.; Cao, Subing; Li, Cui; Hu, Jiyao; Guo, Xinyue; Burow, Matthew E.; Shan, Bin

    2010-02-19

    Histone deacetylase 6 (HDAC6) belongs to the family of class IIb HDACs and predominantly deacetylates non-histone proteins in the cytoplasm via the C-terminal deacetylase domain of its two tandem deacetylase domains. HDAC6 modulates fundamental cellular processes via deacetylation of {alpha}-tubulin, cortactin, molecular chaperones, and other peptides. Our previous study indicates that HDAC6 mediates TGF-{beta}1-induced epithelial-mesenchymal transition (EMT) in A549 cells. In the current study, we identify a novel splicing variant of human HDAC6, hHDAC6p114. The hHDAC6p114 mRNA arises from incomplete splicing and encodes a truncated isoform of the hHDAC6p114 protein of 114 kDa when compared to the major isoform hHDAC6p131. The hHDAC6p114 protein lacks the first 152 amino acids from N-terminus in the hHDAC6p131 protein, which harbors a nuclear export signal peptide and 76 amino acids of the N-terminal deacetylase domain. hHDAC6p114 is intact in its deacetylase activity against {alpha}-tubulin. The expression hHDAC6p114 is elevated in a MCF-7 derivative that exhibits an EMT-like phenotype. Moreover, hHDAC6p114 is required for TGF-{beta}1-activated gene expression associated with EMT in A549 cells. Taken together, our results implicate that expression and function of hHDAC6p114 is differentially regulated when compared to hHDAC6p131.

  19. Unraveling 14-3-3 proteins in C4 panicoids with emphasis on model plant Setaria italica reveals phosphorylation-dependent subcellular localization of RS splicing factor.

    PubMed

    Kumar, Karunesh; Muthamilarasan, Mehanathan; Bonthala, Venkata Suresh; Roy, Riti; Prasad, Manoj

    2015-01-01

    14-3-3 proteins are a large multigenic family of regulatory proteins ubiquitously found in eukaryotes. In plants, 14-3-3 proteins are reported to play significant role in both development and response to stress stimuli. Therefore, considering their importance, genome-wide analyses have been performed in many plants including Arabidopsis, rice and soybean. But, till date, no comprehensive investigation has been conducted in any C4 panicoid crops. In view of this, the present study was performed to identify 8, 5 and 26 potential 14-3-3 gene family members in foxtail millet (Si14-3-3), sorghum (Sb14-3-3) and maize (Zm14-3-3), respectively. In silico characterization revealed large variations in their gene structures; segmental and tandem duplications have played a major role in expansion of these genes in foxtail millet and maize. Gene ontology annotation showed the participation of 14-3-3 proteins in diverse biological processes and molecular functions, and in silico expression profiling indicated their higher expression in all the investigated tissues. Comparative mapping was performed to derive the orthologous relationships between 14-3-3 genes of foxtail millet and other Poaceae members, which showed a higher, as well as similar percentage of orthology among these crops. Expression profiling of Si14-3-3 genes during different time-points of abiotic stress and hormonal treatments showed a differential expression pattern of these genes, and sub-cellular localization studies revealed the site of action of Si14-3-3 proteins within the cells. Further downstream characterization indicated the interaction of Si14-3-3 with a nucleocytoplasmic shuttling phosphoprotein (SiRSZ21A) in a phosphorylation-dependent manner, and this demonstrates that Si14-3-3 might regulate the splicing events by binding with phosphorylated SiRSZ21A. Taken together, the present study is a comprehensive analysis of 14-3-3 gene family members in foxtail millet, sorghum and maize, which provides

  20. Unraveling 14-3-3 Proteins in C4 Panicoids with Emphasis on Model Plant Setaria italica Reveals Phosphorylation-Dependent Subcellular Localization of RS Splicing Factor

    PubMed Central

    Kumar, Karunesh; Muthamilarasan, Mehanathan; Bonthala, Venkata Suresh; Roy, Riti; Prasad, Manoj

    2015-01-01

    14-3-3 proteins are a large multigenic family of regulatory proteins ubiquitously found in eukaryotes. In plants, 14-3-3 proteins are reported to play significant role in both development and response to stress stimuli. Therefore, considering their importance, genome-wide analyses have been performed in many plants including Arabidopsis, rice and soybean. But, till date, no comprehensive investigation has been conducted in any C4 panicoid crops. In view of this, the present study was performed to identify 8, 5 and 26 potential 14-3-3 gene family members in foxtail millet (Si14-3-3), sorghum (Sb14-3-3) and maize (Zm14-3-3), respectively. In silico characterization revealed large variations in their gene structures; segmental and tandem duplications have played a major role in expansion of these genes in foxtail millet and maize. Gene ontology annotation showed the participation of 14-3-3 proteins in diverse biological processes and molecular functions, and in silico expression profiling indicated their higher expression in all the investigated tissues. Comparative mapping was performed to derive the orthologous relationships between 14-3-3 genes of foxtail millet and other Poaceae members, which showed a higher, as well as similar percentage of orthology among these crops. Expression profiling of Si14-3-3 genes during different time-points of abiotic stress and hormonal treatments showed a differential expression pattern of these genes, and sub-cellular localization studies revealed the site of action of Si14-3-3 proteins within the cells. Further downstream characterization indicated the interaction of Si14-3-3 with a nucleocytoplasmic shuttling phosphoprotein (SiRSZ21A) in a phosphorylation-dependent manner, and this demonstrates that Si14-3-3 might regulate the splicing events by binding with phosphorylated SiRSZ21A. Taken together, the present study is a comprehensive analysis of 14-3-3 gene family members in foxtail millet, sorghum and maize, which provides

  1. Spinach 14-3-3 protein interacts with the plasma membrane H(+)-ATPase and nitrate reductase in response to excess nitrate stress.

    PubMed

    Xu, Huini; Zhao, Xiuling; Guo, Chuanlong; Chen, Limei; Li, Kunzhi

    2016-09-01

    To investigate the function of 14-3-3 protein in response to excess nitrate stress, a 14-3-3 protein, designated as So14-3-3, was isolated from spinach. Phylogenetic analysis demonstrated that So14-3-3 belongs to non-ε group of 14-3-3 superfamily. Real time-quantitative RT-PCR and western blot analysis showed that So14-3-3 was induced by excess nitrate stress in spinach roots and leaves. After nitrate treatment, the phosphorylated H(+)-ATPase and nitrate reductase (NR) increased and decreased respectively. Co-Immunoprecipitation (Co-IP) suggested that the interaction of So14-3-3 with the phosphorylated H(+)-ATPase enhanced, but reduced with phosphorylated NR in spinach roots after nitrate treatment. Besides, 5 proteins interacted with So14-3-3 were found by Co-IP and LC-MS/MS analysis. So14-3-3 overexpressing transgenic tobacco plants showed enhanced tolerance to nitrate treatment at the germination and young seedlings stage. The transgenic plants showed longer root length, lower malondialdehyde (MDA), H2O2, protein carbonyl contents, relatively higher soluble sugar and protein contents, than the WT plants after nitrate treatment. The phosphorylation levels of H(+)-ATPase in transgenic plants were higher than the WT plants after nitrate treatment, whereas NR were lower. Additionally, in transgenic plants, the interaction of So14-3-3 with phosphorylated H(+)-ATPase and NR increased and decreased more than the WT plants under nitrate stress, leading to higher H(+)-ATPase and NR activities in transgenic plants. These data suggested that So14-3-3 might be involved in nitrate stress response by interacting with H(+)-ATPase and NR. PMID:27161584

  2. Alternative application of Tau protein in Creutzfeldt-Jakob disease diagnosis: Improvement for weakly positive 14-3-3 protein in the laboratory.

    PubMed

    Hyeon, Jae Wook; Kim, Su Yeon; Lee, Jeongmin; Park, Jun Sun; Hwang, Kyu Jam; Lee, Sol Moe; An, SeongSoo A; Lee, Myung Koo; Ju, Young Ran

    2015-01-01

    The 14-3-3 protein has been used as a biomarker for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD). However, weakly positive 14-3-3 leads to false positive results and an incorrect diagnosis. We attempted to use quantitative data for tau protein to provide an accurate diagnosis based on weak 14-3-3 protein. Sixty-two patients with sCJD, including pathologically confirmed, clinically definite, and probable cases, and 89 non-CJD patients were investigated based on a Korean population. Among them, 20 sCJD and 14 non-CJD showed weakly positive 14-3-3. The total tau (t-tau) and phosphorylated tau (p-tau) protein levels were measured by ELISA, and the p-tau to t-tau ratio (p/t ratio) was calculated. The combined use of the 14-3-3 protein assay, t-tau levels, and p/t ratio improved the specificity of diagnosis compared with the use of the 14-3-3 protein assay alone (47% for 14-3-3 alone; 85.94% for 14-3-3 combined with t-tau; 90.62% for 14-3-3 combined with the p/t ratio). In addition, 18 of 20 sCJD and 12 of 14 non-CJD who were weakly positive for 14-3-3 were positive for the p/t ratio and negative for the p/t ratio, respectively. When used in combination with the 14-3-3 protein, the tau protein is useful as a biomarker for the precise diagnosis of sCJD. PMID:26507666

  3. Alternative application of Tau protein in Creutzfeldt-Jakob disease diagnosis: Improvement for weakly positive 14-3-3 protein in the laboratory.

    PubMed

    Hyeon, Jae Wook; Kim, Su Yeon; Lee, Jeongmin; Park, Jun Sun; Hwang, Kyu Jam; Lee, Sol Moe; An, SeongSoo A; Lee, Myung Koo; Ju, Young Ran

    2015-10-28

    The 14-3-3 protein has been used as a biomarker for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD). However, weakly positive 14-3-3 leads to false positive results and an incorrect diagnosis. We attempted to use quantitative data for tau protein to provide an accurate diagnosis based on weak 14-3-3 protein. Sixty-two patients with sCJD, including pathologically confirmed, clinically definite, and probable cases, and 89 non-CJD patients were investigated based on a Korean population. Among them, 20 sCJD and 14 non-CJD showed weakly positive 14-3-3. The total tau (t-tau) and phosphorylated tau (p-tau) protein levels were measured by ELISA, and the p-tau to t-tau ratio (p/t ratio) was calculated. The combined use of the 14-3-3 protein assay, t-tau levels, and p/t ratio improved the specificity of diagnosis compared with the use of the 14-3-3 protein assay alone (47% for 14-3-3 alone; 85.94% for 14-3-3 combined with t-tau; 90.62% for 14-3-3 combined with the p/t ratio). In addition, 18 of 20 sCJD and 12 of 14 non-CJD who were weakly positive for 14-3-3 were positive for the p/t ratio and negative for the p/t ratio, respectively. When used in combination with the 14-3-3 protein, the tau protein is useful as a biomarker for the precise diagnosis of sCJD.

  4. Phosphorylation of Arabidopsis Ubiquitin Ligase ATL31 Is Critical for Plant Carbon/Nitrogen Nutrient Balance Response and Controls the Stability of 14-3-3 Proteins*

    PubMed Central

    Yasuda, Shigetaka; Sato, Takeo; Maekawa, Shugo; Aoyama, Shoki; Fukao, Yoichiro; Yamaguchi, Junji

    2014-01-01

    Ubiquitin ligase plays a fundamental role in regulating multiple cellular events in eukaryotes by fine-tuning the stability and activity of specific target proteins. We have previously shown that ubiquitin ligase ATL31 regulates plant growth in response to nutrient balance between carbon and nitrogen (C/N) in Arabidopsis. Subsequent study demonstrated that ATL31 targets 14-3-3 proteins for ubiquitination and modulates the protein abundance in response to C/N-nutrient status. However, the underlying mechanism for the targeting of ATL31 to 14-3-3 proteins remains unclear. Here, we show that ATL31 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. We identified Thr209, Ser247, Ser270, and Ser303 as putative 14-3-3 binding sites on ATL31 by motif analysis. Mutation of these Ser/Thr residues to Ala in ATL31 inhibited the interaction with 14-3-3 proteins, as demonstrated by yeast two-hybrid and co-immunoprecipitation analyses. Additionally, we identified in vivo phosphorylation of Thr209 and Ser247 on ATL31 by MS analysis. A peptide competition assay showed that the application of synthetic phospho-Thr209 peptide, but not the corresponding unphosphorylated peptide, suppresses the interaction between ATL31 and 14-3-3 proteins. Moreover, Arabidopsis plants overexpressing mutated ATL31, which could not bind to 14-3-3 proteins, showed accumulation of 14-3-3 proteins and growth arrest in disrupted C/N-nutrient conditions similar to wild-type plants, although overexpression of intact ATL31 resulted in repression of 14-3-3 accumulation and tolerance to the conditions. Together, these results demonstrate that the physiological role of phosphorylation at 14-3-3 binding sites on ATL31 is to modulate the binding ability and stability of 14-3-3 proteins to control plant C/N-nutrient response. PMID:24722992

  5. The effect of Ku on telomere replication time is mediated by telomere length but is independent of histone tail acetylation

    PubMed Central

    Lian, Hui-Yong; Robertson, E. Douglas; Hiraga, Shin-ichiro; Alvino, Gina M.; Collingwood, David; McCune, Heather J.; Sridhar, Akila; Brewer, Bonita J.; Raghuraman, M. K.; Donaldson, Anne D.

    2011-01-01

    DNA replication in Saccharomyces cerevisiae proceeds according to a temporal program. We have investigated the role of the telomere-binding Ku complex in specifying late replication of telomere-proximal sequences. Genome-wide analysis shows that regions extending up to 80 kb from telomeres replicate abnormally early in a yku70 mutant. We find that Ku does not appear to regulate replication time by binding replication origins directly, nor is its effect on telomere replication timing mediated by histone tail acetylation. We show that Ku instead regulates replication timing through its effect on telomere length, because deletion of the telomerase regulator Pif1 largely reverses the short telomere defect of a yku70 mutant and simultaneously rescues its replication timing defect. Consistent with this conclusion, deleting the genome integrity component Elg1 partially rescued both length and replication timing of yku70 telomeres. Telomere length–mediated control of replication timing requires the TG1–3 repeat-counting component Rif1, because a rif1 mutant replicates telomeric regions early, despite having extended TG1–3 tracts. Overall, our results suggest that the effect of Ku on telomere replication timing results from its impact on TG1–3 repeat length and support a model in which Rif1 measures telomere repeat length to ensure that telomere replication timing is correctly programmed. PMID:21441303

  6. Histone chaperone NAP1 mediates sister chromatid resolution by counteracting protein phosphatase 2A.

    PubMed

    Moshkin, Yuri M; Doyen, Cecile M; Kan, Tsung-Wai; Chalkley, Gillian E; Sap, Karen; Bezstarosti, Karel; Demmers, Jeroen A; Ozgur, Zeliha; van Ijcken, Wilfred F J; Verrijzer, C Peter

    2013-01-01

    Chromosome duplication and transmission into daughter cells requires the precisely orchestrated binding and release of cohesin. We found that the Drosophila histone chaperone NAP1 is required for cohesin release and sister chromatid resolution during mitosis. Genome-wide surveys revealed that NAP1 and cohesin co-localize at multiple genomic loci. Proteomic and biochemical analysis established that NAP1 associates with the full cohesin complex, but it also forms a separate complex with the cohesin subunit stromalin (SA). NAP1 binding to cohesin is cell-cycle regulated and increases during G2/M phase. This causes the dissociation of protein phosphatase 2A (PP2A) from cohesin, increased phosphorylation of SA and cohesin removal in early mitosis. PP2A depletion led to a loss of centromeric cohesion. The distinct mitotic phenotypes caused by the loss of either PP2A or NAP1, were both rescued by their concomitant depletion. We conclude that the balanced antagonism between NAP1 and PP2A controls cohesin dissociation during mitosis. PMID:24086141

  7. PARP1 orchestrates variant histone exchange in signal-mediated transcriptional activation.

    PubMed

    O'Donnell, Amanda; Yang, Shen-Hsi; Sharrocks, Andrew D

    2013-12-01

    Transcriptional activation is accompanied by multiple molecular events that remodel the local chromatin environment in promoter regions. These molecular events are often orchestrated in response to the activation of signalling pathways, as exemplified by the response of immediate early genes such as FOS to ERK MAP kinase signalling. Here, we demonstrate that inducible NFI recruitment permits PARP1 binding to the FOS promoter by a mutually reinforcing loop. PARP1 and its poly(ADP-ribosyl)ation activity are required for maintaining FOS activation kinetics. We also show that the histone variant H2A.Z associates with the FOS promoter and acts in a transcription-suppressive manner. However, in response to ERK pathway signalling, H2A.Z is replaced by H2A; PARP1 activity is required to promote this exchange. Thus, our work has revealed an additional facet of PARP1 function in promoting dynamic remodelling of promoter-associated nucleosomes to allow transcriptional activation in response to cellular signalling.

  8. The Crystal Structure of Giardia duodenalis 14-3-3 in the Apo Form: When Protein Post-Translational Modifications Make the Difference

    PubMed Central

    Fiorillo, Annarita; di Marino, Daniele; Bertuccini, Lucia; Via, Allegra; Pozio, Edoardo; Camerini, Serena; Ilari, Andrea; Lalle, Marco

    2014-01-01

    The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3. PMID:24658679

  9. The epsilon isoform of 14-3-3 protein is a component of the prion protein amyloid deposits of Gerstmann-Sträussler-Scheinker disease.

    PubMed

    Di Fede, Giuseppe; Giaccone, Giorgio; Limido, Lucia; Mangieri, Michela; Suardi, Silvia; Puoti, Gianfranco; Morbin, Michela; Mazzoleni, Giulia; Ghetti, Bernardino; Tagliavini, Fabrizio

    2007-02-01

    The 14-3-3 proteins are highly conserved, ubiquitous molecules involved in a variety of biologic events, such as transduction pathway modulation, cell cycle control, and apoptosis. Seven isoforms have been identified that are abundant in the brain, preferentially localized in neurons. Remarkable increases in 14-3-3 are seen in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease (CJD), and it has been found in pathologic inclusions of several neurodegenerative diseases. Moreover, the zeta isoform has been detected in prion protein (PrP) amyloid deposits of CJD patients. To further investigate the cerebral distribution of 14-3-3 in prion-related encephalopathies, we carried out an immunohistochemical and biochemical analysis of brain tissue from patients with Gerstmann-Sträussler-Scheinker disease (GSS) and sporadic, familial and acquired forms of CJD, using specific antibodies against the seven 14-3-3 isoforms. The study showed a strong immunoreactivity of PrP amyloid plaques of GSS patients for the 14-3-3 epsilon isoform, but not for the other isoforms. The epsilon isoform of 14-3-3 was not found in PrP deposits of CJD. These results indicate that the epsilon isoform of 14-3-3 is a component of PrP amyloid deposits of GSS and suggest that this is the sole 14-3-3 isoform specifically involved in the neuropathologic changes associated with this disorder.

  10. Transcription variants of the prostate-specific PrLZ gene and their interaction with 14-3-3 proteins

    SciTech Connect

    Wang, Ruoxiang; He, Hui; Sun, Xiaojuan; Xu, Jianchun; Marshall, Fray F.; Zhau, Haiyen; Chung, Leland W.K.; Fu, Haian; He, Dalin

    2009-11-20

    We have reported isolation and characterization of the prostate-specific and androgen-regulated PrLZ gene abnormally expressed in prostate cancer. PrLZ is a potential biomarker for prostate cancer and a candidate oncogene promoting cell proliferation and survival in prostate cancer cells. A full delineation of the PrLZ gene and its gene products may provide clues to the mechanisms regulating its expression and function. In this report, we identified three additional exons in the PrLZ gene and recognized five transcript variants from alternative splicing that could be detected by RT-PCR and Western blotting. Structural comparison demonstrated that the PrLZ proteins are highly conserved among species. PrLZ contains multiple potential sites for interaction with other proteins. We used mammalian two-hybrid assays to demonstrate that PrLZ isoforms interact with 14-3-3 proteins, and multiple sites in the PrLZ may be involved in the interaction. Alternative splicing may contribute to abnormally enhanced PrLZ levels in prostate cancer, and interaction with 14-3-3 proteins may be a mechanism by which PrLZ promotes cell proliferation and survival during prostate cancer development and progression. This information is a valuable addition to the investigation of the oncogenic properties of the PrLZ gene.

  11. Ustilago maydis Rho1 and 14-3-3 homologues participate in pathways controlling cell separation and cell polarity.

    PubMed

    Pham, Cau D; Yu, Zhanyang; Sandrock, Björn; Bölker, Michael; Gold, Scott E; Perlin, Michael H

    2009-07-01

    Proteins of the 14-3-3 and Rho-GTPase families are functionally conserved eukaryotic proteins that participate in many important cellular processes such as signal transduction, cell cycle regulation, malignant transformation, stress response, and apoptosis. However, the exact role(s) of these proteins in these processes is not entirely understood. Using the fungal maize pathogen, Ustilago maydis, we were able to demonstrate a functional connection between Pdc1 and Rho1, the U. maydis homologues of 14-3-3epsilon and Rho1, respectively. Our experiments suggest that Pdc1 regulates viability, cytokinesis, chromosome condensation, and vacuole formation. Similarly, U. maydis Rho1 is also involved in these three essential processes and exerts an additional function during mating and filamentation. Intriguingly, yeast two-hybrid and epistasis experiments suggest that both Pdc1 and Rho1 could be constituents of the same regulatory cascade(s) controlling cell growth and filamentation in U. maydis. Overexpression of rho1 ameliorated the defects of cells depleted for Pdc1. Furthermore, we found that another small G protein, Rac1, was a suppressor of lethality for both Pdc1 and Rho1. In addition, deletion of cla4, encoding a Rac1 effector kinase, could also rescue cells with Pdc1 depleted. Inferring from these data, we propose a model for Rho1 and Pdc1 functions in U. maydis.

  12. Selective 14-3-3γ induction quenches p-β-catenin Ser37/Bax-enhanced cell death in cerebral cortical neurons during ischemia.

    PubMed

    Lai, X J; Ye, S Q; Zheng, L; Li, L; Liu, Q R; Yu, S B; Pang, Y; Jin, S; Li, Q; Yu, A C H; Chen, X Q

    2014-01-01

    Ischemia-induced cell death is a major cause of disability or death after stroke. Identifying the key intrinsic protective mechanisms induced by ischemia is critical for the development of effective stroke treatment. Here, we reported that 14-3-3γ was a selective ischemia-inducible survival factor in cerebral cortical neurons reducing cell death by downregulating Bax depend direct 14-3-3γ/p-β-catenin Ser37 interactions in the nucleus. 14-3-3γ, but not other 14-3-3 isoforms, was upregulated in primary cerebral cortical neurons upon oxygen-glucose deprivation (OGD) as measured by quantitative PCR, western blot and fluorescent immunostaining. The selective induction of 14-3-3γ in cortical neurons by OGD was verified by the in vivo ischemic stroke model. Knocking down 14-3-3γ alone or inhibiting 14-3-3/client interactions was sufficient to induce cell death in normal cultured neurons and exacerbate OGD-induced neuronal death. Ectopic overexpression of 14-3-3γ significantly reduced OGD-induced cell death in cultured neurons. Co-immunoprecipitation and fluorescence resonance energy transfer demonstrated that endogenous 14-3-3γ bound directly to more p-β-catenin Ser37 but not p-Bad, p-Ask-1, p-p53 and Bax. During OGD, p-β-catenin Ser37 but not p-β-catenin Ser45 was increased prominently, which correlated with Bax elevation in cortical neurons. OGD promoted the entry of 14-3-3γ into the nuclei, in correlation with the increase of nuclear p-β-catenin Ser37 in neurons. Overexpression of 14-3-3γ significantly reduced Bax expression, whereas knockdown of 14-3-3γ increased Bax in cortical neurons. Abolishing β-catenin phosphorylation at Ser37 (S37A) significantly reduced Bax and cell death in neurons upon OGD. Finally, 14-3-3γ overexpression completely suppressed β-catenin-enhanced Bax and cell death in neurons upon OGD. Based on these data, we propose that the 14-3-3γ/p-β-catenin Ser37/Bax axis determines cell survival or death of neurons during ischemia

  13. Vpr Protein of Human Immunodeficiency Virus Type 1 Binds to 14-3-3 Proteins and Facilitates Complex Formation with Cdc25C: Implications for Cell Cycle Arrest

    PubMed Central

    Kino, Tomoshige; Gragerov, Alexander; Valentin, Antonio; Tsopanomihalou, Maria; Ilyina-Gragerova, Galina; Erwin-Cohen, Rebecca; Chrousos, George P.; Pavlakis, George N.

    2005-01-01

    Vpr and selected mutants were used in a Saccharomyces cerevisiae two-hybrid screen to identify cellular interactors. We found Vpr interacted with 14-3-3 proteins, a family regulating a multitude of proteins in the cell. Vpr mutant R80A, which is inactive in cell cycle arrest, did not interact with 14-3-3. 14-3-3 proteins regulate the G2/M transition by inactivating Cdc25C phosphatase via binding to the phosphorylated serine residue at position 216 of Cdc25C. 14-3-3 overexpression in human cells synergized with Vpr in the arrest of cell cycle. Vpr did not arrest efficiently cells not expressing 14-3-3σ. This indicated that a full complement of 14-3-3 proteins is necessary for optimal Vpr function on the cell cycle. Mutational analysis showed that the C-terminal portion of Vpr, known to harbor its cell cycle-arresting activity, bound directly to the C-terminal part of 14-3-3, outside of its phosphopeptide-binding pocket. Vpr expression shifted localization of the mutant Cdc25C S216A to the cytoplasm, indicating that Vpr promotes the association of 14-3-3 and Cdc25C, independently of the presence of serine 216. Immunoprecipitations of cell extracts indicated the presence of triple complexes (Vpr/14-3-3/Cdc25C). These results indicate that Vpr promotes cell cycle arrest at the G2/M phase by facilitating association of 14-3-3 and Cdc25C independently of the latter's phosphorylation status. PMID:15708996

  14. Ablation of the 14-3-3gamma Protein Results in Neuronal Migration Delay and Morphological Defects in the Developing Cerebral Cortex.

    PubMed

    Wachi, Tomoka; Cornell, Brett; Marshall, Courtney; Zhukarev, Vladimir; Baas, Peter W; Toyo-oka, Kazuhito

    2016-06-01

    14-3-3 proteins are ubiquitously-expressed and multifunctional proteins. There are seven isoforms in mammals with a high level of homology, suggesting potential functional redundancy. We previously found that two of seven isoforms, 14-3-3epsilon and 14-3-3zeta, are important for brain development, in particular, radial migration of pyramidal neurons in the developing cerebral cortex. In this work, we analyzed the function of another isoform, the protein 14-3-3gamma, with respect to neuronal migration in the developing cortex. We found that in utero 14-3-3gamma-deficiency resulted in delays in neuronal migration as well as morphological defects. Migrating neurons deficient in 14-3-3gamma displayed a thicker leading process stem, and the basal ends of neurons were not able to reach the boundary between the cortical plate and the marginal zone. Consistent with the results obtained from in utero electroporation, time-lapse live imaging of brain slices revealed that the ablation of the 14-3-3gamma proteins in pyramidal neurons slowed down their migration. In addition, the 14-3-3gamma deficient neurons showed morphological abnormalities, including increased multipolar neurons with a thicker leading processes stem during migration. These results indicate that the 14-3-3gamma proteins play an important role in radial migration by regulating the morphology of migrating neurons in the cerebral cortex. The findings underscore the pathological phenotypes of brain development associated with the disruption of different 14-3-3 proteins and will advance the preclinical data regarding disorders caused by neuronal migration defects.

  15. Tumor suppressor SMAR1 mediates cyclin D1 repression by recruitment of the SIN3/histone deacetylase 1 complex.

    PubMed

    Rampalli, Shravanti; Pavithra, L; Bhatt, Altaf; Kundu, Tapas K; Chattopadhyay, Samit

    2005-10-01

    Matrix attachment region binding proteins have been shown to play an important role in gene regulation by altering chromatin in a stage- and tissue-specific manner. Our previous studies report that SMAR1, a matrix-associated protein, regresses B16-F1-induced tumors in mice. Here we show SMAR1 targets the cyclin D1 promoter, a gene product whose dysregulation is attributed to breast malignancies. Our studies reveal that SMAR1 represses cyclin D1 gene expression, which can be reversed by small interfering RNA specific to SMAR1. We demonstrate that SMAR1 interacts with histone deacetylation complex 1, SIN3, and pocket retinoblastomas to form a multiprotein repressor complex. This interaction is mediated by the SMAR1(160-350) domain. Our data suggest SMAR1 recruits a repressor complex to the cyclin D1 promoter that results in deacetylation of chromatin at that locus, which spreads to a distance of at least the 5 kb studied upstream of the cyclin D1 promoter. Interestingly, we find that the high induction of cyclin D1 in breast cancer cell lines can be correlated to the decreased levels of SMAR1 in these lines. Our results establish the molecular mechanism exhibited by SMAR1 to regulate cyclin D1 by modification of chromatin. PMID:16166625

  16. HUMAN PAPILLOMAVIRUS E7 ENHANCES HYPOXIA-INDUCIBLE FACTOR 1 MEDIATED TRANSCRIPTION BY INHIBITING BINDING OF HISTONE DEACETYLASES

    PubMed Central

    Bodily, Jason M.; Mehta, Kavi P. M.; Laimins, Laimonis A.

    2010-01-01

    Infection by human papillomaviruses (HPVs) leads to the formation of benign lesions, warts, and in some cases, cervical cancer. The formation of these lesions is dependent upon increased expression of pro-angiogenic factors. Angiogenesis is linked to tissue hypoxia through the activity of the oxygen sensitive hypoxia inducible factor 1α (HIF-1α). Our studies indicate that the HPV E7 protein enhances HIF-1 transcriptional activity while E6 functions to counteract the repressive effects of p53. Both high and low risk HPV E7 proteins were found to bind to HIF-1α through a domain located in the the N terminus. Importantly, the ability of E7 to enhance HIF-1 activity mapped to the C terminus and correlated with the displacement of the histone deacetylases HDAC1, HDAC4, and HDAC7 from HIF-1α by E7. Our findings describe a novel role of the E7 oncoprotein in activating the function of a key transcription factor mediating hypoxic responses by blocking the binding of HDACs. PMID:21148070

  17. EGCG-mediated autophagy flux has a neuroprotection effect via a class III histone deacetylase in primary neuron cells

    PubMed Central

    Lee, Ju-Hee; Moon, Ji-Hong; Kim, Sung-Wook; Jeong, Jae-Kyo; Nazim, Uddin MD; Lee, You-Jin; Seol, Jae-Won; Park, Sang-Youel

    2015-01-01

    Prion diseases caused by aggregated misfolded prion protein (PrP) are transmissible neurodegenerative disorders that occur in both humans and animals. Epigallocatechin-3-gallate (EGCG) has preventive effects on prion disease; however, the mechanisms related to preventing prion diseases are unclear. We investigated whether EGCG, the main polyphenol in green tea, prevents neuron cell damage induced by the human prion protein. We also studied the neuroprotective mechanisms and proper signals mediated by EGCG. The results showed that EGCG protects the neuronal cells against human prion protein-induced damage through inhibiting Bax and cytochrome c translocation and autophagic pathways by increasing LC3-II and reducing and blocking p62 by using ATG5 small interfering (si) RNA and autophagy inhibitors. We further demonstrated that the neuroprotective effects of EGCG were exhibited by a class III histone deacetylase; sirt1 activation and the neuroprotective effects attenuated by sirt1 inactivation using sirt1 siRNA and sirtinol. We demonstrated that EGCG activated the autophagic pathways by inducing sirt1, and had protective effects against human prion protein-induced neuronal cell toxicity. These results suggest that EGCG may be a therapeutic agent for treatment of neurodegenerative disorders including prion diseases. PMID:25991666

  18. Inhibition of Different Histone Acetyltransferases (HATs) Uncovers Transcription-Dependent and -Independent Acetylation-Mediated Mechanisms in Memory Formation

    ERIC Educational Resources Information Center

    Merschbaecher, Katja; Hatko, Lucyna; Folz, Jennifer; Mueller, Uli

    2016-01-01

    Acetylation of histones changes the efficiency of the transcription processes and thus contributes to the formation of long-term memory (LTM). In our comparative study, we used two inhibitors to characterize the contribution of different histone acetyl transferases (HATs) to appetitive associative learning in the honeybee. For one we applied…

  19. 14-3-3ζ deficient mice in the BALB/c background display behavioural and anatomical defects associated with neurodevelopmental disorders

    PubMed Central

    Xu, Xiangjun; Jaehne, Emily J.; Greenberg, Zarina; McCarthy, Peter; Saleh, Eiman; Parish, Clare L.; Camera, Daria; Heng, Julian; Haas, Matilda; Baune, Bernhard T.; Ratnayake, Udani; Buuse, Maarten van den; Lopez, Angel F.; Ramshaw, Hayley S.; Schwarz, Quenten

    2015-01-01

    Sequencing and expression analyses implicate 14-3-3ζ as a genetic risk factor for neurodevelopmental disorders such as schizophrenia and autism. In support of this notion, we recently found that 14-3-3ζ−/− mice in the Sv/129 background display schizophrenia-like defects. As epistatic interactions play a significant role in disease pathogenesis we generated a new congenic strain in the BALB/c background to determine the impact of genetic interactions on the 14-3-3ζ−/− phenotype. In addition to replicating defects such as aberrant mossy fibre connectivity and impaired spatial memory, our analysis of 14-3-3ζ−/− BALB/c mice identified enlarged lateral ventricles, reduced synaptic density and ectopically positioned pyramidal neurons in all subfields of the hippocampus. In contrast to our previous analyses, 14-3-3ζ−/− BALB/c mice lacked locomotor hyperactivity that was underscored by normal levels of the dopamine transporter (DAT) and dopamine signalling. Taken together, our results demonstrate that dysfunction of 14-3-3ζ gives rise to many of the pathological hallmarks associated with the human condition. 14-3-3ζ-deficient BALB/c mice therefore provide a novel model to address the underlying biology of structural defects affecting the hippocampus and ventricle, and cognitive defects such as hippocampal-dependent learning and memory. PMID:26207352

  20. Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR.

    PubMed

    Stevers, Loes M; Lam, Chan V; Leysen, Seppe F R; Meijer, Femke A; van Scheppingen, Daphne S; de Vries, Rens M J M; Carlile, Graeme W; Milroy, Lech G; Thomas, David Y; Brunsveld, Luc; Ottmann, Christian

    2016-03-01

    Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein-protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3-CFTR interface might offer an approach for cystic fibrosis therapeutics. PMID:26888287

  1. Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR.

    PubMed

    Stevers, Loes M; Lam, Chan V; Leysen, Seppe F R; Meijer, Femke A; van Scheppingen, Daphne S; de Vries, Rens M J M; Carlile, Graeme W; Milroy, Lech G; Thomas, David Y; Brunsveld, Luc; Ottmann, Christian

    2016-03-01

    Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein-protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3-CFTR interface might offer an approach for cystic fibrosis therapeutics.

  2. The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase.

    PubMed Central

    Jahn, T; Fuglsang, A T; Olsson, A; Brüntrup, I M; Collinge, D B; Volkmann, D; Sommarin, M; Palmgren, M G; Larsson, C

    1997-01-01

    Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H(+)-ATPase isolated from fusicoccin-treated maize shoots was copurified with the 14-3-3 protein (as determined by protein gel blotting), and the H(+)-ATPase was recovered in an activated state. In the absence of fusicoccin treatment, H(+)-ATPase and the 14-3-3 protein were well separated, and the H(+)-ATPase was recovered in a nonactivated form. Trypsin treatment removed the 10-kD C-terminal region from the H(+)-ATPase as well as the 14-3-3 protein. Using the yeast two-hybrid system, we could show a direct interaction between Arabidopsis 14-3-3 GF14-phi and the last 98 C-terminal amino acids of the Arabidopsis AHA2 plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase. PMID:9368417

  3. 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

  4. Phosphorylation of Thr-948 at the C terminus of the plasma membrane H(+)-ATPase creates a binding site for the regulatory 14-3-3 protein.

    PubMed Central

    Svennelid, F; Olsson, A; Piotrowski, M; Rosenquist, M; Ottman, C; Larsson, C; Oecking, C; Sommarin, M

    1999-01-01

    The plant plasma membrane H(+)-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H(+)-ATPase-14-3-3 complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, QQXYpT(948)V, at the C terminus of the H(+)-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H(+)-ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H(+)-ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H(+)-ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin. Furthermore, binding of 14-3-3 to the H(+)-ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H(+)-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H(+)-ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H(+)-ATPase in vivo. Indeed, replacing Thr-948 in the plant H(+)-ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H(+)-ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H(+)-ATPase activity in the plant and thus for plant growth. PMID:10590165

  5. Structural Basis for the 14-3-3 Protein-dependent Inhibition of the Regulator of G Protein Signaling 3 (RGS3) Function*

    PubMed Central

    Rezabkova, Lenka; Man, Petr; Novak, Petr; Herman, Petr; Vecer, Jaroslav; Obsilova, Veronika; Obsil, Tomas

    2011-01-01

    Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins for the α-subunit of heterotrimeric G proteins. The function of certain RGS proteins is negatively regulated by 14-3-3 proteins, a family of highly conserved regulatory molecules expressed in all eukaryotes. In this study, we provide a structural mechanism for 14-3-3-dependent inhibition of RGS3-Gα interaction. We have used small angle x-ray scattering, hydrogen/deuterium exchange kinetics, and Förster resonance energy transfer measurements to determine the low-resolution solution structure of the 14-3-3ζ·RGS3 complex. The structure shows the RGS domain of RGS3 bound to the 14-3-3ζ dimer in an as-yet-unrecognized manner interacting with less conserved regions on the outer surface of the 14-3-3 dimer outside its central channel. Our results suggest that the 14-3-3 protein binding affects the structure of the Gα interaction portion of RGS3 as well as sterically blocks the interaction between the RGS domain and the Gα subunit of heterotrimeric G proteins. PMID:22027839

  6. A Biotin Switch-Based Proteomics Approach Identifies 14-3-3ζ as a Target of Sirt1 in the Metabolic Regulation of Caspase-2

    PubMed Central

    Andersen, Joshua L.; Thompson, J. Will; Lindblom, Kelly R.; Johnson, Erika S.; Yang, Chih-Sheng; Lilley, Lauren R.; Freel, Christopher D.; Moseley, M. Arthur; Kornbluth, Sally

    2011-01-01

    While lysine acetylation in the nucleus is well characterized, comparatively little is known about its significance in cytoplasmic signaling. Here we show that inhibition of the Sirt1 deacetylase, which is primarily cytoplasmic in cancer cell lines, sensitizes these cells to caspase-2-dependent death. To identify relevant Sirt1 substrates, we developed a novel proteomics strategy, enabling the identification of a range of putative substrates, including 14-3-3ζ, a known direct regulator of caspase-2. We show here that inhibition of Sirtuin activity accelerates caspase activation and overrides caspase-2 suppression by nutrient abundance. Furthermore, 14-3-3ζ is acetylated prior to caspase activation, and supplementation of Xenopus egg extract with glucose-6-phosphate, which promotes caspase-2/14-3-3ζ binding, enhances 14-3-3ζ-directed Sirtuin activity. Conversely, inhibiting Sirtuin activity promotes 14-3-3ζ dissociation from caspase-2 in both egg extract and human cell lines. These data reveal a role for Sirt1 in modulating apoptotic sensitivity, in response to metabolic changes, by antagonizing 14-3-3ζ acetylation. PMID:21884983

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

    PubMed Central

    Zhang, Xi; Peng, Danni; Xi, Yuanxin; Yuan, Chao; Sagum, Cari A.; Klein, Brianna J.; Tanaka, Kaori; Wen, Hong; Kutateladze, Tatiana G.; Li, Wei; Bedford, Mark T.; Shi, Xiaobing

    2016-01-01

    The euchromatin histone methyltransferase 2 (also known as G9a) methylates histone H3K9 to repress gene expression, but it also acts as a coactivator for some nuclear receptors. The molecular mechanisms underlying this activation remain elusive. Here we show that G9a functions as a coactivator of the endogenous oestrogen receptor α (ERα) in breast cancer cells in a histone methylation-independent manner. G9a dimethylates ERα at K235 both in vitro and in cells. Dimethylation of ERαK235 is recognized by the Tudor domain of PHF20, which recruits the MOF histone acetyltransferase (HAT) complex to ERα target gene promoters to deposit histone H4K16 acetylation promoting active transcription. Together, our data suggest the molecular mechanism by which G9a functions as an ERα coactivator. Along with the PHF20/MOF complex, G9a links the crosstalk between ERα methylation and histone acetylation that governs the epigenetic regulation of hormonal gene expression. PMID:26960573

  8. Erasure of histone acetylation by Arabidopsis HDA6 mediates large-scale gene silencing in nucleolar dominance

    PubMed Central

    Earley, Keith; Lawrence, Richard J.; Pontes, Olga; Reuther, Rachel; Enciso, Angel J.; Silva, Manuela; Neves, Nuno; Gross, Michael; Viegas, Wanda; Pikaard, Craig S.

    2006-01-01

    Nucleolar dominance describes the silencing of one parental set of ribosomal RNA (rRNA) genes in a genetic hybrid, an epigenetic phenomenon that occurs on a scale second only to X-chromosome inactivation in mammals. An RNA interference (RNAi) knockdown screen revealed that the predicted Arabidopsis histone deacetylase, HDA6, is required for rRNA gene silencing in nucleolar dominance. In vivo, derepression of silenced rRNA genes upon knockdown of HDA6 is accompanied by nucleolus organizer region (NOR) decondensation, loss of promoter cytosine methylation, and replacement of histone H3 Lys 9 (H3K9) dimethylation with H3K4 trimethylation, H3K9 acetylation, H3K14 acetylation, and histone H4 tetra-acetylation. Consistent with these in vivo results, purified HDA6 deacetylates lysines modified by histone acetyltransferases whose substrates include H3K14, H4K5, and H4K12. HDA6 localizes, in part, to the nucleolus, supporting a model whereby HDA6 erases histone acetylation as a key step in an epigenetic switch mechanism that silences rRNA genes through concerted histone and DNA modifications. PMID:16648464

  9. 14-3-3 isoforms bind directly exon B of the 5′-UTR of human surfactant protein A2 mRNA

    PubMed Central

    Noutsios, Georgios T.; Ghattas, Paul; Bennett, Stephanie

    2015-01-01

    Human surfactant protein (SP) A (SP-A), an innate immunity molecule, is encoded by two genes, SFTPA1 and SFTPA2. The 5′-untranslated splice variant of SP-A2 (ABD), but not SP-A1 (AD), contains exon B (eB). eB is an enhancer for transcription and translation and contains cis-regulatory elements. Specific trans-acting factors, including 14-3-3, bind eB. The 14-3-3 protein family contains seven isoforms that have been found by mass spectrometry in eB electromobility shift assays (Noutsios et al. Am J Physiol Lung Cell Mol Physiol 304: L722–L735, 2013). We used four different approaches to investigate whether 14-3-3 isoforms bind directly to eB. 1) eB RNA pulldown assays showed that 14-3-3 isoforms specifically bind eB. 2) RNA electromobility shift assay complexes were formed using purified 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, with wild-type eB RNA. 3 and 4) RNA affinity chromatography assays and surface plasmon resonance analysis showed that 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, specifically and directly bind eB. Inhibition of 14-3-3 isoforms γ, ε, η, and τ/θ with shRNAs in NCI-H441 cells resulted in downregulation of SP-A2 levels but did not affect SP-A1 levels. However, inhibition of 14-3-3 isoform σ was correlated with lower levels of SP-A1 and SP-A2. Inhibition of 14-3-3 isoform ζ/δ, which does not bind eB, had no effect on expression levels of SP-A1 and SP-A2. In conclusion, the 14-3-3 protein family affects differential regulation of SP-A1 and SP-A2 by binding directly to SP-A2 5′-UTR mRNA. PMID:26001776

  10. Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR

    PubMed Central

    Stevers, Loes M.; Lam, Chan V.; Leysen, Seppe F. R.; Meijer, Femke A.; van Scheppingen, Daphne S.; de Vries, Rens M. J. M.; Carlile, Graeme W.; Milroy, Lech G.; Thomas, David Y.; Brunsveld, Luc; Ottmann, Christian

    2016-01-01

    Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein–protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3–CFTR interface might offer an approach for cystic fibrosis therapeutics. PMID:26888287

  11. Evolution of the 14-3-3 protein family: does the large number of isoforms in multicellular organisms reflect functional specificity?

    PubMed

    Rosenquist, M; Sehnke, P; Ferl, R J; Sommarin, M; Larsson, C

    2000-11-01

    14-3-3 proteins constitute a family of eukaryotic proteins that are key regulators of a large number of processes ranging from mitosis to apoptosis. 14-3-3s function as dimers and bind to particular motifs in their target proteins. To date, 14-3-3s have been implicated in regulation or stabilization of more than 35 different proteins. This number is probably only a fraction of the number of proteins that 14-3-3s bind to, as reports of new target proteins have become more frequent. An examination of 14-3-3 entries in the public databases reveals 153 isoforms, including alleloforms, reported in 48 different species. The number of isoforms range from 2, in the unicellular organism Saccharomyces cerevisiae, to 12 in the multicellular organism Arabidopsis thaliana. A phylogenetic analysis reveals that there are four major evolutionary lineages: Viridiplantae (plants), Fungi, Alveolata, and Metazoa (animals). A close examination of the aligned amino acid sequences identifies conserved amino acid residues and regions of importance for monomer stabilization, dimer formation, target protein binding, and the nuclear export function. Given the fact that 53% of the protein is conserved, including all amino acid residues in the target binding groove of the 14-3-3 monomer, one might expect little to no isoform specificity for target protein binding. However, using surface plasmon resonance we show that there are large differences in affinity between nine 14-3-3 isoforms of A. thaliana and a target peptide representing a novel binding motif present in the C terminus of the plant plasma membrane H(+)ATPase. Thus, our data suggest that one reason for the large number of isoforms found in multicellular organisms is isoform-specific functions. PMID:11080367

  12. 14-3-3γ regulates cell viability and milk fat synthesis in lipopolysaccharide-induced dairy cow mammary epithelial cells

    PubMed Central

    LIU, LIXIN; ZHANG, LI; LIN, YE; BIAN, YANJIE; GAO, XUEJUN; QU, BO; LI, QINGZHANG

    2016-01-01

    Our previous study demonstrated that 14-3-3γ overexpression was able to inhibit the production of lipopolysaccharide (LPS)-induced cytokines in dairy cow mammary epithelial cells (DCMECs) by inhibiting the activation of nuclear factor-κB (NF-κB) signaling pathways. However, the association between 14-3-3γ overexpression and milk fat synthesis in LPS-induced DCMECs remains unclear. Therefore, the present study investigated the effect of 14-3-3γ on cell viability and milk fat synthesis in LPS-induced DCMECs. The results of the MTT assay and lactate dehydrogenase activity assay demonstrated that 14-3-3γ overexpression was able to attenuate LPS-induced cytotoxicity in DCMECs, and increase the viability of the cells. In addition, the results of reverse transcription-quantitative polymerase chain reaction suggested that mRNA expression levels of genes associated with milk fat synthesis, including sterol regulatory element binding protein (SREBP1), peroxisome proliferator-activated receptor-γ (PPARG), cluster of differentiation 36, acetyl-coA carboxylase (ACC), fatty acid synthase (FAS) and fatty acid binding protein-3, were significantly upregulated in cells overexpressing the 14-3-3γ protein. In addition, as compared with the LPS-treated group, the activities of FAS and ACC were significantly increased. Furthermore, western blotting demonstrated that 14-3-3γ overexpression enhanced the protein expression levels of phosphorylated SREBP1 and PPARG. These results suggested that high levels of 14-3-3γ protein were able to attenuate LPS-induced cell damage and promote milk fat synthesis in LPS-induced DCMECs by increasing the cell viability and upregulating the expression levels of transcription factors associated with milk fat synthesis. PMID:27073437

  13. Upregulation of lactate dehydrogenase a by 14-3-3ζ leads to increased glycolysis critical for breast cancer initiation and progression

    PubMed Central

    Chang, Chia-Chi; Zhang, Chenyu; Zhang, Qingling; Sahin, Ozgur; Wang, Hai; Xu, Jia; Xiao, Yi; Zhang, Jian; Rehman, Sumaiyah K.; Li, Ping; Hung, Mien-Chie; Behbod, Fariba; Yu, Dihua

    2016-01-01

    Metabolic reprogramming is a hallmark of cancer. Elevated glycolysis in cancer cells switches the cellular metabolic flux to produce more biological building blocks, thereby sustaining rapid proliferation. Recently, new evidence has emerged that metabolic dysregulation may occur at early-stages of neoplasia and critically contribute to cancer initiation. Here, our bioinformatics analysis of microarray data from early-stages breast neoplastic lesions revealed that 14-3-3ζ expression is strongly correlated with the expression of canonical glycolytic genes, particularly lactate dehydrogenase A (LDHA). Experimentally, increasing 14-3-3ζ expression in human mammary epithelial cells (hMECs) up-regulated LDHA expression, elevated glycolytic activity, and promoted early transformation. Knockdown of LDHA in the 14-3-3ζ-overexpressing hMECs significantly reduced glycolytic activity and inhibited transformation. Mechanistically, 14-3-3ζ overexpression activates the MEK-ERK-CREB axis, which subsequently up-regulates LDHA. In vivo, inhibiting the activated the MEK/ERK pathway in 14-3-3ζ-overexpressing hMEC-derived MCF10DCIS.COM lesions led to effective inhibition of tumor growth. Therefore, targeting the MEK/ERK pathway could be an effective strategy for intervention of 14-3-3ζ-overexpressing early breast lesions. Together, our data demonstrate that overexpression of 14-3-3ζ in early stage pre-cancerous breast epithelial cells may trigger an elevated glycolysis and transcriptionally up-regulating LDHA, thereby contributes to human breast cancer initiation. PMID:27150057

  14. Trypanosomatid histones.

    PubMed

    Alsford, Sam; Horn, David

    2004-07-01

    The histones are responsible for packaging and regulating access to eukaryotic genomes. Trypanosomatids are flagellated protists that diverged early from the eukaryotic lineage and include parasites that cause disease in humans and other mammals. Here, we review the properties of histones in parasitic trypanosomatids, from gene organization and sequence to expression, post-translational modification and function within chromatin. Phylogenetic and experimental analysis indicates that certain specifically conserved histone sequence motifs, particularly within the N-terminal 'tail' domains, possibly represent functionally important modification substrates conserved throughout the eukaryotic lineage. For example, histone H3 contains a highly conserved methylation substrate. Trypanosomatids also possess at least three variant histones. Among these is an orthologue of H2A.Z, a histone involved in protecting 'active' chromatin from silencing in yeast. Histones provide docking platforms for a variety of regulatory factors. The presence of histone modification and variant histones in trypanosomatids therefore represents evidence for a network that provides the discrimination required to regulate transcription, recombination, repair and chromosome replication and segregation.

  15. Nuclear localization and interaction of RolB with plant 14-3-3 proteins correlates with induction of adventitious roots by the oncogene rolB.

    PubMed

    Moriuchi, Hiroshi; Okamoto, Chiho; Nishihama, Ryuichi; Yamashita, Ichiro; Machida, Yasunori; Tanaka, Nobukazu

    2004-04-01

    The rooting-locus gene B (rolB) on the T-DNA of the root-inducing (Ri) plasmid in Agrobacterium rhizogenes is responsible for the induction of transformed adventitious roots, although the root induction mechanism is unknown. We report here that the RolB protein of pRi1724 (1724RolB) is associated with Nicotianatabacum14-3-3-like protein omegaII (Nt14-3-3 omegaII) in tobacco bright yellow (BY)-2 cells. Nt14-3-3 omegaII directly interacts with 1724RolB protein. Green fluorescent protein (GFP)-fused 1724RolB is localized to the nucleus. GFP-fused mutant 1724RolB proteins having a deletion or amino acid substitution are unable to interact with Nt14-3-3 omegaII and also show impaired nuclear localization. Moreover, these 1724RolB mutants show decreased capacity for adventitious root induction. These results suggest that adventitious root induction by 1724RolB protein correlates with its interaction with Nt14-3-3 omegaII and the nuclear localization of 1724RolB protein. PMID:15078329

  16. leptin-induced growth stimulation of breast cancer cells involves recruitment of histone acetyltransferases and mediator complex to CYCLIN D1 promoter via activation of Stat3.

    PubMed

    Saxena, Neeraj K; Vertino, Paula M; Anania, Frank A; Sharma, Dipali

    2007-05-01

    Numerous epidemiological studies documented that obesity is a risk factor for breast cancer development in postmenopausal women. Leptin, the key player in the regulation of energy balance and body weight control also acts as a growth factor on certain organs in both normal and disease state. In this study, we analyzed the role of leptin and the molecular mechanism(s) underlying its action in breast cancer cells that express both short and long isoforms of leptin receptor. Leptin increased MCF7 cell population in the S-phase of the cell cycle along with a robust increase in CYCLIN D1 expression. Also, leptin induced Stat3-phosphorylation-dependent proliferation of MCF7 cells as blocking Stat3 phosphorylation with a specific inhibitor, AG490, abolished leptin-induced proliferation. Using deletion constructs of CYCLIN D1 promoter and chromatin immunoprecipitation assay, we show that leptin induced increase in CYCLIN D1 promoter activity is mediated through binding of activated Stat3 at the Stat binding sites and changes in histone acetylation and methylation. We also show specific involvement of coactivator molecules, histone acetyltransferase SRC1, and mediator complex in leptin-mediated regulation of CYCLIN D1 promoter. Importantly, silencing of SRC1 and Med1 abolished the leptin induced increase in CYCLIN D1 expression and MCF7 cell proliferation. Intriguingly, recruitment of both SRC1 and Med1 was dependent on phosphorylated Stat3 as AG490 treatment inhibited leptin-induced recruitment of these coactivators to CYCLIN D1 promoter. Our data suggest that CYCLIN D1 may be a target gene for leptin mediated growth stimulation of breast cancer cells and molecular mechanisms involve activated Stat3-mediated recruitment of distinct coactivator complexes.

  17. `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.

  18. `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

  19. Histone Methyltransferase NSD2/MMSET Mediates Constitutive NF-κB Signaling for Cancer Cell Proliferation, Survival, and Tumor Growth via a Feed-Forward Loop

    PubMed Central

    Yang, Ping; Guo, Linlang; Duan, Zhijian J.; Tepper, Clifford G.; Xue, Ling; Chen, Xinbin; Kung, Hsing-Jien; Gao, Allen C.

    2012-01-01

    Constitutive NF-κB activation by proinflammatory cytokines plays a major role in cancer progression. However, the underlying mechanism is still unclear. We report here that histone methyltransferase NSD2 (also known as MMSET or WHSC1), a target of bromodomain protein ANCCA/ATAD2, acts as a strong coactivator of NF-κB by directly interacting with NF-κB for activation of target genes, including those for interleukin-6 (IL-6), IL-8, vascular endothelial growth factor A (VEGFA), cyclin D, Bcl-2, and survivin, in castration-resistant prostate cancer (CRPC) cells. NSD2 is recruited to the target gene promoters upon induction and mediates NF-κB activation-associated elevation of histone H3K36me2 and H3K36me3 marks at the promoter, which involves its methylase activity. Interestingly, we found that NSD2 is also critical for cytokine-induced recruitment of NF-κB and acetyltransferase p300 and histone hyperacetylation. Importantly, NSD2 is overexpressed in prostate cancer tumors, and its overexpression correlates with NF-κB activation. Furthermore, NSD2 expression is strongly induced by tumor necrosis factor alpha (TNF-α) and IL-6 via NF-κB and plays a crucial role in tumor growth. These results identify NSD2 to be a key chromatin regulator of NF-κB and mediator of the cytokine autocrine loop for constitutive NF-κB activation and emphasize the important roles played by NSD2 in cancer cell proliferation and survival and tumor growth. PMID:22645312

  20. Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 α-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein.

    PubMed

    Llopis-Torregrosa, Vicent; Ferri-Blázquez, Alba; Adam-Artigues, Anna; Deffontaines, Emilie; van Heusden, G Paul H; Yenush, Lynne

    2016-07-15

    Cell viability requires adaptation to changing environmental conditions. Ubiquitin-mediated endocytosis plays a crucial role in this process, because it provides a mechanism to remove transport proteins from the membrane. Arrestin-related trafficking proteins are important regulators of the endocytic pathway in yeast, facilitating selective ubiquitylation of target proteins by the E3 ubiquitin ligase, Rsp5. Specifically, Rod1 (Art4) has been reported to regulate the endocytosis of both the Hxt1, Hxt3, and Hxt6 glucose transporters and the Jen1 lactate transporter. Also, the AMP kinase homologue, Snf1, and 14-3-3 proteins have been shown to regulate Jen1 via Rod1. Here, we further characterized the role of Rod1, Snf1, and 14-3-3 in the signal transduction route involved in the endocytic regulation of the Hxt6 high affinity glucose transporter by showing that Snf1 interacts specifically with Rod1 and Rog3 (Art7), that the interaction between the Bmh2 and several arrestin-related trafficking proteins may be modulated by carbon source, and that both the 14-3-3 protein Bmh2 and the Snf1 regulatory domain interact with the arrestin-like domain containing the N-terminal half of Rod1 (amino acids 1-395). Finally, using both co-immunoprecipitation and bimolecular fluorescence complementation, we demonstrated the interaction of Rod1 with Hxt6 and showed that the localization of the Rod1-Hxt6 complex at the plasma membrane is affected by carbon source and is reduced upon overexpression of SNF1 and BMH2. PMID:27261460

  1. The Tomato 14-3-3 protein TFT4 modulates H+ efflux, basipetal auxin transport, and the PKS5-J3 pathway in the root growth response to alkaline stress.

    PubMed

    Xu, Weifeng; Jia, Liguo; Shi, Weiming; Baluska, Frantisek; Kronzucker, Herbert J; Liang, Jiansheng; Zhang, Jianhua

    2013-12-01

    Alkaline stress is a common environmental stress, in particular in salinized soils. Plant roots respond to a variety of soil stresses by regulating their growth, but the nature of the regulatory pathways engaged in the alkaline stress response (ASR) is not yet understood. Previous studies show that PIN-FORMED2, an auxin (indole-3-acetic acid [IAA]) efflux transporter, PKS5, a protein kinase, and DNAJ HOMOLOG3 (J3), a chaperone, play key roles in root H(+) secretion by regulating plasma membrane (PM) H(+)-ATPases directly or by targeting 14-3-3 proteins. Here, we investigated the expression of all 14-3-3 gene family members (TOMATO 14-3-3 PROTEIN1 [TFT1]-TFT12) in tomato (Solanum lycopersicum) under ASR, showing the involvement of four of them, TFT1, TFT4, TFT6, and TFT7. When these genes were separately introduced into Arabidopsis (Arabidopsis thaliana) and overexpressed, only the growth of TFT4 overexpressors was significantly enhanced when compared with the wild type under stress. H(+) efflux and the activity of PM H(+)-ATPase were significantly enhanced in the root tips of TFT4 overexpressors. Microarray analysis and pharmacological examination of the overexpressor and mutant plants revealed that overexpression of TFT4 maintains primary root elongation by modulating PM H(+)-ATPase-mediated H(+) efflux and basipetal IAA transport in root tips under alkaline stress. TFT4 further plays important roles in the PKS5-J3 signaling pathway. Our study demonstrates that TFT4 acts as a regulator in the integration of H(+) efflux, basipetal IAA transport, and the PKS5-J3 pathway in the ASR of roots and coordinates root apex responses to alkaline stress for the maintenance of primary root elongation. PMID:24134886

  2. Ethanol induced acetylation of histone at G9a exon1 and G9a-mediated histone H3 dimethylation leads to neurodegeneration in neonatal mice.

    PubMed

    Subbanna, S; Nagre, N N; Shivakumar, M; Umapathy, N S; Psychoyos, D; Basavarajappa, B S

    2014-01-31

    The transient exposure of immature rodents to ethanol during postnatal day 7 (P7), comparable to a time point within the third trimester of human pregnancy, induces neurodegeneration. However, the molecular mechanisms underlying the deleterious effects of ethanol on the developing brain are poorly understood. In our previous study, we showed that a high dose administration of ethanol at P7 enhances G9a and leads to caspase-3-mediated degradation of dimethylated H3 on lysine 9 (H3K9me2). In this study, we investigated the potential role of epigenetic changes at G9a exon1, G9a-mediated H3 dimethylation on neurodegeneration and G9a-associated proteins in the P7 brain following exposure to a low dose of ethanol. We found that a low dose of ethanol induces mild neurodegeneration in P7 mice, enhances specific acetylation of H3 on lysine 14 (H3K14ace) at G9a exon1, G9a protein levels, augments the dimethylation of H3K9 and H3 lysine 27 (H3K27me2). However, neither dimethylated H3K9 nor K27 underwent degradation. Pharmacological inhibition of G9a activity prior to ethanol treatment prevented H3 dimethylation and neurodegeneration. Further, our immunoprecipitation data suggest that G9a directly associates with DNA methyltransferase (DNMT3A) and methyl-CpG-binding protein 2 (MeCP2). In addition, DNMT3A and MeCP2 protein levels were enhanced by a low dose of ethanol that was shown to induce mild neurodegeneration. Collectively, these epigenetic alterations lead to association of G9a, DNMT3A and MeCP2 to form a larger repressive complex and have a significant role in low-dose ethanol-induced neurodegeneration in the developing brain.

  3. Induction of androgen formation in the male by a TAT-VDAC1 fusion peptide blocking 14-3-3ɛ protein adaptor and mitochondrial VDAC1 interactions.

    PubMed

    Aghazadeh, Yasaman; Martinez-Arguelles, Daniel B; Fan, Jinjiang; Culty, Martine; Papadopoulos, Vassilios

    2014-10-01

    Low testosterone (T), a major cause of male hypogonadism and infertility, is linked to mood changes, fatigue, osteoporosis, reduced bone-mass index, and aging. The treatment of choice, T replacement therapy, has been linked with increased risk for prostate cancer and luteinizing hormone (LH) suppression, and shown to lead to infertility, cardiovascular diseases, and obesity. Alternate methods to induce T with lower side effects are desirable. In search of the mechanisms regulating T synthesis in the testes, we identified the 14-3-3ɛ protein adaptor as a negative regulator of steroidogenesis. Steroidogenesis begins in mitochondria. 14-3-3ɛ interacts with the outer mitochondrial membrane voltage-dependent anion channel (VDAC1) protein, forming a scaffold that limits the availability of cholesterol for steroidogenesis. We report the development of a tool able to induce endogenous T formation. Peptides able to penetrate testes conjugated to 14-3-3ɛ site of interaction with VDAC1 blocked 14-3-3ɛ-VDAC1 interactions while at the same time increased VDAC1-translocator protein (18 kDa) interactions that induced steroid formation in rat testes, leading to increased serum T levels. These peptides rescued intratesticular and serum T formation in adult male rats treated with gonadotropin-releasing hormone antagonist, which dampened LH and T production.

  4. Bacteria-induced natural product formation in the fungus Aspergillus nidulans requires Saga/Ada-mediated histone acetylation.

    PubMed

    Nützmann, Hans-Wilhelm; Reyes-Dominguez, Yazmid; Scherlach, Kirstin; Schroeckh, Volker; Horn, Fabian; Gacek, Agnieszka; Schümann, Julia; Hertweck, Christian; Strauss, Joseph; Brakhage, Axel A

    2011-08-23

    Sequence analyses of fungal genomes have revealed that the potential of fungi to produce secondary metabolites is greatly underestimated. In fact, most gene clusters coding for the biosynthesis of antibiotics, toxins, or pigments are silent under standard laboratory conditions. Hence, it is one of the major challenges in microbiology to uncover the mechanisms required for pathway activation. Recently, we discovered that intimate physical interaction of the important model fungus Aspergillus nidulans with the soil-dwelling bacterium Streptomyces rapamycinicus specifically activated silent fungal secondary metabolism genes, resulting in the production of the archetypal polyketide orsellinic acid and its derivatives. Here, we report that the streptomycete triggers modification of fungal histones. Deletion analysis of 36 of 40 acetyltransferases, including histone acetyltransferases (HATs) of A. nidulans, demonstrated that the Saga/Ada complex containing the HAT GcnE and the AdaB protein is required for induction of the orsellinic acid gene cluster by the bacterium. We also showed that Saga/Ada plays a major role for specific induction of other biosynthesis gene clusters, such as sterigmatocystin, terrequinone, and penicillin. Chromatin immunoprecipitation showed that the Saga/Ada-dependent increase of histone 3 acetylation at lysine 9 and 14 occurs during interaction of fungus and bacterium. Furthermore, the production of secondary metabolites in A. nidulans is accompanied by a global increase in H3K14 acetylation. Increased H3K9 acetylation, however, was only found within gene clusters. This report provides previously undescribed evidence of Saga/Ada dependent histone acetylation triggered by prokaryotes. PMID:21825172

  5. Exon B of human surfactant protein A2 mRNA, alone or within its surrounding sequences, interacts with 14-3-3; role of cis-elements and secondary structure

    PubMed Central

    Noutsios, Georgios T.; Silveyra, Patricia; Bhatti, Faizah

    2013-01-01

    Human surfactant protein A, an innate immunity molecule, is encoded by two genes: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The 5′ untranslated (5′UTR) splice variant of SP-A2 (ABD), but not of SP-A1 (AD), contains exon B (eB), which is an enhancer for transcription and translation. We investigated whether eB contains cis-regulatory elements that bind trans-acting factors in a sequence-specific manner as well as the role of the eB mRNA secondary structure. Binding of cytoplasmic NCI-H441 proteins to wild-type eB, eB mutant, AD, and ABD 5′UTR mRNAs were studied by RNA electromobility shift assays (REMSAs). The bound proteins were identified by mass spectroscopy and specific antibodies (Abs). We found that 1) proteins bind eB mRNA in a sequence-specific manner, with two cis-elements identified within eB to be important; 2) eB secondary structure is necessary for binding; 3) mass spectroscopy and specific Abs in REMSAs identified 14-3-3 proteins to bind (directly or indirectly) eB and the natural SP-A2 (ABD) splice variant but not the SP-A1 (AD) splice variant; 4) other ribosomal and cytoskeletal proteins, and translation factors, are also present in the eB mRNA-protein complex; 5) knockdown of 14-3-3 β/α isoform resulted in a downregulation of SP-A2 expression. In conclusion, proteins including the 14-3-3 family bind two cis-elements within eB of hSP-A2 mRNA in a sequence- and secondary structure-specific manner. Differential regulation of SP-A1 and SP-A2 is mediated by the 14-3-3 protein family as well as by a number of other proteins that bind UTRs with or without eB mRNA. PMID:23525782

  6. Identification of candidate genes for psychosis in rat models, and possible association between schizophrenia and the 14-3-3eta gene.

    PubMed

    Wong, A H C; Macciardi, F; Klempan, T; Kawczynski, W; Barr, C L; Lakatoo, S; Wong, M; Buckle, C; Trakalo, J; Boffa, E; Oak, J; Azevedo, M-H; Dourado, A; Coelho, I; Macedo, A; Vicente, A; Valente, J; Ferreira, C P; Pato, M T; Pato, C N; Kennedy, J L; Van Tol, H H M

    2003-02-01

    Although the genetic contribution to schizophrenia is substantial, positive findings in whole-genome linkage scans have not been consistently replicated. We analyzed gene expression in various rat conditions to identify novel candidate genes for schizophrenia. Suppression subtraction hybridization (SSH), with polyA mRNA from temporal and frontal cortex of rats, was used to identify differentially expressed genes. Expression of mRNA was compared between adult Lewis and Fischer 344 (F344) rats, adult and postnatal day 6 (d6) F344, and adult F344 treated with haloperidol or control vehicle. These groups were chosen because each highlights a particular aspect of schizophrenia: differences in strain vulnerability to behavioral analogs of psychosis; factors that may relate to disease onset in relation to CNS development; and improvement of symptoms by haloperidol. The 14-3-3 gene family, as represented by 14-3-3gamma and 14-3-3zeta isoforms in the SSH study, and SNAP-25 were among the candidate genes. Genetic association between schizophrenia and the 14-3-3eta gene, positioned close to a genomic locus implicated in schizophrenia, and SNAP-25 genes was analyzed in 168 schizophrenia probands and their families. These findings address three different genes in the 14-3-3 family. We find a significant association with schizophrenia for two polymorphisms in the 14-3-3eta gene: a 7 bp variable number of tandem repeats in the 5' noncoding region (P=0.036, 1 df), and a 3' untranslated region SNP (753G/A) that is an RFLP visualized with Ava II (P=0.028). There was no significant genetic association with SNAP-25. The candidate genes identified may be of functional importance in the etiology, pathophysiology or treatment response of schizophrenia or psychotic symptoms. This is to our knowledge the first report of a significant association between the 14-3-3eta-chain gene and schizophrenia in a family-based sample, strengthening prior association reports in case-control studies and

  7. An embryonic stage–specific enhancer within the murine β-globin locus mediates domain-wide histone hyperacetylation

    PubMed Central

    Fromm, George; Cadiz-Rivera, Brenda; de Vries, Christina; Getman, Michael; McGrath, Kathleen E.; Kingsley, Paul D.; Fields, Jennifer; Fiering, Steven

    2011-01-01

    In mammalian nuclei, a select number of tissue-specific gene loci exhibit broadly distributed patterns of histone modifications, such as histone hyperacetylation, that are normally associated with active gene promoters. Previously, we characterized such hyperacetylated domains within mammalian β-globin gene loci, and determined that within the murine locus, neither the β-globin locus control region nor the gene promoters were required for domain formation. Here, we identify a developmentally specific erythroid enhancer, hypersensitive site-embryonic 1 (HS-E1), located within the embryonic β-globin domain in mouse, which is homologous to a region located downstream of the human embryonic ϵ-globin gene. This sequence exhibits nuclease hypersensitivity in primitive erythroid cells and acts as an enhancer in gain-of-function assays. Deletion of HS-E1 from the endogenous murine β-globin locus results in significant decrease in the expression of the embryonic β-globin genes and loss of the domain-wide pattern of histone hyperacetylation. The data suggest that HS-E1 is an enhancer that is uniquely required for β-like globin expression in primitive erythroid cells, and that it defines a novel class of enhancer that works in part by domain-wide modulation of chromatin structure. PMID:21321362

  8. The Histone Deacetylase Sirt6 Controls Embryonic Stem Cell Fate Via Tet-Mediated Production of 5-Hydroxymethylcytosine

    PubMed Central

    Etchegaray, Jean-Pierre; Chavez, Lukas; Huang, Yun; Ross, Kenneth N.; Choi, Jiho; Martinez-Pastor, Barbara; Walsh, Ryan M.; Sommer, Cesar A.; Lienhard, Matthias; Kugel, Sita; Silberman, Dafne M.; Ramaswamy, Sridhar; Mostoslavsky, Gustavo; Hochedlinger, Konrad; Goren, Alon; Rao, Anjana; Mostoslavsky, Raul

    2015-01-01

    How embryonic stem cells (ESC) commit to specific cell lineages and ultimately yield all cell types of a fully formed organism remains a major question. ESC differentiation is accompanied by large-scale histone and DNA modifications, but the relations between these two categories of epigenetic changes are not understood. Here we demonstrate the hierarchical interplay between the histone deacetylase, sirtuin 6 (Sirt6), which targets acetylated histone H3 at lysines 9 and 56 (H3K9ac and H3K56ac), and the Tet (Ten-eleven translocation) enzymes, which convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). ESCs derived from Sirt6 knockout (S6KO) mice are skewed towards neuroectoderm development. This phenotype is associated with derepression of Oct4, Sox2 and Nanog, which in turn causes an upregulation of Tet enzymes and elevated production of 5hmC. Genome-wide analysis revealed an upregulation of neuroectoderm genes marked with 5hmC in S6KO ESCs, thereby implicating Tet enzymes in the neuroectoderm-skewed differentiation phenotype of S6KO ESCs, which is fully rescued upon knockdown of Tets. We demonstrate a new role for Sirt6 as a chromatin regulator safeguarding the balance between pluripotency and differentiation through Tet-dependent regulation of 5hmC levels. PMID:25915124

  9. Histone Deacetylases

    PubMed Central

    Parbin, Sabnam; Kar, Swayamsiddha; Shilpi, Arunima; Sengupta, Dipta; Deb, Moonmoon; Rath, Sandip Kumar

    2014-01-01

    In the current era of genomic medicine, diseases are identified as manifestations of anomalous patterns of gene expression. Cancer is the principal example among such maladies. Although remarkable progress has been achieved in the understanding of the molecular mechanisms involved in the genesis and progression of cancer, its epigenetic regulation, particularly histone deacetylation, demands further studies. Histone deacetylases (HDACs) are one of the key players in the gene expression regulation network in cancer because of their repressive role on tumor suppressor genes. Higher expression and function of deacetylases disrupt the finely tuned acetylation homeostasis in both histone and non-histone target proteins. This brings about alterations in the genes implicated in the regulation of cell proliferation, differentiation, apoptosis and other cellular processes. Moreover, the reversible nature of epigenetic modulation by HDACs makes them attractive targets for cancer remedy. This review summarizes the current knowledge of HDACs in tumorigenesis and tumor progression as well as their contribution to the hallmarks of cancer. The present report also describes briefly various assays to detect histone deacetylase activity and discusses the potential role of histone deacetylase inhibitors as emerging epigenetic drugs to cure cancer. PMID:24051359

  10. Brainstem deficiency of the 14-3-3 regulator of serotonin synthesis: a proteomics analysis in the sudden infant death syndrome.

    PubMed

    Broadbelt, Kevin G; Rivera, Keith D; Paterson, David S; Duncan, Jhodie R; Trachtenberg, Felicia L; Paulo, Joao A; Stapels, Martha D; Borenstein, Natalia S; Belliveau, Richard A; Haas, Elisabeth A; Stanley, Christina; Krous, Henry F; Steen, Hanno; Kinney, Hannah C

    2012-01-01

    Impaired brainstem responses to homeostatic challenges during sleep may result in the sudden infant death syndrome (SIDS). Previously we reported a deficiency of serotonin (5-HT) and its key biosynthetic enzyme, tryptophan hydroxylase (TPH2), in SIDS infants in the medullary 5-HT system that modulates homeostatic responses during sleep. Yet, the underlying basis of the TPH2 and 5-HT deficiency is unknown. In this study, we tested the hypothesis that proteomics would uncover previously unrecognized abnormal levels of proteins related to TPH2 and 5-HT regulation in SIDS cases compared with controls, which could provide novel insight into the basis of their deficiency. We first performed a discovery proteomic analysis of the gigantocellularis of the medullary 5-HT system in the same data set with deficiencies of TPH2 and 5-HT levels. Analysis in 6 SIDS cases and 4 controls revealed a 42-75% reduction in abundance in 5 of the 6 isoforms identified of the 14-3-3 signal transduction family, which is known to influence TPH2 activity (p < 0.07). These findings were corroborated in an additional SIDS and control sample using an orthogonal MS(E)-based quantitative proteomic strategy. To confirm these proteomics results in a larger data set (38 SIDS, 11 controls), we applied Western blot analysis in the gigantocellularis and found that 4/7 14-3-3 isoforms identified were significantly reduced in SIDS cases (p ≤ 0.02), with a 43% reduction in all 14-3-3 isoforms combined (p < 0.001). Abnormalities in 5-HT and TPH2 levels and 5-HT(1A) receptor binding were associated with the 14-3-3 deficits in the same SIDS cases. These data suggest a potential molecular defect in SIDS related to TPH2 regulation, as 14-3-3 is critical in this process. PMID:21976671

  11. Brainstem Deficiency of the 14-3-3 Regulator of Serotonin Synthesis: A Proteomics Analysis in the Sudden Infant Death Syndrome*

    PubMed Central

    Broadbelt, Kevin G.; Rivera, Keith D.; Paterson, David S.; Duncan, Jhodie R.; Trachtenberg, Felicia L.; Paulo, Joao A.; Stapels, Martha D.; Borenstein, Natalia S.; Belliveau, Richard A.; Haas, Elisabeth A.; Stanley, Christina; Krous, Henry F.; Steen, Hanno; Kinney, Hannah C.

    2012-01-01

    Impaired brainstem responses to homeostatic challenges during sleep may result in the sudden infant death syndrome (SIDS). Previously we reported a deficiency of serotonin (5-HT) and its key biosynthetic enzyme, tryptophan hydroxylase (TPH2), in SIDS infants in the medullary 5-HT system that modulates homeostatic responses during sleep. Yet, the underlying basis of the TPH2 and 5-HT deficiency is unknown. In this study, we tested the hypothesis that proteomics would uncover previously unrecognized abnormal levels of proteins related to TPH2 and 5-HT regulation in SIDS cases compared with controls, which could provide novel insight into the basis of their deficiency. We first performed a discovery proteomic analysis of the gigantocellularis of the medullary 5-HT system in the same data set with deficiencies of TPH2 and 5-HT levels. Analysis in 6 SIDS cases and 4 controls revealed a 42–75% reduction in abundance in 5 of the 6 isoforms identified of the 14-3-3 signal transduction family, which is known to influence TPH2 activity (p < 0.07). These findings were corroborated in an additional SIDS and control sample using an orthogonal MSE-based quantitative proteomic strategy. To confirm these proteomics results in a larger data set (38 SIDS, 11 controls), we applied Western blot analysis in the gigantocellularis and found that 4/7 14-3-3 isoforms identified were significantly reduced in SIDS cases (p ≤ 0.02), with a 43% reduction in all 14-3-3 isoforms combined (p < 0.001). Abnormalities in 5-HT and TPH2 levels and 5-HT1A receptor binding were associated with the 14-3-3 deficits in the same SIDS cases. These data suggest a potential molecular defect in SIDS related to TPH2 regulation, as 14-3-3 is critical in this process. PMID:21976671

  12. Demethylation of IGFBP5 by Histone Demethylase KDM6B Promotes Mesenchymal Stem Cell-Mediated Periodontal Tissue Regeneration by Enhancing Osteogenic Differentiation and Anti-Inflammation Potentials.

    PubMed

    Liu, Dayong; Wang, Yuejun; Jia, Zhi; Wang, Liping; Wang, Jinsong; Yang, Dongmei; Song, Jianqiu; Wang, Songlin; Fan, Zhipeng

    2015-08-01

    Mesenchymal stem cell (MSC)-mediated periodontal tissue regeneration is considered a promising method for periodontitis treatment. The molecular mechanism underlying directed differentiation and anti-inflammatory actions remains unclear, thus limiting potential MSC application. We previously found that insulin-like growth factor binding protein 5 (IGFBP5) is highly expressed in dental tissue-derived MSCs compared with in non-dental tissue-derived MSCs. IGFBP5 is mainly involved in regulating biological activity of insulin-like growth factors, and its functions in human MSCs and tissue regeneration are unclear. In this study, we performed gain- and loss-of-function assays to test whether IGFBP5 could regulate the osteogenic differentiation and anti-inflammatory potential in MSCs. We found that IGFBP5 expression was upregulated upon osteogenic induction, and that IGFBP5 enhanced osteogenic differentiation in MSCs. We further showed that IGFBP5 prompted the anti-inflammation effect of MSCs via negative regulation of NFκB signaling. Depletion of the histone demethylase lysine (K)-specific demethylase 6B (KDM6B) downregulated IGFBP5 expression by increasing histone K27 methylation in the IGFBP5 promoter. Moreover, IGFBP5 expression in periodontal tissues was downregulated in individuals with periodontitis compared with in healthy people, and IGFBP5 enhanced MSC-mediated periodontal tissue regeneration and alleviated local inflammation in a swine model of periodontitis. In conclusion, our present results reveal a new function for IGFBP5, provide insight into the mechanism underlying the directed differentiation and anti-inflammation capacities of MSCs, and identify a potential target mediator for improving tissue regeneration.

  13. Single cell analysis of RNA-mediated histone H3.3 recruitment to a cytomegalovirus promoter-regulated transcription site.

    PubMed

    Newhart, Alyshia; Rafalska-Metcalf, Ilona U; Yang, Tian; Joo, Lucy M; Powers, Sara Lawrence; Kossenkov, Andrew V; Lopez-Jones, Melissa; Singer, Robert H; Showe, Louise C; Skordalakes, Emmanuel; Janicki, Susan M

    2013-07-01

    Unlike the core histones, which are incorporated into nucleosomes concomitant with DNA replication, histone H3.3 is synthesized throughout the cell cycle and utilized for replication-independent (RI) chromatin assembly. The RI incorporation of H3.3 into nucleosomes is highly conserved and occurs at both euchromatin and heterochromatin. However, neither the mechanism of H3.3 recruitment nor its essential function is well understood. Several different chaperones regulate H3.3 assembly at distinct sites. The H3.3 chaperone, Daxx, and the chromatin-remodeling factor, ATRX, are required for H3.3 incorporation and heterochromatic silencing at telomeres, pericentromeres, and the cytomegalovirus (CMV) promoter. By evaluating H3.3 dynamics at a CMV promoter-regulated transcription site in a genetic background in which RI chromatin assembly is blocked, we have been able to decipher the regulatory events upstream of RI nucleosomal deposition. We find that at the activated transcription site, H3.3 accumulates with sense and antisense RNA, suggesting that it is recruited through an RNA-mediated mechanism. Sense and antisense transcription also increases after H3.3 knockdown, suggesting that the RNA signal is amplified when chromatin assembly is blocked and attenuated by nucleosomal deposition. Additionally, we find that H3.3 is still recruited after Daxx knockdown, supporting a chaperone-independent recruitment mechanism. Sequences in the H3.3 N-terminal tail and αN helix mediate both its recruitment to RNA at the activated transcription site and its interaction with double-stranded RNA in vitro. Interestingly, the H3.3 gain-of-function pediatric glioblastoma mutations, G34R and K27M, differentially affect H3.3 affinity in these assays, suggesting that disruption of an RNA-mediated regulatory event could drive malignant transformation. PMID:23689370

  14. Adjudin disrupts spermatogenesis via the action of some unlikely partners: Eps8, Arp2/3 complex, drebrin E, PAR6 and 14-3-3.

    PubMed

    Cheng, C Yan; Lie, Pearl Py; Wong, Elissa Wp; Mruk, Dolores D; Silvestrini, Bruno

    2011-10-01

    expression of PAR6 (partitioning defective protein 6) and 14-3-3 (also known as PAR5) considerably at the apical ES, disrupting the homeostasis of endocytic vesicle-mediated protein trafficking, which in turn leads to an increase in protein endocytosis. The net result of these changes destabilizes cell adhesion and induces degeneration of the apical ES, causing premature release of spermatids, mimicking spermiation.

  15. Histone deacetylases inhibitor sodium butyrate inhibits JAK2/STAT signaling through upregulation of SOCS1 and SOCS3 mediated by HDAC8 inhibition in myeloproliferative neoplasms.

    PubMed

    Gao, Shen-meng; Chen, Chi-qi; Wang, Lu-yao; Hong, Li-li; Wu, Jian-bo; Dong, Pei-hong; Yu, Fu-jun

    2013-03-01

    Constitutive activation of Janus kinase 2/signal transducers and activators of transcription (JAK2/STAT) signaling has an important role in the oncogenesis of myeloproliferative neoplasms (MPNs) and leukemia. Histone deacetylases (HDACs) inhibitors have been reported to possess anticancer activity through different mechanisms. However, whether HDACs inhibitors suppress JAK2/STAT signaling in MPNs is still unknown. In this study, we show that the HDAC inhibitor sodium butyrate (SB) inhibited JAK2/STAT signaling and increased the expression of suppressors of cytokine signaling 1 (SOCS1) and SOCS3, both of which are the potent feedback inhibitors of JAK2/STAT signaling. SB upregulated the expression of SOCS1 and SOCS3 by triggering the promoter-associated histone acetylation of SOCS1 and SOCS3 in K562 and HEL cell lines. Importantly, we found that upon knockdown of each class I HDACs, only knockdown of HDAC8 resulted in the increased expression of SOCS1 and SOCS3. Moreover, overexpression of SOCS1 and SOCS3 significantly inhibited cell growth and suppressed JAK2/STAT signaling in K562 and HEL cells. Furthermore, SB increased the transcript levels of SOCS1 and SOCS3 and inhibited the clonogenic activity of hematopoietic progenitors from patients with MPNs. Taken together, these data establish a new anticancer mechanism that SB inhibits JAK2/STAT signaling through HDAC8-mediated upregulation of SOCS1 and SOCS3. Thus, HDACs inhibitors may have therapeutic potential for the treatment of MPNs.

  16. The histone deacetylase inhibitor sodium valproate causes limited transcriptional change in mouse embryonic stem cells but selectively overrides Polycomb-mediated Hoxb silencing

    PubMed Central

    2013-01-01

    Background Histone deacetylase inhibitors (HDACi) cause histone hyperacetylation and H3K4 hypermethylation in various cell types. They find clinical application as anti-epileptics and chemotherapeutic agents, but the pathways through which they operate remain unclear. Surprisingly, changes in gene expression caused by HDACi are often limited in extent and can be positive or negative. Here we have explored the ability of the clinically important HDACi valproic acid (VPA) to alter histone modification and gene expression, both globally and at specific genes, in mouse embryonic stem (ES) cells. Results Microarray expression analysis of ES cells exposed to VPA (1 mM, 8 h), showed that only 2.4% of genes showed a significant, >1.5-fold transcriptional change. Of these, 33% were down-regulated. There was no correlation between gene expression and VPA-induced changes in histone acetylation or H3K4 methylation at gene promoters, which were usually minimal. In contrast, all Hoxb genes showed increased levels of H3K9ac after exposure to VPA, but much less change in other modifications showing bulk increases. VPA-induced changes were lost within 24 h of inhibitor removal. VPA significantly increased the low transcription of Hoxb4 and Hoxb7, but not other Hoxb genes. Expression of Hoxb genes increased in ES cells lacking functional Polycomb silencing complexes PRC1 and PRC2. Surprisingly, VPA caused no further increase in Hoxb transcription in these cells, except for Hoxb1, whose expression increased several fold. Retinoic acid (RA) increased transcription of all Hoxb genes in differentiating ES cells within 24 h, but thereafter transcription remained the same, increased progressively or fell progressively in a locus-specific manner. Conclusions Hoxb genes in ES cells are unusual in being sensitive to VPA, with effects on both cluster-wide and locus-specific processes. VPA increases H3K9ac at all Hoxb loci but significantly overrides PRC-mediated silencing only at Hoxb4 and

  17. The EFF-1A Cytoplasmic Domain Influences Hypodermal Cell Fusions in C. elegans But Is Not Dependent on 14-3-3 Proteins

    PubMed Central

    Shinn-Thomas, Jessica H.; del Campo, Jacob J.; Wang, Jianjun; Mohler, William A.

    2016-01-01

    Background Regulatory and biophysical mechanisms of cell-cell fusion are largely unknown despite the fundamental requirement for fused cells in eukaryotic development. Only two cellular fusogens that are not of clear recent viral origin have been identified to date, both in nematodes. One of these, EFF-1, is necessary for most cell fusions in Caenorhabditis elegans. Unregulated EFF-1 expression causes lethality due to ectopic fusion between cells not developmentally programmed to fuse, highlighting the necessity of tight fusogen regulation for proper development. Identifying factors that regulate EFF-1 and its paralog AFF-1 could lead to discovery of molecular mechanisms that control cell fusion upstream of the action of a membrane fusogen. Bioinformatic analysis of the EFF-1A isoform’s predicted cytoplasmic domain (endodomain) previously revealed two motifs that have high probabilities of interacting with 14-3-3 proteins when phosphorylated. Mutation of predicted phosphorylation sites within these motifs caused measurable loss of eff-1 gene function in cell fusion in vivo. Moreover, a human 14-3-3 isoform bound to EFF-1::GFP in vitro. We hypothesized that the two 14-3-3 proteins in C. elegans, PAR-5 and FTT-2, may regulate either localization or fusion-inducing activity of EFF-1. Methodology/Principal Findings Timing of fusion events was slightly but significantly delayed in animals unable to produce full-length EFF-1A. Yet, mutagenesis and live imaging showed that phosphoserines in putative 14-3-3 binding sites are not essential for EFF-1::GFP accumulation at the membrane contact between fusion partner cells. Moreover, although the EFF-1A endodomain was required for normal rates of eff-1-dependent epidermal cell fusions, reduced levels of FTT-2 and PAR-5 did not visibly affect the function of wild-type EFF-1 in the hypodermis. Conclusions/Significance Deletion of the EFF-1A endodomain noticeably affects the timing of hypodermal cell fusions in vivo. However

  18. The docking domain of histone H2A is required for H1 binding and RSC-mediated nucleosome remodeling.

    PubMed

    Shukla, Manu Shubhdarshan; Syed, Sajad Hussain; Goutte-Gattat, Damien; Richard, John Lalith Charles; Montel, Fabien; Hamiche, Ali; Travers, Andrew; Faivre-Moskalenko, Cendrine; Bednar, Jan; Hayes, Jeffrey J; Angelov, Dimitar; Dimitrov, Stefan

    2011-04-01

    Histone variants within the H2A family show high divergences in their C-terminal regions. In this work, we have studied how these divergences and in particular, how a part of the H2A COOH-terminus, the docking domain, is implicated in both structural and functional properties of the nucleosome. Using biochemical methods in combination with Atomic Force Microscopy and Electron Cryo-Microscopy, we show that the H2A-docking domain is a key structural feature within the nucleosome. Deletion of this domain or replacement with the incomplete docking domain from the variant H2A.Bbd results in significant structural alterations in the nucleosome, including an increase in overall accessibility to nucleases, un-wrapping of ∼10 bp of DNA from each end of the nucleosome and associated changes in the entry/exit angle of DNA ends. These structural alterations are associated with a reduced ability of the chromatin remodeler RSC to both remodel and mobilize the nucleosomes. Linker histone H1 binding is also abrogated in nucleosomes containing the incomplete docking domain of H2A.Bbd. Our data illustrate the unique role of the H2A-docking domain in coordinating the structural-functional aspects of the nucleosome properties. Moreover, our data suggest that incorporation of a 'defective' docking domain may be a primary structural role of H2A.Bbd in chromatin.

  19. The docking domain of histone H2A is required for H1 binding and RSC-mediated nucleosome remodeling

    PubMed Central

    Shukla, Manu Shubhdarshan; Syed, Sajad Hussain; Goutte-Gattat, Damien; Richard, John Lalith Charles; Montel, Fabien; Hamiche, Ali; Travers, Andrew; Faivre-Moskalenko, Cendrine; Bednar, Jan; Hayes, Jeffrey J.; Angelov, Dimitar; Dimitrov, Stefan

    2011-01-01

    Histone variants within the H2A family show high divergences in their C-terminal regions. In this work, we have studied how these divergences and in particular, how a part of the H2A COOH-terminus, the docking domain, is implicated in both structural and functional properties of the nucleosome. Using biochemical methods in combination with Atomic Force Microscopy and Electron Cryo-Microscopy, we show that the H2A-docking domain is a key structural feature within the nucleosome. Deletion of this domain or replacement with the incomplete docking domain from the variant H2A.Bbd results in significant structural alterations in the nucleosome, including an increase in overall accessibility to nucleases, un-wrapping of ∼10 bp of DNA from each end of the nucleosome and associated changes in the entry/exit angle of DNA ends. These structural alterations are associated with a reduced ability of the chromatin remodeler RSC to both remodel and mobilize the nucleosomes. Linker histone H1 binding is also abrogated in nucleosomes containing the incomplete docking domain of H2A.Bbd. Our data illustrate the unique role of the H2A-docking domain in coordinating the structural-functional aspects of the nucleosome properties. Moreover, our data suggest that incorporation of a ‘defective’ docking domain may be a primary structural role of H2A.Bbd in chromatin. PMID:21131284

  20. A rare case of rapidly progressive dementia with elevated RT-QuIC and negative 14-3-3 and tau proteins.

    PubMed

    Trikamji, Bhavesh; Hamlin, Clive; Baldwin, Kelly J

    2016-05-01

    Creutzfeldt-Jakob disease (CJD) is characterized by rapidly progressing dementia with death usually occurring within 6 months. There is no verified disease-specific pre-mortem diagnostic test besides brain biopsy. We describe a 66 y old previously high functioning male who presented with a 5 month history of rapidly progressive dementia. Neurological examination revealed a score of 19/30 on MOCA testing. An extensive workup into various causes of dementia including electroencephalography and imaging studies was unremarkable. The cerebrospinal fluid was sent to National Prion Disease Center and it revealed elevated RT-QuIC levels with negative 14-3-3 and T tau proteins. Based on literature review, our case is one of few living subjects with elevated RT-QuIC levels and negative 14-3-3 and tau proteins. PMID:27249661

  1. A Glycine soja 14-3-3 protein GsGF14o participates in stomatal and root hair development and drought tolerance in Arabidopsis thaliana.

    PubMed

    Sun, Xiaoli; Luo, Xiao; Sun, Mingzhe; Chen, Chao; Ding, Xiaodong; Wang, Xuedong; Yang, Shanshan; Yu, Qingyue; Jia, Bowei; Ji, Wei; Cai, Hua; Zhu, Yanming

    2014-01-01

    It is well established that 14-3-3 proteins are key regulators of multiple stress signal transduction cascades. However, the biological functions of soybean 14-3-3 proteins, especially in plant drought response, are not yet known. In this study, we characterized a Glycine soja 14-3-3 gene, GsGF14o, which is involved in plant development and drought response. GsGF14o expression was greatly induced by drought stress, as evidenced by the quantitative real-time PCR and β-glucuronidase (GUS) activity analysis. GsGF14o overexpression in Arabidopsis thaliana resulted in decreased drought tolerance during seed germination and seedling growth. Furthermore, silencing of AtGF14µ, the most homologous 14-3-3 gene of GsGF14o, led to enhanced drought tolerance at both the seed germination and seedling stage. Unexpectedly, GsGF14o transgenic lines showed reduced water loss and transpiration rates compared with wild-type plants, which was demonstrated to be the consequence of the decreased stomatal size. At the same time, the smaller stomata due to GsGF14o overexpression led to a relatively slow net photosynthesis rate, which led to a growth penalty under drought stress. We further demonstrated that GsGF14o overexpression caused deficits in root hair formation and development, and thereby reduced the water intake capacity of the transgenic root system. In addition, GsGF14o overexpression down-regulated the transcript levels of drought-responsive marker genes. Finally, we also investigated the tissue-specific accumulation of GsGF14o by using a GUS activity assay. Collectively, the results presented here confirm that GsGF14o plays a dual role in drought stress responses through its involvement in the regulation of stomatal size and root hair development.

  2. p38- and MK2-dependent signalling promotes stress-induced centriolar satellite remodelling via 14-3-3-dependent sequestration of CEP131/AZI1

    PubMed Central

    Tollenaere, Maxim A. X.; Villumsen, Bine H.; Blasius, Melanie; Nielsen, Julie C.; Wagner, Sebastian A.; Bartek, Jiri; Beli, Petra; Mailand, Niels; Bekker-Jensen, Simon

    2015-01-01

    Centriolar satellites (CS) are small granular structures that cluster in the vicinity of centrosomes. CS are highly susceptible to stress stimuli, triggering abrupt displacement of key CS factors. Here we discover a linear p38-MK2-14-3-3 signalling pathway that specifically targets CEP131 to trigger CS remodelling after cell stress. We identify CEP131 as a substrate of the p38 effector kinase MK2 and pinpoint S47 and S78 as critical MK2 phosphorylation sites in CEP131. Ultraviolet-induced phosphorylation of these residues generates direct binding sites for 14-3-3 proteins, which sequester CEP131 in the cytoplasm to block formation of new CS, thereby leading to rapid depletion of these structures. Mutating S47 and S78 in CEP131 is sufficient to abolish stress-induced CS reorganization, demonstrating that CEP131 is the key regulatory target of MK2 and 14-3-3 in these structures. Our findings reveal the molecular mechanism underlying dynamic CS remodelling to modulate centrosome functions on cell stress. PMID:26616734

  3. Proteomics Profiling Reveals Carbohydrate Metabolic Enzymes and 14-3-3 Proteins Play Important Roles for Starch Accumulation during Cassava Root Tuberization

    PubMed Central

    Wang, Xuchu; Chang, Lili; Tong, Zheng; Wang, Dongyang; Yin, Qi; Wang, Dan; Jin, Xiang; Yang, Qian; Wang, Liming; Sun, Yong; Huang, Qixing; Guo, Anping; Peng, Ming

    2016-01-01

    Cassava is one of the most important root crops as a reliable source of food and carbohydrates. Carbohydrate metabolism and starch accumulation in cassava storage root is a cascade process that includes large amounts of proteins and cofactors. Here, comparative proteomics were conducted in cassava root at nine developmental stages. A total of 154 identified proteins were found to be differentially expressed during starch accumulation and root tuberization. Many enzymes involved in starch and sucrose metabolism were significantly up-regulated, and functional classification of the differentially expressed proteins demonstrated that the majority were binding-related enzymes. Many proteins were took part in carbohydrate metabolism to produce energy. Among them, three 14-3-3 isoforms were induced to be clearly phosphorylated during storage root enlargement. Overexpression of a cassava 14-3-3 gene in Arabidopsis thaliana confirmed that the older leaves of these transgenic plants contained higher sugar and starch contents than the wild-type leaves. The 14-3-3 proteins and their binding enzymes may play important roles in carbohydrate metabolism and starch accumulation during cassava root tuberization. These results not only deepened our understanding of the tuberous root proteome, but also uncovered new insights into carbohydrate metabolism and starch accumulation during cassava root enlargement. PMID:26791570

  4. Proteomics Profiling Reveals Carbohydrate Metabolic Enzymes and 14-3-3 Proteins Play Important Roles for Starch Accumulation during Cassava Root Tuberization.

    PubMed

    Wang, Xuchu; Chang, Lili; Tong, Zheng; Wang, Dongyang; Yin, Qi; Wang, Dan; Jin, Xiang; Yang, Qian; Wang, Liming; Sun, Yong; Huang, Qixing; Guo, Anping; Peng, Ming

    2016-01-21

    Cassava is one of the most important root crops as a reliable source of food and carbohydrates. Carbohydrate metabolism and starch accumulation in cassava storage root is a cascade process that includes large amounts of proteins and cofactors. Here, comparative proteomics were conducted in cassava root at nine developmental stages. A total of 154 identified proteins were found to be differentially expressed during starch accumulation and root tuberization. Many enzymes involved in starch and sucrose metabolism were significantly up-regulated, and functional classification of the differentially expressed proteins demonstrated that the majority were binding-related enzymes. Many proteins were took part in carbohydrate metabolism to produce energy. Among them, three 14-3-3 isoforms were induced to be clearly phosphorylated during storage root enlargement. Overexpression of a cassava 14-3-3 gene in Arabidopsis thaliana confirmed that the older leaves of these transgenic plants contained higher sugar and starch contents than the wild-type leaves. The 14-3-3 proteins and their binding enzymes may play important roles in carbohydrate metabolism and starch accumulation during cassava root tuberization. These results not only deepened our understanding of the tuberous root proteome, but also uncovered new insights into carbohydrate metabolism and starch accumulation during cassava root enlargement.

  5. Identification and interrogation of combinatorial histone modifications.

    PubMed

    Karch, Kelly R; Denizio, Jamie E; Black, Ben E; Garcia, Benjamin A

    2013-01-01

    Histone proteins are dynamically modified to mediate a variety of cellular processes including gene transcription, DNA damage repair, and apoptosis. Regulation of these processes occurs through the recruitment of non-histone proteins to chromatin by specific combinations of histone post-translational modifications (PTMs). Mass spectrometry has emerged as an essential tool to discover and quantify histone PTMs both within and between samples in an unbiased manner. Developments in mass spectrometry that allow for characterization of large histone peptides or intact protein has made it possible to determine which modifications occur simultaneously on a single histone polypeptide. A variety of techniques from biochemistry, biophysics, and chemical biology have been employed to determine the biological relevance of discovered combinatorial codes. This review first describes advancements in the field of mass spectrometry that have facilitated histone PTM analysis and then covers notable approaches to probe the biological relevance of these modifications in their nucleosomal context.

  6. Histone Octamer

    NASA Technical Reports Server (NTRS)

    1997-01-01

    This is a large 2 mm crystal of histone octamer, grown on STS-81. A very dynamic structure which functions in many aspects of gene regulation from control of gene activity to the more subtle mechanisms of genetic imprinting. Principle Investigator is Dan Carter of New Century Pharmaceuticals.

  7. Histone Octamer

    NASA Technical Reports Server (NTRS)

    1997-01-01

    1 mm histone octamer crystal grown on STS-81. A very dynamic structure which functions in many aspects of gene regulation from control of gene activity to the more subtle mechanisms of genetic imprinting. Principle Investigator is Dan Carter of New Century Pharmaceuticals.

  8. Proteomic Identification of 14-3-3ζ as an Adapter for IGF-1 and Akt/GSK-3β Signaling and Survival of Renal Mesangial Cells

    PubMed Central

    Singh, Lalit P.; Jiang, Yan; Cheng, Davis W.

    2007-01-01

    Recently we demonstrated that IGF-1 expression is increased in the diabetic kidney and that it may involve in renal hypertrophy and extracellular matrix protein (ECM) accumulation in mesangial cells as seen in diabetic glomerulopathy. The present study investigates the molecular mechanism(s) of IGF-1 and Akt/glycogen synthase kinase-3beta (GSK-3β) signaling pathway in the regulation of fibronectin and cyclin D1 expression and survival of renal mesangial cells. A proteomic approach is also employed to identify protein targets of IGF-1 signaling via GSK-3β inhibition in mesangial cells. We show that IGF-1 (100 ng/ml) significantly increases the protein kinase Akt/PKB activity (1.5-2-fold, p<0.05) within 1-5 minutes, which is completely blocked by the presence of 100 nM Wortmannin (phosphatidyl-inositol 3-kinase inhibitor). Akt activation is coupled with Ser9 phosphorylation and inactivation of its down-stream target GSK-3β. IGF-1 increases the cyclic AMP-responsive element (CRE) binding transcription factor CREB phosphorylation at Ser 133 and CRE-binding activity in mesangial cells, which parallels cyclin D1 and fibronectin expressions. Both proteins are known to have CRE-sequences in their promoter regions upstream of the transcription start site. Suppression of GSK-3β by SB216763 (100 nM) increases CREB phosphorylation, cyclin D1 and fibronectin levels. Two dimensional gel electrophoresis followed by MALDI-TOF mass spectrometric analysis of mesangial proteins reveals that IGF-1 treatment or an inhibition of GSK-3β increases the expression of the phosphorylated Ser/Thr binding signal adapter protein 14-3-3ζ. Immuno-precipitation of 14-3-3ζ followed by Western blotting validates the association of phosphorylated GSK-3β with 14-3-3ζ in renal mesangial cells. Stable expression of a constitutively active GSK-3β(Ser9Ala) induces cell death while overexpression of HA-tagged 14-3-3ζ increases cell viability as measured by MTT assays. These results indicate that

  9. Aurora B-dependent regulation of class IIa histone deacetylases by mitotic nuclear localization signal phosphorylation.

    PubMed

    Guise, Amanda J; Greco, Todd M; Zhang, Irene Y; Yu, Fang; Cristea, Ileana M

    2012-11-01

    Class IIa histone deacetylases (HDACs 4/5/7/9) are transcriptional regulators with critical roles in cardiac disease and cancer. HDAC inhibitors are promising anticancer agents, and although they are known to disrupt mitotic progression, the underlying mechanisms of mitotic regulation by HDACs are not fully understood. Here we provide the first identification of histone deacetylases as substrates of Aurora B kinase (AurB). Our study identifies class IIa HDACs as a novel family of AurB targets and provides the first evidence that HDACs are temporally and spatially regulated by phosphorylation during the cell cycle. We define the precise site of AurB-mediated phosphorylation as a conserved serine within the nuclear localization signals of HDAC4, HDAC5, and HDAC9 at Ser265, Ser278, and Ser242, respectively. We establish that AurB interacts with these HDACs in vivo, and that this association increases upon disruption of 14-3-3 binding. We observe colocalization of endogenous, phosphorylated HDACs with AurB at the mitotic midzone in late anaphase and the midbody during cytokinesis, complemented by a reduction in HDAC interactions with components of the nuclear corepressor complex. We propose that AurB-dependent phosphorylation of HDACs induces sequestration within a phosphorylation gradient at the midzone, maintaining separation from re-forming nuclei and contributing to transcriptional control.

  10. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

    SciTech Connect

    Si, Lina; Shi, Jin; Gao, Wenqun; Zheng, Min; Liu, Lingjuan; Zhu, Jing; Tian, Jie

    2014-07-18

    Highlights: • BMP2 can upregulated cardiac related gene GATA4, Nkx2.5, MEF2c and Tbx5. • Inhibition of Smad4 decreased BMP2-induced hyperacetylation of histone H3. • Inhibition of Smad4 diminished BMP2-induced overexpression of GATA4 and Nkx2.5. • Inhibition of Smad4 decreased hyperacetylated H3 in the promoter of GATA4 and Nkx2.5. • Smad4 is essential for BMP2 induced hyperacetylated histone H3. - Abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of

  11. Histone deacetylase 3 unconventional splicing mediates endothelial-to-mesenchymal transition through transforming growth factor β2.

    PubMed

    Zeng, Lingfang; Wang, Gang; Ummarino, Dario; Margariti, Andriana; Xu, Qihe; Xiao, Qingzhong; Wang, Wen; Zhang, Zhongyi; Yin, Xiaoke; Mayr, Manuel; Cockerill, Gillian; Li, Julie Yi-shuan; Chien, Shu; Hu, Yanhua; Xu, Qingbo

    2013-11-01

    Histone deacetylase 3 (HDAC3) plays a critical role in the maintenance of endothelial integrity and other physiological processes. In this study, we demonstrated that HDAC3 undergoes unconventional splicing during stem cell differentiation. Four different splicing variants have been identified, designated as HD3α, -β, -γ, and -δ, respectively. HD3α was confirmed in stem cell differentiation by specific antibody against the sequences from intron 12. Immunofluorescence staining indicated that the HD3α isoform co-localized with CD31-positive or α-smooth muscle actin-positive cells at different developmental stages of mouse embryos. Overexpression of HD3α reprogrammed human aortic endothelial cells into mesenchymal cells featuring an endothelial-to-mesenchymal transition (EndMT) phenotype. HD3α directly interacts with HDAC3 and Akt1 and selectively activates transforming growth factor β2 (TGFβ2) secretion and cleavage. TGFβ2 functioned as an autocrine and/or paracrine EndMT factor. The HD3α-induced EndMT was both PI3K/Akt- and TGFβ2-dependent. This study provides the first evidence of the role of HDAC3 splicing in the maintenance of endothelial integrity.

  12. Positive 14-3-3 and tau proteins in a sporadic Creutzfeldt-Jakob disease case and a brief perspective of prion diseases in Colombia.

    PubMed

    Escandón-Vargas, Kevin; Zorrilla-Vaca, Andrés; Corral-Prado, Raúl Heli

    2016-01-01

    Prion diseases are rare neurodegenerative disorders occurring worldwide and affecting both humans and animals. Herein, we present the case of a patient diagnosed with definite sporadic Creutzfeldt-Jakob disease in Cali, Colombia. Besides neurological examination, 14-3-3 and tau proteins were valuable tools supporting the diagnosis. We also present a brief perspective of the prion diseases reported in Colombia to date. Although the incidence of prion diseases is unknown in Colombia, our literature review revealed that one case of scrapie in 1981 and 29 human sporadic cases of Creutzfeldt-Jakob disease have been documented and published in our country. PMID:27622622

  13. Positive 14-3-3 and tau proteins in a sporadic Creutzfeldt-Jakob disease case and a brief perspective of prion diseases in Colombia.

    PubMed

    Escandón-Vargas, Kevin; Zorrilla-Vaca, Andrés; Corral-Prado, Raúl Heli

    2016-02-24

    Prion diseases are rare neurodegenerative disorders occurring worldwide and affecting both humans and animals. Herein, we present the case of a patient diagnosed with definite sporadic Creutzfeldt-Jakob disease in Cali, Colombia. Besides neurological examination, 14-3-3 and tau proteins were valuable tools supporting the diagnosis. We also present a brief perspective of the prion diseases reported in Colombia to date. Although the incidence of prion diseases is unknown in Colombia, our literature review revealed that one case of scrapie in 1981 and 29 human sporadic cases of Creutzfeldt-Jakob disease have been documented and published in our country.

  14. Histone Methyltransferase Enhancer of Zeste Homolog 2-Mediated ABCA1 Promoter DNA Methylation Contributes to the Progression of Atherosclerosis

    PubMed Central

    Wan, Wei; Yao, Feng; He, Ping-Ping; Xie, Wei; Mo, Zhong-Cheng; Shi, Jin-Feng; Wu, Jian-Feng; Peng, Juan; Liu, Dan; Cayabyab, Francisco S.; Zheng, Xi-Long; Tang, Xiang-Yang; Ouyang, Xin-Ping; Tang, Chao-Ke

    2016-01-01

    ATP-binding cassette transporter A1 (ABCA1) plays a critical role in maintaining cellular cholesterol homeostasis. The purpose of this study is to identify the molecular mechanism(s) underlying ABCA1 epigenetic modification and determine its potential impact on ABCA1 expression in macrophage-derived foam cell formation and atherosclerosis development. DNA methylation induced foam cell formation from macrophages and promoted atherosclerosis in apolipoprotein E-deficient (apoE−/−) mice. Bioinformatics analyses revealed a large CpG island (CGI) located in the promoter region of ABCA1. Histone methyltransferase enhancer of zeste homolog 2 (EZH2) downregulated ABCA1 mRNA and protein expression in THP-1 and RAW264.7 macrophage-derived foam cells. Pharmacological inhibition of DNA methyltransferase 1 (DNMT1) with 5-Aza-dC or knockdown of DNMT1 prevented the downregulation of macrophage ABCA1 expression, suggesting a role of DNA methylation in ABCA1 expression. Polycomb protein EZH2 induced DNMT1 expression and methyl-CpG-binding protein-2 (MeCP2) recruitment, and stimulated the binding of DNMT1 and MeCP2 to ABCA1 promoter, thereby promoting ABCA1 gene DNA methylation and atherosclerosis. Knockdown of DNMT1 inhibited EZH2-induced downregulation of ABCA1 in macrophages. Conversely, EZH2 overexpression stimulated DNMT1-induced ABCA1 gene promoter methylation and atherosclerosis. EZH2-induced downregulation of ABCA1 gene expression promotes foam cell formation and the development of atherosclerosis by DNA methylation of ABCA1 gene promoter. PMID:27295295

  15. Wig-1 regulates cell cycle arrest and cell death through the p53 targets FAS and 14-3-3σ

    PubMed Central

    Bersani, C; Xu, L-D; Vilborg, A; Lui, W-O; Wiman, K G

    2014-01-01

    Wig-1, also known as ZMAT3, is a p53 target gene that encodes an RNA-binding zinc-finger protein involved in the regulation of mRNA stability through binding to AU-rich elements (AREs). We have used microarray analysis to identify novel Wig-1 target mRNAs. We identified 2447 transcripts with >fourfold differential expression between Wig-1 and control small interfering (si)RNA-treated HCT116 cells. Several p53 target genes were among the deregulated transcripts. We found that Wig-1 regulates FAS and 14-3-3σ mRNA independently of p53. We show that Wig-1 binds to FAS mRNA 3′-UTR and decreases its stability through an ARE in the 3′-UTR. Depletion of Wig-1 was associated with increased cell death and reduced cell cycle arrest upon DNA damage. Our results suggest a role of Wig-1 as a survival factor that directs the p53 stress response toward cell cycle arrest rather than apoptosis through the regulation of FAS and 14-3-3σ mRNA levels. PMID:24469038

  16. Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

    SciTech Connect

    Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.; E-mail: andy.blakely@vanderbilt.edu

    2005-08-05

    The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH{sub 2}-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking.

  17. Echinococcus multilocularis laminated-layer components and the E14t 14-3-3 recombinant protein decrease NO production by activated rat macrophages in vitro.

    PubMed

    Andrade, M Amparo; Siles-Lucas, Mar; Espinoza, Elsa; Pérez Arellano, José Luis; Gottstein, Bruno; Muro, Antonio

    2004-05-01

    Echinococcus multilocularis and Echinococcus granulosus cause alveolar and cystic (unilocular) echinococcosis, respectively, in humans and animals. It is known that these parasites can affect, among other molecules, nitric oxide (NO) production by periparasitic host cells. Nevertheless, detailed dissection of parasite components specifically affecting cell NO production has not been done to date. We compare the effect of E. granulosus and E. multilocularis defined metacestode structural (laminated-layer associated) and metabolic (14-3-3 protein, potentially related with E. multilocularis metacestode tumor-like growth) components on the NO production by rat alveolar macrophages in vitro. Our results showed that none of these antigens could stimulate macrophage NO production in vitro. However, a reversed effect of some Echinococcus antigens on NO in vitro production was found when cells were previously exposed to LPS stimulation. This inhibitory effect was found when E. multilocularis laminated-layer (LL) or cyst wall (CW) soluble components from both species were used. Pre-stimulation of cells with LPS also resulted in a strong, dose-dependent reduction of NO and iNOS mRNA production after incubation of cells with the E14t protein. Thus, the E. multilocularis 14-3-3 protein appears to be one of the components accounting for the suppressive effect of the CW and LL metacestode extracts.

  18. Involvement of 14-3-3 protein GRF9 in root growth and response under polyethylene glycol-induced water stress.

    PubMed

    He, Yuchi; Wu, Jingjing; Lv, Bing; Li, Jia; Gao, Zhiping; Xu, Weifeng; Baluška, František; Shi, Weiming; Shaw, Pang Chui; Zhang, Jianhua

    2015-04-01

    Plant 14-3-3 proteins are phosphoserine-binding proteins that regulate a wide array of targets via direct protein-protein interactions. In this study, the role of a 14-3-3 protein, GRF9, in plant response to water stress was investigated. Arabidopsis wild-type, GRF9-deficient mutant (grf9), and GRF9-overexpressing (OE) plants were treated with polyethylene glycol (PEG) to induce mild water stress. OE plant showed better whole-plant growth and root growth than the wild type under normal or water stress conditions while the grf9 mutant showed worse growth. In OE plants, GRF9 favours the allocation of shoot carbon to roots. In addition, GRF9 enhanced proton extrusion, mainly in the root elongation zone and root hair zone, and maintained root growth under mild water stress. Grafting among the wild type, OE, and grf9 plants showed that when OE plants were used as the scion and GRF9 was overexpressed in the shoot, it enhanced sucrose transport into the root, and when OE plants were used as rootstock and GRF9 was overexpressed in the root, it caused more release of protons into the root surface under water stress. Taken together, the results suggest that under PEG-induced water stress, GRF9 is involved in allocating more carbon from the shoot to the root and enhancing proton secretion in the root growing zone, and this process is important for root response to mild water stress.

  19. Protein Kinase CK2 Interacts at the Neuromuscular Synapse with Rapsyn, Rac1, 14-3-3γ, and Dok-7 Proteins and Phosphorylates the Latter Two*

    PubMed Central

    Herrmann, Dustin; Straubinger, Marion; Hashemolhosseini, Said

    2015-01-01

    Previously, we demonstrated that the protein kinase CK2 associates with and phosphorylates the receptor tyrosine kinase MuSK (muscle specific receptor tyrosine kinase) at the neuromuscular junction (NMJ), thereby preventing fragmentation of the NMJs (Cheusova, T., Khan, M. A., Schubert, S. W., Gavin, A. C., Buchou, T., Jacob, G., Sticht, H., Allende, J., Boldyreff, B., Brenner, H. R., and Hashemolhosseini, S. (2006) Genes Dev. 20, 1800–1816). Here, we asked whether CK2 interacts with other proteins involved in processes at the NMJ, which would be consistent with the previous observation that CK2 appears enriched at the NMJ. We identified the following proteins to interact with protein kinase CK2: (a) the α and β subunits of the nicotinic acetylcholine receptors with weak interaction, (b) dishevelled (Dsh), and (c) another four proteins, Rapsyn, Rac1, 14-3-3γ, and Dok-7, with strong interaction. CK2 phosphorylated 14-3-3γ at serine residue 235 and Dok-7 at several serine residues but does not phosphorylate Rapsyn or Rac1. Furthermore, phosphomimetic Dok-7 mutants aggregated nicotinic acetylcholine receptors in C2C12 myotubes with significantly higher frequency than wild type Dok-7. Additionally, we mapped the interacting epitopes of all four binding partners to CK2 and thereby gained insights into the potential role of the CK2/Rapsyn interaction. PMID:26198629

  20. Protein kinase CK2 interacts at the neuromuscular synapse with Rapsyn, Rac1, 14-3-3γ, and Dok-7 proteins and phosphorylates the latter two.

    PubMed

    Herrmann, Dustin; Straubinger, Marion; Hashemolhosseini, Said

    2015-09-11

    Previously, we demonstrated that the protein kinase CK2 associates with and phosphorylates the receptor tyrosine kinase MuSK (muscle specific receptor tyrosine kinase) at the neuromuscular junction (NMJ), thereby preventing fragmentation of the NMJs (Cheusova, T., Khan, M. A., Schubert, S. W., Gavin, A. C., Buchou, T., Jacob, G., Sticht, H., Allende, J., Boldyreff, B., Brenner, H. R., and Hashemolhosseini, S. (2006) Genes Dev. 20, 1800-1816). Here, we asked whether CK2 interacts with other proteins involved in processes at the NMJ, which would be consistent with the previous observation that CK2 appears enriched at the NMJ. We identified the following proteins to interact with protein kinase CK2: (a) the α and β subunits of the nicotinic acetylcholine receptors with weak interaction, (b) dishevelled (Dsh), and (c) another four proteins, Rapsyn, Rac1, 14-3-3γ, and Dok-7, with strong interaction. CK2 phosphorylated 14-3-3γ at serine residue 235 and Dok-7 at several serine residues but does not phosphorylate Rapsyn or Rac1. Furthermore, phosphomimetic Dok-7 mutants aggregated nicotinic acetylcholine receptors in C2C12 myotubes with significantly higher frequency than wild type Dok-7. Additionally, we mapped the interacting epitopes of all four binding partners to CK2 and thereby gained insights into the potential role of the CK2/Rapsyn interaction.

  1. A calcium and free fatty acid-modulated protein kinase as putative effector of the fusicoccin 14-3-3 receptor.

    PubMed Central

    van der Hoeven, P C; Siderius, M; Korthout, H A; Drabkin, A V; de Boer, A H

    1996-01-01

    A protein kinase that is activated by calcium and cis-unsaturated fatty acids has been characterized from oat (Avena sativa L.) root plasma membranes. The kinase phosphorylates a synthetic peptide with a motif (-R-T-L-S-) that can be phosphorylated by both protein kinase C (PKC) and calcium-dependent protein kinase (CDPK)-type kinases. Calphostin C and chelerythrine, two PKC inhibitors, completely inhibited the kinase activity with values of inhibitor concentration for 50% inhibition of 0.7 and 30 microns, respectively. At low Ca2+ concentrations cis-unsaturated fatty acids (linolenic acid, linoleic acid, arachidonic acid, and oleic acid) stimulated the kinase activity almost 10-fold. The two inhibitors of the kinase, calphostin C and chelerythrin, strongly reduced the fusicoccin (FC)-induced H+ extrusion, and the activators of the kinase, the cis-unsaturated fatty acids, prevented [3H]FC binding to the FC 14-3-3 receptor. CDPK antibodies cross-reacted with a 43-kD band in the plasma membrane and in a purified FC receptor fraction. A polypeptide with the same apparent molecular mass was recognized by a synthetic peptide that has a sequence homologous to the annexin-like domain from barely 14-3-3. The possibility of the involvement of a kinase, with properties from both CDPK and PKC, and a phospholipase A2 in the FC Signal transduction pathway is discussed. PMID:8754686

  2. Involvement of 14-3-3 protein GRF9 in root growth and response under polyethylene glycol-induced water stress

    PubMed Central

    He, Yuchi; Wu, Jingjing; Lv, Bing; Li, Jia; Gao, Zhiping; Xu, Weifeng; Baluška, František; Shi, Weiming; Shaw, Pang Chui; Zhang, Jianhua

    2015-01-01

    Plant 14-3-3 proteins are phosphoserine-binding proteins that regulate a wide array of targets via direct protein–protein interactions. In this study, the role of a 14-3-3 protein, GRF9, in plant response to water stress was investigated. Arabidopsis wild-type, GRF9-deficient mutant (grf9), and GRF9-overexpressing (OE) plants were treated with polyethylene glycol (PEG) to induce mild water stress. OE plant showed better whole-plant growth and root growth than the wild type under normal or water stress conditions while the grf9 mutant showed worse growth. In OE plants, GRF9 favours the allocation of shoot carbon to roots. In addition, GRF9 enhanced proton extrusion, mainly in the root elongation zone and root hair zone, and maintained root growth under mild water stress. Grafting among the wild type, OE, and grf9 plants showed that when OE plants were used as the scion and GRF9 was overexpressed in the shoot, it enhanced sucrose transport into the root, and when OE plants were used as rootstock and GRF9 was overexpressed in the root, it caused more release of protons into the root surface under water stress. Taken together, the results suggest that under PEG-induced water stress, GRF9 is involved in allocating more carbon from the shoot to the root and enhancing proton secretion in the root growing zone, and this process is important for root response to mild water stress. PMID:25873671

  3. Intellectual disability-associated dBRWD3 regulates gene expression through inhibition of HIRA/YEM-mediated chromatin deposition of histone H3.3.

    PubMed

    Chen, Wei-Yu; Shih, Hsueh-Tzu; Liu, Kwei-Yan; Liu, Kuei-Yan; Shih, Zong-Siou; Chen, Li-Kai; Tsai, Tsung-Han; Chen, Mei-Ju; Liu, Hsuan; Tan, Bertrand Chin-Ming; Chen, Chien-Yu; Lee, Hsiu-Hsiang; Loppin, Benjamin; Aït-Ahmed, Ounissa; Wu, June-Tai

    2015-04-01

    Many causal mutations of intellectual disability have been found in genes involved in epigenetic regulations. Replication-independent deposition of the histone H3.3 variant by the HIRA complex is a prominent nucleosome replacement mechanism affecting gene transcription, especially in postmitotic neurons. However, how HIRA-mediated H3.3 deposition is regulated in these cells remains unclear. Here, we report that dBRWD3, the Drosophila ortholog of the intellectual disability gene BRWD3, regulates gene expression through H3.3, HIRA, and its associated chaperone Yemanuclein (YEM), the fly ortholog of mammalian Ubinuclein1. In dBRWD3 mutants, increased H3.3 levels disrupt gene expression, dendritic morphogenesis, and sensory organ differentiation. Inactivation of yem or H3.3 remarkably suppresses the global transcriptome changes and various developmental defects caused by dBRWD3 mutations. Our work thus establishes a previously unknown negative regulation of H3.3 and advances our understanding of BRWD3-dependent intellectual disability. PMID:25666827

  4. Intellectual disability-associated dBRWD3 regulates gene expression through inhibition of HIRA/YEM-mediated chromatin deposition of histone H3.3

    PubMed Central

    Chen, Wei-Yu; Shih, Hsueh-Tzu; Liu, Kuei-Yan; Shih, Zong-Siou; Chen, Li-Kai; Tsai, Tsung-Han; Chen, Mei-Ju; Liu, Hsuan; Tan, Bertrand Chin-Ming; Chen, Chien-Yu; Lee, Hsiu-Hsiang; Loppin, Benjamin; Aït-Ahmed, Ounissa; Wu, June-Tai

    2015-01-01

    Many causal mutations of intellectual disability have been found in genes involved in epigenetic regulations. Replication-independent deposition of the histone H3.3 variant by the HIRA complex is a prominent nucleosome replacement mechanism affecting gene transcription, especially in postmitotic neurons. However, how HIRA-mediated H3.3 deposition is regulated in these cells remains unclear. Here, we report that dBRWD3, the Drosophila ortholog of the intellectual disability gene BRWD3, regulates gene expression through H3.3, HIRA, and its associated chaperone Yemanuclein (YEM), the fly ortholog of mammalian Ubinuclein1. In dBRWD3 mutants, increased H3.3 levels disrupt gene expression, dendritic morphogenesis, and sensory organ differentiation. Inactivation of yem or H3.3 remarkably suppresses the global transcriptome changes and various developmental defects caused by dBRWD3 mutations. Our work thus establishes a previously unknown negative regulation of H3.3 and advances our understanding of BRWD3-dependent intellectual disability. PMID:25666827

  5. AIF-mediated caspase-independent necroptosis requires ATM and DNA-PK-induced histone H2AX Ser139 phosphorylation

    PubMed Central

    Baritaud, M; Cabon, L; Delavallée, L; Galán-Malo, P; Gilles, M-E; Brunelle-Navas, M-N; Susin, S A

    2012-01-01

    The alkylating DNA-damage agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces a form of caspase-independent necroptosis implicating the mitochondrial flavoprotein apoptosis-inducing factor (AIF). Following the activation of PARP-1 (poly(ADP-ribose) polymerase-1), calpains, BID (BH3 interacting domain death agonist), and BAX (Bcl-2-associated X protein), the apoptogenic form of AIF (tAIF) is translocated to the nucleus where, associated with Ser139-phosphorylated histone H2AX (γH2AX), it creates a DNA-degrading complex that provokes chromatinolysis and cell death by necroptosis. The generation of γH2AX is crucial for this form of cell death, as mutation of H2AX Ser139 to Ala or genetic ablation of H2AX abolish both chromatinolysis and necroptosis. On the contrary, reintroduction of H2AX-wt or the phosphomimetic H2AX mutant (H2AX-S139E) into H2AX−/− cells resensitizes to MNNG-triggered necroptosis. Employing a pharmacological approach and gene knockout cells, we also demonstrate in this paper that the phosphatidylinositol-3-OH kinase-related kinases (PIKKs) ATM (ataxia telangiectasia mutated) and DNA-dependent protein kinase (DNA-PK) mediate γH2AX generation and, consequently, MNNG-induced necroptosis. By contrast, H2AX phosphorylation is not regulated by ATR or other H2AX-related kinases, such as JNK. Interestingly, ATM and DNA-PK phosphorylate H2AX at Ser139 in a synergistic manner with different kinetics of activation. Early after MNNG treatment, ATM generates γH2AX. Further, DNA-PK contributes to H2AX Ser139 phosphorylation. In revealing the pivotal role of PIKKs in MNNG-induced cell death, our data uncover a milestone in the mechanisms regulating AIF-mediated caspase-independent necroptosis. PMID:22972376

  6. Histones in protistan evolution.

    PubMed

    Rizzo, P J

    1985-01-01

    The potential of comparative studies on histones for use in protistan evolution is discussed, using algal histones as specific examples. A basic premise for the importance of histones in protistan evolution is the observation that these proteins are completely absent in prokaryotes (and cytoplasmic organelles), but with few exceptions, the same five major histone types are found in all higher plants and animals. Since the histone content of the algae and other protists is not constant, some of these organisms may represent transition forms between the prokaryotic and eukaryotic modes of packaging the genetic material. Comparative studies of protistan histones may thus be of help in determining evolutionary relationships. However, several problems are encounter with protistan histones, including difficulties in isolating nuclei, proteolytic degradation, anomalous gel migration of histones, and difficulties in histone identification. Because of the above problems, and the observed variability in protistan histones, it is suggested that several criteria be employed for histone identification in protists.

  7. Spatial coordination of chloroplast and plasma membrane activities in Chara cells and its disruption through inactivation of 14-3-3 proteins.

    PubMed

    Bulychev, A A; van den Wijngaard, P W J; de Boer, A H

    2005-01-01

    In Chara corallina cells exposed to continuous light, external pH (pH(o)) and photosystem II (PSII) photochemical yield show correlated banding patterns. Photosynthetic activity is low in cell regions producing alkaline zones and high in the acid regions. We addressed the question whether (and how) photosynthetic activity and plasma membrane (PM) H+-pumping and H+-conductance are coupled in the different bands. First, PM H+-pump activity was stimulated with fusicoccin. This resulted in a more acidic pH in the acid bands without disturbing the correlation of photosynthetic electron transport and H+ fluxes across the PM. Next, H+-pump activity was reduced through microinjection of a phosphorylated peptide matching the canonical 14-3-3 binding motif RSTpSTP in the acid cell region. Microinjection induced a rapid (~5 min) rise in pH(o) by ca. 1.0 unit near the injection site, whereas the injection of the non-phosphorylated peptide had no effect. This pH rise confirms the supposed inhibition of the H+-pump upon the detachment of 14-3-3 proteins from the H+-ATPase. However, the PSII yield in the cell regions corresponding to the new alkaline peak remained high, which violated the normal inverse relations between the pH(o) and PSII photochemical yield. We conclude that the injection of the competitive inhibitor of the H+-ATPase disrupts the balanced operation of PM H+-transport and photosynthetic electron flow and promotes electron flow through alternative pathways.

  8. Specific interactions with TBP and TFIIB in vitro suggest that 14-3-3 proteins may participate in the regulation of transcription when part of a DNA binding complex.

    PubMed

    Pan, S; Sehnke, P C; Ferl, R J; Gurley, W B

    1999-08-01

    The 14-3-3 family of multifunctional proteins is highly conserved among animals, plants, and yeast. Several studies have shown that these proteins are associated with a G-box DNA binding complex and are present in the nucleus in several plant and animal species. In this study, 14-3-3 proteins are shown to bind the TATA box binding protein (TBP), transcription factor IIB (TFIIB), and the human TBP-associated factor hTAF(II)32 in vitro but not hTAF(II)55. The interactions with TBP and TFIIB were highly specific, requiring amino acid residues in the box 1 domain of the 14-3-3 protein. These interactions do not require formation of the 14-3-3 dimer and are not dependent on known 14-3-3 recognition motifs containing phosphoserine. The 14-3-3-TFIIB interaction appears to occur within the same domain of TFIIB that binds the human herpes simplex virus transcriptional activator VP16, because VP16 and 14-3-3 were able to compete for interaction with TFIIB in vitro. In a plant transient expression system, 14-3-3 was able to activate GAL4-dependent beta-glucuronidase reporter gene expression at low levels when translationally fused with the GAL4 DNA binding domain. The in vitro binding with general transcription factors TBP and TFIIB together with its nuclear location provide evidence supporting a role for 14-3-3 proteins as transcriptional activators or coactivators when part of a DNA binding complex. PMID:10449590

  9. The histone demethylase JMJD1A regulates adrenomedullin-mediated cell proliferation in hepatocellular carcinoma under hypoxia

    SciTech Connect

    Park, Seong-Joon; Kim, Joong-Gook; Son, Tae Gen; Yi, Joo Mi; Kim, Nam Deuk; Yang, Kwangmo; Heo, Kyu

    2013-05-17

    Highlights: •Hypoxia stimulates HepG2 and Hep3B cell proliferation. •The JMJD1A and ADM expressions are enhanced by hypoxia in HCCs. •Increased JMJD1A expression reduces a H3K9 di-methylation in the ADM promoter region. •Knock-down of JMJD1A abrogates the hypoxia-induced HepG2 and Hep3B cell growth. -- Abstract: We studied the roles of JMJD1A and its target gene ADM in the growth of hepatocellular carcinomas (HCCs) and breast cancer cells under hypoxic conditions. Hypoxia stimulated HepG2 and Hep3B cell proliferation but had no effect on MDA-MB-231 cell proliferation. Interestingly, the JMJD1A and ADM expressions were enhanced by hypoxia only in HepG2 and Hep3B cells. Our ChIP results showed that hypoxia-induced HepG2 and Hep3B cell proliferation is mediated by JMJD1A upregulation and subsequent decrease in methylation in the ADM promoter region. Furthermore, JMJD1A gene silencing abrogated the hypoxia-induced ADM expression and inhibited HepG2 and Hep3B cell growth. These data suggest that JMJD1A might function as a proliferation regulator in some cancer cell types.

  10. Caenorhabditis elegans Histone Deacetylase hda-1 Is Required for Morphogenesis of the Vulva and LIN-12/Notch-Mediated Specification of Uterine Cell Fates

    PubMed Central

    Ranawade, Ayush Vasant; Cumbo, Philip; Gupta, Bhagwati P.

    2013-01-01

    Chromatin modification genes play crucial roles in development and disease. In Caenorhabditis elegans, the class I histone deacetylase family member hda-1, a component of the nucleosome remodeling and deacetylation complex, has been shown to control cell proliferation. We recovered hda-1 in an RNA interference screen for genes involved in the morphogenesis of the egg-laying system. We found that hda-1 mutants have abnormal vulva morphology and vulval-uterine connections (i.e., no uterine-seam cell). We characterized the vulval defects by using cell fate-specific markers and found that hda-1 is necessary for the specification of all seven vulval cell types. The analysis of the vulval-uterine connection defect revealed that hda-1 is required for the differentiation of the gonadal anchor cell (AC), which in turn induces ventral uterine granddaughters to adopt π fates, leading to the formation of the uterine-seam cell. Consistent with these results, hda-1 is expressed in the vulva and AC. A search for hda-1 target genes revealed that fos-1 (fos proto-oncogene family) acts downstream of hda-1 in vulval cells, whereas egl-43 (evi1 proto-oncogene family) and nhr-67 (tailless homolog, NHR family) mediate hda-1 function in the AC. Furthermore, we showed that AC expression of hda-1 plays a crucial role in the regulation of the lin-12/Notch ligand lag-2 to specify π cell fates. These results demonstrate the pivotal role of hda-1 in the formation of the vulva and the vulval-uterine connection. Given that hda-1 homologs are conserved across the phyla, our findings are likely to provide a better understanding of HDAC1 function in development and disease. PMID:23797102

  11. Disruption of IkappaB kinase (IKK)-mediated RelA serine 536 phosphorylation sensitizes human multiple myeloma cells to histone deacetylase (HDAC) inhibitors.

    PubMed

    Dai, Yun; Chen, Shuang; Wang, Li; Pei, Xin-Yan; Funk, Vanessa L; Kramer, Lora B; Dent, Paul; Grant, Steven

    2011-09-30

    Post-translational modifications of RelA play an important role in regulation of NF-κB activation. We previously demonstrated that in malignant hematopoietic cells, histone deacetylase inhibitors (HDACIs) induced RelA hyperacetylation and NF-κB activation, attenuating lethality. We now present evidence that IκB kinase (IKK) β-mediated RelA Ser-536 phosphorylation plays a significant functional role in promoting RelA acetylation, inducing NF-κB activation, and limiting HDACI lethality in human multiple myeloma (MM) cells. Immunoblot profiling revealed that although basal RelA phosphorylation varied in MM cells, Ser-536 phosphorylation correlated with IKK activity. Exposure to the pan-HDACIs vorinostat or LBH-589 induced phosphorylation of IKKα/β (Ser-180/Ser-181) and RelA (Ser-536) in MM cells, including cells expressing an IκBα "super-repressor," accompanied by increased RelA nuclear translocation, acetylation, DNA binding, and transactivation activity. These events were substantially blocked by either pan-IKK or IKKβ-selective inhibitors, resulting in marked apoptosis. Consistent with these events, inhibitory peptides targeting either the NF-κB essential modulator (NEMO) binding domain for IKK complex formation or RelA phosphorylation sites also significantly increased HDACI lethality. Moreover, IKKβ knockdown by shRNA prevented Ser-536 phosphorylation and significantly enhanced HDACI susceptibility. Finally, introduction of a nonphosphorylatable RelA mutant S536A, which failed to undergo acetylation in response to HDACIs, impaired NF-κB activation and increased cell death. These findings indicate that HDACIs induce Ser-536 phosphorylation of the NF-κB subunit RelA through an IKKβ-dependent mechanism, an action that is functionally involved in activation of the cytoprotective NF-κB signaling cascade primarily through facilitation of RelA acetylation rather than nuclear translocation. PMID:21816815

  12. Inhibition of class I histone deacetylases by romidepsin potently induces Epstein-Barr virus lytic cycle and mediates enhanced cell death with ganciclovir.

    PubMed

    Hui, Kwai Fung; Cheung, Arthur Kwok Leung; Choi, Chung King; Yeung, Po Ling; Middeldorp, Jaap M; Lung, Maria Li; Tsao, Sai Wah; Chiang, Alan Kwok Shing

    2016-01-01

    Pan-histone deacetylase (HDAC) inhibitors, which inhibit 11 HDAC isoforms, are widely used to induce Epstein-Barr virus (EBV) lytic cycle in EBV-associated cancers in vitro and in clinical trials. Here, we hypothesized that inhibition of one or several specific HDAC isoforms by selective HDAC inhibitors could potently induce EBV lytic cycle in EBV-associated malignancies such as nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). We found that inhibition of class I HDACs, particularly HDAC-1, -2 and -3, was sufficient to induce EBV lytic cycle in NPC and GC cells in vitro and in vivo. Among a panel of selective HDAC inhibitors, the FDA-approved HDAC inhibitor romidepsin was found to be the most potent lytic inducer, which could activate EBV lytic cycle at ∼0.5 to 5 nM (versus ∼800 nM achievable concentration in patients' plasma) in more than 75% of cells. Upregulation of p21(WAF1) , which is negatively regulated by class I HDACs, was observed before the induction of EBV lytic cycle. The upregulation of p21(WAF1) and induction of lytic cycle were abrogated by a specific inhibitor of PKC-δ but not the inhibitors of PI3K, MEK, p38 MAPK, JNK or ATM pathways. Interestingly, inhibition of HDAC-1, -2 and -3 by romidepsin or shRNA knockdown could confer susceptibility of EBV-positive epithelial cells to the treatment with ganciclovir (GCV). In conclusion, we demonstrated that inhibition of class I HDACs by romidepsin could potently induce EBV lytic cycle and mediate enhanced cell death with GCV, suggesting potential application of romidepsin for the treatment of EBV-associated cancers.

  13. Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation

    PubMed Central

    Snyder, Nathaniel W.; Wei, Shuanzeng; Venneti, Sriram; Worth, Andrew J.; Yuan, Zuo-Fei; Lim, Hee-Woong; Liu, Shichong; Jackson, Ellen; Aiello, Nicole M.; Haas, Naomi B.; Rebbeck, Timothy R.; Judkins, Alexander; Won, Kyoung-Jae; Chodosh, Lewis A.; Garcia, Benjamin A.; Stanger, Ben Z.; Feldman, Michael D.; Blair, Ian A.; Wellen, Kathryn E.

    2014-01-01

    SUMMARY Histone acetylation plays important roles in gene regulation, DNA replication, and the response to DNA damage, and it is frequently deregulated in tumors. We postulated that tumor cell histone acetylation levels are determined in part by changes in acetyl-CoA availability mediated by oncogenic metabolic reprogramming. Here, we demonstrate that acetyl-CoA is dynamically regulated by glucose availability in cancer cells and that the ratio of acetyl-CoA: coenzyme A within the nucleus modulates global histone acetylation levels. In vivo, expression of oncogenic Kras or Akt stimulates histone acetylation changes that precede tumor development. Furthermore, we show that Akt's effects on histone acetylation are mediated through the metabolic enzyme ATP-citrate lyase (ACLY), and that pAkt(Ser473) levels correlate significantly with histone acetylation marks in human gliomas and prostate tumors. The data implicate acetyl-CoA metabolism as a key determinant of histone acetylation levels in cancer cells. PMID:24998913

  14. 14-3-3ζ up-regulates hypoxia-inducible factor-1α in hepatocellular carcinoma via activation of PI3K/Akt/NF-кB signal transduction pathway

    PubMed Central

    Tang, Yufu; Lv, Pengfei; Sun, Zhongyi; Han, Lei; Luo, Bichao; Zhou, Wenping

    2015-01-01

    14-3-3ζ protein, a member of 14-3-3 family, plays important roles in multiple cellular processes. Our previous study showed that 14-3-3ζ could bind to regulate the expression of hypoxia-inducible factor-1α (HIF-1α), which is induced by hypoxia and a crucial factor for induction of tumor metastasis. Moreover, we also have confirmed the response of 14-3-3ζ to hypoxia in our unpublished data as well. Thus, in the present study, we attempted to reveal that whether the regulation effect of 14-3-3ζ on HIF-1α functioned in a similar pattern as hypoxia. Stable regulation of 14-3-3ζ in human HCC cell line SMMC-772 and HCC-LM3 was achieved. The regulation of 14-3-3ζ on HIF-1α mRNA transcription was evaluated by luciferase activity assay and quantitative real-time PCR (qPCR). The effect of 14-3-3ζ on the production of HIF-1α and pathways determining HIF-1α’s response to hypoxia was assessed using western blotting assay. Our results showed that regulation of 14-3-3ζ expression influenced the activity of HIF-1α, phosphatidyl inositol 3-kinase (PI3K), Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), and nuclear factor kappa B (NF-кB). Blocking of these pathways using indicated inhibitors revealed that 14-3-3ζ enhanced the production of HIF-1α via the activation of PI3K/Akt/NF-кB pathway, which was identical to hypoxia induced HIF-1α expression. For the first time, our study described the key role of 14-3-3ζ in the HIF-1α production in HCC cells. And the molecule exerted its function on HIF-1α both by directly binding to it and via PI3K/Akt/NF-кB signal transduction pathway. PMID:26884855

  15. The histone chaperones Nap1 and Vps75 bind histones H3 and H4 in a tetrameric conformation.

    PubMed

    Bowman, Andrew; Ward, Richard; Wiechens, Nicola; Singh, Vijender; El-Mkami, Hassane; Norman, David George; Owen-Hughes, Tom

    2011-02-18

    Histone chaperones physically interact with histones to direct proper assembly and disassembly of nucleosomes regulating diverse nuclear processes such as DNA replication, promoter remodeling, transcription elongation, DNA damage, and histone variant exchange. Currently, the best-characterized chaperone-histone interaction is that between the ubiquitous chaperone Asf1 and a dimer of H3 and H4. Nucleosome assembly proteins (Nap proteins) represent a distinct class of histone chaperone. Using pulsed electron double resonance (PELDOR) measurements and protein crosslinking, we show that two members of this class, Nap1 and Vps75, bind histones in the tetrameric conformation also observed when they are sequestered within the nucleosome. Furthermore, H3 and H4 trapped in their tetrameric state can be used as substrates in nucleosome assembly and chaperone-mediated lysine acetylation. This alternate mode of histone interaction provides a potential means of maintaining the integrity of the histone tetramer during cycles of nucleosome reassembly.

  16. In-vivo administration of clozapine affects behaviour but does not reverse dendritic spine deficits in the 14-3-3ζ KO mouse model of schizophrenia-like disorders.

    PubMed

    Jaehne, Emily J; Ramshaw, Hayley; Xu, Xiangjun; Saleh, Eiman; Clark, Scott R; Schubert, Klaus Oliver; Lopez, Angel; Schwarz, Quenten; Baune, Bernhard T

    2015-11-01

    Clozapine is an atypical antipsychotic drug used in the treatment of schizophrenia, which has been shown to reverse behavioural and dendritic spine deficits in mice. It has recently been shown that deficiency of 14-3-3ζ has an association with schizophrenia, and that a mouse model lacking this protein displays several schizophrenia-like behavioural deficits. To test the effect of clozapine in this mouse model, 14-3-3ζ KO mice were administered clozapine (5mg/kg) for two weeks prior to being analysed in a test battery of cognition, anxiety, and despair (depression-like) behaviours. Following behavioural testing brain samples were collected for analysis of specific anatomical defects and dendritic spine formation. We found that clozapine reduced despair behaviour of 14-3-3ζ KO mice in the forced swim test (FST) and altered the behaviour of wild types and 14-3-3ζ KO mice in the Y-maze task. In contrast, clozapine had no effects on hippocampal laminar defects or decreased dendritic spine density observed in 14-3-3ζ KO mice. Our results suggest that clozapine may have beneficial effects on clinical behaviours associated with deficiencies in the 14-3-3ζ molecular pathway, despite having no effects on morphological defects. These findings may provide mechanistic insight to the action of this drug.

  17. Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells.

    PubMed

    Silva, Julhiany de Fátima da; Vicentim, Juliana; Oliveira, Haroldo Cesar de; Marcos, Caroline Maria; Assato, Patricia Akemi; Andreotti, Patrícia Ferrari; Silva, Juliana Leal Monteiro da; Soares, Christiane Pienna; Benard, Gil; Almeida, Ana Marisa Fusco; Mendes-Giannini, Maria José Soares

    2015-06-01

    The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis. PMID:26038961

  18. Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

    PubMed Central

    da Silva, Julhiany de Fátima; Vicentim, Juliana; de Oliveira, Haroldo Cesar; Marcos, Caroline Maria; Assato, Patricia Akemi; Andreotti, Patrícia Ferrari; da Silva, Juliana Leal Monteiro; Soares, Christiane Pienna; Benard, Gil; Almeida, Ana Marisa Fusco; Mendes-Giannini, Maria José Soares

    2015-01-01

    The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis. PMID:26038961

  19. Proteomic screening for Rho-kinase substrates by combining kinase and phosphatase inhibitors with 14-3-3ζ affinity chromatography.

    PubMed

    Nishioka, Tomoki; Nakayama, Masanori; Amano, Mutsuki; Kaibuchi, Kozo

    2012-01-01

    The small GTPase RhoA is a molecular switch in various extracellular signals. Rho-kinase/ROCK/ROK, a major effector of RhoA, regulates diverse cellular functions by phosphorylating cytoskeletal proteins, endocytic proteins, and polarity proteins. More than twenty Rho-kinase substrates have been reported, but the known substrates do not fully explain the Rho-kinase functions. Herein, we describe the comprehensive screening for Rho-kinase substrates by treating HeLa cells with Rho-kinase and phosphatase inhibitors. The cell lysates containing the phosphorylated substrates were then subjected to affinity chromatography using beads coated with 14-3-3 protein, which interacts with proteins containing phosphorylated serine or threonine residues, to enrich the phosphorylated proteins. The identities of the molecules and phosphorylation sites were determined by liquid chromatography tandem mass spectrometry (LC/MS/MS) after tryptic digestion and phosphopeptide enrichment. The phosphorylated proteins whose phosphopeptide ion peaks were suppressed by treatment with the Rho-kinase inhibitor were regarded as candidate substrates. We identified 121 proteins as candidate substrates. We also identified phosphorylation sites in Partitioning defective 3 homolog (Par-3) at Ser143 and Ser144. We found that Rho-kinase phosphorylated Par-3 at Ser144 both in vitro and in vivo. The method used in this study would be applicable and useful to identify novel substrates of other kinases.

  20. 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.

  1. 14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling

    PubMed Central

    Liu, Lixin; Lin, Ye; Liu, Lili; Bian, Yanjie; Zhang, Li; Gao, Xuejun; Li, Qingzhang

    2015-01-01

    As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-κB (IκB) phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPAR

  2. Up-regulation and interaction of the plasma membrane H(+)-ATPase and the 14-3-3 protein are involved in the regulation of citrate exudation from the broad bean (Vicia faba L.) under Al stress.

    PubMed

    Chen, Qi; Guo, Chuan-Long; Wang, Ping; Chen, Xuan-Qin; Wu, Kong-Huan; Li, Kui-Zhi; Yu, Yong-Xiong; Chen, Li-Mei

    2013-09-01

    Our previous study showed that citrate excretion coupled with a concomitant release of protons was involved in aluminum (Al) resistance in the broad bean. Furthermore, genes encoding plasma membrane (PM) H(+)-ATPase (vha2) and the 14-3-3 protein (vf14-3-3b) were up-regulated by Al in Al-resistant (YD) broad bean roots. In this study, the roles of PM H(+)-ATPase (E.C. 3.6.3.6) and the 14-3-3 protein in the regulation of citrate secretion were further investigated in Al-resistant (YD) and Al-sensitive (AD) broad bean cultivars under Al stress. The results showed that greater citrate exudation was positively correlated with higher activities of PM H(+)-ATPase in roots of YD than AD. Real-time RT-PCR analysis revealed that vha2 was clearly up-regulated by Al in YD but not in AD roots, whereas the transcription levels of vf14-3-3b were elevated in a time-dependent manner in both YD and AD roots. Immunoprecipitation and Western analysis suggested that phosphorylation and interaction with the vf14-3-3b protein of the VHA2 were enhanced in YD roots but not in AD roots with increasing Al treatment time. Fusicoccin or adenosine 5'-monophosphate increased or decreased the interaction between the phosphorylated VHA2 and the vf14-3-3b protein, followed by an enhancement or reduction of the PM H(+)-ATPase activity and citrate exudation in both cultivars under Al stress conditions, respectively. Taken together, these results suggested that Al enhanced the expression and interaction of the PM H(+)-ATPase and the 14-3-3 protein, which thereby led to higher activity of the PM H(+)-ATPase and more citrate exudation from YD plants.

  3. Histone H2AX Is Involved in FoxO3a-Mediated Transcriptional Responses to Ionizing Radiation to Maintain Genome Stability

    PubMed Central

    Tarrade, Stephane; Bhardwaj, Tanya; Flegal, Matthew; Bertrand, Lindsey; Velegzhaninov, Ilya; Moskalev, Alexey; Klokov, Dmitry

    2015-01-01

    Histone H2AX plays a crucial role in molecular and cellular responses to DNA damage and in the maintenance of genome stability. It is downstream of ataxia telangiectasia mutated (ATM) damage signaling pathway and there is an emerging role of the transcription factor FoxO3a, a regulator of a variety of other pathways, in activating this signaling. We asked whether H2AX may feedback to FoxO3a to affect respective FoxO3a-dependent pathways. We used a genetically matched pair of mouse embryonic fibroblast H2AX+/+ and H2AX−/− cell lines to carry out comprehensive time-course and dose-response experiments and to show that the expression of several FoxO3a-regulated genes was altered in H2AX−/− compared to H2AX+/+ cells at both basal and irradiated conditions. Hspa1b and Gadd45a were down-regulated four- to five-fold and Ddit3, Cdkn1a and Sod2 were up-regulated 2–3-fold in H2AX−/− cells. Using the luciferase reporter assay, we directly demonstrated that transcriptional activity of FoxoO3a was reduced in H2AX−/− cells. FoxO3a localization within the nuclear phospho-ATM (Ser1981) foci in irradiated cells was affected by the H2AX status, as well as its posttranslational modification (phospho-Thr32). These differences were associated with genomic instability and radiosensitivity in H2AX−/− cells. Finally, knockdown of H2AX in H2AX+/+ cells resulted in FoxO3a-dependent gene expression patterns and increased radiosensitivity that partially mimicked those found in H2AX−/− cells. Taken together, our data suggest a role for FoxO3a in the maintenance of genome integrity in response to DNA damage that is mediated by H2AX via yet unknown mechanisms. PMID:26694365

  4. Sensing core histone phosphorylation — A matter of perfect timing☆

    PubMed Central

    Sawicka, Anna; Seiser, Christian

    2014-01-01

    Systematic analysis of histone modifications has revealed a plethora of posttranslational modifications that mediate changes in chromatin structure and gene expression. Histone phosphorylation is a transient histone modification that becomes induced by extracellular signals, DNA damage or entry into mitosis. Importantly, phosphorylation of histone proteins does lead not only to the binding of specific reader proteins but also to changes in the affinity for readers or writers of other histone modifications. This induces a cross-talk between different chromatin modifications that allows the spatio-temporal control of chromatin-associated events. In this review we will summarize the progress in our current knowledge of factors sensing reversible histone phosphorylation in different biological scenarios. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function. PMID:24747175

  5. Lysine-specific demethylase 1-mediated demethylation of histone H3 lysine 9 contributes to interleukin 1β-induced microsomal prostaglandin E synthase 1 expression in human osteoarthritic chondrocytes

    PubMed Central

    2014-01-01

    Introduction Microsomal prostaglandin E synthase 1 (mPGES-1) catalyzes the terminal step in the biosynthesis of PGE2, a critical mediator in the pathophysiology of osteoarthritis (OA). Histone methylation plays an important role in epigenetic gene regulation. In this study, we investigated the roles of histone H3 lysine 9 (H3K9) methylation in interleukin 1β (IL-1β)-induced mPGES-1 expression in human chondrocytes. Methods Chondrocytes were stimulated with IL-1β, and the expression of mPGES-1 mRNA was evaluated using real-time RT-PCR. H3K9 methylation and the recruitment of the histone demethylase lysine-specific demethylase 1 (LSD1) to the mPGES-1 promoter were evaluated using chromatin immunoprecipitation assays. The role of LSD1 was further evaluated using the pharmacological inhibitors tranylcypromine and pargyline and small interfering RNA (siRNA)-mediated gene silencing. The LSD1 level in cartilage was determined by RT-PCR and immunohistochemistry. Results The induction of mPGES-1 expression by IL-1β correlated with decreased levels of mono- and dimethylated H3K9 at the mPGES-1 promoter. These changes were concomitant with the recruitment of the histone demethylase LSD1. Treatment with tranylcypromine and pargyline, which are potent inhibitors of LSD1, prevented IL-1β-induced H3K9 demethylation at the mPGES-1 promoter and expression of mPGES-1. Consistently, LSD1 gene silencing with siRNA prevented IL-1β-induced H3K9 demethylation and mPGES-1 expression, suggesting that LSD1 mediates IL-1β-induced mPGES-1 expression via H3K9 demethylation. We show that the level of LSD1 was elevated in OA compared to normal cartilage. Conclusion These results indicate that H3K9 demethylation by LSD1 contributes to IL-1β-induced mPGES-1 expression and suggest that this pathway could be a potential target for pharmacological intervention in the treatment of OA and possibly other arthritic conditions. PMID:24886859

  6. Development of primer sets that can verify the enrichment of histone modifications, and their application to examining vernalization-mediated chromatin changes in Brassica rapa L.

    PubMed

    Kawanabe, Takahiro; Osabe, Kenji; Itabashi, Etsuko; Okazaki, Keiichi; Dennis, Elizabeth S; Fujimoto, Ryo

    2016-07-20

    Epigenetic regulation is crucial for the development of plants and for adaptation to a changing environment. Recently, genome-wide profiles of histone modifications have been determined by a combination of chromatin immunoprecipitation (ChIP) and genomic tiling arrays (ChIP on chip) or ChIP and high-throughput sequencing (ChIP-seq) in species including Arabidopsis thaliana, rice and maize. Validation of ChIP analysis by PCR or qPCR using positive and negative regions of histone modification is necessary. In contrast, information about histone modifications is limited in Chinese cabbage, Brassica rapa. The aim of this study was to develop positive and negative control primer sets for H3K4me3 (trimethylation of the 4(th) lysine of H3), H3K9me2, H3K27me3 and H3K36me3 in B. rapa. The expression and histone modification of four FLC paralogs in B. rapa, before and after vernalization, were examined using the method developed here. After vernalization, expression of all four BrFLC genes was reduced, and accumulation of H3K27me3 was observed in three of them. As with A. thaliana, the vernalization response and stability of FLC repression correlated with the accumulation of H3K27me3. These results suggest that the epigenetic state during vernalization is important for high bolting resistance in B. rapa. The positive and negative control primer sets developed here revealed positive and negative histone modifications in B. rapa that can be used as a control for future studies. PMID:27074983

  7. Development of primer sets that can verify the enrichment of histone modifications, and their application to examining vernalization-mediated chromatin changes in Brassica rapa L.

    PubMed

    Kawanabe, Takahiro; Osabe, Kenji; Itabashi, Etsuko; Okazaki, Keiichi; Dennis, Elizabeth S; Fujimoto, Ryo

    2016-07-20

    Epigenetic regulation is crucial for the development of plants and for adaptation to a changing environment. Recently, genome-wide profiles of histone modifications have been determined by a combination of chromatin immunoprecipitation (ChIP) and genomic tiling arrays (ChIP on chip) or ChIP and high-throughput sequencing (ChIP-seq) in species including Arabidopsis thaliana, rice and maize. Validation of ChIP analysis by PCR or qPCR using positive and negative regions of histone modification is necessary. In contrast, information about histone modifications is limited in Chinese cabbage, Brassica rapa. The aim of this study was to develop positive and negative control primer sets for H3K4me3 (trimethylation of the 4(th) lysine of H3), H3K9me2, H3K27me3 and H3K36me3 in B. rapa. The expression and histone modification of four FLC paralogs in B. rapa, before and after vernalization, were examined using the method developed here. After vernalization, expression of all four BrFLC genes was reduced, and accumulation of H3K27me3 was observed in three of them. As with A. thaliana, the vernalization response and stability of FLC repression correlated with the accumulation of H3K27me3. These results suggest that the epigenetic state during vernalization is important for high bolting resistance in B. rapa. The positive and negative control primer sets developed here revealed positive and negative histone modifications in B. rapa that can be used as a control for future studies.

  8. Histone H3 lysine 27 and 9 hypermethylation within the Bad promoter region mediates 5-Aza-2'-deoxycytidine-induced Leydig cell apoptosis: implications of 5-Aza-2'-deoxycytidine toxicity to male reproduction.

    PubMed

    Choi, Ji-Young; Lee, Sangmi; Hwang, Soojin; Jo, Sangmee Ahn; Kim, Miji; Kim, Young Ju; Pang, Myung-Geol; Jo, Inho

    2013-01-01

    5-Aza-2'-deoxycitidine (5-Aza), an anticancer agent, results in substantial toxicity to male reproduction, causing a decline in sperm quality associated with reduced testosterone. Here, we report that 5-Aza increased the apoptotic protein Bad epigenetically in the testosterone-producing mouse TM3 Leydig cell line. 5-Aza decreased cell viability in a dose- and time-dependent manner with concomitant increase in Bad protein. This increase is accompanied by increased cleavages of both poly ADP ribose polymerase and caspase-3. Flow cytometric analysis further supported 5-Aza-derived apoptosis in TM3 cells. Bisulfite sequencing analysis failed to identify putative methylcytosine site(s) in CpG islands of the Bad promoter. A chromatin immunoprecipitation assay revealed decreased levels of trimethylation at lysine 27 of histone H3 (H3K27-3me) and H3K9-3me in the Bad promoter region in response to 5-Aza treatment. Knock-down by siRNA of enhancer of zeste homologue 2 (EZH2), a histone methyltransferase responsible for H3K27-3me, or demethylation of H3K9-3me by BIX-01294 showed significantly increased levels in Bad expression and consequent Leydig cell apoptosis. In conclusion, our results demonstrate for the first time that Bad expression is regulated at least by EZH2-mediated H3K27-3me or G9a-like protein/euchromatic histone methyltransferase 1 (GLP/Eu-HMTase1)-mediated H3K9-3me in mouse TM3 Leydig cells, which may be implicated in 5-Aza-derived toxicity to male reproduction.

  9. Extracellular histones in tissue injury and inflammation.

    PubMed

    Allam, Ramanjaneyulu; Kumar, Santhosh V R; Darisipudi, Murthy N; Anders, Hans-Joachim

    2014-05-01

    Neutrophil NETosis is an important element of host defense as it catapults chromatin out of the cell to trap bacteria, which then are killed, e.g., by the chromatin's histone component. Also, during sterile inflammation TNF-alpha and other mediators trigger NETosis, which elicits cytotoxic effects on host cells. The same mechanism should apply to other forms of regulated necrosis including pyroptosis, necroptosis, ferroptosis, and cyclophilin D-mediated regulated necrosis. Beyond these toxic effects, extracellular histones also trigger thrombus formation and innate immunity by activating Toll-like receptors and the NLRP3 inflammasome. Thereby, extracellular histones contribute to the microvascular complications of sepsis, major trauma, small vessel vasculitis as well as acute liver, kidney, brain, and lung injury. Finally, histones prevent the degradation of extracellular DNA, which promotes autoimmunization, anti-nuclear antibody formation, and autoimmunity in susceptible individuals. Here, we review the current evidence on the pathogenic role of extracellular histones in disease and discuss how to target extracellular histones to improve disease outcomes. PMID:24706102

  10. Extracellular histones in tissue injury and inflammation.

    PubMed

    Allam, Ramanjaneyulu; Kumar, Santhosh V R; Darisipudi, Murthy N; Anders, Hans-Joachim

    2014-05-01

    Neutrophil NETosis is an important element of host defense as it catapults chromatin out of the cell to trap bacteria, which then are killed, e.g., by the chromatin's histone component. Also, during sterile inflammation TNF-alpha and other mediators trigger NETosis, which elicits cytotoxic effects on host cells. The same mechanism should apply to other forms of regulated necrosis including pyroptosis, necroptosis, ferroptosis, and cyclophilin D-mediated regulated necrosis. Beyond these toxic effects, extracellular histones also trigger thrombus formation and innate immunity by activating Toll-like receptors and the NLRP3 inflammasome. Thereby, extracellular histones contribute to the microvascular complications of sepsis, major trauma, small vessel vasculitis as well as acute liver, kidney, brain, and lung injury. Finally, histones prevent the degradation of extracellular DNA, which promotes autoimmunization, anti-nuclear antibody formation, and autoimmunity in susceptible individuals. Here, we review the current evidence on the pathogenic role of extracellular histones in disease and discuss how to target extracellular histones to improve disease outcomes.

  11. Histone chaperones: assisting histone traffic and nucleosome dynamics.

    PubMed

    Gurard-Levin, Zachary A; Quivy, Jean-Pierre; Almouzni, Geneviève

    2014-01-01

    The functional organization of eukaryotic DNA into chromatin uses histones as components of its building block, the nucleosome. Histone chaperones, which are proteins that escort histones throughout their cellular life, are key actors in all facets of histone metabolism; they regulate the supply and dynamics of histones at chromatin for its assembly and disassembly. Histone chaperones can also participate in the distribution of histone variants, thereby defining distinct chromatin landscapes of importance for genome function, stability, and cell identity. Here, we discuss our current knowledge of the known histone chaperones and their histone partners, focusing on histone H3 and its variants. We then place them into an escort network that distributes these histones in various deposition pathways. Through their distinct interfaces, we show how they affect dynamics during DNA replication, DNA damage, and transcription, and how they maintain genome integrity. Finally, we discuss the importance of histone chaperones during development and describe how misregulation of the histone flow can link to disease.

  12. Novel curcumin analog C66 prevents diabetic nephropathy via JNK pathway with the involvement of p300/CBP-mediated histone acetylation.

    PubMed

    Wang, Yangwei; Wang, Yonggang; Luo, Manyu; Wu, Hao; Kong, Lili; Xin, Ying; Cui, Wenpeng; Zhao, Yunjie; Wang, Jingying; Liang, Guang; Miao, Lining; Cai, Lu

    2015-01-01

    Glomerulosclerosis and interstitial fibrosis represent the key events in development of diabetic nephropathy (DN), with connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1) and fibronectin 1 (FN-1) playing important roles in these pathogenic processes. To investigate whether the plant metabolite curcumin, which exerts epigenetic modulatory properties when applied as a pharmacological agent, may prevent DN via inhibition of the JNK pathway and epigenetic histone acetylation, diabetic and age-matched non-diabetic control mice were administered a 3-month course of curcumin analogue (C66), c-Jun N-terminal kinase inhibitor (JNKi, sp600125), or vehicle alone. At treatment end, half of the mice were sacrificed for analysis and the other half were maintained without treatment for an additional 3 months. Renal JNK phosphorylation was found to be significantly increased in the vehicle-treated diabetic mice, but not the C66- and JNKi-treated diabetic mice, at both the 3-month and 6-month time points. C66 and JNKi treatment also significantly prevented diabetes-induced renal fibrosis and dysfunction. Diabetes-related increases in histone acetylation, histone acetyl transferases' (HATs) activity, and the p300/CBP HAT expression were also significantly attenuated by C66 or JNKi treatment. Chromatin immunoprecipitation assays showed that C66 and JNKi treatments decreased H3-lysine9/14-acetylation (H3K9/14Ac) level and p300/CBP occupancy at the CTGF, PAI-1 and FN-1 gene promoters. Thus, C66 may significantly and persistently prevent renal injury and dysfunction in diabetic mice via down-regulation of diabetes-related JNK activation and consequent suppression of the diabetes-related increases in HAT activity, p300/CBP expression, and histone acetylation.

  13. Arabidopsis Histone Methylase CAU1/PRMT5/SKB1 Acts as an Epigenetic Suppressor of the Calcium Signaling Gene CAS to Mediate Stomatal Closure in Response to Extracellular Calcium[W

    PubMed Central

    Fu, Yan-Lei; Zhang, Guo-Bin; Lv, Xin-Fang; Guan, Yuan; Yi, Hong-Ying; Gong, Ji-Ming

    2013-01-01

    Elevations in extracellular calcium ([Ca2+]o) are known to stimulate cytosolic calcium ([Ca2+]cyt) oscillations to close stomata. However, the underlying mechanisms regulating this process remain largely to be determined. Here, through the functional characterization of the calcium underaccumulation mutant cau1, we report that the epigenetic regulation of CAS, a putative Ca2+ binding protein proposed to be an external Ca2+ sensor, is involved in this process. cau1 mutant plants display increased drought tolerance and stomatal closure. A mutation in CAU1 significantly increased the expression level of the calcium signaling gene CAS, and functional disruption of CAS abolished the enhanced drought tolerance and stomatal [Ca2+]o signaling in cau1. Map-based cloning revealed that CAU1 encodes the H4R3sme2 (for histone H4 Arg 3 with symmetric dimethylation)-type histone methylase protein arginine methytransferase5/Shk1 binding protein1. Chromatin immunoprecipitation assays showed that CAU1 binds to the CAS promoter and modulates the H4R3sme2-type histone methylation of the CAS chromatin. When exposed to elevated [Ca2+]o, the protein levels of CAU1 decreased and less CAU1 bound to the CAS promoter. In addition, the methylation level of H4R3sme2 decreased in the CAS chromatin. Together, these data suggest that in response to increases in [Ca2+]o, fewer CAU1 protein molecules bind to the CAS promoter, leading to decreased H4R3sme2 methylation and consequent derepression of the expression of CAS to mediate stomatal closure and drought tolerance. PMID:23943859

  14. IκB kinase-induced interaction of TPL-2 kinase with 14-3-3 is essential for Toll-like receptor activation of ERK-1 and -2 MAP kinases.

    PubMed

    Ben-Addi, Abduelhakem; Mambole-Dema, Agnes; Brender, Christine; Martin, Stephen R; Janzen, Julia; Kjaer, Sven; Smerdon, Stephen J; Ley, Steven C

    2014-06-10

    The MEK-1/2 kinase TPL-2 is critical for Toll-like receptor activation of the ERK-1/2 MAP kinase pathway during inflammatory responses, but it can transform cells following C-terminal truncation. IκB kinase (IKK) complex phosphorylation of the TPL-2 C terminus regulates full-length TPL-2 activation of ERK-1/2 by a mechanism that has remained obscure. Here, we show that TPL-2 Ser-400 phosphorylation by IKK and TPL-2 Ser-443 autophosphorylation cooperated to trigger TPL-2 association with 14-3-3. Recruitment of 14-3-3 to the phosphorylated C terminus stimulated TPL-2 MEK-1 kinase activity, which was essential for TPL-2 activation of ERK-1/2. The binding of 14-3-3 to TPL-2 was also indispensible for lipopolysaccharide-induced production of tumor necrosis factor by macrophages, which is regulated by TPL-2 independently of ERK-1/2 activation. Our data identify a key step in the activation of TPL-2 signaling and provide a mechanistic insight into how C-terminal deletion triggers the oncogenic potential of TPL-2 by rendering its kinase activity independent of 14-3-3 binding.

  15. Inter-α inhibitor protein and its associated glycosaminoglycans protect against histone-induced injury

    PubMed Central

    Chaaban, Hala; Keshari, Ravi S.; Silasi-Mansat, Robert; Popescu, Narcis I.; Mehta-D’Souza, Padmaja; Lim, Yow-Pin

    2015-01-01

    Extracellular histones are mediators of tissue injury and organ dysfunction; therefore they constitute potential therapeutic targets in sepsis, inflammation, and thrombosis. Histone cytotoxicity in vitro decreases in the presence of plasma. Here, we demonstrate that plasma inter-α inhibitor protein (IAIP) neutralizes the cytotoxic effects of histones and decreases histone-induced platelet aggregation. These effects are mediated through the negatively charged glycosaminoglycans (GAGs) chondroitin sulfate and high-molecular-weight hyaluronan (HMW-HA) associated with IAIP. Cell surface anionic glycosaminoglycans heparan sulfate and HA protect the cells against histone-mediated damage in vitro. Surface plasmon resonance showed that both IAIP and HMW-HA directly bind to recombinant histone H4. In vivo neutralization of histones with IAIP and HMW-HA prevented histone-induced thrombocytopenia, bleeding, and lung microvascular thrombosis, decreased neutrophil activation, and averted histone-induced production of inflammatory cytokines and chemokines. IAIP and HMW-HA colocalized with histones in necrotic tissues and areas that displayed neutrophil extracellular traps. Increasing amounts of IAIP-histone complexes detected in the plasma of septic baboons correlated with increase in histones and/or nucleosomes and consumption of plasma IAIP. Our data suggest that IAIP, chondroitin sulfate, and HMW-HA are potential therapeutic agents to protect against histone-induced cytotoxicity, coagulopathy, systemic inflammation, and organ damage during inflammatory conditions such as sepsis and trauma. PMID:25631771

  16. Inter-α inhibitor protein and its associated glycosaminoglycans protect against histone-induced injury.

    PubMed

    Chaaban, Hala; Keshari, Ravi S; Silasi-Mansat, Robert; Popescu, Narcis I; Mehta-D'Souza, Padmaja; Lim, Yow-Pin; Lupu, Florea

    2015-04-01

    Extracellular histones are mediators of tissue injury and organ dysfunction; therefore they constitute potential therapeutic targets in sepsis, inflammation, and thrombosis. Histone cytotoxicity in vitro decreases in the presence of plasma. Here, we demonstrate that plasma inter-α inhibitor protein (IAIP) neutralizes the cytotoxic effects of histones and decreases histone-induced platelet aggregation. These effects are mediated through the negatively charged glycosaminoglycans (GAGs) chondroitin sulfate and high-molecular-weight hyaluronan (HMW-HA) associated with IAIP. Cell surface anionic glycosaminoglycans heparan sulfate and HA protect the cells against histone-mediated damage in vitro. Surface plasmon resonance showed that both IAIP and HMW-HA directly bind to recombinant histone H4. In vivo neutralization of histones with IAIP and HMW-HA prevented histone-induced thrombocytopenia, bleeding, and lung microvascular thrombosis, decreased neutrophil activation, and averted histone-induced production of inflammatory cytokines and chemokines. IAIP and HMW-HA colocalized with histones in necrotic tissues and areas that displayed neutrophil extracellular traps. Increasing amounts of IAIP-histone complexes detected in the plasma of septic baboons correlated with increase in histones and/or nucleosomes and consumption of plasma IAIP. Our data suggest that IAIP, chondroitin sulfate, and HMW-HA are potential therapeutic agents to protect against histone-induced cytotoxicity, coagulopathy, systemic inflammation, and organ damage during inflammatory conditions such as sepsis and trauma.

  17. Metabolic regulation of histone post-translational modifications

    PubMed Central

    Fan, Jing; Krautkramer, Kimberly A.; Feldman, Jessica L.; Denu, John M.

    2015-01-01

    Histone post-translational modifications regulate transcription and other DNA-templated functions. This process is dynamically regulated by specific modifying enzymes whose activities require metabolites that either serve as co-substrates or act as activators/inhibitors. Therefore, metabolism can influence histone modification by changing local concentrations of key metabolites. Physiologically, the epigenetic response to metabolism is important for nutrient sensing and environment adaption. In pathologic states, the connection between metabolism and histone modification mediates epigenetic abnormality in complex disease. In this review, we summarize recent studies of the molecular mechanisms involved in metabolic regulation of histone modifications and discuss their biological significance. PMID:25562692

  18. Histone variants--the structure behind the function.

    PubMed

    Ausió, Juan

    2006-09-01

    In recent years, the chromatin field has witnessed a renewed interest in histone variants as pertaining to their structural role, but mainly because of the functional specificity they impart to chromatin. In this review, I am going to discuss several of the most recent structural studies on core histone (H2A.Bbd, H2A.Z, H2A.X, macroH2A, H3.3, CENP-A) and linker histone variants (histone H1 microheterogeneity) focusing on their role in nucleosome stability and chromatin fibre dynamics with special emphasis on their possible functional implications. The data accumulated to date indicates that histone variability plays an important role in the histone-mediated regulation of chromatin metabolism. Understanding and deciphering the underlying structural amino acid code behind such variability remains one of the most exciting future challenges in chromatin research.

  19. Structure of the histone chaperone CIA/ASF1-double bromodomain complex linking histone modifications and site-specific histone eviction.

    PubMed

    Akai, Yusuke; Adachi, Naruhiko; Hayashi, Yohei; Eitoku, Masamitsu; Sano, Norihiko; Natsume, Ryo; Kudo, Norio; Tanokura, Masaru; Senda, Toshiya; Horikoshi, Masami

    2010-05-01

    Nucleosomes around the promoter region are disassembled for transcription in response to various signals, such as acetylation and methylation of histones. Although the interactions between histone-acetylation-recognizing bromodomains and factors involved in nucleosome disassembly have been reported, no structural basis connecting histone modifications and nucleosome disassembly has been obtained. Here, we determined at 3.3 A resolution the crystal structure of histone chaperone cell cycle gene 1 (CCG1) interacting factor A/antisilencing function 1 (CIA/ASF1) in complex with the double bromodomain in the CCG1/TAF1/TAF(II)250 subunit of transcription factor IID. Structural, biochemical, and biological studies suggested that interaction between double bromodomain and CIA/ASF1 is required for their colocalization, histone eviction, and pol II entry at active promoter regions. Furthermore, the present crystal structure has characteristics that can connect histone acetylation and CIA/ASF1-mediated histone eviction. These findings suggest that the molecular complex between CIA/ASF1 and the double bromodomain plays a key role in site-specific histone eviction at active promoter regions. The model we propose here is the initial structure-based model of the biological signaling from histone modifications to structural change of the nucleosome (hi-MOST model).

  20. Disclosing the in vivo organization of a viral histone-like protein in Bacillus subtilis mediated by its capacity to recognize the viral genome.

    PubMed

    Holguera, Isabel; Ballesteros-Plaza, David; Muñoz-Espín, Daniel; Salas, Margarita

    2012-04-10

    Organization of replicating prokaryotic genomes requires architectural elements that, similarly to eukaryotic systems, induce topological changes such as DNA supercoiling. Bacteriophage 29 protein p6 has been described as a histone-like protein that compacts the viral genome by forming a nucleoprotein complex and plays a key role in the initiation of protein-primed DNA replication. In this work, we analyze the subcellular localization of protein p6 by immunofluorescence microscopy and show that, at early infection stages, it localizes in a peripheral helix-like configuration. Later, at middle infection stages, protein p6 is recruited to the bacterial nucleoid. This migrating process is shown to depend on the synthesis of components of the 29 DNA replication machinery (i.e., terminal protein and DNA polymerase) needed for the replication of viral DNA, which is required to recruit the bulk of protein p6. Importantly, the double-stranded DNA-binding capacity of protein p6 is essential for its relocalization at the nucleoid. Altogether, the results disclose the in vivo organization of a viral histone-like protein in bacteria.

  1. Maternal Betaine Supplementation throughout Gestation and Lactation Modifies Hepatic Cholesterol Metabolic Genes in Weaning Piglets via AMPK/LXR-Mediated Pathway and Histone Modification

    PubMed Central

    Cai, Demin; Yuan, Mengjie; Liu, Haoyu; Pan, Shifeng; Ma, Wenqiang; Hong, Jian; Zhao, Ruqian

    2016-01-01

    Betaine serves as an animal and human nutrient which has been heavily investigated in glucose and lipid metabolic regulation, yet the underlying mechanisms are still elusive. In this study, feeding sows with betaine-supplemented diets during pregnancy and lactation increased cholesterol content and low-density lipoprotein receptor (LDLR) and scavenger receptor class B type I (SR-BI) gene expression, but decreasing bile acids content and cholesterol-7a-hydroxylase (CYP7a1) expression in the liver of weaning piglets. This was associated with the significantly elevated serum betaine and methionine levels and hepatic S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) content. Concurrently, the hepatic nuclear transcription factor liver X receptor LXR was downregulated along with activated signal protein AMP-activated protein kinase (AMPK). Moreover, a chromatin immunoprecipitation assay showed lower LXR binding on CYP7a1 gene promoter and more enriched activation histone marker H3K4me3 on LDLR and SR-BI promoters. These results suggest that gestational and lactational betaine supplementation modulates hepatic gene expression involved in cholesterol metabolism via an AMPK/LXR pathway and histone modification in the weaning offspring. PMID:27763549

  2. HD2C histone deacetylase and a SWI/SNF chromatin remodelling complex interact and both are involved in mediating the heat stress response in Arabidopsis.

    PubMed

    Buszewicz, Daniel; Archacki, Rafał; Palusiński, Antoni; Kotliński, Maciej; Fogtman, Anna; Iwanicka-Nowicka, Roksana; Sosnowska, Katarzyna; Kuciński, Jan; Pupel, Piotr; Olędzki, Jacek; Dadlez, Michał; Misicka, Aleksandra; Jerzmanowski, Andrzej; Koblowska, Marta Kamila

    2016-10-01

    Studies in yeast and animals have revealed that histone deacetylases (HDACs) often act as components of multiprotein complexes, including chromatin remodelling complexes (CRCs). However, interactions between HDACs and CRCs in plants have yet to be demonstrated. Here, we present evidence for the interaction between Arabidopsis HD2C deacetylase and a BRM-containing SWI/SNF CRC. Moreover, we reveal a novel function of HD2C as a regulator of the heat stress response. HD2C transcript levels were strongly induced in plants subjected to heat treatment, and the expression of selected heat-responsive genes was up-regulated in heat-stressed hd2c mutant, suggesting that HD2C acts to down-regulate heat-activated genes. In keeping with the HDAC activity of HD2C, the altered expression of HD2C-regulated genes coincided in most cases with increased histone acetylation at their loci. Microarray transcriptome analysis of hd2c and brm mutants identified a subset of commonly regulated heat-responsive genes, and the effect of the brm hd2c double mutation on the expression of these genes was non-additive. Moreover, heat-treated 3-week-old hd2c, brm and brm hd2c mutants displayed similar rates of growth retardation. Taken together, our findings suggest that HD2C and BRM act in a common genetic pathway to regulate the Arabidopsis heat stress response.

  3. The transcription factor GATA1 and the histone methyltransferase SET7 interact to promote VEGF-mediated angiogenesis and tumor growth and predict clinical outcome of breast cancer

    PubMed Central

    Zhang, Yanan; Liu, Jie; Lin, Jing; Zhou, Lei; Song, Yuhua; Wei, Bo; Luo, Xiaoli; Chen, Zhida; Chen, Yingjie; Xiong, Jiaxiu; Xu, Xiaojie; Ding, Lihua; Ye, Qinong

    2016-01-01

    Angiogenesis is essential for tumor growth. Vascular endothelial growth factor (VEGF) is the most important regulator of tumor angiogenesis. However, how transcription factors interact with histone-modifying enzymes to regulate VEGF transcription and tumor angiogenesis remains unclear. Here, we show that transcription factor GATA1 associates with the histone methyltransferase SET7 to promote VEGF transcription and breast tumor angiogenesis. Using chromatin immunoprecipitation assay, we found that GATA1 was required for recruitment of SET7, RNA polymerase II and transcription factor II B to VEGF core promoter. GATA1 enhanced breast cancer cell (MCF7, ZR75-1 and MDA-MB-231)-secreted VEGF via SET7, which promoted vascular endothelial cell (HUVEC) proliferation, migration and tube formation. SET7 was required for GATA1-induced breast tumor angiogenesis and growth in nude mice. Immunohistochemical staining showed that expression of GATA1 and SET7 was upregulated and positively correlated with VEGF expression and microvessel number in 80 breast cancer patients. GATA1 and SET7 are independent poor prognostic factors in breast cancer. Our data provide novel insights into VEGF transcriptional regulation and suggest GATA1/SET7 as cancer therapeutic targets. PMID:26848522

  4. The Histone Database: an integrated resource for histones and histone fold-containing proteins.

    PubMed

    Mariño-Ramírez, Leonardo; Levine, Kevin M; Morales, Mario; Zhang, Suiyuan; Moreland, R Travis; Baxevanis, Andreas D; Landsman, David

    2011-01-01

    Eukaryotic chromatin is composed of DNA and protein components-core histones-that act to compactly pack the DNA into nucleosomes, the fundamental building blocks of chromatin. These nucleosomes are connected to adjacent nucleosomes by linker histones. Nucleosomes are highly dynamic and, through various core histone post-translational modifications and incorporation of diverse histone variants, can serve as epigenetic marks to control processes such as gene expression and recombination. The Histone Sequence Database is a curated collection of sequences and structures of histones and non-histone proteins containing histone folds, assembled from major public databases. Here, we report a substantial increase in the number of sequences and taxonomic coverage for histone and histone fold-containing proteins available in the database. Additionally, the database now contains an expanded dataset that includes archaeal histone sequences. The database also provides comprehensive multiple sequence alignments for each of the four core histones (H2A, H2B, H3 and H4), the linker histones (H1/H5) and the archaeal histones. The database also includes current information on solved histone fold-containing structures. The Histone Sequence Database is an inclusive resource for the analysis of chromatin structure and function focused on histones and histone fold-containing proteins.

  5. Computerized video time lapse study of cell cycle delay and arrest, mitotic catastrophe, apoptosis and clonogenic survival in irradiated 14-3-3sigma and CDKN1A (p21) knockout cell lines.

    PubMed

    Chu, Kenneth; Teele, Noella; Dewey, Michael W; Albright, Norman; Dewey, William C

    2004-09-01

    Computerized video time lapse (CVTL) microscopy was used to observe cellular events induced by ionizing radiation (10-12 Gy) in nonclonogenic cells of the wild-type HCT116 colorectal carcinoma cell line and its three isogenic derivative lines in which p21 (CDKN1A), 14-3-3sigma or both checkpoint genes (double-knockout) had been knocked out. Cells that fused after mitosis or failed to complete mitosis were classified together as cells that underwent mitotic catastrophe. Seventeen percent of the wild-type cells and 34-47% of the knockout cells underwent mitotic catastrophe to enter generation 1 with a 4N content of DNA, i.e., the same DNA content as irradiated cells arrested in G(2) at the end of generation 0. Radiation caused a transient division delay in generation 0 before the cells divided or underwent mitotic catastrophe. Compared with the division delay for wild-type cells that express CDKN1A and 14-3-3sigma, knocking out CDKN1A reduced the delay the most for cells irradiated in G(1) (from approximately 15 h to approximately 3- 5 h), while knocking out 14-3-3sigma reduced the delay the most for cells irradiated in late S and G(2) (from approximately 18 h to approximately 3-4 h). However, 27% of wild-type cells and 17% of 14-3-3sigma(-/-) cells were arrested at 96 h in generation 0 compared with less than 1% for CDKN1A(-/-) and double-knockout cells. Thus expression of CDKN1A is necessary for the prolonged delay or arrest in generation 0. Furthermore, CDKN1A plays a crucial role in generation 1, greatly inhibiting progression into subsequent generations of both diploid cells and polyploid cells produced by mitotic catastrophe. Thus, in CDKN1A-deficient cell lines, a series of mitotic catastrophe events occurred to produce highly polyploid progeny during generations 3 and 4. Most importantly, the polyploid progeny produced by mitotic catastrophe events did not die sooner than the progeny of dividing cells. Death was identified as loss of cell movement, i

  6. Histone Deacetylation Mediates the Rejuvenation of Osteoblastogenesis by the Combination of 25(OH)D3 and Parathyroid Hormone in MSCs from Elders

    PubMed Central

    Zhou, Shuanhu; Geng, Shuo; Glowacki, Julie

    2012-01-01

    Vitamin D metabolites are important effectors of bone and mineral homeostasis. Human bone marrow stromal cells (hMSCs) are targets of 1α,25-dihydroxyvitamin D [1α, 25(OH)2D] action to promote their differentiation to osteoblasts. Osteoblastogenesis is also stimulated by 25-hydroxyvitamin D [25(OH)D], an effect that requires conversion to 1α, 25(OH)2D3 by 25-hydroxyvitamin D3 1α-hydroxylase (CYP27B1). These findings support an autocrine/paracrine role of vitamin D metabolism in osteoblastogenesis of hMSCs. In this study, we assessed whether and by what mechanisms osteoblastogenesis could be rejuvenated with hMSCs from elders. First, knockdown studies with VDR-siRNA showed that both the pro-differentiation and anti-proliferative effects of 1α, 25(OH)2D3 required VDR. Second, 100 nM 25(OH)D3 (p<0.01 vs. control, ANOVA) and 100 nM PTH1-34 (p<0.05) significantly stimulated alkaline phosphatase activity (a measure of osteoblastogenesis), with a synergistic effect when combined (p<0.001). Scriptaid, an inhibitor of histone deacetylase, blocked the effect of 25(OH)D3 and PTH on osteoblastogenesis. Scriptaid alone downregulated VDR in hMSCs. These data demonstrate that histone deacetylation is required for the synergistic effect of 25(OH)D3 and PTH on osteoblastogenesis in hMSCs. Both VDR siRNA and Scriptaid dowregulated VDR mRNA and inhibited osteoblastogenesis. Thus, epigenetic regulation of the VDR may be central to rejuvenating osteoblastogenesis in hMSCs from elders. PMID:22982627

  7. UVB-induced COX-2 expression requires histone H3 phosphorylation at Ser10 and Ser28

    PubMed Central

    Keum, Young-Sam; Kim, Hong-Gyum; Bode, Ann M.; Surh, Young-Joon; Dong, Zigang

    2012-01-01

    Cyclooxygenase-2 (COX-2) is an inducible enzyme that contributes to the generation of chronic inflammation in response to chemical carcinogens and environmental stresses, including ultraviolet B (UVB) irradiation. Although post-translational histone modifications are believed to play an important role in modulating transcriptional regulation of UVB-induced COX-2, the underlying biochemical mechanisms are completely unknown. Here, we show that UVB activates the p38 MAPK/MSK1 kinase cascade to phosphorylate histone H3 at Ser10 and Ser28, contributing to UVB-induced COX-2 expression. UVB has no effect on the global trimethylation level of histone H3 (H3K4me3, H3K9me3, and H3K27me3). We observed that selected mammalian 14-3-3 proteins bind to UVB-induced phosphorylated histone H3 (Ser10 and Ser28). In particular, 14-3-3ε is critical for recruiting MSK1 and Cdk9 to the chromatin and subsequently phosphorylating the C-terminal domain (CTD) of RNA polymerase II in the cox-2 promoter. We propose that histone H3 phosphorylation at Ser10 and Ser28 serve as critical switches to promote cox-2 gene expression by facilitating the recruitment of MSK1 and Cdk9 to the cox-2 promoter, thereby promoting RNA polymerase II phosphorylation. PMID:22391560

  8. The Arabidopsis 14-3-3 Protein RARE COLD INDUCIBLE 1A Links Low-Temperature Response and Ethylene Biosynthesis to Regulate Freezing Tolerance and Cold Acclimation[C][W

    PubMed Central

    Catalá, Rafael; López-Cobollo, Rosa; Mar Castellano, M.; Angosto, Trinidad; Alonso, José M.; Ecker, Joseph R.; Salinas, Julio

    2014-01-01

    In plants, the expression of 14-3-3 genes reacts to various adverse environmental conditions, including cold, high salt, and drought. Although these results suggest that 14-3-3 proteins have the potential to regulate plant responses to abiotic stresses, their role in such responses remains poorly understood. Previously, we showed that the RARE COLD INDUCIBLE 1A (RCI1A) gene encodes the 14-3-3 psi isoform. Here, we present genetic and molecular evidence implicating RCI1A in the response to low temperature. Our results demonstrate that RCI1A functions as a negative regulator of constitutive freezing tolerance and cold acclimation in Arabidopsis thaliana by controlling cold-induced gene expression. Interestingly, this control is partially performed through an ethylene (ET)-dependent pathway involving physical interaction with different ACC SYNTHASE (ACS) isoforms and a decreased ACS stability. We show that, consequently, RCI1A restrains ET biosynthesis, contributing to establish adequate levels of this hormone in Arabidopsis under both standard and low-temperature conditions. We further show that these levels are required to promote proper cold-induced gene expression and freezing tolerance before and after cold acclimation. All these data indicate that RCI1A connects the low-temperature response with ET biosynthesis to modulate constitutive freezing tolerance and cold acclimation in Arabidopsis. PMID:25122152

  9. Characteristics of Korean patients with suspected Creutzfeldt-Jakob disease with 14-3-3 protein in cerebrospinal fluid: Preliminary study of the Korean Creutzfeldt-Jakob disease active surveillance program.

    PubMed

    Lim, Jae-Sung; Kwon, Hyung-Min; Jang, Jae-Won; Ju, Young-Ran; Kim, SuYeon; Park, Young Ho; Park, So Young; Kim, SangYun

    2015-01-01

    Although Korea had a national surveillance system for Creutzfeldt-Jakob disease (CJD), it was mainly dependent on attending physician's reports. Thus, little prospective data about the epidemiology, characteristics, and final diagnoses of suspected patients were available. We have established a nationwide network for the active surveillance of patients with suspected CJD. When the requested cerebrospinal fluid (CSF) samples tested positive for 14-3-3 protein, we investigated the clinical characteristics of the corresponding patients and followed them until their final diagnoses were confirmed. A total of 218 samples were requested for CSF assays from May 2010 to August 2012, and 106 (48.6%) were positive for 14-3-3 protein. In 89 patients with complete clinical data, 38 (42.7%) were diagnosed with probable CJD and the estimated annual occurrence of CJD was 16.3 persons-per-year. The most common diagnoses of the remainder were central nervous system infection and any-cause encephalopathy. Non-CJD subjects showed worse initial consciousness levels than CJD patients. This preliminary study showed that the number of reported cases of CJD and the true positivity rates of CSF 14-3-3 protein assays were both low in Korea. An active surveillance system is urgently needed to provide the latest nationwide epidemiological data of CJD.

  10. Characteristics of Korean patients with suspected Creutzfeldt-Jakob disease with 14-3-3 protein in cerebrospinal fluid: Preliminary study of the Korean Creutzfeldt-Jakob disease active surveillance program

    PubMed Central

    Lim, Jae-Sung; Kwon, Hyung-Min; Jang, Jae-Won; Ju, Young-Ran; Kim, SuYeon; Park, Young Ho; Park, So Young; Kim, SangYun

    2015-01-01

    Abstract Although Korea had a national surveillance system for Creutzfeldt-Jakob disease (CJD), it was mainly dependent on attending physician's reports. Thus, little prospective data about the epidemiology, characteristics, and final diagnoses of suspected patients were available. We have established a nationwide network for the active surveillance of patients with suspected CJD. When the requested cerebrospinal fluid (CSF) samples tested positive for 14-3-3 protein, we investigated the clinical characteristics of the corresponding patients and followed them until their final diagnoses were confirmed. A total of 218 samples were requested for CSF assays from May 2010 to August 2012, and 106 (48.6%) were positive for 14-3-3 protein. In 89 patients with complete clinical data, 38 (42.7%) were diagnosed with probable CJD and the estimated annual occurrence of CJD was 16.3 persons-per-year. The most common diagnoses of the remainder were central nervous system infection and any-cause encephalopathy. Non-CJD subjects showed worse initial consciousness levels than CJD patients. This preliminary study showed that the number of reported cases of CJD and the true positivity rates of CSF 14-3-3 protein assays were both low in Korea. An active surveillance system is urgently needed to provide the latest nationwide epidemiological data of CJD. PMID:25996401

  11. PI 3-kinase-dependent phosphorylation of Plk1–Ser99 promotes association with 14-3-3γ and is required for metaphase–anaphase transition

    PubMed Central

    Kasahara, Kousuke; Goto, Hidemasa; Izawa, Ichiro; Kiyono, Tohru; Watanabe, Nobumoto; Elowe, Sabine; Nigg, Erich A; Inagaki, Masaki

    2013-01-01

    Polo-like kinase 1 (Plk1) controls multiple aspects of mitosis and is activated through its phosphorylation at Thr210. Here we identify Ser99 on Plk1 as a novel mitosis-specific phosphorylation site, which operates independently of Plk1–Thr210 phosphorylation. Plk1–Ser99 phosphorylation creates a docking site for 14-3-3γ, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3γ or replacement of wild-type (WT) Plk1 by a Ser99-phospho-blocking mutant leads to a prometaphase/metaphase-like arrest due to the activation of the spindle assembly checkpoint. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1–Ser99 phosphorylation and delays metaphase to anaphase transition. Plk1–Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner. Therefore, mitotic Plk1 activity is regulated not only by Plk1–Thr210 phosphorylation, but also by Plk1 binding to 14-3-3γ following Plk1–Ser99 phosphorylation downstream of the PI3K–Akt signalling pathway. This novel Plk1 activation pathway controls proper progression from metaphase to anaphase. PMID:23695676

  12. Destabilization of the Outer and Inner Mitochondrial Membranes by Core and Linker Histones

    PubMed Central

    Cascone, Annunziata; Bruelle, Celine; Lindholm, Dan; Bernardi, Paolo; Eriksson, Ove

    2012-01-01

    Background Extensive DNA damage leads to apoptosis. Histones play a central role in DNA damage sensing and may mediate signals of genotoxic damage to cytosolic effectors including mitochondria. Methodology/Principal Findings We have investigated the effects of histones on mitochondrial function and membrane integrity. We demonstrate that both linker histone H1 and core histones H2A, H2B, H3, and H4 bind strongly to isolated mitochondria. All histones caused a rapid and massive release of the pro-apoptotic intermembrane space proteins cytochrome c and Smac/Diablo, indicating that they permeabilize the outer mitochondrial membrane. In addition, linker histone H1, but not core histones, permeabilized the inner membrane with a collapse of the membrane potential, release of pyridine nucleotides, and mitochondrial fragmentation. Conclusions We conclude that histones destabilize the mitochondrial membranes, a mechanism that may convey genotoxic signals to mitochondria and promote apoptosis following DNA damage. PMID:22523586

  13. Epigenetic Modifications of Histones in Periodontal Disease.

    PubMed

    Martins, M D; Jiao, Y; Larsson, L; Almeida, L O; Garaicoa-Pazmino, C; Le, J M; Squarize, C H; Inohara, N; Giannobile, W V; Castilho, R M

    2016-02-01

    Periodontitis is a chronic infectious disease driven by dysbiosis, an imbalance between commensal bacteria and the host organism. Periodontitis is a leading cause of tooth loss in adults and occurs in about 50% of the US population. In addition to the clinical challenges associated with treating periodontitis, the progression and chronic nature of this disease seriously affect human health. Emerging evidence suggests that periodontitis is associated with mechanisms beyond bacteria-induced protein and tissue degradation. Here, we hypothesize that bacteria are able to induce epigenetic modifications in oral epithelial cells mediated by histone modifications. In this study, we found that dysbiosis in vivo led to epigenetic modifications, including acetylation of histones and downregulation of DNA methyltransferase 1. In addition, in vitro exposure of oral epithelial cells to lipopolysaccharides resulted in histone modifications, activation of transcriptional coactivators, such as p300/CBP, and accumulation of nuclear factor-κB (NF-κB). Given that oral epithelial cells are the first line of defense for the periodontium against bacteria, we also evaluated whether activation of pathogen recognition receptors induced histone modifications. We found that activation of the Toll-like receptors 1, 2, and 4 and the nucleotide-binding oligomerization domain protein 1 induced histone acetylation in oral epithelial cells. Our findings corroborate the emerging concept that epigenetic modifications play a role in the development of periodontitis. PMID:26496800

  14. Histone profiles in cancer.

    PubMed

    Riedel, Simone S; Neff, Tobias; Bernt, Kathrin M

    2015-10-01

    While DNA abnormalities have long been recognized as the cause of cancer, the contribution of chromatin is a relatively recent discovery. Excitement in the field of cancer epigenetics is driven by 3 key elements: 1. Chromatin may play an active and often critical role in controlling gene expression, DNA stability and cell identity. 2. Chromatin modifiers are frequent targets of DNA aberrations, in some cancers reaching near 100%. Particularly in cancers with low rates of DNA mutations, the key "driver" of malignancy is often a chromatin modifier. 3. Cancer-associated aberrant chromatin is amenable to pharmacologic modulation. This has sparked the rapidly expanding development of small molecules targeting chromatin modifiers or reader domains, several of which have shown promise in clinical trials. In parallel, technical advances have greatly enhanced our ability to perform comprehensive chromatin/histone profiling. Despite the discovery that distinct histone profiles are associated with prognostic subgroups, and in some instances may point towards an underlying aberration that can be targeted, histone profiling has not entered clinical diagnostics. Even eligibility for clinical trials targeting chromatin hinges on traditional histologic or DNA-based molecular criteria rather than chromatin profiles. This review will give an overview of the philosophical debate around the role of histones in controlling or modulating gene expression and discuss the most common techniques for histone profiling. In addition, we will provide prominent examples of aberrantly expressed or mutated chromatin modifiers that result in either globally or locally aberrant histone profiles, and that may be promising therapeutic targets.

  15. Human GRK4γ142V Variant Promotes Angiotensin II Type I Receptor-Mediated Hypertension via Renal Histone Deacetylase Type 1 Inhibition.

    PubMed

    Wang, Zheng; Zeng, Chunyu; Villar, Van Anthony M; Chen, Shi-You; Konkalmatt, Prasad; Wang, Xiaoyan; Asico, Laureano D; Jones, John E; Yang, Yu; Sanada, Hironobu; Felder, Robin A; Eisner, Gilbert M; Weir, Matthew R; Armando, Ines; Jose, Pedro A

    2016-02-01

    The influence of a single gene on the pathogenesis of essential hypertension may be difficult to ascertain, unless the gene interacts with other genes that are germane to blood pressure regulation. G-protein-coupled receptor kinase type 4 (GRK4) is one such gene. We have reported that the expression of its variant hGRK4γ(142V) in mice results in hypertension because of impaired dopamine D1 receptor. Signaling through dopamine D1 receptor and angiotensin II type I receptor (AT1R) reciprocally modulates renal sodium excretion and blood pressure. Here, we demonstrate the ability of the hGRK4γ(142V) to increase the expression and activity of the AT1R. We show that hGRK4γ(142V) phosphorylates histone deacetylase type 1 and promotes its nuclear export to the cytoplasm, resulting in increased AT1R expression and greater pressor response to angiotensin II. AT1R blockade and the deletion of the Agtr1a gene normalize the hypertension in hGRK4γ(142V) mice. These findings illustrate the unique role of GRK4 by targeting receptors with opposite physiological activity for the same goal of maintaining blood pressure homeostasis, and thus making the GRK4 a relevant therapeutic target to control blood pressure.

  16. The interaction of mycobacterial protein Rv2966c with host chromatin is mediated through non-CpG methylation and histone H3/H4 binding.

    PubMed

    Sharma, Garima; Upadhyay, Sandeep; Srilalitha, M; Nandicoori, Vinay K; Khosla, Sanjeev

    2015-04-30

    To effectively modulate the gene expression within an infected mammalian cell, the pathogen Mycobacterium tuberculosis would need to bring about epigenetic modifications at appropriate genomic loci. Working on this hypothesis, we show in this study that the mycobacterial protein Rv2966c is a 5-methylcytosine-specific DNA methyltransferase that is secreted out from the mycobacterium and gets localized to the nucleus in addition to the cytoplasm inside the host cell. Importantly, Rv2966c binds to specific DNA sequences, methylates cytosines predominantly in a non-CpG context and its methylation activity is positively influenced by phosphorylation. Interestingly, like the mammalian DNA methyltransferase, DNMT3L, Rv2966c can also interact with histone proteins. Ours is the first study that identifies a protein from a pathogenic bacteria with potential to influence host DNA methylation in a non-canonical manner providing the pathogen with a novel mechanism to alter the host epigenetic machinery. This contention is supported by repression of host genes upon M. tuberculosis infection correlated with Rv2966c binding and non-CpG methylation.

  17. HIGH NITROGEN INSENSITIVE 9 (HNI9)-mediated systemic repression of root NO3− uptake is associated with changes in histone methylation

    PubMed Central

    Widiez, Thomas; El Kafafi, El Sayed; Girin, Thomas; Berr, Alexandre; Ruffel, Sandrine; Krouk, Gabriel; Vayssières, Alice; Shen, Wen-Hui; Coruzzi, Gloria M.; Gojon, Alain; Lepetit, Marc

    2011-01-01

    In plants, root nitrate uptake systems are under systemic feedback repression by the N satiety of the whole organism, thus adjusting the N acquisition capacity to the N demand for growth; however, the underlying molecular mechanisms are largely unknown. We previously isolated the Arabidopsis high nitrogen-insensitive 9-1 (hni9-1) mutant, impaired in the systemic feedback repression of the root nitrate transporter NRT2.1 by high N supply. Here, we show that HNI9 encodes Arabidopsis INTERACT WITH SPT6 (AtIWS1), an evolutionary conserved component of the RNA polymerase II complex. HNI9/AtIWS1 acts in roots to repress NRT2.1 transcription in response to high N supply. At a genomic level, HNI9/AtIWS1 is shown to play a broader role in N signaling by regulating several hundred N-responsive genes in roots. Repression of NRT2.1 transcription by high N supply is associated with an HNI9/AtIWS1-dependent increase in histone H3 lysine 27 trimethylation at the NRT2.1 locus. Our findings highlight the hypothesis that posttranslational chromatin modifications control nutrient acquisition in plants. PMID:21788519

  18. The interaction of mycobacterial protein Rv2966c with host chromatin is mediated through non-CpG methylation and histone H3/H4 binding

    PubMed Central

    Sharma, Garima; Upadhyay, Sandeep; Srilalitha, M.; Nandicoori, Vinay K.; Khosla, Sanjeev

    2015-01-01

    To effectively modulate the gene expression within an infected mammalian cell, the pathogen Mycobacterium tuberculosis would need to bring about epigenetic modifications at appropriate genomic loci. Working on this hypothesis, we show in this study that the mycobacterial protein Rv2966c is a 5-methylcytosine-specific DNA methyltransferase that is secreted out from the mycobacterium and gets localized to the nucleus in addition to the cytoplasm inside the host cell. Importantly, Rv2966c binds to specific DNA sequences, methylates cytosines predominantly in a non-CpG context and its methylation activity is positively influenced by phosphorylation. Interestingly, like the mammalian DNA methyltransferase, DNMT3L, Rv2966c can also interact with histone proteins. Ours is the first study that identifies a protein from a pathogenic bacteria with potential to influence host DNA methylation in a non-canonical manner providing the pathogen with a novel mechanism to alter the host epigenetic machinery. This contention is supported by repression of host genes upon M. tuberculosis infection correlated with Rv2966c binding and non-CpG methylation. PMID:25824946

  19. Dynamic interplay between histone H3 modifications and protein interpreters: emerging evidence for a "histone language".

    PubMed

    Oliver, Samuel S; Denu, John M

    2011-01-24

    Histone proteins organize DNA into dynamic chromatin structures and regulate processes such as transcription, repair, and replication. Control of chromatin function and structure is mediated in part by reversible post-translational modifications (PTMs) on histones. The most N-terminal region of histone H3 contains a high density of modifiable residues. Here we focus on the dynamic interplay between histone modification states on the H3 N terminus and the binding modules that recognize these states. Specifically, we discuss the effect of auxiliary modifications to H3K4unmod/me3 binding modules (specifically H3R2 methylation, H3T3 phosphorylation, and H3T6 phosphorylation). Emerging evidence suggests that histone PTMs behave less like a strict "code", but more like a "language", which better illustrates the importance of context. Using androgen-receptor-mediated gene activation as an example, we propose a model of how the combinatorial natures of PTMs on the H3 N terminus and the complexes that recognize these epigenetic modifications control gene expression. PMID:21243717

  20. Roles for Histone Acetylation in Regulation of Telomere Elongation and Two-cell State in Mouse ES Cells.

    PubMed

    Dan, Jiameng; Yang, Jiao; Liu, Yifei; Xiao, Andrew; Liu, Lin

    2015-10-01

    Mammalian telomeres and subtelomeres are marked by heterochromatic epigenetic modifications, including repressive DNA methylation and histone methylation (e.g., H3K9me3 and H4K20me3). Loss of these epigenetic marks results in increased rates of telomere recombination and elongation. Other than these repressive epigenetic marks, telomeric and subtelomeric H3 and H4 are underacetylated. Yet, whether histone acetylation also regulates telomere length has not been directly addressed. We thought to test the effects of histone acetylation levels on telomere length using histone deacetylase (HDAC) inhibitor (sodium butyrate, NaB) that mediates histone hyperacetylation and histone acetyltransferase (HAT) inhibitor (C646) that mediates histone hypoacetylation. We show that histone hyperacetylation dramatically elongates telomeres in wild-type ES cells, and only slightly elongates telomeres in Terc(-/-) ES cells, suggesting that Terc is involved in histone acetylation-induced telomere elongation. In contrast, histone hypoacetylation shortens telomeres in both wild-type and Terc(-/-) ES cells. Additionally, histone hyperacetylation activates 2-cell (2C) specific genes including Zscan4, which is involved in telomere recombination and elongation, whereas histone hypoacetylation represses Zscan4 and 2C genes. These data suggest that histone acetylation levels affect the heterochromatic state at telomeres and subtelomeres, and regulate gene expression at subtelomeres, linking histone acetylation to telomere length maintenance.

  1. Structure and function of the histone chaperone CIA/ASF1 complexed with histones H3 and H4.

    PubMed

    Natsume, Ryo; Eitoku, Masamitsu; Akai, Yusuke; Sano, Norihiko; Horikoshi, Masami; Senda, Toshiya

    2007-03-15

    CIA (CCG1-interacting factor A)/ASF1, which is the most conserved histone chaperone among the eukaryotes, was genetically identified as a factor for an anti-silencing function (Asf1) by yeast genetic screening. Shortly after that, the CIA-histone-H3-H4 complex was isolated from Drosophila as a histone chaperone CAF-1 stimulator. Human CIA-I/II (ASF1a/b) was identified as a histone chaperone that interacts with the bromodomain-an acetylated-histone-recognizing domain-of CCG1, in the general transcription initiation factor TFIID. Intensive studies have revealed that CIA/ASF1 mediates nucleosome assembly by forming a complex with another histone chaperone in human cells and yeast, and is involved in DNA replication, transcription, DNA repair and silencing/anti-silencing in yeast. CIA/ASF1 was shown as a major storage chaperone for soluble histones in proliferating human cells. Despite all these biochemical and biological functional analyses, the structure-function relationship of the nucleosome assembly/disassembly activity of CIA/ASF1 has remained elusive. Here we report the crystal structure, at 2.7 A resolution, of CIA-I in complex with histones H3 and H4. The structure shows the histone H3-H4 dimer's mutually exclusive interactions with another histone H3-H4 dimer and CIA-I. The carboxy-terminal beta-strand of histone H4 changes its partner from the beta-strand in histone H2A to that of CIA-I through large conformational change. In vitro functional analysis demonstrated that CIA-I has a histone H3-H4 tetramer-disrupting activity. Mutants with weak histone H3-H4 dimer binding activity showed critical functional effects on cellular processes related to transcription. The histone H3-H4 tetramer-disrupting activity of CIA/ASF1 and the crystal structure of the CIA/ASF1-histone-H3-H4 dimer complex should give insights into mechanisms of both nucleosome assembly/disassembly and nucleosome semi-conservative replication.

  2. Heparanase-mediated Loss of Nuclear Syndecan-1 Enhances Histone Acetyltransferase (HAT) Activity to Promote Expression of Genes That Drive an Aggressive Tumor Phenotype*

    PubMed Central

    Purushothaman, Anurag; Hurst, Douglas R.; Pisano, Claudio; Mizumoto, Shuji; Sugahara, Kazuyuki; Sanderson, Ralph D.

    2011-01-01

    Heparanase acts as a master regulator of the aggressive tumor phenotype in part by enhancing expression of proteins known to drive tumor progression (e.g. VEGF, MMP-9, hepatocyte growth factor (HGF), and RANKL). However, the mechanism whereby this enzyme regulates gene expression remains unknown. We previously reported that elevation of heparanase levels in myeloma cells causes a dramatic reduction in the amount of syndecan-1 in the nucleus. Because syndecan-1 has heparan sulfate chains and because exogenous heparan sulfate has been shown to inhibit the activity of histone acetyltransferase (HAT) enzymes in vitro, we hypothesized that the reduction in nuclear syndecan-1 in cells expressing high levels of heparanase would result in increased HAT activity leading to stimulation of protein transcription. We found that myeloma cells or tumors expressing high levels of heparanase and low levels of nuclear syndecan-1 had significantly higher levels of HAT activity when compared with cells or tumors expressing low levels of heparanase. High levels of HAT activity in heparanase-high cells were blocked by SST0001, an inhibitor of heparanase. Restoration of high syndecan-1 levels in heparanase-high cells diminished nuclear HAT activity, establishing syndecan-1 as a potent inhibitor of HAT. Exposure of heparanase-high cells to anacardic acid, an inhibitor of HAT activity, significantly suppressed their expression of VEGF and MMP-9, two genes known to be up-regulated following elevation of heparanase. These results reveal a novel mechanistic pathway driven by heparanase expression, which leads to decreased nuclear syndecan-1, increased HAT activity, and up-regulation of transcription of multiple genes that drive an aggressive tumor phenotype. PMID:21757697

  3. Chimeric NKG2D CAR-Expressing T Cell-Mediated Attack of Human Ovarian Cancer Is Enhanced by Histone Deacetylase Inhibition

    PubMed Central

    Song, De-Gang; Ye, Qunrui; Santoro, Stephen; Fang, Chongyun; Best, Andrew

    2013-01-01

    Abstract NKG2D ligands (NKG2DLs) are widely expressed on ovarian cancers to various degrees, making them attractive targets for immunotherapy. Here, we applied a chimeric antigen receptor (CAR) approach for the targeting of NKG2DLs expressed on human ovarian cancer cells and evaluated the impact of pharmacological upregulation of NKG2DLs on immune recognition. Various NKG2DLs, including MICA/B and ULBP-1, -2, -3, and -4, were expressed at various levels on the surface of all established ovarian cancer cell lines and primary ovarian cancer samples tested. To redirect human T cells against NKG2DLs, an NKG2DL-specific CAR was generated by fusing the extracellular domain of the NKG2D receptor to the 4-1BB costimulatory and CD3-ζ chain signaling domains. In vitro expansion of chimeric NKG2D CAR T cells was delayed compared with untransduced T cells and control CAR T cells; the likely result of fratricide among activated T cells expressing NKG2DLs. However, NKG2D CAR T cells did expand and were selectively enriched during prolonged culture. In coculture, CD4+ and CD8+ NKG2D CAR T cells specifically recognized and killed NKG2DL-expressing ovarian cancer cell lines but not NKG2DL-negative cells. Notably, pretreatment of ovarian cancer cells expressing moderate to low levels of NKG2DLs with the histone deacetylase inhibitor sodium valproate (VPA) upregulated NKG2DL cell surface expression and consequently enhanced their immune recognition by chimeric NKG2D CAR T cells. Our results demonstrate that VPA-induced upregulation of NKG2DL expression enhances the immune recognition of ovarian cancer cells by engineered NKG2D CAR T cells, and rationalizes the use of VPA in combination with NKG2DL-targeted immunotherapy in ovarian cancer. PMID:23297870

  4. Structure-Function Analysis of PPP1R3D, a Protein Phosphatase 1 Targeting Subunit, Reveals a Binding Motif for 14-3-3 Proteins which Regulates its Glycogenic Properties.

    PubMed

    Rubio-Villena, Carla; Sanz, Pascual; Garcia-Gimeno, Maria Adelaida

    2015-01-01

    Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. It plays a key role in regulating glycogen synthesis, by dephosphorylating crucial enzymes involved in glycogen homeostasis such as glycogen synthase (GS) and glycogen phosphorylase (GP). To play this role, PP1 binds to specific glycogen targeting subunits that, on one hand recognize the substrates to be dephosphorylated and on the other hand recruit PP1 to glycogen particles. In this work we have analyzed the functionality of the different protein binding domains of one of these glycogen targeting subunits, namely PPP1R3D (R6) and studied how binding properties of different domains affect its glycogenic properties. We have found that the PP1 binding domain of R6 comprises a conserved RVXF motif (R102VRF) located at the N-terminus of the protein. We have also identified a region located at the C-terminus of R6 (W267DNND) that is involved in binding to the PP1 glycogenic substrates. Our results indicate that although binding to PP1 and glycogenic substrates are independent processes, impairment of any of them results in lack of glycogenic activity of R6. In addition, we have characterized a novel site of regulation in R6 that is involved in binding to 14-3-3 proteins (RARS74LP). We present evidence indicating that when binding of R6 to 14-3-3 proteins is prevented, R6 displays hyper-glycogenic activity although is rapidly degraded by the lysosomal pathway. These results define binding to 14-3-3 proteins as an additional pathway in the control of the glycogenic properties of R6.

  5. The Histone Database: a comprehensive resource for histones and histone fold-containing proteins.

    PubMed

    Mariño-Ramírez, Leonardo; Hsu, Benjamin; Baxevanis, Andreas D; Landsman, David

    2006-03-01

    The Histone Database is a curated and searchable collection of full-length sequences and structures of histones and nonhistone proteins containing histone-like folds, compiled from major public databases. Several new histone fold-containing proteins have been identified, including the huntingtin-interacting protein HYPM. Additionally, based on the recent crystal structure of the Son of Sevenless protein, an interpretation of the sequence analysis of the histone fold domain is presented. The database contains an updated collection of multiple sequence alignments for the four core histones (H2A, H2B, H3, and H4) and the linker histones (H1/H5) from a total of 975 organisms. The database also contains information on the human histone gene complement and provides links to three-dimensional structures of histone and histone fold-containing proteins. The Histone Database is a comprehensive bioinformatics resource for the study of structure and function of histones and histone fold-containing proteins. The database is available at http://research.nhgri.nih.gov/histones/.

  6. Arginine-rich histones have strong antiviral activity for influenza A viruses.

    PubMed

    Hoeksema, Marloes; Tripathi, Shweta; White, Mitchell; Qi, Li; Taubenberger, Jeffery; van Eijk, Martin; Haagsman, Henk; Hartshorn, Kevan L

    2015-10-01

    While histones are best known for DNA binding and transcription-regulating properties, they also have antimicrobial activity against a broad range of potentially pathogenic organisms. Histones are abundant in neutrophil extracellular traps, where they play an important role in NET-mediated antimicrobial killing. Here, we show anti-influenza activity of histones against both seasonal H3N2 and H1N1, but not pandemic H1N1. The arginine rich histones, H3 and H4, had greater neutralizing and viral aggregating activity than the lysine rich histones, H2A and H2B. Of all core histones, histone H4 is most potent in neutralizing IAV, and incubation with IAV with histone H4 results in a decrease in uptake and viral replication by epithelial cells when measured by qRT-PCR. The antiviral activity of histone H4 is mediated principally by direct effects on viral particles. Histone H4 binds to IAV as assessed by ELISA and co-sedimentation of H4 with IAV. H4 also induces aggregation, as assessed by confocal microscopy and light transmission assays. Despite strong antiviral activity against the seasonal IAV strains, H4 was inactive against pandemic H1N1. These findings indicate a possible role for histones in the innate immune response against IAV.

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

    PubMed

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

    2007-03-13

    The forkhead family protein FOXP3 acts as a repressor of transcription and is both an essential and sufficient regulator of the development and function of regulatory T cells. The molecular mechanism by which FOXP3-mediated transcriptional repression occurs remains unclear. Here, we report that transcriptional repression by FOXP3 involves a histone acetyltransferase-deacetylase complex that includes histone acetyltransferase TIP60 (Tat-interactive protein, 60 kDa) and class II histone deacetylases HDAC7 and HDAC9. The N-terminal 106-190 aa of FOXP3 are required for TIP60-FOXP3, HDAC7-FOXP3 association, as well as for the transcriptional repression of FOXP3 via its forkhead domain. FOXP3 can be acetylated in primary human regulatory T cells, and TIP60 promotes FOXP3 acetylation in vivo. Overexpression of TIP60 but not its histone acetyltransferase-deficient mutant promotes, whereas knockdown of endogenous TIP60 relieved, FOXP3-mediated transcriptional repression. A minimum FOXP3 ensemble containing native TIP60 and HDAC7 is necessary for IL-2 production regulation in T cells. Moreover, FOXP3 association with HDAC9 is antagonized by T cell stimulation and can be restored by the protein deacetylation inhibitor trichostatin A, indicating a complex dynamic aspect of T suppressor cell regulation. These findings identify a previously uncharacterized complex-based mechanism by which FOXP3 actively mediates transcriptional repression. PMID:17360565

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

    PubMed Central

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

    2007-01-01

    The forkhead family protein FOXP3 acts as a repressor of transcription and is both an essential and sufficient regulator of the development and function of regulatory T cells. The molecular mechanism by which FOXP3-mediated transcriptional repression occurs remains unclear. Here, we report that transcriptional repression by FOXP3 involves a histone acetyltransferase–deacetylase complex that includes histone acetyltransferase TIP60 (Tat-interactive protein, 60 kDa) and class II histone deacetylases HDAC7 and HDAC9. The N-terminal 106–190 aa of FOXP3 are required for TIP60–FOXP3, HDAC7–FOXP3 association, as well as for the transcriptional repression of FOXP3 via its forkhead domain. FOXP3 can be acetylated in primary human regulatory T cells, and TIP60 promotes FOXP3 acetylation in vivo. Overexpression of TIP60 but not its histone acetyltransferase-deficient mutant promotes, whereas knockdown of endogenous TIP60 relieved, FOXP3-mediated transcriptional repression. A minimum FOXP3 ensemble containing native TIP60 and HDAC7 is necessary for IL-2 production regulation in T cells. Moreover, FOXP3 association with HDAC9 is antagonized by T cell stimulation and can be restored by the protein deacetylation inhibitor trichostatin A, indicating a complex dynamic aspect of T suppressor cell regulation. These findings identify a previously uncharacterized complex-based mechanism by which FOXP3 actively mediates transcriptional repression. PMID:17360565

  9. Phosphorylation-dependent inhibition of Cdc42 GEF Gef1 by 14-3-3 protein Rad24 spatially regulates Cdc42 GTPase activity and oscillatory dynamics during cell morphogenesis

    PubMed Central

    Das, Maitreyi; Nuñez, Illyce; Rodriguez, Marbelys; Wiley, David J.; Rodriguez, Juan; Sarkeshik, Ali; Yates, John R.; Buchwald, Peter; Verde, Fulvia

    2015-01-01

    Active Cdc42 GTPase, a key regulator of cell polarity, displays oscillatory dynamics that are anticorrelated at the two cell tips in fission yeast. Anticorrelation suggests competition for active Cdc42 or for its effectors. Here we show how 14-3-3 protein Rad24 associates with Cdc42 guanine exchange factor (GEF) Gef1, limiting Gef1 availability to promote Cdc42 activation. Phosphorylation of Gef1 by conserved NDR kinase Orb6 promotes Gef1 binding to Rad24. Loss of Rad24–Gef1 interaction increases Gef1 protein localization and Cdc42 activation at the cell tips and reduces the anticorrelation of active Cdc42 oscillations. Increased Cdc42 activation promotes precocious bipolar growth activation, bypassing the normal requirement for an intact microtubule cytoskeleton and for microtubule-dependent polarity landmark Tea4-PP1. Further, increased Cdc42 activation by Gef1 widens cell diameter and alters tip curvature, countering the effects of Cdc42 GTPase-activating protein Rga4. The respective levels of Gef1 and Rga4 proteins at the membrane define dynamically the growing area at each cell tip. Our findings show how the 14-3-3 protein Rad24 modulates the availability of Cdc42 GEF Gef1, a homologue of mammalian Cdc42 GEF DNMBP/TUBA, to spatially control Cdc42 GTPase activity and promote cell polarization and cell shape emergence. PMID:26246599

  10. Characterization of the Interactome of the Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 2 Reveals the Hyper Variable Region as a Binding Platform for Association with 14-3-3 Proteins.

    PubMed

    Xiao, Yihong; Wu, Weining; Gao, Jiming; Smith, Nikki; Burkard, Christine; Xia, Dong; Zhang, Minxia; Wang, Chengbao; Archibald, Alan; Digard, Paul; Zhou, En-Min; Hiscox, Julian A

    2016-05-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry worldwide and hence global food security, exacerbated by a newly emerged highly pathogenic (HP-PRRSV) strain from China. PRRSV nonstructural protein 2 (nsp2) is a multifunctional polypeptide with strain-dependent influences on pathogenicity. A number of discrete functional regions have been identified on the protein. Quantitative label free proteomics was used to identify cellular binding partners of nsp2 expressed by HP-PRRSV. This allowed the identification of potential cellular interacting partners and the discrimination of nonspecific interactions. The interactome data were further investigated and validated using biological replicates and also compared with nsp2 from a low pathogenic (LP) strain of PRRSV. Validation included both forward and reverse pulldowns and confocal microscopy. The data indicated that nsp2 interacted with a number of cellular proteins including 14-3-3, CD2AP, and other components of cellular aggresomes. The hyper-variable region of nsp2 protein was identified as a binding platform for association with 14-3-3 proteins. PMID:26709850

  11. Analysis of histones and histone variants in plants.

    PubMed

    Trivedi, Ila; Rai, Krishan Mohan; Singh, Sunil Kumar; Kumar, Verandra; Singh, Mala; Ranjan, Amol; Lodhi, Niraj; Sawant, Samir V

    2012-01-01

    Histone proteins are the major protein components of chromatin - the physiologically relevant form of the genome (or epigenome) in all eukaryotic cells. For many years, histones were considered passive structural components of eukaryotic chromatin. In recent years, it has been demonstrated that dynamic association of histones and their variants to the genome plays a very important role in gene regulation. Histones are extensively modified during posttranslation viz. acetylation, methylation, phosphorylation, ubiquitylation, etc., and the identification of these covalent marks on canonical and variant histones is crucial for the understanding of their biological significance. Different biochemical techniques have been developed to purify and separate histone proteins; here, we describe techniques for analysis of histones from plant tissues.

  12. Molecular and biochemical mining of heat-shock and 14-3-3 proteins in drug-induced protoscolices of Echinococcus granulosus and the detection of a candidate gene for anthelmintic resistance.

    PubMed

    Pan, D; Das, S; Bera, A K; Bandyopadhyay, S; Bandyopadhyay, S; De, S; Rana, T; Das, S K; Suryanaryana, V V; Deb, J; Bhattacharya, D

    2011-06-01

    Cystic echinococcosis (CE) caused by the larval stage of Echinococcus granulosus is a disease that affects both humans and animals. In humans the disease is treated by surgery with a supplementary option of chemotherapy with a benzimidazole compound. During the present study heat-shock protein 60 (HSP 60) was identified as one of the most frequently expressed biomolecules by E. granulosus after albendazole treatment. Data were correlated with 14-3-3 protein signature, and overexpression of this molecule after albendazole induction was an indicator of cell survival and signal transduction during in vitro maintenance of E. granulosus for up to 72 h. This observation was further correlated with a uniform expression pattern of a housekeeping gene (actin II). Out of three β-tubulin gene isoforms of E. granulosus, β-tubulin gene isoform 2 showed a conserved point mutation indicative of benzimidazole resistance.

  13. The FAD-dependent glycerol-3-phosphate dehydrogenase of Giardia duodenalis: an unconventional enzyme that interacts with the g14-3-3 and it is a target of the antitumoral compound NBDHEX

    PubMed Central

    Lalle, Marco; Camerini, Serena; Cecchetti, Serena; Finelli, Renata; Sferra, Gabriella; Müller, Joachim; Ricci, Giorgio; Pozio, Edoardo

    2015-01-01

    The flagellated protozoan Giardia duodenalis is a worldwide parasite causing giardiasis, an acute and chronic diarrheal disease. Metabolism in G. duodenalis has a limited complexity thus making metabolic enzymes ideal targets for drug development. However, only few metabolic pathways (i.e., carbohydrates) have been described so far. Recently, the parasite homolog of the mitochondrial-like glycerol-3-phosphate dehydrogenase (gG3PD) has been identified among the interactors of the g14-3-3 protein. G3PD is involved in glycolysis, electron transport, glycerophospholipids metabolism, and hyperosmotic stress response, and is emerging as promising target in tumor treatment. In this work, we demonstrate that gG3PD is a functional flavoenzyme able to convert glycerol-3-phosphate into dihydroxyacetone phosphate and that its activity and the intracellular glycerol level increase during encystation. Taking advantage of co-immunoprecipitation assays and deletion mutants, we provide evidence that gG3PD and g14-3-3 interact at the trophozoite stage, the intracellular localization of gG3PD is stage dependent and it partially co-localizes with mitosomes during cyst development. Finally, we demonstrate that the gG3PD activity is affected by the antitumoral compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, that results more effective in vitro at killing G. duodenalis trophozoites than the reference drug metronidazole. Overall, our results highlight the involvement of gG3PD in processes crucial for the parasite survival thus proposing this enzyme as target for novel antigiardial interventions. PMID:26082764

  14. Actin and DNA Protect Histones from Degradation by Bacterial Proteases but Inhibit Their Antimicrobial Activity.

    PubMed

    Sol, Asaf; Skvirsky, Yaniv; Blotnick, Edna; Bachrach, Gilad; Muhlrad, Andras

    2016-01-01

    Histones are small polycationic proteins located in the cell nucleus. Together, DNA and histones are integral constituents of the nucleosomes. Upon apoptosis, necrosis, and infection - induced cell death, histones are released from the cell. The extracellular histones have strong antimicrobial activity but are also cytotoxic and thought as mediators of cell death in sepsis. The antimicrobial activity of the cationic extracellular histones is inhibited by the polyanionic DNA and F-actin, which also become extracellular upon cell death. DNA and F-actin protect histones from degradation by the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis. However, though the integrity of the histones is protected, the activity of histones as antibacterial agents is lost. The inhibition of the histone's antibacterial activity and their protection from proteolysis by DNA and F-actin indicate a tight electrostatic interaction between the positively charged histones and negatively charged DNA and F-actin, which may have physiological significance in maintaining the equilibrium between the beneficial antimicrobial activity of extracellular histones and their cytotoxic effects. PMID:27555840

  15. Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

    PubMed Central

    Sidoli, Simone; Bhanu, Natarajan V.; Karch, Kelly R.; Wang, Xiaoshi; Garcia, Benjamin A.

    2016-01-01

    Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude. PMID:27286567

  16. Identification and quantification of histone PTMs using high-resolution mass spectrometry

    PubMed Central

    Karch, Kelly R.; Sidoli, Simone; Garcia, Benjamin A.

    2016-01-01

    DNA is organized into nucleosomes, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins including H2A, H2B, H3, and H4. Histones are critical regulators of many nuclear processes, including transcription, DNA damage repair, and higher order chromatin structure. Much of their function is mediated through extensive and dynamic post-translational modification (PTM) by nuclear enzymes. Histone PTMs are thought to form a code, where combinations of PTMs are responsible for specific biological functions. Here, we present protocols to identify and quantify histone PTMs using nano-flow liquid chromatography (nLC) coupled to mass spectrometry (MS). We first describe how to purify histones and prepare them for MS. We then describe three MS platforms for histone PTM analysis, including bottom up, middle down, and top down approaches, and explain the relative benefits and pitfalls of each approach. We also include tips to increase the throughput of large experiments. PMID:27423855

  17. Actin and DNA Protect Histones from Degradation by Bacterial Proteases but Inhibit Their Antimicrobial Activity

    PubMed Central

    Sol, Asaf; Skvirsky, Yaniv; Blotnick, Edna; Bachrach, Gilad; Muhlrad, Andras

    2016-01-01

    Histones are small polycationic proteins located in the cell nucleus. Together, DNA and histones are integral constituents of the nucleosomes. Upon apoptosis, necrosis, and infection – induced cell death, histones are released from the cell. The extracellular histones have strong antimicrobial activity but are also cytotoxic and thought as mediators of cell death in sepsis. The antimicrobial activity of the cationic extracellular histones is inhibited by the polyanionic DNA and F-actin, which also become extracellular upon cell death. DNA and F-actin protect histones from degradation by the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis. However, though the integrity of the histones is protected, the activity of histones as antibacterial agents is lost. The inhibition of the histone’s antibacterial activity and their protection from proteolysis by DNA and F-actin indicate a tight electrostatic interaction between the positively charged histones and negatively charged DNA and F-actin, which may have physiological significance in maintaining the equilibrium between the beneficial antimicrobial activity of extracellular histones and their cytotoxic effects. PMID:27555840

  18. Histone lysine methylation: critical regulator of memory and behavior.

    PubMed

    Jarome, Timothy J; Lubin, Farah D

    2013-01-01

    Histone lysine methylation is a well-established transcriptional mechanism for the regulation of gene expression changes in eukaryotic cells and is now believed to function in neurons of the central nervous system to mediate the process of memory formation and behavior. In mature neurons, methylation of histone proteins can serve to both activate and repress gene transcription. This is in stark contrast to other epigenetic modifications, including histone acetylation and DNA methylation, which have largely been associated with one transcriptional state in the brain. In this review, we discuss the evidence for histone methylation mechanisms in the coordination of complex cognitive processes such as long-term memory formation and storage. In addition, we address the current literature highlighting the role of histone methylation in intellectual disability, addiction, schizophrenia, autism, depression, and neurodegeneration. Further, we discuss histone methylation within the context of other epigenetic modifications and the potential advantages of exploring this newly identified mechanism of cognition, emphasizing the possibility that this molecular process may provide an alternative locus for intervention in long-term psychopathologies that cannot be clearly linked to genes or environment alone.

  19. Histone deacetylases and atherosclerosis.

    PubMed

    Zheng, Xia-xia; Zhou, Tian; Wang, Xin-An; Tong, Xiao-hong; Ding, Jia-wang

    2015-06-01

    Atherosclerosis is the most common pathological process that leads to cardiovascular diseases, a disease of large- and medium-sized arteries that is characterized by a formation of atherosclerotic plaques consisting of necrotic cores, calcified regions, accumulated modified lipids, smooth muscle cells (SMCs), endothelial cells, leukocytes, and foam cells. Recently, the question about how to suppress the occurrence of atherosclerosis and alleviate the progress of cardiovascular disease becomes the hot topic. Accumulating evidence suggests that histone deacetylases(HDACs) play crucial roles in arteriosclerosis. This review summarizes the effect of HDACs and HDAC inhibitors(HDACi) on the progress of atherosclerosis.

  20. Trout-liver histones

    PubMed Central

    Palau, J.; Butler, J. A. V.

    1966-01-01

    1. The histones of trout liver were examined and four main fractions (f1, f2b, f2a and f3) were isolated and characterized. 2. The amino acid analyses, N-terminal group analyses and starch-gel electrophoresis patterns are remarkably similar to the corresponding fractions of calf thymus. 3. The group f2a was also separated into two subfractions, f2al and f2a2, which are similar to those of calf thymus. ImagesFig. 1. PMID:5969291

  1. Proteomic pathway analysis of the hippocampus in schizophrenia and bipolar affective disorder implicates 14-3-3 signaling, aryl hydrocarbon receptor signaling, and glucose metabolism: potential roles in GABAergic interneuron pathology.

    PubMed

    Schubert, Klaus Oliver; Föcking, Melanie; Cotter, David R

    2015-09-01

    Neuropathological changes of the hippocampus have been associated with psychotic disorders such as schizophrenia and bipolar disorder. Recent work has particularly implicated hippocampal GABAergic interneurons in the pathophysiology of these diseases. However, the molecular mechanisms underlying structural and cellular hippocampal pathology remain poorly understood. We used data from comprehensive difference-in-gel electrophoresis (2-D DIGE) investigations of postmortem human hippocampus of people with schizophrenia and bipolar disorder, covering the acidic (isoelectric point (pI) between pH4 and 7) and, separately, the basic (pI between pH6 and 11) sub-proteome, for Ingenuity Pathway Analysis (IPA) of implicated protein networks and pathways. Comparing disease and control cases, we identified 58 unique differentially expressed proteins in schizophrenia, and 70 differentially expressed proteins in bipolar disorder, using mass spectrometry. IPA implicated, most prominently, 14-3-3 and aryl hydrocarbon receptor signaling in schizophrenia, and gluconeogenesis/glycolysis in bipolar disorder. Both disorders were characterized by alterations of proteins involved in the oxidative stress response, mitochondrial function, and protein-endocytosis, -trafficking, -degradation, and -ubiquitination. These findings are interpreted with a focus on GABAergic interneuron pathology in the hippocampus.

  2. Histones Differentially Modulate the Anticoagulant and Profibrinolytic Activities of Heparin, Heparin Derivatives, and Dabigatran.

    PubMed

    Ammollo, Concetta Tiziana; Semeraro, Nicola; Carratù, Maria Rosaria; Colucci, Mario; Semeraro, Fabrizio

    2016-02-01

    The antithrombin activity of unfractionated heparin (UFH) is offset by extracellular histones, which, along with DNA, represent a novel mediator of thrombosis and a structural component of thrombi. Here, we systematically evaluated the effect of histones, DNA, and histone-DNA complexes on the anticoagulant and profibrinolytic activities of UFH, its derivatives enoxaparin and fondaparinux, and the direct thrombin inhibitor dabigatran. Thrombin generation was assessed by calibrated automated thrombinography, inhibition of factor Xa and thrombin by synthetic substrates, tissue plasminogen activator-mediated clot lysis by turbidimetry, and thrombin-activatable fibrinolysis inhibitor (TAFI) activation by a functional assay. Histones alone delayed coagulation and slightly stimulated fibrinolysis. The anticoagulant activity of UFH and enoxaparin was markedly inhibited by histones, whereas that of fondaparinux was enhanced. Histones neutralized both the anti-Xa and anti-IIa activities of UFH and preferentially blocked the anti-IIa activity of enoxaparin. The anti-Xa activity of fondaparinux was not influenced by histones when analyzed by chromogenic substrates, but was potentiated in a plasma prothrombinase assay. Histones inhibited the profibrinolytic activity of UFH and enoxaparin and enhanced that of fondaparinux by acting on the modulation of TAFI activation by anticoagulants. Histone H1 was mainly responsible for these effects. Histone-DNA complexes, as well as intact neutrophil extracellular traps, impaired the activities of UFH, enoxaparin, and fondaparinux. Dabigatran was not noticeably affected by histones and/or DNA, whatever the assay performed. In conclusion, histones and DNA present in the forming clot may variably influence the antithrombotic activities of anticoagulants, suggesting a potential therapeutic advantage of dabigatran and fondaparinux over heparins.

  3. Histone acetylation: truth of consequences?

    PubMed

    Choi, Jennifer K; Howe, Leann J

    2009-02-01

    Eukaryotic DNA is packaged into a nucleoprotein structure known as chromatin, which is comprised of DNA, histones, and nonhistone proteins. Chromatin structure is highly dynamic, and can shift from a transcriptionally inactive state to an active form in response to intra- and extracellular signals. A major factor in chromatin architecture is the covalent modification of histones through the addition of chemical moieties, such as acetyl, methyl, ubiquitin, and phosphate groups. The acetylation of the amino-terminal tails of histones is a process that is highly conserved in eukaryotes, and was one of the earliest histone modifications characterized. Since its identification in 1964, a large body of evidence has accumulated demonstrating that histone acetylation plays an important role in transcription. Despite our ever-growing understanding of the nuclear processes involved in nucleosome acetylation, however, the exact biochemical mechanisms underlying the downstream effects of histone acetylation have yet to be fully elucidated. To date, histone acetylation has been proposed to function in 2 nonmutually exclusive manners: by directly altering chromatin structure, and by acting as a molecular tag for the recruitment of chromatin-modifying complexes. Here, we discuss recent research focusing on these 2 potential roles of histone acetylation and clarify what we actually know about the function of this modification.

  4. Chatting histone modifications in mammals

    PubMed Central

    Izzo, Annalisa

    2010-01-01

    Eukaryotic chromatin can be highly dynamic and can continuously exchange between an open transcriptionally active conformation and a compacted silenced one. Post-translational modifications of histones have a pivotal role in regulating chromatin states, thus influencing all chromatin dependent processes. Methylation is currently one of the best characterized histone modification and occurs on arginine and lysine residues. Histone methylation can regulate other modifications (e.g. acetylation, phosphorylation and ubiquitination) in order to define a precise functional chromatin environment. In this review we focus on histone methylation and demethylation, as well as on the enzymes responsible for setting these marks. In particular we are describing novel concepts on the interdependence of histone modifications marks and discussing the molecular mechanisms governing this cross-talks. PMID:21266346

  5. Dynamic interplay between histone H3 modifications and protein interpreters: emerging evidence for a ‘histone language’

    PubMed Central

    Oliver, Samuel S.; Denu, John M.

    2010-01-01

    Histone proteins organize DNA into dynamic chromatin structures and regulate processes such as transcription, repair and replication. Control of chromatin function and structure is mediated in part by reversible posttranslational modifications (PTMs) on histones. The most N-terminal region of histone H3 contains a high density of modifiable residues. In this review, we focus on the dynamic interplay between histone modification states on the H3 N-terminus and the binding modules that recognize these states. Specifically, we will discuss the effect of auxiliary modifications to H3K4unmod/me3 binding modules (specifically H3R2 methylation, H3T3 phosphorylation and H3T6 phosphorylation). Emerging evidence suggests that histone PTMs behave less like a strict ‘code’, but rather like a ‘language’, which better illustrates the importance of context. Using androgen receptor-mediated gene activation as an example, we propose a model for how the combinatorial nature of PTMs on the H3 N-terminus and the complexes that recognize these epigenetic modifications control gene expression. PMID:21243717

  6. Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: Lessons learned from Rtt109 histone acetyltransferases.

    PubMed

    Dahlin, Jayme L; Chen, Xiaoyue; Walters, Michael A; Zhang, Zhiguo

    2015-01-01

    During DNA replication, nucleosomes ahead of replication forks are disassembled to accommodate replication machinery. Following DNA replication, nucleosomes are then reassembled onto replicated DNA using both parental and newly synthesized histones. This process, termed DNA replication-coupled nucleosome assembly (RCNA), is critical for maintaining genome integrity and for the propagation of epigenetic information, dysfunctions of which have been implicated in cancers and aging. In recent years, it has been shown that RCNA is carefully orchestrated by a series of histone modifications, histone chaperones and histone-modifying enzymes. Interestingly, many features of RCNA are also found in processes involving DNA replication-independent nucleosome assembly like histone exchange and gene transcription. In yeast, histone H3 lysine K56 acetylation (H3K56ac) is found in newly synthesized histone H3 and is critical for proper nucleosome assembly and for maintaining genomic stability. The histone acetyltransferase (HAT) regulator of Ty1 transposition 109 (Rtt109) is the sole enzyme responsible for H3K56ac in yeast. Much research has centered on this particular histone modification and histone-modifying enzyme. This Critical Review summarizes much of our current understanding of nucleosome assembly and highlights many important insights learned from studying Rtt109 HATs in fungi. We highlight some seminal features in nucleosome assembly conserved in mammalian systems and describe some of the lingering questions in the field. Further studying fungal and mammalian chromatin assembly may have important public health implications, including deeper understandings of human cancers and aging as well as the pursuit of novel anti-fungal therapies.

  7. Histone-histone interactions in a lower eukaryote, Tetrahymena thermophila.

    PubMed

    Glover, C V; Gorovsky, M A

    1978-12-26

    The six pairwise interactions of Tetrahymena macronuclear histones H2A (formerly HX), H2B, H3, and H4 have been studied using the techniques of circular dichroism and continuous variation. Parallel experiments have been performed with calf thymus histones as controls, and the 12 possible interspecies pairs have also been examined. The behavior of three of the ciliate histones, H2A, H2B, and H3 is virtually identical with that of their vertebrate counterparts. Tetrahymena H4 exhibits a pattern of interactions identical with that of calf thymus H4, but displays significant quantitative differences in the strength of those interactions. Although we have not entirely eliminated the possibility that these differences may be artifactual, we suggest that they reflect the unique primary structure of Tetrahymena H4. These results extend the pattern of histone-histone interactions defined for plants, animals, and fungi to include the protists as well and imply the strong evolutionary conservatism of this pattern in spite of the electrophoretic and compositional differences which exist between homologous histones of different organisms. In addition, they demonstrate conclusively that histone HX should be redefined as Tetrahymena H2A.

  8. DNA methylation pathways and their crosstalk with histone methylation

    PubMed Central

    Du, Jiamu; Johnson, Lianna M.; Jacobsen, Steven E.; Patel, Dinshaw J.

    2015-01-01

    Methylation of DNA and of histone 3 at Lys 9 (H3K9) are highly correlated with gene silencing in eukaryotes from fungi to humans. Both of these epigenetic marks need to be established at specific regions of the genome and then maintained at these sites through cell division. Protein structural domains that specifically recognize methylated DNA and methylated histones are key for targeting enzymes that catalyse these marks to appropriate genome sites. Genetic, genomic, structural and biochemical data reveal connections between these two epigenetic marks, and these domains mediate much of the crosstalk. PMID:26296162

  9. Targeted chromatin binding and histone acetylation in vivo by thyroid hormone receptor during amphibian development.

    PubMed

    Sachs, L M; Shi, Y B

    2000-11-21

    Amphibian metamorphosis is marked by dramatic, thyroid hormone (TH)-induced changes involving gene regulation by TH receptor (TR). It has been postulated that TR-mediated gene regulation involves chromatin remodeling. In the absence of ligand, TR can repress gene expression by recruiting a histone deacetylase complex, whereas liganded TR recruits a histone acetylase complex for gene activation. Earlier studies have led us to propose a dual function model for TR during development. In premetamorphic tadpoles, unliganded TR represses transcription involving histone deacetylation. During metamorphosis, endogenous TH allows TR to activate gene expression through histone acetylation. Here using chromatin immunoprecipitation assay, we directly demonstrate TR binding to TH response genes constitutively in vivo in premetamorphic tadpoles. We further show that TH treatment leads to histone deacetylase release from TH response gene promoters. Interestingly, in whole animals, changes in histone acetylation show little correlation with the expression of TH response genes. On the other hand, in the intestine and tail, where TH response genes are known to be up-regulated more dramatically by TH than in most other organs, we demonstrate that TH treatment induces gene activation and histone H4 acetylation. These data argue for a role of histone acetylation in transcriptional regulation by TRs during amphibian development in some tissues, whereas in others changes in histone acetylation levels may play no or only a minor role, supporting the existence of important alternative mechanisms in gene regulation by TR.

  10. Histone deacetylase 3 indirectly modulates tubulin acetylation.

    PubMed

    Bacon, Travis; Seiler, Caroline; Wolny, Marcin; Hughes, Ruth; Watson, Peter; Schwabe, John; Grigg, Ronald; Peckham, Michelle

    2015-12-15

    Histone deacetylase 3 (HDAC3), a member of the Class I subfamily of HDACs, is found in both the nucleus and the cytoplasm. Its roles in the nucleus have been well characterized, but its cytoplasmic roles are still not elucidated fully. We found that blocking HDAC3 activity using MI192, a compound specific for HDAC3, modulated tubulin acetylation in the human prostate cancer cell line PC3. A brief 1 h treatment of PC3 cells with MI192 significantly increased levels of tubulin acetylation and ablated the dynamic behaviour of microtubules in live cells. siRNA-mediated knockdown (KD) of HDAC3 in PC3 cells, significantly increased levels of tubulin acetylation, and overexpression reduced it. However, the active HDAC3-silencing mediator of retinoic and thyroid receptors (SMRT)-deacetylase-activating domain (DAD) complex did not directly deacetylate tubulin in vitro. These data suggest that HDAC3 indirectly modulates tubulin acetylation.

  11. Ordered histone modifications are associated with transcriptional poising and activation of the phaseolin promoter.

    PubMed

    Ng, Danny W-K; Chandrasekharan, Mahesh B; Hall, Timothy C

    2006-01-01

    The phaseolin (phas) promoter drives copious production of transcripts encoding the protein phaseolin during seed embryogenesis but is silent in vegetative tissues, in which a nucleosome is positioned over its three-phased TATA boxes. Transition from the inactive state in transgenic Arabidopsis thaliana leaves was accomplished by ectopic expression of the transcription factor Phaseolus vulgaris ABI3-like factor (ALF) and application of abscisic acid (ABA). Placement of hemagglutinin-tagged ALF expression under the control of an estradiol-inducible promoter permitted chromatin immunoprecipitation analysis of chronological changes in histone modifications, notably increased acetylation of H3-K9 and H4-K12, as phas chromatin was remodeled (potentiated). A different array of changes, including acetylation of H3-K14 and methylation of H3-K4, was found to be associated with ABA-mediated activation. Thus, temporal separation of phas potentiation from activation revealed that histone H3 and H4 Lys residues are not globally hyperacetylated during phas expression. Whereas decreases in histone H3 and H4 levels were detected during ALF-mediated remodeling, slight increases occurred after ABA-mediated activation, suggesting the restoration of histone-phas interactions or the replacement of histones in the phas chromatin. The observed histone modifications provide insight into factors involved in the euchromatinization and activation of a plant gene and expand the evidence for histone code conservation among eukaryotes. PMID:16326929

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

    PubMed Central

    Chang, Le; Takada, Shoji

    2016-01-01

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

  13. Histone Deacetylase Inhibitors Stimulate Histone H3 Lysine 4 Methylation in Part Via Transcriptional Repression of Histone H3 Lysine 4 DemethylasesS⃞

    PubMed Central

    Huang, Po-Hsien; Chen, Chun-Han; Chou, Chih-Chien; Sargeant, Aaron M.; Kulp, Samuel K.; Teng, Che-Ming; Byrd, John C.

    2011-01-01

    This study investigates the mechanism by which histone deacetylase (HDAC) inhibitors up-regulate histone H3 lysine 4 (H3K4) methylation. Exposure of LNCaP prostate cancer cells and the prostate tissue of transgenic adenocarcinoma of the mouse prostate mice to the pan- and class I HDAC inhibitors (S)-(+)-N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)-benzamide (AR42), N-(2-aminophenyl)-4-[N-(pyridine-3-yl-methoxycarbonyl)-aminomethyl]-benzamide (MS-275), and vorinostat led to differential increases in H3K4 methylation. Chromatin immunoprecipitation shows that this accumulation of methylated H3K4 occurred in conjunction with decreases in the amount of the H3K4 demethylase RBP2 at the promoter of genes associated with tumor suppression and differentiation, including KLF4 and E-cadherin. This finding, together with the HDAC inhibitor-induced up-regulation of KLF4 and E-cadherin, suggests that HDAC inhibitors could activate the expression of these genes through changes in histone methylation status. Evidence indicates that this up-regulation of H3K4 methylation was attributable to the suppressive effect of these HDAC inhibitors on the expression of RBP2 and other JARID1 family histone demethylases, including PLU-1, SMCX, and LSD1, via the down-regulation of Sp1 expression. Moreover, shRNA-mediated silencing of the class I HDAC isozymes 1, 2, 3, and 8, but not that of the class II isozyme HDAC6, mimicked the drug effects on H3K4 methylation and H3K4 demethylases, which could be reversed by ectopic Sp1 expression. These data suggest a cross-talk mechanism between HDACs and H3K4 demethylases via Sp1-mediated transcriptional regulation, which underlies the complexity of the functional role of HDACs in the regulation of histone modifications. PMID:20959362

  14. Histone deacetylase inhibitors stimulate histone H3 lysine 4 methylation in part via transcriptional repression of histone H3 lysine 4 demethylases.

    PubMed

    Huang, Po-Hsien; Chen, Chun-Han; Chou, Chih-Chien; Sargeant, Aaron M; Kulp, Samuel K; Teng, Che-Ming; Byrd, John C; Chen, Ching-Shih

    2011-01-01

    This study investigates the mechanism by which histone deacetylase (HDAC) inhibitors up-regulate histone H3 lysine 4 (H3K4) methylation. Exposure of LNCaP prostate cancer cells and the prostate tissue of transgenic adenocarcinoma of the mouse prostate mice to the pan- and class I HDAC inhibitors (S)-(+)-N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)-benzamide (AR42), N-(2-aminophenyl)-4-[N-(pyridine-3-yl-methoxycarbonyl)-aminomethyl]-benzamide (MS-275), and vorinostat led to differential increases in H3K4 methylation. Chromatin immunoprecipitation shows that this accumulation of methylated H3K4 occurred in conjunction with decreases in the amount of the H3K4 demethylase RBP2 at the promoter of genes associated with tumor suppression and differentiation, including KLF4 and E-cadherin. This finding, together with the HDAC inhibitor-induced up-regulation of KLF4 and E-cadherin, suggests that HDAC inhibitors could activate the expression of these genes through changes in histone methylation status. Evidence indicates that this up-regulation of H3K4 methylation was attributable to the suppressive effect of these HDAC inhibitors on the expression of RBP2 and other JARID1 family histone demethylases, including PLU-1, SMCX, and LSD1, via the down-regulation of Sp1 expression. Moreover, shRNA-mediated silencing of the class I HDAC isozymes 1, 2, 3, and 8, but not that of the class II isozyme HDAC6, mimicked the drug effects on H3K4 methylation and H3K4 demethylases, which could be reversed by ectopic Sp1 expression. These data suggest a cross-talk mechanism between HDACs and H3K4 demethylases via Sp1-mediated transcriptional regulation, which underlies the complexity of the functional role of HDACs in the regulation of histone modifications. PMID:20959362

  15. Exercise-induced histone modifications in human skeletal muscle

    PubMed Central

    McGee, Sean L; Fairlie, Erin; Garnham, Andrew P; Hargreaves, Mark

    2009-01-01

    Skeletal muscle adaptations to exercise confer many of the health benefits of physical activity and occur partly through alterations in skeletal muscle gene expression. The exact mechanisms mediating altered skeletal muscle gene expression in response to exercise are unknown. However, in recent years, chromatin remodelling through epigenetic histone modifications has emerged as a key regulatory mechanism controlling gene expression in general. The purpose of this study was to examine the effect of exercise on global histone modifications that mediate chromatin remodelling and transcriptional activation in human skeletal muscle in response to exercise. In addition, we sought to examine the signalling mechanisms regulating these processes. Following 60 min of cycling, global histone 3 acetylation at lysine 9 and 14, a modification associated with transcriptional initiation, was unchanged from basal levels, but was increased at lysine 36, a site associated with transcriptional elongation. We examined the regulation of the class IIa histone deacetylases (HDACs), which are enzymes that suppress histone acetylation and have been implicated in the adaptations to exercise. While we found no evidence of proteasomal degradation of the class IIa HDACs, we found that HDAC4 and 5 were exported from the nucleus during exercise, thereby removing their transcriptional repressive function. We also observed activation of the AMP-activated protein kinase (AMPK) and the calcium–calmodulin-dependent protein kinase II (CaMKII) in response to exercise, which are two kinases that induce phosphorylation-dependent class IIa HDAC nuclear export. These data delineate a signalling pathway that might mediate skeletal muscle adaptations in response to exercise. PMID:19884317

  16. A novel, enigmatic histone modification: biotinylation of histones by holocarboxylase synthetase.

    PubMed

    Hassan, Yousef I; Zempleni, Janos

    2008-12-01

    Holocarboxylase synthetase catalyzes the covalent binding of biotin to histones in humans and other eukaryotes. Eleven biotinylation sites have been identified in histones H2A, H3, and H4. K12-biotinylated histone H4 is enriched in heterochromatin, repeat regions, and plays a role in gene repression. About 30% of the histone H4 molecules are biotinylated at K12 in histone H4 in human fibroblast telomeres. The abundance of biotinylated histones at distinct genomic loci depends on biotin availability. Decreased histone biotinylation decreases life span and stress resistance in Drosophila. Low enrichment of biotinylated histones at transposable elements impairs repression of these elements.

  17. Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation.

    PubMed

    Strickfaden, Hilmar; McDonald, Darin; Kruhlak, Michael J; Haince, Jean-Francois; Th'ng, John P H; Rouleau, Michele; Ishibashi, Toytaka; Corry, Gareth N; Ausio, Juan; Underhill, D Alan; Poirier, Guy G; Hendzel, Michael J

    2016-01-22

    Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.

  18. Characteristics and expression patterns of histone-modifying enzyme systems in the migratory locust.

    PubMed

    Guo, Siyuan; Jiang, Feng; Yang, Pengcheng; Liu, Qing; Wang, Xianhui; Kang, Le

    2016-09-01

    The density-dependent phase polyphenism in locusts offers an excellent model to investigate the epigenetic regulatory mechanisms underlying phenotypic plasticity. In this study, we identified histone-modifying enzymes mediating histone post-translational modifications, which serve as a major regulatory mechanism of epigenetic processes, on the basis of the whole genome sequence of the migratory locust, Locusta migratoria. We confirmed the existence of various functional histone modifications in the locusts. Compared with other sequenced insect genomes, the locust genome contains a richer repertoire of histone-modifying enzymes. Several locust histone-modifying enzymes display vertebrate-like characteristics, such as the presence of a Sirt3-like gene and multiple alternative splicing of GCN5 gene. Most histone-modifying enzymes are highly expressed in the eggs or in the testis tissues of male adults. Several histone deacetylases and H3K4-specific methyltransferases exhibit differential expression patterns in brain tissues between solitarious and gregarious locusts. These results reveal the main characteristics of histone-modifying enzymes and provide important cues for understanding the epigenetic mechanisms underlying phase polyphenism in locusts. PMID:27343382

  19. Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation.

    PubMed

    Strickfaden, Hilmar; McDonald, Darin; Kruhlak, Michael J; Haince, Jean-Francois; Th'ng, John P H; Rouleau, Michele; Ishibashi, Toytaka; Corry, Gareth N; Ausio, Juan; Underhill, D Alan; Poirier, Guy G; Hendzel, Michael J

    2016-01-22

    Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement. PMID:26559976

  20. Mechanical Stability and Fibrinolytic Resistance of Clots Containing Fibrin, DNA, and Histones*

    PubMed Central

    Longstaff, Colin; Varjú, Imre; Sótonyi, Péter; Szabó, László; Krumrey, Michael; Hoell, Armin; Bóta, Attila; Varga, Zoltán; Komorowicz, Erzsébet; Kolev, Krasimir

    2013-01-01

    Neutrophil extracellular traps are networks of DNA and associated proteins produced by nucleosome release from activated neutrophils in response to infection stimuli and have recently been identified as key mediators between innate immunity, inflammation, and hemostasis. The interaction of DNA and histones with a number of hemostatic factors has been shown to promote clotting and is associated with increased thrombosis, but little is known about the effects of DNA and histones on the regulation of fibrin stability and fibrinolysis. Here we demonstrate that the addition of histone-DNA complexes to fibrin results in thicker fibers (increase in median diameter from 84 to 123 nm according to scanning electron microscopy data) accompanied by improved stability and rigidity (the critical shear stress causing loss of fibrin viscosity increases from 150 to 376 Pa whereas the storage modulus of the gel increases from 62 to 82 pascals according to oscillation rheometric data). The effects of DNA and histones alone are subtle and suggest that histones affect clot structure whereas DNA changes the way clots are lysed. The combination of histones + DNA significantly prolongs clot lysis. Isothermal titration and confocal microscopy studies suggest that histones and DNA bind large fibrin degradation products with 191 and 136 nm dissociation constants, respectively, interactions that inhibit clot lysis. Heparin, which is known to interfere with the formation of neutrophil extracellular traps, appears to prolong lysis time at a concentration favoring ternary histone-DNA-heparin complex formation, and DNase effectively promotes clot lysis in combination with tissue plasminogen activator. PMID:23293023

  1. A phosphorylation switch regulates the transcriptional activation of cell cycle regulator p21 by histone deacetylase inhibitors.

    PubMed

    Simboeck, Elisabeth; Sawicka, Anna; Zupkovitz, Gordin; Senese, Silvia; Winter, Stefan; Dequiedt, Franck; Ogris, Egon; Di Croce, Luciano; Chiocca, Susanna; Seiser, Christian

    2010-12-24

    Histone deacetylase inhibitors induce cell cycle arrest and apoptosis in tumor cells and are, therefore, promising anti-cancer drugs. The cyclin-dependent kinase inhibitor p21 is activated in histone deacetylase (HDAC) inhibitor-treated tumor cells, and its growth-inhibitory function contributes to the anti-tumorigenic effect of HDAC inhibitors. We show here that induction of p21 by trichostatin A involves MAP kinase signaling. Activation of the MAP kinase signaling pathway by growth factors or stress signals results in histone H3 serine 10 phosphorylation at the p21 promoter and is crucial for acetylation of the neighboring lysine 14 and recruitment of activated RNA polymerase II in response to trichostatin A treatment. In non-induced cells, the protein phosphatase PP2A is associated with the p21 gene and counteracts its activation. Induction of p21 is linked to simultaneous acetylation and phosphorylation of histone H3. The dual modification mark H3S10phK14ac at the activated p21 promoter is recognized by the phospho-binding protein 14-3-3ζ, which protects the phosphoacetylation mark from being processed by PP2A. Taken together we have revealed a cross-talk of reversible phosphorylation and acetylation signals that controls the activation of p21 by HDAC inhibitors and identify the phosphatase PP2A as chromatin-associated transcriptional repressor in mammalian cells.

  2. Solution structure of histone chaperone ANP32B: interaction with core histones H3-H4 through its acidic concave domain.

    PubMed

    Tochio, Naoya; Umehara, Takashi; Munemasa, Yoshiko; Suzuki, Toru; Sato, Shin; Tsuda, Kengo; Koshiba, Seizo; Kigawa, Takanori; Nagai, Ryozo; Yokoyama, Shigeyuki

    2010-08-01

    Eukaryotic gene expression is regulated by histone deposition onto and eviction from nucleosomes, which are mediated by several chromatin-modulating factors. Among them, histone chaperones are key factors that facilitate nucleosome assembly. Acidic nuclear phosphoprotein 32B (ANP32B) belongs to the ANP32 family, which shares N-terminal leucine-rich repeats (LRRs) and a C-terminal variable anionic region. The C-terminal region functions as an inhibitor of histone acetylation, but the functional roles of the LRR domain in chromatin regulation have remained elusive. Here, we report that the LRR domain of ANP32B possesses histone chaperone activity and forms a curved structure with a parallel beta-sheet on the concave side and mostly helical elements on the convex side. Our analyses revealed that the interaction of ANP32B with the core histones H3-H4 occurs on its concave side, and both the acidic and hydrophobic residues that compose the concave surface are critical for histone binding. These results provide a structural framework for understanding the functional mechanisms of acidic histone chaperones.

  3. DAXX envelops a histone H3.3-H4 dimer for H3.3-specific recognition

    SciTech Connect

    Elsässer, Simon J; Huang, Hongda; Lewis, Peter W; Chin, Jason W; Allis, C David; Patel, Dinshaw J

    2013-01-24

    Histone chaperones represent a structurally and functionally diverse family of histone-binding proteins that prevent promiscuous interactions of histones before their assembly into chromatin. DAXX is a metazoan histone chaperone specific to the evolutionarily conserved histone variant H3.3. Here we report the crystal structures of the DAXX histone-binding domain with a histone H3.3–H4 dimer, including mutants within DAXX and H3.3, together with in vitro and in vivo functional studies that elucidate the principles underlying H3.3 recognition specificity. Occupying 40% of the histone surface-accessible area, DAXX wraps around the H3.3–H4 dimer, with complex formation accompanied by structural transitions in the H3.3–H4 histone fold. DAXX uses an extended α-helical conformation to compete with major inter-histone, DNA and ASF1 interaction sites. Our structural studies identify recognition elements that read out H3.3-specific residues, and functional studies address the contributions of Gly90 in H3.3 and Glu225 in DAXX to chaperone-mediated H3.3 variant recognition specificity.

  4. Impact of Histone H1 on the Progression of Allergic Rhinitis and Its Suppression by Neutralizing Antibody in Mice

    PubMed Central

    Nakano, Toshiaki; Kamei, Rikiya; Fujimura, Takashi; Takaoka, Yuki; Hori, Ayane; Lai, Chia-Yun; Chiang, Kuei-Chen; Shimada, Yayoi; Ohmori, Naoya; Goto, Takeshi; Ono, Kazuhisa; Chen, Chao-Long; Goto, Shigeru; Kawamoto, Seiji

    2016-01-01

    Nuclear antigens are known to trigger off innate and adaptive immune responses. Recent studies have found that the complex of nucleic acids and core histones that are derived from damaged cells may regulate allergic responses. However, no fundamental study has been performed concerning the role of linker histone H1 in mast cell-mediated type I hyperreactivity. In this study, we explored the impact of histone H1 on mast cell-mediated allergic responses both in vitro and in vivo. In the course of a bona-fide experimental allergen sensitization model upon co-injection with alum adjuvant, ovalbumin (OVA), but not PBS, induced elevated levels of circulating histone H1. Intranasal challenge with histone H1 to OVA/alum- (but not PBS/alum)-sensitized mice induced significantly severer symptoms of allergic rhinitis than those in mice sensitized and challenged with OVA. A monoclonal antibody against histone H1 not only suppressed mast cell degranulation, but also ameliorated OVA-induced nasal hyperreactivity and IgE-mediated passive cutaneous anaphylaxis. Our present data suggest that nuclear histone H1 represents an alarmin-like endogenous mediator acting on mast cells, and that its blockage has a therapeutic potential for mast cell-mediated type I hyperreactivity. PMID:27088594

  5. Impact of Histone H1 on the Progression of Allergic Rhinitis and Its Suppression by Neutralizing Antibody in Mice.

    PubMed

    Nakano, Toshiaki; Kamei, Rikiya; Fujimura, Takashi; Takaoka, Yuki; Hori, Ayane; Lai, Chia-Yun; Chiang, Kuei-Chen; Shimada, Yayoi; Ohmori, Naoya; Goto, Takeshi; Ono, Kazuhisa; Chen, Chao-Long; Goto, Shigeru; Kawamoto, Seiji

    2016-01-01

    Nuclear antigens are known to trigger off innate and adaptive immune responses. Recent studies have found that the complex of nucleic acids and core histones that are derived from damaged cells may regulate allergic responses. However, no fundamental study has been performed concerning the role of linker histone H1 in mast cell-mediated type I hyperreactivity. In this study, we explored the impact of histone H1 on mast cell-mediated allergic responses both in vitro and in vivo. In the course of a bona-fide experimental allergen sensitization model upon co-injection with alum adjuvant, ovalbumin (OVA), but not PBS, induced elevated levels of circulating histone H1. Intranasal challenge with histone H1 to OVA/alum- (but not PBS/alum)-sensitized mice induced significantly severer symptoms of allergic rhinitis than those in mice sensitized and challenged with OVA. A monoclonal antibody against histone H1 not only suppressed mast cell degranulation, but also ameliorated OVA-induced nasal hyperreactivity and IgE-mediated passive cutaneous anaphylaxis. Our present data suggest that nuclear histone H1 represents an alarmin-like endogenous mediator acting on mast cells, and that its blockage has a therapeutic potential for mast cell-mediated type I hyperreactivity. PMID:27088594

  6. Discovery and Mechanism of Natural Products as Modulators of Histone Acetylation

    PubMed Central

    Salvador, Lilibeth A.; Luesch, Hendrik

    2013-01-01

    Small molecules that modulate histone acetylation by targeting key enzymes mediating this posttranslational modification – histone acetyltransferases and histone deacetylases – are validated chemotherapeutic agents for the treatment of cancer. This area of research has seen a rapid increase in interest in the past decade, with the structurally diverse natural products-derived compounds at its forefront. These secondary metabolites from various biological sources target this epigenetic modification through distinct mechanisms of enzyme regulation by utilizing a diverse array of pharmacophores. We review the discovery of these compounds and discuss their modes of inhibition together with their downstream biological effects. PMID:22594471

  7. Histone variants: key players of chromatin.

    PubMed

    Biterge, Burcu; Schneider, Robert

    2014-06-01

    Histones are fundamental structural components of chromatin. Eukaryotic DNA is wound around an octamer of the core histones H2A, H2B, H3, and H4. Binding of linker histone H1 promotes higher order chromatin organization. In addition to their structural role, histones impact chromatin function and dynamics by, e.g., post-translational histone modifications or the presence of specific histone variants. Histone variants exhibit differential expression timings (DNA replication-independent) and mRNA characteristics compared to canonical histones. Replacement of canonical histones with histone variants can affect nucleosome stability and help to create functionally distinct chromatin domains. In line with this, several histone variants have been implicated in the regulation of cellular processes such as DNA repair and transcriptional activity. In this review, we focus on recent progress in the study of core histone variants H2A.X, H2A.Z, macroH2A, H3.3, and CENP-A, as well as linker histone H1 variants, their functions and their links to development and disease.

  8. The SANT domain of Ada2 is required for normal acetylation of histones by the yeast SAGA complex.

    PubMed

    Sterner, David E; Wang, Xun; Bloom, Melissa H; Simon, Gabriel M; Berger, Shelley L

    2002-03-01

    Transcription is regulated through chromatin remodeling and histone modification, mediated by large protein complexes. Histone and nucleosome interaction has been shown to be mediated by specific chromatin domains called bromodomains and chromodomains. Here we provide evidence for a similar function of two additional domains within the yeast SAGA complex, containing the histone acetyltransferase Gcn5. We have analyzed deletion and substitution mutations within Gcn5 and Ada2, an interacting protein within SAGA, and have identified substrate recognition functions within the SANT domain of Ada2 and regions of the histone acetyltransferase domain of Gcn5 that are distinct from catalytic function itself. These results suggest that histone and nucleosomal substrate recognition by SAGA involves multiple conserved domains and proteins, beyond those that have been previously identified. PMID:11777910

  9. Histone variants: emerging players in cancer biology

    PubMed Central

    Vardabasso, Chiara; Hasson, Dan; Ratnakumar, Kajan; Chung, Chi-Yeh; Duarte, Luis F.

    2014-01-01

    Histone variants are key players in shaping chromatin structure, and, thus, in regulating fundamental cellular processes such as chromosome segregation and gene expression. Emerging evidence points towards a role for histone variants in contributing to tumor progression, and, recently, the first cancer-associated mutation in a histone variant-encoding gene was reported. In addition, genetic alterations of the histone chaperones that specifically regulate chromatin incorporation of histone variants are rapidly being uncovered in numerous cancers. Collectively, these findings implicate histone variants as potential drivers of cancer initiation and/or progression, and, therefore, targeting histone deposition or the chromatin remodeling machinery may be of therapeutic value. Here, we review the mammalian histone variants of the H2A and H3 families in their respective cellular functions, and their involvement in tumor biology. PMID:23652611

  10. Quantitative Histone Mass Spectrometry Identifies Elevated Histone H3 Lysine 27 (Lys27) Trimethylation in Melanoma.

    PubMed

    Sengupta, Deepanwita; Byrum, Stephanie D; Avaritt, Nathan L; Davis, Lauren; Shields, Bradley; Mahmoud, Fade; Reynolds, Matthew; Orr, Lisa M; Mackintosh, Samuel G; Shalin, Sara C; Tackett, Alan J

    2016-03-01

    Normal cell growth is characterized by a regulated epigenetic program that drives cellular activities such as gene transcription, DNA replication, and DNA damage repair. Perturbation of this epigenetic program can lead to events such as mis-regulation of gene transcription and diseases such as cancer. To begin to understand the epigenetic program correlated to the development of melanoma, we performed a novel quantitative mass spectrometric analysis of histone post-translational modifications mis-regulated in melanoma cell culture as well as patient tumors. Aggressive melanoma cell lines as well as metastatic melanoma were found to have elevated histone H3 Lys(27) trimethylation (H3K27me3) accompanied by overexpressed methyltransferase EZH2 that adds the specific modification. The altered epigenetic program that led to elevated H3K27me3 in melanoma cell culture was found to directly silence transcription of the tumor suppressor genes RUNX3 and E-cadherin. The EZH2-mediated silencing of RUNX3 and E-cadherin transcription was also validated in advanced stage human melanoma tissues. This is the first study focusing on the detailed epigenetic mechanisms leading to EZH2-mediated silencing of RUNX3 and E-cadherin tumor suppressors in melanoma. This study underscores the utility of using high resolution mass spectrometry to identify mis-regulated epigenetic programs in diseases such as cancer, which could ultimately lead to the identification of biological markers for diagnostic and prognostic applications. PMID:26621846

  11. [WEE1, histone and tumor].

    PubMed

    Liu, Bin

    2015-07-01

    WEE1 is an important factor for histone transcription, chromosome condensation and regulation of cell cycle progression. WEE1 kinase can phosphorylate Cdc2 and down-regulate Cdc2 kinase activity. It can regulate G2 to M phase transition and cell mitosis. It plays a key role in chromosome condensation delay and histone synthesis, suggesting the important functions of WEE1 in the occurrence and development in cancer. At present, a multiple WEE1 inhibitors have been discovered. A great progress has been made in combination of WEE1 inhibitors with DNA damage treatment (chemotherapy or radiotherapy), which makes WEE1 an important target in cancer treatment. PMID:26267696

  12. Diversity and Divergence of Dinoflagellate Histone Proteins

    PubMed Central

    Marinov, Georgi K.; Lynch, Michael

    2015-01-01

    Histone proteins and the nucleosomal organization of chromatin are near-universal eukaroytic features, with the exception of dinoflagellates. Previous studies have suggested that histones do not play a major role in the packaging of dinoflagellate genomes, although several genomic and transcriptomic surveys have detected a full set of core histone genes. Here, transcriptomic and genomic sequence data from multiple dinoflagellate lineages are analyzed, and the diversity of histone proteins and their variants characterized, with particular focus on their potential post-translational modifications and the conservation of the histone code. In addition, the set of putative epigenetic mark readers and writers, chromatin remodelers and histone chaperones are examined. Dinoflagellates clearly express the most derived set of histones among all autonomous eukaryote nuclei, consistent with a combination of relaxation of sequence constraints imposed by the histone code and the presence of numerous specialized histone variants. The histone code itself appears to have diverged significantly in some of its components, yet others are conserved, implying conservation of the associated biochemical processes. Specifically, and with major implications for the function of histones in dinoflagellates, the results presented here strongly suggest that transcription through nucleosomal arrays happens in dinoflagellates. Finally, the plausible roles of histones in dinoflagellate nuclei are discussed. PMID:26646152

  13. Diversity and Divergence of Dinoflagellate Histone Proteins.

    PubMed

    Marinov, Georgi K; Lynch, Michael

    2015-12-08

    Histone proteins and the nucleosomal organization of chromatin are near-universal eukaroytic features, with the exception of dinoflagellates. Previous studies have suggested that histones do not play a major role in the packaging of dinoflagellate genomes, although several genomic and transcriptomic surveys have detected a full set of core histone genes. Here, transcriptomic and genomic sequence data from multiple dinoflagellate lineages are analyzed, and the diversity of histone proteins and their variants characterized, with particular focus on their potential post-translational modifications and the conservation of the histone code. In addition, the set of putative epigenetic mark readers and writers, chromatin remodelers and histone chaperones are examined. Dinoflagellates clearly express the most derived set of histones among all autonomous eukaryote nuclei, consistent with a combination of relaxation of sequence constraints imposed by the histone code and the presence of numerous specialized histone variants. The histone code itself appears to have diverged significantly in some of its components, yet others are conserved, implying conservation of the associated biochemical processes. Specifically, and with major implications for the function of histones in dinoflagellates, the results presented here strongly suggest that transcription through nucleosomal arrays happens in dinoflagellates. Finally, the plausible roles of histones in dinoflagellate nuclei are discussed.

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

    SciTech Connect

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

    2007-02-28

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

  15. Undetectable histone O-GlcNAcylation in mammalian cells.

    PubMed

    Gagnon, Jessica; Daou, Salima; Zamorano, Natalia; Iannantuono, Nicholas V G; Hammond-Martel, Ian; Mashtalir, Nazar; Bonneil, Eric; Wurtele, Hugo; Thibault, Pierre; Affar, El Bachir

    2015-01-01

    O-GlcNAcylation is a posttranslational modification catalyzed by the O-Linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and reversed by O-GlcNAcase (OGA). Numerous transcriptional regulators, including chromatin modifying enzymes, transcription factors, and co-factors, are targeted by O-GlcNAcylation, indicating that this modification is central for chromatin-associated processes. Recently, OGT-mediated O-GlcNAcylation was reported to be a novel histone modification, suggesting a potential role in directly coordinating chromatin structure and function. In contrast, using multiple biochemical approaches, we report here that histone O-GlcNAcylation is undetectable in mammalian cells. Conversely, O-GlcNAcylation of the transcription regulators Host Cell Factor-1 (HCF-1) and Ten-Eleven Translocation protein 2 (TET2) could be readily observed. Our study raises questions on the occurrence and abundance of O-GlcNAcylation as a histone modification in mammalian cells and reveals technical complications regarding the detection of genuine protein O-GlcNAcylation. Therefore, the identification of the specific contexts in which histone O-GlcNAcylation might occur is still to be established. PMID:26075789

  16. Beyond Histone and Deacetylase: An Overview of Cytoplasmic Histone Deacetylases and Their Nonhistone Substrates

    PubMed Central

    Yao, Ya-Li; Yang, Wen-Ming

    2011-01-01

    Acetylation of lysines is a prominent form of modification in mammalian proteins. Deacetylation of proteins is catalyzed by histone deacetylases, traditionally named after their role in histone deacetylation, transcriptional modulation, and epigenetic regulation. Despite the link between histone deacetylases and chromatin structure, some of the histone deacetylases reside in various compartments in the cytoplasm. Here, we review how these cytoplasmic histone deacetylases are regulated, the identification of nonhistone substrates, and the functional implications of their nondeacetylase enzymatic activities. PMID:21234400

  17. Regulation of mouse steroidogenesis by WHISTLE and JMJD1C through histone methylation balance.

    PubMed

    Kim, Sung-Mi; Kim, Ji-Young; Choe, Nak-Won; Cho, Ick-Hyun; Kim, Ju-Ryoung; Kim, Dong-Wook; Seol, Jin-Ee; Lee, Song Eun; Kook, Hoon; Nam, Kwang-Il; Kook, Hyun; Bhak, Young-Yil; Seo, Sang-Beom

    2010-10-01

    The dynamic exchange of histone lysine methylation status by histone methyltransferases and demethylases has been previously implicated as an important factor in chromatin structure and transcriptional regulation. Using immunoaffinity TAP analysis, we purified the WHISTLE-interacting protein complexes, which include the heat shock protein HSP90α and the jumonji C-domain harboring the histone demethylase JMJD1C. In this study, we demonstrate that JMJD1C specifically demethylates histone H3K9 mono- and di-methylation, and mediates transcriptional activation. We also provide evidence suggesting that both WHISTLE and JMJD1C performs functions in the development of mouse testes by regulating the expression of the steroidogenesis marker, p450c17, via SF-1-mediated transcription. Furthermore, we demonstrate that WHISTLE is recruited to the p450c17 promoter via SF-1 and represses the transcription of prepubertal stages of steroidogenesis, after which JMJD1C replaces WHISTLE and activates the expression of target genes via SF-1-mediated interactions. Our results demonstrate that the histone methylation balance mediated by HMTase WHISTLE and demethylase JMJD1C perform a transcriptional regulatory function in mouse testis development.

  18. A Quantitative Characterization of Nucleoplasmin/Histone Complexes Reveals Chaperone Versatility

    PubMed Central

    Fernández-Rivero, Noelia; Franco, Aitor; Velázquez-Campoy, Adrian; Alonso, Edurne; Muga, Arturo; Prado, Adelina

    2016-01-01

    Nucleoplasmin (NP) is an abundant histone chaperone in vertebrate oocytes and embryos involved in storing and releasing maternal histones to establish and maintain the zygotic epigenome. NP has been considered a H2A–H2B histone chaperone, and recently it has been shown that it can also interact with H3-H4. However, its interaction with different types of histones has not been quantitatively studied so far. We show here that NP binds H2A–H2B, H3-H4 and linker histones with Kd values in the subnanomolar range, forming different complexes. Post-translational modifications of NP regulate exposure of the polyGlu tract at the disordered distal face of the protein and induce an increase in chaperone affinity for all histones. The relative affinity of NP for H2A–H2B and linker histones and the fact that they interact with the distal face of the chaperone could explain their competition for chaperone binding, a relevant process in NP-mediated sperm chromatin remodelling during fertilization. Our data show that NP binds H3-H4 tetramers in a nucleosomal conformation and dimers, transferring them to DNA to form disomes and tetrasomes. This finding might be relevant to elucidate the role of NP in chromatin disassembly and assembly during replication and transcription. PMID:27558753

  19. 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

  20. Enzymatic immuno-assembly of gold nanoparticles for visualized activity screening of histone-modifying enzymes.

    PubMed

    Zhen, Zhen; Tang, Li-Juan; Long, Haoxu; Jiang, Jian-Hui

    2012-04-17

    Activity screening of histone-modifying enzymes is of paramount importance for epigenetic research as well as clinical diagnostics and therapeutics. A novel biosensing strategy has been developed for sensitive and selective detection of histone-modifying enzymes as well as their inhibitors. This strategy relies on the antibody-mediated assembly of gold nanoparticles (AuNPs) decorated with substrate peptides that are subjected to enzymatic modifications by the histone-modifying enzymes. This design allows a visual and homogeneous assay of the enzyme activity using antibodies without any labels, which circumvents the requirements to prefunctionalize the antibody and affords improved assay simplicity and throughput. Additionally, the use of antibody-based recognition of modified peptides could offer improved specificity as compared with existing techniques based on the enzyme coupled assay. We have demonstrated this strategy using a histone methyltransferase acting on histone H3 (Lys 4) and a histone acetyltransferase acting on histone H3 (Lys 14). The results reveal that the absorption peak characteristic for AuNPs decreases dynamically with increasing activity of the enzymes with concomitant visualizable color attenuation, and subnanomolar detection limits are readily achieved for both enzymes. The developed strategy can thus offer a robust and convenient visualized platform for screening the enzyme activities and their inhibitors with high sensitivity and selectivity.

  1. Mass Spectrometric Analysis of Histone Proteoforms

    NASA Astrophysics Data System (ADS)

    Yuan, Zuo-Fei; Arnaudo, Anna M.; Garcia, Benjamin A.

    2014-06-01

    Histones play important roles in chromatin, in the forms of various posttranslational modifications (PTMs) and sequence variants, which are called histone proteoforms. Investigating modifications and variants is an ongoing challenge. Previous methods are based on antibodies, and because they usually detect only one modification at a time, they are not suitable for studying the various combinations of modifications on histones. Fortunately, mass spectrometry (MS) has emerged as a high-throughput technology for histone analysis and does not require prior knowledge about any modifications. From the data generated by mass spectrometers, both identification and quantification of modifications, as well as variants, can be obtained easily. On the basis of this information, the functions of histones in various cellular contexts can be revealed. Therefore, MS continues to play an important role in the study of histone proteoforms. In this review, we discuss the analysis strategies of MS, their applications on histones, and some key remaining challenges.

  2. Mass Spectrometric Analysis of Histone Proteoforms

    PubMed Central

    Yuan, Zuo-Fei; Arnaudo, Anna M.; Garcia, Benjamin A.

    2014-01-01

    Histones play important roles in chromatin, due to various post-translational modifications and sequence variants, which are called histone proteoforms. Investigating modifications and variants is an on-going challenge. Previous methods are based on antibodies and because they usually detect only one modification at a time, they are not suitable to study the various combinations of modifications on histones. Fortunately, mass spectrometry has emerged as a high-throughput technology for histone analysis and does not require prior knowledge about any modifications. From the data generated by mass spectrometers, both identification and quantification of modifications and variants can be easily obtained. Based on this information, the functions of histones in various cellular contexts can be revealed. Therefore, mass spectrometry continues to play an important role in the study of histone proteoforms. In this review, we will discuss the analysis strategies of mass spectrometry, their applications on histones, and some key remaining challenges. PMID:24896311

  3. Histone H3.3 and its proteolytically processed form drive a cellular senescence program

    PubMed Central

    Duarte, Luis F.; Young, Andrew R. J.; Wang, Zichen; Wu, Hsan-Au; Panda, Taniya; Kou, Yan; Kapoor, Avnish; Hasson, Dan; Mills, Nicholas R.; Ma’ayan, Avi; Narita, Masashi; Bernstein, Emily

    2014-01-01

    The process of cellular senescence generates a repressive chromatin environment, however, the role of histone variants and histone proteolytic cleavage in senescence remains unclear. Using models of oncogene-induced and replicative senescence, here we report novel histone H3 tail cleavage events mediated by the protease Cathepsin L. We find that cleaved forms of H3 are nucleosomal and the histone variant H3.3 is the preferred cleaved form of H3. Ectopic expression of H3.3 and its cleavage product (H3.3cs1), which lacks the first twenty-one amino acids of the H3 tail, is sufficient to induce senescence. Further, H3.3cs1 chromatin incorporation is mediated by the HUCA histone chaperone complex. Genome-wide transcriptional profiling revealed that H3.3cs1 facilitates transcriptional silencing of cell cycle regulators including RB/E2F target genes, likely via the permanent removal of H3K4me3. Collectively, our study identifies histone H3.3 and its proteolytically processed forms as key regulators of cellular senescence. PMID:25394905

  4. Histone H2A.Z subunit exchange controls consolidation of recent and remote memory.

    PubMed

    Zovkic, Iva B; Paulukaitis, Brynna S; Day, Jeremy J; Etikala, Deepa M; Sweatt, J David

    2014-11-27

    Memory formation is a multi-stage process that initially requires cellular consolidation in the hippocampus, after which memories are downloaded to the cortex for maintenance, in a process termed systems consolidation. Epigenetic mechanisms regulate both types of consolidation, but histone variant exchange, in which canonical histones are replaced with their variant counterparts, is an entire branch of epigenetics that has received limited attention in the brain and has never, to our knowledge, been studied in relation to cognitive function. Here we show that histone H2A.Z, a variant of histone H2A, is actively exchanged in response to fear conditioning in the hippocampus and the cortex, where it mediates gene expression and restrains the formation of recent and remote memory. Our data provide evidence for H2A.Z involvement in cognitive function and specifically implicate H2A.Z as a negative regulator of hippocampal consolidation and systems consolidation, probably through downstream effects on gene expression. Moreover, alterations in H2A.Z binding at later stages of systems consolidation suggest that this histone has the capacity to mediate stable molecular modifications required for memory retention. Overall, our data introduce histone variant exchange as a novel mechanism contributing to the molecular basis of cognitive function and implicate H2A.Z as a potential therapeutic target for memory disorders. PMID:25219850

  5. Nitric Oxide Regulates Gene Expression in Cancers by Controlling Histone Posttranslational Modifications.

    PubMed

    Vasudevan, Divya; Hickok, Jason R; Bovee, Rhea C; Pham, Vy; Mantell, Lin L; Bahroos, Neil; Kanabar, Pinal; Cao, Xing-Jun; Maienschein-Cline, Mark; Garcia, Benjamin A; Thomas, Douglas D

    2015-12-15

    Altered nitric oxide (•NO) metabolism underlies cancer pathology, but mechanisms explaining many •NO-associated phenotypes remain unclear. We have found that cellular exposure to •NO changes histone posttranslational modifications (PTM) by directly inhibiting the catalytic activity of JmjC-domain containing histone demethylases. Herein, we describe how •NO exposure links modulation of histone PTMs to gene expression changes that promote oncogenesis. Through high-resolution mass spectrometry, we generated an extensive map of •NO-mediated histone PTM changes at 15 critical lysine residues on the core histones H3 and H4. Concomitant microarray analysis demonstrated that exposure to physiologic •NO resulted in the differential expression of over 6,500 genes in breast cancer cells. Measurements of the association of H3K9me2 and H3K9ac across genomic loci revealed that differential distribution of these particular PTMs correlated with changes in the level of expression of numerous oncogenes, consistent with epigenetic code. Our results establish that •NO functions as an epigenetic regulator of gene expression mediated by changes in histone PTMs. PMID:26542213

  6. Chromatin remodelers Isw1 and Chd1 maintain chromatin structure during transcription by preventing histone exchange

    PubMed Central

    Smolle, Michaela; Venkatesh, Swaminathan; Gogol, Madelaine M.; Li, Hua; Zhang, Ying; Florens, Laurence; Washburn, Michael P.; Workman, Jerry L.

    2012-01-01

    Set2-mediated methylation of histone H3 Lys36 (H3K36) is a mark associated with the coding sequences of actively transcribed genes, yet plays a negative role during transcription elongation. It prevents trans-histone exchange over coding regions and signals for histone deacetylation in the wake of RNA polymerase II (RNAPII) passage. We have found that in Saccharomyces cerevisiae the Isw1b chromatin-remodeling complex is specifically recruited to open reading frames (ORFs) by H3K36 methylation through the PWWP domain of its Ioc4 subunit in vivo and in vitro. Isw1b acts in conjunction with Chd1 to regulate chromatin structure by preventing trans-histone exchange from taking place over coding regions and thus maintains chromatin integrity during transcription elongation by RNA polymerase II. PMID:22922743

  7. The evolutionary differentiation of two histone H2A.Z variants in chordates (H2A.Z-1 and H2A.Z-2) is mediated by a stepwise mutation process that affects three amino acid residues

    PubMed Central

    Eirín-López, José M; González-Romero, Rodrigo; Dryhurst, Deanna; Ishibashi, Toyotaka; Ausió, Juan

    2009-01-01

    Background The histone H2A family encompasses the greatest number of core histone variants of which the replacement variant H2A.Z is currently one of the most heavily studied. No clear mechanism for the functional variability that H2A.Z imparts to chromatin has yet been proposed. While most of the past studies have referred to H2A.Z generically as a single protein, in vertebrates it is a mixture of two protein forms H2A.Z-1 (previously H2A.Z) and H2A.Z-2 (previously H2A.F/Z or H2A.V) that differ by three amino acids. Results We have performed an extensive study on the long-term evolution of H2A.Z across metazoans with special emphasis on the possible selective mechanisms responsible for the differentiation between H2A.Z-1 and H2A.Z-2. Our results reveal a common origin of both forms early in chordate evolution. The evolutionary process responsible for the differentiation involves refined stepwise mutation change within the codons of the three differential residues. This eventually led to differences in the intensity of the selective constraints acting upon the different H2A.Z forms in vertebrates. Conclusion The results presented in this work definitively reveal that the existence of H2A.Z-1 and H2A.Z-2 is not a whim of random genetic drift. Our analyses demonstrate that H2A.Z-2 is not only subject to a strong purifying selection but it is significantly more evolutionarily constrained than H2A.Z-1. Whether or not the evolutionary drift between H2A.Z-1 and H2A.Z-2 has resulted in a functional diversification of these proteins awaits further research. Nevertheless, the present work suggests that in the process of their differently constrained evolutionary pathways, these two forms may have acquired new or complementary functions. PMID:19193230

  8. Histone deacetylase 3 indirectly modulates tubulin acetylation

    PubMed Central

    Bacon, Travis; Seiler, Caroline; Wolny, Marcin; Hughes, Ruth; Watson, Peter; Schwabe, John; Grigg, Ronald; Peckham, Michelle

    2015-01-01

    Histone deacetylase 3 (HDAC3), a member of the Class I subfamily of HDACs, is found in both the nucleus and the cytoplasm. Its roles in the nucleus have been well characterized, but its cytoplasmic roles are still not elucidated fully. We found that blocking HDAC3 activity using MI192, a compound specific for HDAC3, modulated tubulin acetylation in the human prostate cancer cell line PC3. A brief 1 h treatment of PC3 cells with MI192 significantly increased levels of tubulin acetylation and ablated the dynamic behaviour of microtubules in live cells. siRNA-mediated knockdown (KD) of HDAC3 in PC3 cells, significantly increased levels of tubulin acetylation, and overexpression reduced it. However, the active HDAC3–silencing mediator of retinoic and thyroid receptors (SMRT)–deacetylase-activating domain (DAD) complex did not directly deacetylate tubulin in vitro. These data suggest that HDAC3 indirectly modulates tubulin acetylation. PMID:26450925

  9. Writers and Readers of Histone Acetylation: Structure, Mechanism, and Inhibition

    PubMed Central

    Marmorstein, Ronen; Zhou, Ming-Ming

    2014-01-01

    Histone acetylation marks are written by histone acetyltransferases (HATs) and read by bromodomains (BrDs), and less commonly by other protein modules. These proteins regulate many transcription-mediated biological processes, and their aberrant activities are correlated with several human diseases. Consequently, small molecule HAT and BrD inhibitors with therapeutic potential have been developed. Structural and biochemical studies of HATs and BrDs have revealed that HATs fall into distinct subfamilies containing a structurally related core for cofactor binding, but divergent flanking regions for substrate-specific binding, catalysis, and autoregulation. BrDs adopt a conserved left-handed four-helix bundle to recognize acetyllysine; divergent loop residues contribute to substrate-specific acetyllysine recognition. PMID:24984779

  10. Immunodetection of histone epitopes correlates with early stages of apoptosis in activated human peripheral T lymphocytes.

    PubMed Central

    Zunino, S. J.; Singh, M. K.; Bass, J.; Picker, L. J.

    1996-01-01

    By coupling intracellular staining with terminal deoxynucleotidyl transferase (TdT)-mediated labeling of internucleosomal DNA strand breaks in a flow cytometric assay, we observed a strong correlation between apoptosis-associated DNA strand breaks and immunoreactivity with the monoclonal antibody (MAb) B-F6 in activated human peripheral blood T lymphocytes (PBTs). Although MAb B-F6 has been reported to be specific for the cytokine interleukin-6, Western blot analysis of activated PBT lysates revealed that the predominant protein band detected by this MAb was 17 kd (p17), distinct from the 23-kd core protein and 26- to 30-kd mature glycosylated forms of interleukin-6. Immunoaffinity isolation and amino-terminal amino acid sequence analysis of p17 revealed identity with the histone H2B, a finding confirmed by Western blot analysis of purified histones and by similar staining of activated PBTs with an unrelated anti-histone MAb. Neither histone staining nor DNA strand breakage was observed in freshly isolated PBTs; however, after T cell activation, histone immunoreactivity appeared to precede the appearance of DNA strand breaks, with both increasing to a maximal level by day 3 after activation. Two-parameter confocal immunofluorescence microscopy of histone and DNA staining confirmed a lack of histone immunoreactivity in viable cells and demonstrated co-localization of histone epitopes with abnormally clumped chromatin in apoptotic cells. These data indicate that alteration of histone epitope accessibility is a marker of early apoptosis and suggest that multiparameter flow cytometric analysis of intracellular epitopes may be a powerful tool in the elucidation of intracellular mechanisms of apoptosis. Images Figure 4 Figure 5 Figure 6 Figure 8 PMID:8702003

  11. Chemical and Biochemical Approaches in the Study of Histone Methylation and Demethylation

    PubMed Central

    Li, Keqin Kathy; Luo, Cheng; Wang, Dongxia; Jiang, Hualiang; Zheng, Y. George

    2014-01-01

    Histone methylation represents one of the most critical epigenetic events in DNA function regulation in eukaryotic organisms. Classic molecular biology and genetics tools provide significant knowledge about mechanisms and physiological roles of histone methyltransferases and demethylases in various cellular processes. In addition to this stream line, development and application of chemistry and chemistry-related techniques are increasingly involved in biological study, and provide information otherwise difficulty to obtain by standard molecular biology methods. Herein, we review recent achievements and progress in developing and applying chemical and biochemical approaches in the study of histone methylation, including chromatin immunoprecipitation (ChIP), chemical ligation, mass spectrometry (MS), biochemical assays, and inhibitor development. These technological advances allow histone methylation to be studied from genome-wide level to molecular and atomic levels. With ChIP technology, information can be obtained about precise mapping of histone methylation patterns at specific promoters, genes or other genomic regions. MS is particularly useful in detecting and analyzing methylation marks in histone and nonhistone protein substrates. Chemical approaches that permit site-specific incorporation of methyl groups into histone proteins greatly facilitate the investigation of the biological impacts of methylation at individual modification sites. Discovery and design of selective organic inhibitors of histone methyltransferases and demethylases provide chemical probes to interrogate methylation-mediated cellular pathways. Overall, these chemistry-related technological advances have greatly improved our understanding of the biological functions of histone methylation in normal physiology and diseased states, and also are of great potential to translate basic epigenetics research into diagnostic and therapeutic application in the clinic. PMID:22777714

  12. Chemical and biochemical approaches in the study of histone methylation and demethylation.

    PubMed

    Li, Keqin Kathy; Luo, Cheng; Wang, Dongxia; Jiang, Hualiang; Zheng, Y George

    2012-07-01

    Histone methylation represents one of the most critical epigenetic events in DNA function regulation in eukaryotic organisms. Classic molecular biology and genetics tools provide significant knowledge about mechanisms and physiological roles of histone methyltransferases and demethylases in various cellular processes. In addition to this stream line, development and application of chemistry and chemistry-related techniques are increasingly involved in biological study, and offer information otherwise difficult to obtain by standard molecular biology methods. Herein, we review recent achievements and progress in developing and applying chemical and biochemical approaches in the study of histone methylation, including chromatin immunoprecipitation, chemical ligation, mass spectrometry, biochemical methylation and demethylation assays, and inhibitor development. These technological advances allow histone methylation to be studied from genome-wide level to molecular and atomic levels. With ChIP technology, information can be obtained about precise mapping of histone methylation patterns at specific promoters, genes, or other genomic regions. MS is particularly useful in detecting and analyzing methylation marks in histone and nonhistone protein substrates. Chemical approaches that permit site-specific incorporation of methyl groups into histone proteins greatly facilitate the investigation of biological impacts of methylation at individual modification sites. Discovery and design of selective organic inhibitors of histone methyltransferases and demethylases provide chemical probes to interrogate methylation-mediated cellular pathways. Overall, these chemistry-related technological advances have greatly improved our understanding of the biological functions of histone methylation in normal physiology and diseased states, and also are of great potential to translate basic epigenetics research into diagnostic and therapeutic applications in the clinic.

  13. Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B).

    PubMed

    Haigney, Allison; Ricketts, M Daniel; Marmorstein, Ronen

    2015-12-18

    The HAT-B enzyme complex is responsible for acetylating newly synthesized histone H4 on lysines K5 and K12. HAT-B is a multisubunit complex composed of the histone acetyltransferase 1 (Hat1) catalytic subunit and the Hat2 (rbap46) histone chaperone. Hat1 is predominantly localized in the nucleus as a member of a trimeric NuB4 complex containing Hat1, Hat2, and a histone H3-H4 specific histone chaperone called Hif1 (NASP). In addition to Hif1 and Hat2, Hat1 interacts with Asf1 (anti-silencing function 1), a histone chaperone that has been reported to be involved in both replication-dependent and -independent chromatin assembly. To elucidate the molecular roles of the Hif1 and Asf1 histone chaperones in HAT-B histone binding and acetyltransferase activity, we have characterized the stoichiometry and binding mode of Hif1 and Asf1 to HAT-B and the effect of this binding on the enzymatic activity of HAT-B. We find that Hif1 and Asf1 bind through different modes and independently to HAT-B, whereby Hif1 binds directly to Hat2, and Asf1 is only capable of interactions with HAT-B through contacts with histones H3-H4. We also demonstrate that HAT-B is significantly more active against an intact H3-H4 heterodimer over a histone H4 peptide, independent of either Hif1 or Asf1 binding. Mutational studies further demonstrate that HAT-B binding to the histone tail regions is not sufficient for this enhanced activity. Based on these data, we propose a model for HAT-B/histone chaperone assembly and acetylation of H3-H4 complexes.

  14. Release and activity of histone in diseases.

    PubMed

    Chen, R; Kang, R; Fan, X-G; Tang, D

    2014-01-01

    Histones and their post-translational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage-associated molecular pattern molecules when they are released into the extracellular space. Administration of exogenous histones to animals leads to systemic inflammatory and toxic responses through activating Toll-like receptors and inflammasome pathways. Anti-histone treatment (e.g., neutralizing antibodies, activated protein C, recombinant thrombomodulin, and heparin) protect mice against lethal endotoxemia, sepsis, ischemia/reperfusion injury, trauma, pancreatitis, peritonitis, stroke, coagulation, and thrombosis. In addition, elevated serum histone and nucleosome levels have been implicated in multiple pathophysiological processes and progression of diseases including autoimmune diseases, inflammatory diseases, and cancer. Therefore, extracellular histones could serve as biomarkers and novel therapeutic targets in human diseases. PMID:25118930

  15. Proteasome inhibitor MG-132 enhances histone deacetylase inhibitor SAHA-induced cell death of chronic myeloid leukemia cells by an ROS-mediated mechanism and downregulation of the Bcr-Abl fusion protein

    PubMed Central

    ZHOU, WENJING; ZHU, WEIWEI; MA, LIYA; XIAO, FENG; QIAN, WENBIN

    2015-01-01

    Recently, there has been progress in the treatment of chronic myeloid leukemia (CML). However, novel therapeutic strategies are required in order to address the emerging problem of imatinib resistance. Histone deacetylase inhibitors (HDACi) and proteasome inhibitors are promising alternatives, and may be amenable to integration with current therapeutic approaches. However, the mechanisms underlying the interaction between these two agents remain unclear. The present study assessed the cytotoxic effect of the HDACi, suberoylanilide hydroxamic acid (SAHA), in combination with the proteasome inhibitor, MG-132, in imatinib-sensitive K562 and imatinib-resistant K562G cells, and investigated the mechanism underlying this effect. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and protein expression levels were determined by western blotting. Reactive oxygen species (ROS) generation levels were observed under a fluorescence microscope The results indicated that SAHA and MG-132 act in a synergistic manner to induce cell death in K562 and K562G cells. This effect was associated with Bcr-Abl downregulation and the production of ROS. Notably, the ROS scavenger, N-acetyl-L-cysteine, almost fully reversed the cell death and Bcr-Abl downregulation that was induced by the combination of SAHA and MG-132. By contrast, the pan-caspase inhibitor, z-VAD-fmk, only partially reversed the cell death induced by these two drugs in CML cells. These results indicated that increased intracellular ROS levels are important in the induction of cell death and the downregulation of Bcr-Abl. In conclusion, the present results suggested that combined SAHA and MG-132 may be a promising treatment for CML. PMID:26722260

  16. Origin of H1 linker histones.

    PubMed

    Kasinsky, H E; Lewis, J D; Dacks, J B; Ausió, J

    2001-01-01

    In which taxa did H1 linker histones appear in the course of evolution? Detailed comparative analysis of the histone H1 and histone H1-related sequences available to date suggests that the origin of histone H1 can be traced to bacteria. The data also reveal that the sequence corresponding to the 'winged helix' motif of the globular structural domain, a domain characteristic of all metazoan histone H1 molecules, is evolutionarily conserved and appears separately in several divergent lines of protists. Some protists, however, appear to have only a lysine-rich basic protein, which has compositional similarity to some of the histone H1-like proteins from eubacteria and to the carboxy-terminal domain of the H1 linker histones from animals and plants. No lysine-rich basic proteins have been described in archaebacteria. The data presented in this review provide the surprising conclusion that whereas DNA-condensing H1-related histones may have arisen early in evolution in eubacteria, the appearance of the sequence motif corresponding to the globular domain of metazoan H1s occurred much later in the protists, after and independently of the appearance of the chromosomal core histones in archaebacteria.

  17. Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation

    PubMed Central

    Zhang, Yi; Ng, Huck-Hui; Erdjument-Bromage, Hediye; Tempst, Paul; Bird, Adrian; Reinberg, Danny

    1999-01-01

    ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation. PMID:10444591

  18. Uncoupling histone turnover from transcription-associated histone H3 modifications.

    PubMed

    Ferrari, Paolo; Strubin, Michel

    2015-04-30

    Transcription in eukaryotes is associated with two major changes in chromatin organization. Firstly, nucleosomal histones are continuously replaced by new histones, an event that in yeast occurs predominantly at transcriptionally active promoters. Secondly, histones become modified post-translationally at specific lysine residues. Some modifications, including histone H3 trimethylation at lysine 4 (H3K4me3) and acetylation at lysines 9 (H3K9ac) and 14 (H3K14ac), are specifically enriched at active promoters where histones exchange, suggesting a possible causal relationship. Other modifications accumulate within transcribed regions and one of them, H3K36me3, is thought to prevent histone exchange. Here we explored the relationship between these four H3 modifications and histone turnover at a few selected genes. Using lysine-to-arginine mutants and a histone exchange assay, we found that none of these modifications plays a major role in either promoting or preventing histone turnover. Unexpectedly, mutation of H3K56, whose acetylation occurs prior to chromatin incorporation, had an effect only when introduced into the nucleosomal histone. Furthermore, we used various genetic approaches to show that histone turnover can be experimentally altered with no major consequence on the H3 modifications tested. Together, these results suggest that transcription-associated histone turnover and H3 modification are two correlating but largely independent events.

  19. Chromatin condensation in terminally differentiating mouse erythroblasts does not involve special architectural proteins but depends on histone deacetylation

    SciTech Connect

    Popova, Evgenya Y.; Krauss, Sharon Wald; Short, Sarah A.; Lee, Gloria; Villalobos, Jonathan; Etzell, Joan; Koury, Mark J.; Ney, Paul A.; Chasis, Joel Anne; Grigoryev, Sergei A.

    2008-08-21

    Terminal erythroid differentiation in vertebrates is characterized by progressive heterochromatin formation, chromatin condensation and, in mammals, culminates in nuclear extrusion. To date, although mechanisms regulating avian erythroid chromatin condensation have been identified, little is known regarding this process during mammalian erythropoiesis. To elucidate the molecular basis for mammalian erythroblast chromatin condensation, we used Friend virus-infected murine spleen erythroblasts that undergo terminal differentiation in vitro. Chromatin isolated from early and late stage erythroblasts had similar levels of linker and core histones, only a slight difference in nucleosome repeats, and no significant accumulation of known developmentally-regulated architectural chromatin proteins. However, histone H3(K9) dimethylation markedly increased while histone H4(K12) acetylation dramatically decreased and became segregated from the histone methylation as chromatin condensed. One histone deacetylase, HDAC5, was significantly upregulated during the terminal stages of Friend virus-infected erythroblast differentiation. Treatment with histone deacetylase inhibitor, trichostatin A, blocked both chromatin condensation and nuclear extrusion. Based on our data, we propose a model for a unique mechanism in which extensive histone deacetylation at pericentromeric heterochromatin mediates heterochromatin condensation in vertebrate erythroblasts that would otherwise be mediated by developmentally-regulated architectural proteins in nucleated blood cells.

  20. CHROMATIN CONDENSATION IN TERMINALLY DIFFERENTIATING MOUSE ERYTHROBLASTS DOES NOT INVOLVE SPECIAL ARCHITECTURAL PROTEINS BUT DEPENDS ON HISTONE DEACETYLATION

    PubMed Central

    Popova, Evgenya Y.; Krauss, Sharon Wald; Short, Sarah A.; Lee, Gloria; Villalobos, Jonathan; Etzell, Joan; Koury, Mark J.; Ney, Paul A.; Chasis, Joel Anne; Grigoryev, Sergei A.

    2009-01-01

    Terminal erythroid differentiation in vertebrates is characterized by progressive heterochromatin formation, chromatin condensation and, in mammals, culminates in nuclear extrusion. To date, although mechanisms regulating avian erythroid chromatin condensation have been identified, little is known regarding this process during mammalian erythropoiesis. To elucidate the molecular basis for mammalian erythroblast chromatin condensation, we used Friend virus-infected murine spleen erythroblasts that undergo terminal differentiation in vitro. Chromatin isolated from early and late stage erythroblasts had similar levels of linker and core histones, only a slight difference in nucleosome repeats, and no significant accumulation of known developmentally-regulated architectural chromatin proteins. However, histone H3(K9) dimethylation markedly increased while histone H4(K12) acetylation dramatically decreased and became segregated from the histone methylation as chromatin condensed. One histone deacetylase, HDAC5, was significantly upregulated during the terminal stages of Friend virus-infected erythroblast differentiation. Treatment with histone deacetylase inhibitor, trichostatin A, blocked both chromatin condensation and nuclear extrusion. Based on our data, we propose a model for a unique mechanism in which extensive histone deacetylation at pericentromeric heterochromatin mediates heterochromatin condensation in vertebrate erythroblasts that would otherwise be mediated by developmentally-regulated architectural proteins in nucleated blood cells. PMID:19172406

  1. The top-down, middle-down, and bottom-up mass spectrometry approaches for characterization of histone variants and their post-translational modifications.

    PubMed

    Moradian, Annie; Kalli, Anastasia; Sweredoski, Michael J; Hess, Sonja

    2014-03-01

    Epigenetic regulation of gene expression is, at least in part, mediated by histone modifications. PTMs of histones change chromatin structure and regulate gene transcription, DNA damage repair, and DNA replication. Thus, studying histone variants and their modifications not only elucidates their functional mechanisms in chromatin regulation, but also provides insights into phenotypes and diseases. A challenge in this field is to determine the best approach(es) to identify histone variants and their PTMs using a robust high-throughput analysis. The large number of histone variants and the enormous diversity that can be generated through combinatorial modifications, also known as histone code, makes identification of histone PTMs a laborious task. MS has been proven to be a powerful tool in this regard. Here, we focus on bottom-up, middle-down, and top-down MS approaches, including CID and electron-capture dissociation/electron-transfer dissociation based techniques for characterization of histones and their PTMs. In addition, we discuss advances in chromatographic separation that take advantage of the chemical properties of the specific histone modifications. This review is also unique in its discussion of current bioinformatic strategies for comprehensive histone code analysis.

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

    PubMed

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

    2016-06-01

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

  3. Role for histone deacetylase 1 in human tumor cell proliferation.

    PubMed

    Senese, Silvia; Zaragoza, Katrin; Minardi, Simone; Muradore, Ivan; Ronzoni, Simona; Passafaro, Alfonso; Bernard, Loris; Draetta, Giulio F; Alcalay, Myriam; Seiser, Christian; Chiocca, Susanna

    2007-07-01

    Posttranslational modifications of core histones are central to the regulation of gene expression. Histone deacetylases (HDACs) repress transcription by deacetylating histones, and class I HDACs have a crucial role in mouse, Xenopus laevis, zebra fish, and Caenorhabditis elegans development. The role of individual class I HDACs in tumor cell proliferation was investigated using RNA interference-mediated protein knockdown. We show here that in the absence of HDAC1 cells can arrest either at the G(1) phase of the cell cycle or at the G(2)/M transition, resulting in the loss of mitotic cells, cell growth inhibition, and an increase in the percentage of apoptotic cells. On the contrary, HDAC2 knockdown showed no effect on cell proliferation unless we concurrently knocked down HDAC1. Using gene expression profiling analysis, we found that inactivation of HDAC1 affected the transcription of specific target genes involved in proliferation and apoptosis. Furthermore, HDAC2 downregulation did not cause significant changes compared to control cells, while inactivation of HDAC1, HDAC1 plus HDAC2, or HDAC3 resulted in more distinct clusters. Loss of these HDACs might impair cell cycle progression by affecting not only the transcription of specific target genes but also other biological processes. Our data support the idea that a drug targeting specific HDACs could be highly beneficial in the treatment of cancer.

  4. The role dietary of bioactive compounds on the regulation of histone acetylases and deacetylases: a review.

    PubMed

    Vahid, F; Zand, H; Nosrat-Mirshekarlou, E; Najafi, R; Hekmatdoost, A

    2015-05-10

    Nutrigenomics is an area of epigenomics that explores and defines the rapidly evolving field of diet-genome interactions. Lifestyle and diet can significantly influence epigenetic mechanisms, which cause heritable changes in gene expression without changes in DNA sequence. Nutrient-dependent epigenetic variations can significantly affect genome stability, mRNA and protein expression, and metabolic changes, which in turn influence food absorption and the activity of its constituents. Dietary bioactive compounds can affect epigenetic alterations, which are accumulated over time and are shown to be involved in the pathogenesis of age-related diseases such as diabetes, cancer, and cardiovascular disease. Histone acetylation is an epigenetic modification mediated by histone acetyl transferases (HATs) and histone deacetylases (HDACs) critically involved in regulating affinity binding between the histones and DNA backbone. The HDAC-mediated increase in histone affinity to DNA causes DNA condensation, preventing transcription, whereas HAT-acetylated chromatin is transcriptionally active. HDAC and HAT activities are reported to be associated with signal transduction, cell growth and death, as well as with the pathogenesis of various diseases. The aim of this review was to evaluate the role of diet and dietary bioactive compounds on the regulation of HATs and HDACs in epigenetic diseases. Dietary bioactive compounds such as genistein, phenylisothiocyanate, curcumin, resveratrol, indole-3-carbinol, and epigallocatechin-3-gallate can regulate HDAC and HAT activities and acetylation of histones and non-histone chromatin proteins, and their health benefits are thought to be attributed to these epigenetic mechanisms. The intake of dietary compounds that regulate epigenetic modifications can provide significant health effects and may prevent various pathological processes involved in the development of cancer and other life-threatening diseases.

  5. Chromosomal Protein HMGN1 Modulates the Phosphorylation of Serine 1 in Histone H2A

    PubMed Central

    Postnikov, Yuri V.; Belova, Galina I.; Lim, Jae-Hwan; Bustin, Michael

    2008-01-01

    Here we demonstrate that HMGN1, a nuclear protein that binds specifically to nucleosomes, modulates the level of histone H2A phosphorylation. In Hmgn1-/- cells, loss of HMGN1 elevates the steady-state levels of H2AS1ph throughout the cell cycle. In vitro, HMGN1 reduces the rate of Rsk2- and Msk1-mediated phosphorylation of nucleosomal, but not free, histone H2A. HMGN1 inhibits H2A phosphorylation by binding to nucleosomes since an HMGN mutant, which cannot bind to chromatin, does not inhibit the Rsk2- mediated H2A phosphorylation. HMGN2 also inhibits H2A phosphorylation, suggesting that the inhibition of H2A phosphorylation is not specific to only one member of this protein family. Thus, the present data add modifications of histone H2A to the list of histone modifications affected by HMGN proteins. It supports the suggestion that structural chromatin binding proteins can modify the whole profile of post-translational modifications of core histones. PMID:17154547

  6. Depletion of Histone Demethylase Jarid1A Resulting in Histone Hyperacetylation and Radiation Sensitivity Does Not Affect DNA Double-Strand Break Repair

    PubMed Central

    Penterling, Corina; Drexler, Guido A.; Böhland, Claudia; Stamp, Ramona; Wilke, Christina; Braselmann, Herbert; Caldwell, Randolph B.; Reindl, Judith; Girst, Stefanie; Greubel, Christoph; Siebenwirth, Christian; Mansour, Wael Y.; Borgmann, Kerstin; Dollinger, Günther; Unger, Kristian; Friedl, Anna A.

    2016-01-01

    Histone demethylases have recently gained interest as potential targets in cancer treatment and several histone demethylases have been implicated in the DNA damage response. We investigated the effects of siRNA-mediated depletion of histone demethylase Jarid1A (KDM5A, RBP2), which demethylates transcription activating tri- and dimethylated lysine 4 at histone H3 (H3K4me3/me2), on growth characteristics and cellular response to radiation in several cancer cell lines. In unirradiated cells Jarid1A depletion lead to histone hyperacetylation while not affecting cell growth. In irradiated cells, depletion of Jarid1A significantly increased cellular radiosensitivity. Unexpectedly, the hyperacetylation phenotype did not lead to disturbed accumulation of DNA damage response and repair factors 53BP1, BRCA1, or Rad51 at damage sites, nor did it influence resolution of radiation-induced foci or rejoining of reporter constructs. We conclude that the radiation sensitivity observed following depletion of Jarid1A is not caused by a deficiency in repair of DNA double-strand breaks. PMID:27253695

  7. Presence of histones in Aspergillus nidulans

    PubMed Central

    1976-01-01

    Five major histone proteins have been extracted from chromatin isolated from purified nuclei of the fungus, Aspergillus nidulans. These proteins had chromatographic properties which were similar to reference calf thymus histones and were purified to electrophoretic homegeneity by gel chromatography of Bio-Gel P10, Bio-Gel P60, and Sephadex G-100. Electrophoresis of these proteins in three different systems (urea- starch, urea-acetic acid polyacrylamide, and discontinuous SDS polyacrylamide) showed that the A. nidulans histones H3 and H4 were nearly identical to calf thymus H3 and H4 with respect to net charge and molecular weight criteria, whereas the fungal histones H1, H2a and H2b were similar but not identical to the corresponding calf thymus histones. Amino acid analysis of A. nidulans histones H2a, H2b, and H4 showed them to be closely related to the homologous calf thymus histones. The mobility patterns of A. nidulans ribosomal basic proteins in three different electrophoretic systems were distinctly different from those of the fungal histones. PMID:799641

  8. Canonical and variant histones of protozoan parasites.

    PubMed

    Dalmasso, Maria Carolina; Sullivan, William Joseph; Angel, Sergio Oscar

    2011-06-01

    Protozoan parasites have tremendously diverse lifestyles that require adaptation to a remarkable assortment of different environmental conditions. In order to complete their life cycles, protozoan parasites rely on fine-tuning gene expression. In general, protozoa use novel regulatory elements, transcription factors, and epigenetic mechanisms to regulate their transcriptomes. One of the most surprising findings includes the nature of their histones--these primitive eukaryotes lack some histones yet harbor novel histone variants of unknown function. In this review, we describe the histone components of different protozoan parasites based on literature and database searching. We summarize the key discoveries regarding histones and histone variants and their impact on chromatin regulation in protozoan parasites. In addition, we list histone genes IDs, sequences, and genomic localization of several protozoan parasites and Microsporidia histones, obtained from a thorough search of genome databases. We then compare these findings with those observed in higher eukaryotes, allowing us to highlight some novel aspects of epigenetic regulation in protists and to propose questions to be addressed in the upcoming years.

  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.

  10. 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