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Sample records for activates 14-3-3 protein

  1. Analysis of the cruciform binding activity of recombinant 14-3-3zeta-MBP fusion protein, its heterodimerization profile with endogenous 14-3-3 isoforms, and effect on mammalian DNA replication in vitro.

    Alvarez, David; Callejo, Mario; Shoucri, Rami; Boyer, Lee; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2003-06-17

    The human cruciform binding protein (CBP), a member of the 14-3-3 protein family, has been recently identified as an origin of DNA replication binding protein and involved in DNA replication. Here, pure recombinant 14-3-3zeta tagged with maltose binding protein (r14-3-3zeta-MBP) at its N-terminus was tested for binding to cruciform DNA either in the absence or presence of F(TH), a CBP-enriched fraction, by electromobility shift assay (EMSA), followed by Western blot analysis of the electroeluted CBP-cruciform DNA complex. The r14-3-3zeta-MBP was found to have cruciform binding activity only after preincubation with F(TH). Anti-MBP antibody immunoprecipitation of F(TH) preincubated with r14-3-3zeta-MBP, followed by Western blot analysis with antibodies specific to the beta, gamma, epsilon, zeta, and sigma 14-3-3 isoforms showed that r14-3-3zeta-MBP heterodimerized with the endogenous beta, epsilon, and zeta isoforms present in the F(TH) but not with the gamma or sigma isoforms. Immunoprecipitation of endogenous 14-3-3zeta from nuclear extracts (NE) of HeLa cells that were either serum-starved (s-s) or blocked at the G(1)/S or G(2)/M phases of the cell cycle revealed that at G(1)/S and G(2)/M, the zeta isoform heterodimerized only with the beta and epsilon isoforms, while in s-s extracts, the 14-3-3zeta/epsilon heterodimer was never detected, and the 14-3-3zeta/beta heterodimer was seldom detected. Furthermore, addition of r14-3-3zeta-MBP to HeLa cell extracts used in a mammalian in vitro replication system increased the replication level of p186, a plasmid bearing the minimal 186-bp origin of the monkey origin of DNA replication ors8, by approximately 3.5-fold. The data suggest that specific dimeric combinations of the 14-3-3 isoforms have CBP activity and that upregulation of this activity leads to an increase in DNA replication. PMID:12795617

  2. 14-3-3 proteins as potential therapeutic targets

    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 support...

  3. 14-3-3 proteins-an update

    Paulette MHAWECH

    2005-01-01

    14-3-3 is a highly conserved acidic protein family, composed of seven isoforms in mammals. 14-3-3 protein can interact with over 200 target proteins by phosphoserine-dependent and phosphoserine-independent manners. Little is known about the consequences of these interactions, and thus are the subjects of ongoing studies. 14-3-3 controls cell cycle, cell growth, differentiation, survival, apoptosis, migration and spreading. Recent studies have revealed new mechanisms and new functions of 14-3-3, giving us more insights on this fascinating and complex family of proteins.Of all the seven isoforms, 14-3-3σ seems to be directly involved in human cancer. 14-3-3σ itself is subject to regulation by p53 upon DNA damage and by epigenetic deregulation. Gene silencing of 14-3-3σ by CpG methylation has been found in many human cancer types. This suggests that therapy-targeting 14-3-3σ may be beneficial for future cancer treatment.

  4. Accumulation of Carbohydrate and Regulation of 14-3-3 Protein on Sucrose Phosphate Synthase (SPS) Activity in Two Tomato Species

    WANG Li; CUI Na; ZHAO Xiao-cui; FAN Hai-yan; LI Tian-lai

    2014-01-01

    To explore the differences of carbohydrate metabolism in two tomato species and discuss the possible regulation of 14-3-3 proteins on the sucrose phosphate synthase (SPS) activity, we determined the contents of soluble sugar and starch through high performance liquid chromatography (HPLC). The activities of sugar-metabolizing enzymes were assayed in desalted extract, and the relative expression levels of related genes in sugar metabolism were determined though real-time RT-PCR. The results indicated that glucose and fructose were mainly accumulated during the maturation of the fruit because of the high acid invertase (AI) and neutral invertase (NI) in Micro-Tom (Solanum lycopersicum) fruit, while inSolanum chmielewskii fruit, SPS which went along with the change of sucrose content led to the rapid sucrose increase during the fruit ripening. TFT1 and TFT10, belonging to 14-3-3 protein in tomato, were likely to down-regulated SPS activity during young and intumescence period.

  5. Characterization of 14-3-3 Proteins from Cryptosporidium parvum

    Brokx, Stephen J.; Wernimont, Amy K.; Aiping Dong; Wasney, Gregory A.; Yu-Hui Lin; Jocelyne Lew; Masoud Vedadi; Wen Hwa Lee; Raymond Hui

    2011-01-01

    UNLABELLED: The parasite Cryptosporidium parvum has three 14-3-3 proteins: Cp14ε, Cp14a and Cp14b, with only Cp14ε similar to human 14-3-3 proteins in sequence, peptide-binding properties and structure. Structurally, Cp14a features the classical 14-3-3 dimer but with a uniquely wide pocket and a disoriented RRY triad potentially incapable of binding phosphopeptides. The Cp14b protein deviates from the norm significantly: (i) In one subunit, the phosphorylated C-terminal tail is bound in the b...

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

    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

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

    Wu, Yi-Ju [Institute of Cellular and System Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan 350, Taiwan (China); Jan, Yee-Jee [Department of Pathology and Laboratory Medicine, Taichung Veterans General Hospital, Taichung 407, Taiwan (China); Ko, Bor-Sheng [Department of Internal Medicine, National Taiwan University Hospital, Taipei 100, Taiwan (China); Liang, Shu-Man; Liou, Jun-Yang, E-mail: jliou@nhri.org.tw [Institute of Cellular and System Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan 350, Taiwan (China)

    2015-06-15

    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.

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

    Yi-Ju Wu

    2015-06-01

    Full Text Available 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.

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

    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

  10. A novel pathway down-modulating T cell activation involves HPK-1–dependent recruitment of 14-3-3 proteins on SLP-76

    Bartolo, Vincenzo Di; Montagne, Benjamin; Salek, Mogjiborahman; Jungwirth, Britta; Carrette, Florent; Fourtane, Julien; Sol-Foulon, Nathalie; Michel, Frédérique; Schwartz, Olivier; Lehmann, Wolf D.; Acuto, Oreste

    2007-01-01

    The SH2 domain–containing leukocyte protein of 76 kD (SLP-76) is a pivotal element of the signaling machinery controlling T cell receptor (TCR)-mediated activation. Here, we identify 14-3-3ɛ and ζ proteins as SLP-76 binding partners. This interaction was induced by TCR ligation and required phosphorylation of SLP-76 at serine 376. Ribonucleic acid interference and in vitro phosphorylation experiments showed that serine 376 is the target of the hematopoietic progenitor kinase 1 (HPK-1). Intere...

  11. Role of individual phosphorylation sites for the 14-3-3-protein-dependent activation of yeast neutral trehalase Nth1

    Veisová, Dana; Macáková, Eva; Řežábková, Lenka; Šulc, Miroslav; Vácha, Petr; Sychrová, Hana; Obšil, T.; Obšilová, Veronika

    2012-01-01

    Roč. 443, č. 3 (2012), s. 663-670. ISSN 0264-6021 R&D Projects: GA ČR(CZ) GAP207/11/0455; GA AV ČR(CZ) IAA500110801 Grant ostatní: Univerzita Karlova(CZ) 350111 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z50200510 Keywords : 14-3-3 protein * Bmh * neutral trehalase (Nth1) * enzymatic activity * phosphorylation * Saccharomyces cerevisiae Subject RIV: CE - Biochemistry Impact factor: 4.654, year: 2012

  12. 14-3-3 proteins in guard cell signaling

    Valérie eCotelle

    2016-01-01

    Full Text Available 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.

  13. 14-3-3 Proteins in Guard Cell Signaling

    Cotelle, Valérie; Leonhardt, Nathalie

    2016-01-01

    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. PMID:26858725

  14. Arabidopsis 14-3-3 proteins: fascinating and less fascinating aspects

    Nina eJaspert

    2011-12-01

    Full Text Available 14-3-3 dimers are well known to interact with diverse target proteins throughout eukaryotes. Most notably, association of 14-3-3s commonly requires phosphorylation of a serine or threonine residue within a specific sequence motif of the client protein. Studies with a focus on individual target proteins have unequivocally demonstrated 14-3-3s to be the crucial factors modifying the client’s activity state upon phosphorylation and, thus, finishing the job initiated by a kinase. In this respect, a recent in-depth analysis of the rice transcription factor FLOWERING LOCUS D1 (OsFD1 revealed 14-3-3s to be essential players in floral induction. However, such fascinating discoveries can often be ascribed to the random identification of 14-3-3 as an interaction partner of the favorite protein. In contrast, our understanding of 14-3-3 function in higher organisms is frustratingly limited, mainly due to an overwhelming spectrum of putative targets in combination with the existence of a multigene 14-3-3 family. In this review we will discuss our current understanding of the function of plant 14-3-3 proteins, taking into account surveys of the Arabidopsis 14-3-3 interactome.

  15. Comprehensive proteomic analysis of interphase and mitotic 14-3-3-binding proteins.

    Meek, Sarah E M; Lane, William S; Piwnica-Worms, Helen

    2004-07-30

    14-3-3 proteins regulate the cell division cycle and play a pivotal role in blocking cell cycle advancement after activation of the DNA replication and DNA damage checkpoints. Here we describe a global proteomics analysis to identify proteins that bind to 14-3-3s during interphase and mitosis. 14-3-3-binding proteins were purified from extracts of interphase and mitotic HeLa cells using specific peptide elution from 14-3-3 zeta affinity columns. Proteins that specifically bound and eluted from the affinity columns were identified by microcapillary high pressure liquid chromatography tandem mass spectrometry analysis. Several known and novel 14-3-3-interacting proteins were identified in this screen. Identified proteins are involved in cell cycle regulation, signaling, metabolism, protein synthesis, nucleic acid binding, chromatin structure, protein folding, proteolysis, nucleolar function, and nuclear transport as well as several other cellular processes. In some cases 14-3-3 binding was cell cycle-dependent, whereas in other cases the binding was shown to be cell cycle-independent. This study adds to the growing list of human 14-3-3-binding proteins and implicates a role for 14-3-3 proteins in a plethora of essential biological processes. PMID:15161933

  16. 14-3-3σ induces heat shock protein 70 expression in hepatocellular carcinoma

    14-3-3σ is implicated in promoting tumor development of various malignancies. However, the clinical relevance of 14-3-3σ in hepatocellular carcinoma (HCC) tumor progression and modulation and pathway elucidation remain unclear. We investigated 14-3-3σ expression in 109 HCC tissues by immunohistochemistry. Overexpression and knockdown experiments were performed by transfection with cDNA or siRNA. Protein expression and cell migration were determined by Western blot and Boyden chamber assay. In this study, we found that 14-3-3σ is abundantly expressed in HCC tumors. Stable or transient overexpression of 14-3-3σ induces the expression of heat shock factor-1α (HSF-1α) and heat shock protein 70 (HSP70) in HCC cells. Moreover, expression of 14-3-3σ significantly correlates with HSF-1α/HSP70 in HCC tumors and both 14-3-3σ and HSP70 overexpression are associated with micro-vascular thrombi in HCC patients, suggesting that 14-3-3σ/HSP70 expression is potentially involved in cell migration/invasion. Results of an in vitro migration assay indicate that 14-3-3σ promotes cell migration and that 14-3-3σ-induced cell migration is impaired by siRNA knockdown of HSP70. Finally, 14-3-3σ-induced HSF-1α/HSP70 expression is abolished by the knockdown of β-catenin or activation of GSK-3β. Our findings indicate that 14-3-3σ participates in promoting HCC cell migration and tumor development via β-catenin/HSF-1α/HSP70 pathway regulation. Thus, 14-3-3σ alone or combined with HSP70 are potential prognostic biomarkers for HCC

  17. Characterization of 14-3-3 proteins from Cryptosporidium parvum.

    Stephen J Brokx

    Full Text Available UNLABELLED: The parasite Cryptosporidium parvum has three 14-3-3 proteins: Cp14ε, Cp14a and Cp14b, with only Cp14ε similar to human 14-3-3 proteins in sequence, peptide-binding properties and structure. Structurally, Cp14a features the classical 14-3-3 dimer but with a uniquely wide pocket and a disoriented RRY triad potentially incapable of binding phosphopeptides. The Cp14b protein deviates from the norm significantly: (i In one subunit, the phosphorylated C-terminal tail is bound in the binding groove like a phosphopeptide. This supports our binding study indicating this protein was stabilized by a peptide mimicking its last six residues. (ii The other subunit has eight helices instead of nine, with αA and αB forming a single helix and occluding the peptide-binding cleft. (iii The protein forms a degenerate dimer with the two binding grooves divided and facing opposite directions. These features conspire to block and disrupt the bicameral substrate-binding pocket, suggesting a possible tripartite auto-regulation mechanism that has not been observed previously. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

  18. Role of the EF-hand-like Motif in the 14-3-3 Protein- mediated Activation of Yeast Neutral Trehalase Nth1

    Kopecká, Miroslava; Košek, Dalibor; Kukačka, Zdeněk; Řežábková, Lenka; Man, Petr; Novák, Petr; Obšil, T.; Obšilová, Veronika

    2014-01-01

    Roč. 289, č. 20 (2014), s. 13948-13961. ISSN 0021-9258 R&D Projects: GA ČR(CZ) GAP207/11/0455 Grant ostatní: Univerzita Karlova(CZ) 644313; Univerzita Karlova(CZ) 800413 Institutional support: RVO:67985823 ; RVO:61388971 Keywords : calcium * enzyme mechanisms * mass spectrometry (MS) * protein cross-linking * protein structure * 14-3-3 * Bmh * H/D exchange * neutral trehalase * SAXS Subject RIV: CE - Biochemistry Impact factor: 4.573, year: 2014

  19. 14-3-3 proteins and the p53 family : a study in keratinocytes

    Niemantsverdriet, Maarten

    2008-01-01

    Several associations between 14-3-3 proteins and members of the p53 family have been revealed. However, numerous questions regarding 14-3-3 proteins, p53 family members and the relationships between thetwo families remain. This thesis contributes to answer these questions. Downregulation of 14-3-3ζ

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

    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 (Emory-MED); (GSU); (MCW); (Chinese Aca. Sci.)

    2013-02-14

    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 kD{sub a} 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 14-3-3{zeta} 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.

  1. Differential neuroprotective effects of 14-3-3 proteins in models of Parkinson's disease

    Yacoubian, T A; Slone, S R; Harrington, A J; Hamamichi, S; Schieltz, J M; Caldwell, K A; Caldwell, G A; Standaert, D.G.

    2010-01-01

    14-3-3 proteins are important negative regulators of cell death pathways. Recent studies have revealed alterations in 14-3-3s in Parkinson's disease (PD) and the ability of 14-3-3s to interact with α-synuclein (α-syn), a protein central to PD pathophysiology. In a transgenic α-syn mouse model, we found reduced expression of 14-3-3θ, -ɛ, and -γ. These same isoforms prevent α-syn inclusion formation in an H4 neuroglioma cell model. Using dopaminergic cell lines stably overexpressing each 14-3-3...

  2. 14-3-3 proteins regulate Tctp–Rheb interaction for organ growth in Drosophila

    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

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

    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

  4. Insight into Conformational Change for 14-3-3σ Protein by Molecular Dynamics Simulation

    Guodong Hu

    2014-02-01

    Full Text Available 14-3-3σ is a member of a highly conserved family of 14-3-3 proteins that has a double-edged sword role in human cancers. Former reports have indicated that the 14-3-3 protein may be in an open or closed state. In this work, we found that the apo-14-3-3σ is in an open state compared with the phosphopeptide bound 14-3-3σ complex which is in a more closed state based on our 80 ns molecular dynamics (MD simulations. The interaction between the two monomers of 14-3-3σ in the open state is the same as that in the closed state. In both open and closed states, helices A to D, which are involved in dimerization, are stable. However, large differences are found in helices E and F. The hydrophobic contacts and hydrogen bonds between helices E and G in apo-14-3-3σ are different from those in the bound 14-3-3σ complex. The restrained and the mutated (Arg56 or Arg129 to alanine MD simulations indicate that the conformation of four residues (Lys49, Arg56, Arg129 and Tyr130 may play an important role to keep the 14-3-3σ protein in an open or closed state. These results would be useful to evaluate the 14-3-3σ protein structure-function relationship.

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

    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.

  6. Role 14-3-3 Protein in Regulation Some Cellular Processes

    Nagam Khudhair

    2014-09-01

    Full Text Available The aim of this study to review an overview of the current information on the structure of proteins 14-3-3 and their complexes, in addition to that it provides insights into the mechanisms of their functions. The 14-3-3 proteins are from families maintain regulatory molecules expressed in all eukaryotic cells. It was discovered before thirty years, it is attributes of 14-3-3 proteins are able to connect a large number of signalling proteins are functionally diverse, including kinases, phosphatases and transmembrane receptors. 14-3-3 proteins play an important role in a variety of vital regulatory processes, such as protein regulation, apoptotic cell death and cell cycle control. In this review, we discussed the structural basis of 14-3-3 proteins, common structural features of their complexes, Phosphorylation, Cell cycle and Apoptosis.

  7. Differential neuroprotective effects of 14-3-3 proteins in models of Parkinson's disease.

    Yacoubian, T A; Slone, S R; Harrington, A J; Hamamichi, S; Schieltz, J M; Caldwell, K A; Caldwell, G A; Standaert, D G

    2010-01-01

    14-3-3 proteins are important negative regulators of cell death pathways. Recent studies have revealed alterations in 14-3-3s in Parkinson's disease (PD) and the ability of 14-3-3s to interact with alpha-synuclein (α-syn), a protein central to PD pathophysiology. In a transgenic α-syn mouse model, we found reduced expression of 14-3-3θ, ε, and γ. These same isoforms prevent α-syn inclusion formation in an H4 neuroglioma cell model. Using dopaminergic cell lines stably overexpressing each 14-3-3 isoform, we found that overexpression of 14-3-3θ, ε, or γ led to resistance to both rotenone and 1-methyl-4-phenylpyridinium (MPP(+)), while other isoforms were not protective against both toxins. Inhibition of a single protective isoform, 14-3-3θ, by shRNA did not increase vulnerability to neurotoxic injury, but toxicity was enhanced by broad-based inhibition of 14-3-3 action with the peptide inhibitor difopein. Using a transgenic C. elegans model of PD, we confirmed the ability of both human 14-3-3θ and a C. elegans 14-3-3 homolog (ftt-2) to protect dopaminergic neurons from α-syn toxicity. Collectively, these data show a strong neuroprotective effect of enhanced 14-3-3 expression - particularly of the 14-3-3θ, ε, and γ isoforms - in multiple cellular and animal models of PD, and point to the potential value of these proteins in the development of neuroprotective therapies for human PD. PMID:21152247

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

    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

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

    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

  10. Suppression of apoptosis signal-regulating kinase 1-induced cell death by 14-3-3 proteins

    Zhang, Lixin; Chen, Jing; Fu, Haian

    1999-01-01

    Apoptosis signal-regulating kinase 1 (ASK1) is a pivotal component of a signaling pathway induced by many death stimuli, including tumor necrosis factor α, Fas, and the anticancer drugs cisplatin and paclitaxel. Here we report that ASK1 proapoptotic activity is antagonized by association with 14-3-3 proteins. We found that ASK1 specifically bound 14-3-3 proteins via a site involving Ser-967 of ASK1. Interestingly, overexpression of 14-3-3 in HeLa cells blocked ASK1-induced apoptosis whereas d...

  11. Expression of 14-3-3 protein isoforms in mouse oocytes, eggs and ovarian follicular development

    De Santanu

    2012-01-01

    Full Text Available Abstract Background The 14-3-3 (YWHA proteins are a highly conserved, ubiquitously expressed family of proteins. Seven mammalian isoforms of 14-3-3 are known (β, γ, ε, ζ, η, τ and, σ. These proteins associate with many intracellular proteins involved in a variety of cellular processes including regulation of the cell cycle, metabolism and protein trafficking. We are particularly interested in the role of 14-3-3 in meiosis in mammalian eggs and the role 14-3-3 proteins may play in ovarian function. Therefore, we examined the expression of 14-3-3 proteins in mouse oocyte and egg extracts by Western blotting after polyacrylamide gel electrophoresis, viewed fixed cells by indirect immunofluorescence, and examined mouse ovarian cells by immunohistochemical staining to study the expression of the different 14-3-3 isoforms. Results We have determined that all of the mammalian 14-3-3 isoforms are expressed in mouse eggs and ovarian follicular cells including oocytes. Immunofluorescence confocal microscopy of isolated oocytes and eggs confirmed the presence of all of the isoforms with characteristic differences in some of their intracellular localizations. For example, some isoforms (β, ε, γ, and ζ are expressed more prominently in peripheral cytoplasm compared to the germinal vesicles in oocytes, but are uniformly dispersed within eggs. On the other hand, 14-3-3η is diffusely dispersed in the oocyte, but attains a uniform punctate distribution in the egg with marked accumulation in the region of the meiotic spindle apparatus. Immunohistochemical staining detected all isoforms within ovarian follicles, with some similarities as well as notable differences in relative amounts, localizations and patterns of expression in multiple cell types at various stages of follicular development. Conclusions We found that mouse oocytes, eggs and follicular cells within the ovary express all seven isoforms of the 14-3-3 protein. Examination of the

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

    Řežábková, L.; Kacířová, M.; Šulc, Miroslav; Herman, P.; Večeř, J.; Štěpánek, M.; Obšilová, Veronika; Obšil, T.

    2012-01-01

    Roč. 103, č. 9 (2012), s. 1960-1969. ISSN 0006-3495 Institutional support: RVO:61388971 ; RVO:67985823 Keywords : phosducin * 14-3-3 protein * fluorescence Subject RIV: CE - Biochemistry Impact factor: 3.668, year: 2012

  13. Altered expression of 14-3-3ζ protein in spinal cords of rat fetuses with spina bifida aperta.

    Li-na Wu

    Full Text Available BACKGROUND: A large number of studies have confirmed that excessive apoptosis is one of the reasons for deficient neuronal function in neural tube defects (NTDs. A previous study from our laboratory used 2-D gel electrophoresis to demonstrate that 14-3-3ζ expression was low in the spinal cords of rat fetuses with spina bifida aperta at embryonic day (E 17. As a member of the 14-3-3 protein family, 14-3-3ζ plays a crucial role in the determination of cell fate and anti-apoptotic activity. However, neither the expression of 14-3-3ζ in defective spinal cords, nor the correlation between 14-3-3ζ and excessive apoptosis in NTDs has been fully confirmed. METHODOLOGY/PRINCIPAL FINDINGS: We used immunoblotting and quantitative real-time PCR (qRT-PCR to quantify the expression of 14-3-3ζ and double immunofluorescence to visualize 14-3-3ζ and apoptosis. We found that, compared with controls, 14-3-3ζ was down-regulated in spina bifida between E12 and E15. Excessive apoptotic cells and low expression of 14-3-3ζ were observed in the dorsal region of spinal cords with spina bifida during the same time period. To initially explore the molecular mechanisms of apoptosis in NTDs, we investigated the expression of microRNA-7 (miR-7, microRNA-375 (miR-375 and microRNA-451 (miR-451, which are known to down-regulate 14-3-3ζ in several different cell types. We also investigated the expression of p53, a molecule that is downstream of 14-3-3ζ and can be down-regulated by it. We discovered that, in contrast to the reduction of 14-3-3ζ expression, the expression of miR-451, miR-375 and p53 increased in spina bifida rat fetuses. CONCLUSIONS/SIGNIFICANCE: These data suggest that the reduced expression of 14-3-3ζ plays a role in the excessive apoptosis that occurs in spina bifida and may be partly regulated by the over-expression of miR-451 and miR-375, and the consequent up-regulation of p53 might further promote apoptosis in spina bifida.

  14. Mechanisms of the 14-3-3 protein function: regulation of protein function through conformational modulation

    Obšilová, Veronika; Kopecká, Miroslava; Košek, Dalibor; Kacířová, M.; Kylarová, Salome; Řežábková, L.; Obšil, T.

    2014-01-01

    Roč. 63, Suppl.1 (2014), S155-S164. ISSN 0862-8408 R&D Projects: GA ČR(CZ) GAP207/11/0455 Grant ostatní: GA ČR(CZ) GAP305/11/0708 Institutional support: RVO:67985823 Keywords : 14-3-3 protein * regulator of G-protein signalling (RGS3), * phosducin * neutral trehalase * Bmh1 * conformation * signaling Subject RIV: BO - Biophysics Impact factor: 1.293, year: 2014

  15. 14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling.

    Michael P Grant

    Full Text Available Calcium sensing receptors (CaSR interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium.

  16. Neuroprotective effect of TAT-14-3-3ε fusion protein against cerebral ischemia/reperfusion injury in rats.

    Yuanjun Zhu

    Full Text Available Stroke is the major cause of death and disability worldwide, and the thrombolytic therapy currently available was unsatisfactory. 14-3-3ε is a well characterized member of 14-3-3 family, and has been reported to protect neurons against apoptosis in cerebral ischemia. However, it cannot transverse blood brain barrier (BBB due to its large size. A protein transduction domain (PTD of HIV TAT protein, is capable of delivering a large variety of proteins into the brain. In this study, we generated a fusion protein TAT-14-3-3ε, and evaluated its potential neuroprotective effect in rat focal ischemia/reperfusion (I/R model. Western blot analysis validated the efficient transduction of TAT-14-3-3ε fusion protein into brain via a route of intravenous injection. TAT-14-3-3ε pre-treatment 2 h before ischemia significantly reduced cerebral infarction volume and improved neurologic score, while post-treatment 2 h after ischemia was less effective. Importantly, pre- or post-ischemic treatment with TAT-14-3-3ε significantly increased the number of surviving neurons as determined by Nissl staining, and attenuated I/R-induced neuronal apoptosis as showed by the decrease in apoptotic cell numbers and the inhibition of caspase-3 activity. Moreover, the introduction of 14-3-3ε into brain by TAT-mediated delivering reduced the formation of autophagosome, attenuated LC3B-II upregulation and reversed p62 downregulation induced by ischemic injury. Such inhibition of autophagy was reversed by treatment with an autophagy inducer rapamycin (RAP, which also attenuated the neuroprotective effect of TAT-14-3-3ε. Conversely, autophagy inhibitor 3-methyladenine (3-MA inhibited I/R-induced the increase in autophagic activity, and attenuated I/R-induced brain infarct. These results suggest that TAT-14-3-3ε can be efficiently transduced into brain and exert significantly protective effect against brain ischemic injury through inhibiting neuronal apoptosis and autophagic

  17. Phosphorylation-Dependent Protein Interaction with Trypanosoma brucei 14-3-3 Proteins that Display Atypical Target Recognition

    Inoue, Masahiro; Yasuda, Kouichi; Uemura, Haruki; Yasaka, Natsumi; Inoue, Hiroshi; Sei, Yoshitatsu; Horikoshi, Nobuo; Fukuma, Toshihide

    2010-01-01

    Background The 14-3-3 proteins are structurally conserved throughout eukaryotes and participate in protein kinase signaling. All 14-3-3 proteins are known to bind to evolutionally conserved phosphoserine-containing motifs (modes 1 and/or 2) with high affinity. In Trypanosoma brucei, 14-3-3I and II play pivotal roles in motility, cytokinesis and the cell cycle. However, none of the T. brucei 14-3-3 binding proteins have previously been documented. Methodology/Principal Findings Initially we sh...

  18. Structural Characterization of Phosducin and Its Complex with the 14-3-3 Protein

    Kacířová, Miroslava; Košek, Dalibor; Kádek, Alan; Man, Petr; Večeř, J.; Herman, P.; Obšilová, Veronika; Obšil, Tomáš

    2015-01-01

    Roč. 290, č. 26 (2015), s. 16246-16260. ISSN 0021-9258 Grant ostatní: GA ČR(CZ) GAP305/11/0708 Institutional support: RVO:67985823 ; RVO:61388971 Keywords : Phosducin * 14-3-3 protein * fluorescence spectroscopy * SAXS * hydrogen-deuterium exchange Subject RIV: CE - Biochemistry Impact factor: 4.573, year: 2014

  19. Molecular Dynamics Simulations and Structural Analysis of Giardia duodenalis 14-3-3 Protein-Protein Interactions.

    Cau, Ylenia; Fiorillo, Annarita; Mori, Mattia; Ilari, Andrea; Botta, Maurizo; Lalle, Marco

    2015-12-28

    Giardiasis is a gastrointestinal diarrheal illness caused by the protozoan parasite Giardia duodenalis, which affects annually over 200 million people worldwide. The limited antigiardial drug arsenal and the emergence of clinical cases refractory to standard treatments dictate the need for new chemotherapeutics. The 14-3-3 family of regulatory proteins, extensively involved in protein-protein interactions (PPIs) with pSer/pThr clients, represents a highly promising target. Despite homology with human counterparts, the single 14-3-3 of G. duodenalis (g14-3-3) is characterized by a constitutive phosphorylation in a region critical for target binding, thus affecting the function and the conformation of g14-3-3/clients interaction. However, to approach the design of specific small molecule modulators of g14-3-3 PPIs, structural elucidations are required. Here, we present a detailed computational and crystallographic study exploring the implications of g14-3-3 phosphorylation on protein structure and target binding. Self-Guided Langevin Dynamics and classical molecular dynamics simulations show that phosphorylation affects locally and globally g14-3-3 conformation, inducing a structural rearrangement more suitable for target binding. Profitable features for g14-3-3/clients interaction were highlighted using a hydrophobicity-based descriptor to characterize g14-3-3 client peptides. Finally, the X-ray structure of g14-3-3 in complex with a mode-1 prototype phosphopeptide was solved and combined with structure-based simulations to identify molecular features relevant for clients binding to g14-3-3. The data presented herein provide a further and structural understanding of g14-3-3 features and set the basis for drug design studies. PMID:26551337

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

    Jahn, T.; Fuglsang, A.T.; Olsson, A.;

    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...... 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.......(+)-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...

  1. Regulation of starch accumulation by granule-associated plant 14-3-3 proteins

    Sehnke, Paul C.; Chung, Hwa-Jee; Wu, Ke; Ferl, Robert J.

    2001-01-01

    In higher plants the production of starch is orchestrated by chloroplast-localized biosynthetic enzymes, namely starch synthases, ADP-glucose pyrophosphorylase, and starch branching and debranching enzymes. Diurnal regulation of these enzymes, as well as starch-degrading enzymes, influences both the levels and composition of starch, and is dependent in some instances upon phosphorylation-linked regulation. The phosphoserine/threonine-binding 14-3-3 proteins partici...

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

    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

  3. Does 14-3-3 protein affect conformation FoxO4 DNA-binding domain?

    Šilhán, Jan; Bouřa, Evžen; Vácha, Pavel; Herman, P.; Večeř, J.; Obšil, Tomáš

    2007-01-01

    Roč. 102, Suppl.1 (2007), s. 276-276. ISSN 0022-3042. [Biennial meeting of the International Society for Neurochemistry /21./ and Annual meeting of the American Society for Neurochemistry /38./. 19.08.2007-24.08.2007, Cancun] R&D Projects: GA ČR(CZ) GA204/06/0565; GA MŠk(CZ) LC554; GA AV ČR(CZ) KJB500110601 Institutional research plan: CEZ:AV0Z50110509 Keywords : cpo1 * FoxO4 DNA-Binding-Domain * 14-3-3 protein * molecular anvil Subject RIV: BO - Biophysics

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

    Kim, Yoonseo; Kim, Hyungjin; Jang, Sung-Wuk [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of); Ko, Jesang, E-mail: jesangko@korea.ac.kr [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of)

    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}.

  5. Differential interaction and aggregation of 3-repeat and 4-repeat tau isoforms with 14-3-3ζ protein

    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 14-3-3ζ. We show by surface plasmon resonance studies that the interaction between unphosphorylated 3R-tau and 14-3-3ζ is ∼3-folds higher than that between unphosphorylated 4R-tau and 14-3-3ζ. Phosphorylation of tau by protein kinase A (PKA) increases the affinity of both 3R- and 4R-tau for 14-3-3ζ 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 14-3-3ζ. 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 14-3-3ζ exhibits a similar role for tau isoforms after PKA-phosphorylation, but a differential role for unphosphorylated tau. The significant aggregation of 4R-tau by 14-3-3ζ suggests that 14-3-3 may act as an inducer in the generation of 4R-tau-predominant neurofibrillary tangles in tauopathies.

  6. Using SANS to monitor the interaction of misfolding alcohol dehydrogenase with the molecular chaperone protein 14-3-3ζ

    14-3-3 is a family of acidic, dimeric proteins which are highly conserved across many species. Each monomer is approximately 30kDa in mass and contains 9 α-helices. Dimer formation is initiated at the N-terminal region of the protein as a result of the interaction between several buried polar and hydrophobic residues in this region. 14-3-3 proteins interact with a wide range of proteins to regulate many cellular processes, e.g. apoptosis and mitosis, as well as protein misfolding associated with conformational diseases such as Alzheimer’s and Parkinson’s Disease. A potential role of 14-3-3 in these diseases was discovered with the observation that 14-3-3ζ can act as a molecular chaperone, whereby it stabilises intermediately folded proteins to prevent their aggregation. The binding site and mechanism of the chaperone action of 14-3-3ζ are not known, despite being narrowed down in our NMR study. We produced deuterated 14-3-3ζ and used it in SANS experiments with a model misfolding protein, alcohol dehydrogenase (ADH). Contrast variation allowed us to monitor changes in each component separately after the initiation of ADH misfolding. The Rg and Dmax values of ADH under stress show an increase in size with time, consistent with unfolding and aggregation. In the presence of 14-3-3ζ, the unfolding of ADH is reduced and the protein maintains a globular expanded conformation consistent with an adoption of an intermediately folded (molten globule) state. 14-3-3ζ whilst chaperoning showed a reduction in size, possibly due to dissociation. Ab initio models were also obtained. This is the first instance where conformational changes during chaperoning of either a partly folded target protein, or 14-3-3ζ, have been observed.

  7. Small-Molecule Stabilization of the 14-3-3/Gab2 Protein-Protein Interaction (PPI) Interface.

    Bier, David; Bartel, Maria; Sies, Katharina; Halbach, Sebastian; Higuchi, Yusuke; Haranosono, Yu; Brummer, Tilman; Kato, Nobuo; Ottmann, Christian

    2016-04-19

    Small-molecule modulation of protein-protein interactions (PPIs) is one of the most promising new areas in drug discovery. In the vast majority of cases only inhibition or disruption of PPIs is realized, whereas the complementary strategy of targeted stabilization of PPIs is clearly under-represented. Here, we report the example of a semi-synthetic natural product derivative--ISIR-005--that stabilizes the cancer-relevant interaction of the adaptor protein 14-3-3 and Gab2. The crystal structure of ISIR-005 in complex with 14-3-3 and the binding motif of Gab2 comprising two phosphorylation sites (Gab2pS210pT391) showed how the stabilizing molecule binds to the rim-of-the-interface of the protein complex. Only in the direct vicinity of 14-3-3/Gab2pT391 site is a pre-formed pocket occupied by ISIR-005; binding of the Gab2pS210 motif to 14-3-3 does not create an interface pocket suitable for the molecule. Accordingly, ISIR-005 only stabilizes the binding of the Gab2pT391 but not the Gab2pS210 site. This study represents structural and biochemical proof of the druggability of the 14-3-3/Gab2 PPI interface with important implications for the development of PPI stabilizers. PMID:26644359

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

    Tinti, Michele; Johnson, Catherine; Toth, Rachel; Ferrier, David Ellard Keith; 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 ...

  9. 14-3-3sigma is a cruciform DNA binding protein and associates in vivo with origins of DNA replication.

    Alvarez, David; Novac, Olivia; Callejo, Mario; Ruiz, Marcia T; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2002-01-01

    A human cruciform binding protein (CBP) was previously shown to bind to cruciform DNA in a structure-specific manner and be a member of the 14-3-3 protein family. CBP had been found to contain the 14-3-3 isoforms beta, gamma, epsilon, and zeta. Here, we show by Western blot analysis that the CBP-cruciform DNA complex eluted from band-shift polyacrylamide gels also contains the 14-3-3sigma isoform, which is present in HeLa cell nuclear extracts. An antibody specific for the 14-3-3sigma isoform was able to interfere with the formation of the CBP-cruciform DNA complex. The effect of the same anti-14-3-3sigma antibody in the in vitro replication of p186, a plasmid containing the minimal replication origin of the monkey origin ors8, was also analyzed. Pre-incubation of total HeLa cell extracts with this antibody decreased p186 in vitro replication to approximately 30% of control levels, while non-specific antibodies had no effect. 14-3-3sigma was found to associate in vivo with the monkey origins of DNA replication ors8 and ors12 in a cell cycle-dependent manner, as assayed by a chromatin immunoprecipitation (ChIP) assay that involved formaldehyde cross-linking, followed by immunoprecipitation with anti-14-3-3sigma antibody and quantitative PCR. The association of 14-3-3sigma with the replication origins was maximal at the G(1)/S phase. The results indicate that 14-3-3sigma is an origin binding protein involved in the regulation of DNA replication via cruciform DNA binding. PMID:12244572

  10. The crystal structure of the non-liganded 14-3-3σ protein: insights into determinants of isoform specific ligand binding and dimerization

    Anne BENZINGER; Grzegorz M. POPOWICZ; Joma K. JOY; Sudipta MAJUMDAR; Tad A. HOLAK; Heiko HERMEKING

    2005-01-01

    Seven different, but highly conserved 14-3-3 proteins are involved in diverse signaling pathways in human cells. It is unclear how the 14-3-3σ isoform, a transcriptional target of p53, exerts its inhibitory effect on the cell cycle in the presence of other 14-3-3 isoforms, which are constitutively expressed at high levels. In order to identify structural differences between the 14-3-3 isoforms, we solved the crystal structure of the human 14-3-3σ protein at a resolution of 2.8 A and compared it to the known structures of 14-3-3ζ and 14-3-3τ. The global architecture of the 14-3-3σ fold is similar to the previously determined structures of 14-3-3ζ and 14-3-3τ: two 14-3-3σ molecules form a cup-shaped dimer. Significant differences between these 14-3-3 isoforms were detected adjacent to the amphipathic groove, which mediates the binding to phosphorylated consensus motifs in 14-3-3-1igands. Another specificity determining region is localized between amino-acids 203 to 215. These differences presumably select for the interaction with specific ligands,which may explain the different biological functions of the respective 14-3-3 isoforms. Furthermore, the two 14-3-3σ molecules forming a dimer differ by the spatial position of the ninth helix, which is shifted to the inside of the ligand interaction surface, thus indicating adaptability of this part of the molecule. In addition, 5 non-conserved residues are located at the interface between two 14-3-3σ proteins forming a dimer and represent candidate determinants of homoand hetero-dimerization specificity. The structural differences among the 14-3-3 isoforms described here presumably contribute to isoform-specific interactions and functions.

  11. SGK and 14-3-3 protein areinvolved in the serine/threonine phosphorylationmechanism for TPO/MPLsignal transduction

    2001-01-01

    Thrombopioetin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Yeast two-hybrid screening was performed to isolate the proteins interacting with the cytoplasmic domain of c-Mpl. 48 positive clones were isolated from 5 × 106 independent transformants. The results of sequence analysis demonstrate that they represent 13 different protein encoding sequences. Among them there are a partial coding sequence of serine/threonine protein kinase SGK (serum and glucocorticoid-inducible kinase ) and 14-3-3 theta protein partial coding sequence. GST-pull-down assay and co-immunoprecipitation in mammal cells have confirmed the interaction between these two proteins and c-Mpl. By constructing a series of deleted c-Mpl cytoplasmic domain, the interaction region in c-Mpl cytoplasmic tail was localized in amino acids 523-554. At the same time, the directed interaction between SGK and 14-3-3 proteins also has been verified by yeast two-hybrid assay. The present note is the first time to report that two proteins act with c-Mpl at the same time and put forward that SGK and 14-3-3 protein may be involved in the serine/threonine phosphorylation mechanism for signal transduction.``

  12. Proteomic screen in the simple metazoan Hydra identifies 14-3-3 binding proteins implicated in cellular metabolism, cytoskeletal organisation and Ca2+ signalling

    Pauly, Barbara; Lasi, Margherita; MacKintosh, Carol; Morrice, Nick; Imhof, Axel; Regula, Jörg; Rudd, Stephen; David, Charles N; Böttger, Angelika

    2007-01-01

    Background 14-3-3 proteins have been implicated in many signalling mechanisms due to their interaction with Ser/Thr phosphorylated target proteins. They are evolutionarily well conserved in eukaryotic organisms from single celled protozoans and unicellular algae to plants and humans. A diverse array of target proteins has been found in higher plants and in human cell lines including proteins involved in cellular metabolism, apoptosis, cytoskeletal organisation, secretion and Ca2+ signalling. Results We found that the simple metazoan Hydra has four 14-3-3 isoforms. In order to investigate whether the diversity of 14-3-3 target proteins is also conserved over the whole animal kingdom we isolated 14-3-3 binding proteins from Hydra vulgaris using a 14-3-3-affinity column. We identified 23 proteins that covered most of the above-mentioned groups. We also isolated several novel 14-3-3 binding proteins and the Hydra specific secreted fascin-domain-containing protein PPOD. In addition, we demonstrated that one of the 14-3-3 isoforms, 14-3-3 HyA, interacts with one Hydra-Bcl-2 like protein in vitro. Conclusion Our results indicate that 14-3-3 proteins have been ubiquitous signalling components since the start of metazoan evolution. We also discuss the possibility that they are involved in the regulation of cell numbers in response to food supply in Hydra. PMID:17651497

  13. Proteomic screen in the simple metazoan Hydra identifies 14-3-3 binding proteins implicated in cellular metabolism, cytoskeletal organisation and Ca2+ signalling

    Imhof Axel

    2007-07-01

    Full Text Available Abstract Background 14-3-3 proteins have been implicated in many signalling mechanisms due to their interaction with Ser/Thr phosphorylated target proteins. They are evolutionarily well conserved in eukaryotic organisms from single celled protozoans and unicellular algae to plants and humans. A diverse array of target proteins has been found in higher plants and in human cell lines including proteins involved in cellular metabolism, apoptosis, cytoskeletal organisation, secretion and Ca2+ signalling. Results We found that the simple metazoan Hydra has four 14-3-3 isoforms. In order to investigate whether the diversity of 14-3-3 target proteins is also conserved over the whole animal kingdom we isolated 14-3-3 binding proteins from Hydra vulgaris using a 14-3-3-affinity column. We identified 23 proteins that covered most of the above-mentioned groups. We also isolated several novel 14-3-3 binding proteins and the Hydra specific secreted fascin-domain-containing protein PPOD. In addition, we demonstrated that one of the 14-3-3 isoforms, 14-3-3 HyA, interacts with one Hydra-Bcl-2 like protein in vitro. Conclusion Our results indicate that 14-3-3 proteins have been ubiquitous signalling components since the start of metazoan evolution. We also discuss the possibility that they are involved in the regulation of cell numbers in response to food supply in Hydra.

  14. 14-3-3 protein C-terminal stretch occupies ligand binding groove and is displaced by phosphopeptide binding

    Šilhán, Jan; Obšilová, Veronika; Večeř, J.; Herman, P.; Šulc, Miroslav; Teisinger, Jan; Obšil, Tomáš

    2004-01-01

    Roč. 279, č. 47 (2004), s. 49113-49119. ISSN 0021-9258 R&D Projects: GA ČR GA204/03/0714; GA AV ČR KJB5011308; GA MŠk LZ1K03020 Institutional research plan: CEZ:AV0Z5011922; CEZ:MSM 113100001 Keywords : 14-3-3 protein * FRET * molecular dynamics Subject RIV: BO - Biophysics Impact factor: 6.355, year: 2004

  15. 14-3-3 Protein C-terminal stretch occupies ligand binding groove and is displaced by phosphopeptide binding

    Šilhan, J.; Obšilová, V.; Večeř, J.; Herman, P.; Šulc, Miroslav; Teisinger, J.; Obšil, T.

    2004-01-01

    Roč. 279, č. 47 (2004), s. 49113-49119. ISSN 0021-9258 R&D Projects: GA ČR GA204/03/0714; GA ČR GA309/02/1479; GA AV ČR KJB5011308; GA MŠk LZ1K03020 Keywords : 14-3-3 protein * c-terminal * phosphopeptide Subject RIV: EE - Microbiology, Virology Impact factor: 6.355, year: 2004

  16. The 14-3-3 protein PAR-5 regulates the asymmetric localization of the LET-99 spindle positioning protein.

    Wu, Jui-Ching; Espiritu, Eugenel B; Rose, Lesilee S

    2016-04-15

    PAR proteins play important roles in establishing cytoplasmic polarity as well as regulating spindle positioning during asymmetric division. However, the molecular mechanisms by which the PAR proteins generate asymmetry in different cell types are still being elucidated. Previous studies in Caenorhabditis elegans revealed that PAR-3 and PAR-1 regulate the asymmetric localization of LET-99, which in turn controls spindle positioning by affecting the distribution of the conserved force generating complex. In wild-type embryos, LET-99 is localized in a lateral cortical band pattern, via inhibition at the anterior by PAR-3 and at the posterior by PAR-1. In this report, we show that the 14-3-3 protein PAR-5 is also required for cortical LET-99 asymmetry. PAR-5 associated with LET-99 in pull-down assays, and two PAR-5 binding sites were identified in LET-99 using the yeast two-hybrid assay. Mutation of these sites abolished binding in yeast and altered LET-99 localization in vivo: LET-99 was present at the highest levels at the posterior pole of the embryo instead of a band in par-5 embryos. Together the results indicate that PAR-5 acts in a mechanism with PAR-1 to regulate LET-99 cortical localization. PMID:26921457

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

    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.

  18. Conformational changes of FoxO4-DNA binding domain upon binding to 14-3-3 protein

    Šilhán, Jan; Bouřa, Evžen; Obšil, Tomáš

    2007-01-01

    Roč. 14, č. 1 (2007), s. 46-46. ISSN 1211-5894. [Discussions in Structural Molecular Biology and Bioinformatics /6./. 29.03.2007-31.03.2007, Nové Hrady] R&D Projects: GA ČR(CZ) GA204/06/0565; GA AV ČR(CZ) KJB500110601; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50110509 Keywords : cpo1 * 14-3-3 protein * PKB phosphorylation of FoxO4 * anisotropy decays Subject RIV: BO - Biophysics

  19. Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

    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 NH2-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking

  20. The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference.

    Annarita Fiorillo

    Full Text Available 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.

  1. The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference.

    Fiorillo, Annarita

    2014-03-21

    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.

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

    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. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 600-614, 2016. PMID:26297819

  3. AIP1 mediates TNF-α–induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3

    Zhang, Rong; He, Xiangrong; Liu, Weimin; Lu, Meng; Hsieh, Jer-Tsong; Min, Wang

    2003-01-01

    TNF-α activates ASK1 in part by dissociating 14-3-3 from apoptosis signal–regulating kinase 1 (ASK1). In the present study, we identified a novel Ras GTPase-activating protein (Ras-GAP) as an ASK1-interacting protein (AIP1). AIP1 binds to the C-terminal domain of ASK1 via a lysine-rich cluster within the N-terminal C2 domain. AIP1 exists in a closed form through an intramolecular interaction between the N-terminus and the C-terminus, and TNF-α induces unfolding of AIP1 leading to association ...

  4. 14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif

    Andersen, Sofie Dabros; Keijzers, Guido; Rampakakis, Emmanouil;

    2012-01-01

    Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins...

  5. Heterologous expression of Schistosoma japanicum signal protein 14-3-3 in Pichia pastoris and the subsequent immune response in mice

    Meijuan ZHENG; Jilong SHEN; Yuanhong XU; Qingli LUO

    2008-01-01

    Schistosomiasis japonica, a zoonosis caused by Schistosomajaponicum, is endemic to the Philippines and China. Several vaccine candidates have been identified and tested in different animal models, but it is still unclear which will be optimal for testing in the field. Therefore, new antigens and strategies are necessary for vaccine development against schistosomiasis japonica. The Sj14-3-3 gene was amplified and subcloned into the expression vector pPICZα-B and transformed into P. pastoris X-33 by electroporation. Three transformants were induced with methanol. The cultural supernatant was collected and tested by SDS-PAGE and Western blotting. The pro-tein of rSj14-3-3 was prepared and purified and BALB/c mice were immunized which was followed by a challen-ging infection. The immuno-protection was then evalu-ated. The Sj14-3-3 gene was expressed and secreted into the medium and its molecular weight was about 35000 as determined by SDS-PAGE. Western blotting showed that the protein had a high specificity against mouse-anti-Sj14-3-3 monoclonal antibody and rSj14-3-3 had a promising immune reactivity. The results of the immuno-protective experiments revealed that the worm reduction was 26.0%, 32.2%, and 36.8%, respectively. The number of eggs in liver tissue was reduced by 36.8%, 43.2%, and 46.1%, respectively. The recombinant Sj14-3-3 of eukaryotic expression in Pichia pastoris was successfully harvested. The molecular vaccine of Sj14-3-3 could partially induce resistance to the infection with S. japonicum in BALB/c mice. The recombinant protein Sj14-3-3 has promising immunological potentials for further approach to the dia-gnosis and development of molecular vaccine.

  6. 14-3-3 protein interacts with and affects the structure of RGS domain of regulator of G protein signaling 3 (RGS3)

    Řežábková, L.; Bouřa, E.; Herman, P.; Večeř, J.; Bouřová, Lenka; Šulc, Miroslav; Svoboda, Petr; Obšilová, Veronika; Obšil, T.

    2010-01-01

    Roč. 170, č. 3 (2010), s. 451-461. ISSN 1047-8477 R&D Projects: GA MŠk(CZ) LC554; GA AV ČR(CZ) IAA501110801 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z50200510 Keywords : 14-3-3 protein * time-resolved fluorescence * RGS3 Subject RIV: BO - Biophysics Impact factor: 3.497, year: 2010

  7. Studies on Clinical Aspects, Pathological Changes, Immunohistochemistry, 14-3-3 protein, PrP Gene, and Animal Transmission of Creutzldt-Jakob Disease in China

    Lin Shilie; Zhao Jiexu; Jiang Xinmei; Song Xiaonan; Wang Weimin; Fan Yengyeng; Tao Yuiqin; Chen Xiuyun

    2000-01-01

    Objectives To investigate the clinical manifestations, pathological changes, expression of PrP gene, 14-3-3 protein in cerebrospinal fluid (CSF) and experimental animal transmission of Creuizfeldt-Jakob disease (CJD) in China. Methods Clinical aspects of 24 patients with CJD which was confirmed neuropathological were evaluated. Brain sections of 10 cases of them were given immunostaining with antiserum to a synthetic polypeptide of prioni protein (PrP). PrP gene was analyzed in 10 cases, and 14-3-3 protein in CSF was detected in 5 cases. Experimental mouse transmission was carried out using brain suspension from 7 patients with CJD. Results 1) Nineteen cases with sporadic CJD, 3 cases with iatrogenic CJD, 1 case with inherited CJD and 1 case with coexistence of Alzheimer disease(AD) and CJD were found. 2) The percentage of acute and subacute onset was high up to 96%. The illness duration was shorter in a subacute onset and the brain atrophy was not obvious.3) The synaptic type of PrP deposition was shown in paraffin sections in all -cases by immunostaining.4) 14-3-3 protein was detected in 5 eases in cerebrospinal fluid with CJD 5) Spongiform degeneration and PrP deposition could be shown in the brain sections of experimental mouse transmission. Conclusion There are special characteristics in clinical aspects of CJD in China. The detection of 14-3-3 protein can provide objective evidence for early diagnosis of CJD in order to prevent its transmission

  8. Saccharomyces cerevisiae 14-3-3 proteins Bmh1 and Bmh2 participate in the process of catabolite inactivation of maltose permease

    Mayordomo, I.; Regelmann, J.; Horák, Jaroslav; Sanz, P.

    2003-01-01

    Roč. 544, 1-3 (2003), s. 160-164. ISSN 0014-5793 Grant ostatní: Spanish Ministry of Educaton and Science(ES) PB98-0486 Institutional research plan: CEZ:AV0Z5011922 Keywords : 14-3-3 proteins * maltose permease * catabolite inactivation Subject RIV: CE - Biochemistry Impact factor: 3.609, year: 2003

  9. Elevated levels of 14-3-3 proteins, serotonin, gamma enolase and pyruvate kinase identified in clinical samples from patients diagnosed with colorectal cancer

    Dowling, P.; Hughes, D. J.; Larkin, A.M.; Meiller, J.; Henry, M.; Meleady, P.; Lynch, V.; Pardini, B.; Naccarati, A.; Levý, M.; Vodička, Pavel; Neary, P.; Clynes, M.

    2015-01-01

    Roč. 441, feb. (2015), s. 133-141. ISSN 0009-8981 Institutional support: RVO:68378041 Keywords : biomarkers * colorectal cancer * proteomics * mass spectrometry * 14-3-3 proteins * pyruvate kinase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.824, year: 2014

  10. 14-3-3 protein C-terminal stretch occupies ligand binding groove and is displaced by phosphodipeptide binding

    Šilhán, Jan; Obšilová, Veronika; Večeř, J.; Heřman, P.; Šulc, Miroslav; Teisinger, Jan; Obšil, Tomáš

    2005-01-01

    Roč. 12, č. 1 (2005), s. 41-41. ISSN 1211-5894. [Discussions in Structural Molecular Biology /4./. 10.03.2005-12.03.2005, Nové Hrady] R&D Projects: GA AV ČR(CZ) KJB5011308; GA ČR(CZ) GA204/03/0714; GA ČR(CZ) GA309/02/1479; GA MŠk(CZ) LZ1K03020 Institutional research plan: CEZ:AV0Z5011922 Keywords : 14-3-3 * fluorescence * phosphopeptide Subject RIV: BO - Biophysics

  11. A Time-Resolved Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of 14-3-3 Protein–Protein Interaction Inhibitors

    Du, Yuhong; Fu, Robert W.; Lou, Bin; Zhao, Jing; Qui, Min; Khuri, Fadlo R.; Fu, Haian

    2013-01-01

    Protein–protein interaction networks mediate diverse biological processes by regulating various signaling hubs and clusters. 14-3-3 proteins, a family of phosphoserine/threonine-binding molecules, serve as major interaction hubs in eukaryotic cells and have emerged as promising therapeutic targets for various human diseases. In order to identify chemical probes for mechanistic studies and for potential therapeutic development, we have developed highly sensitive bioassays to monitor the intera...

  12. Effect of 14-3-3σ on Transcriptional Activity of p73 Gene%14-3-3σ对p73基因转录活性的影响

    桑梅香; 耿翠芝; 单保恩

    2007-01-01

    背景与目的:p53基因是14-3-3σ的主要调节基因,活化的p53可以诱导14-3-3σ的表达,反过来14-3-3σ又可以稳定p53的表达并且增加其转录活性.p53基因家族中的其它成员p63和p73也存存着一些与p53基因相似的功能.本研究目的在于探讨14-3-3σ对p73基因转录活性的影响.方法:采用荧光素酶报告剂分析、反转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)及Western blot研究在p53缺失型人类肺癌细胞系H1299中14-3-3σ对p73基因转录活性的调节作用;用集落形成实验研究在p53突变型人类乳腺癌细胞系MDA-MB-436中14-3-3σ对p73基因转录活性的调节作用.结果:荧光素酶报告剂分析结果显示,在H1299细胞系中,转染p73可以使bax和p21WAF1启动子介导的荧光素酶表达增加;共转染p73和14-3-3σ后,bax和p21WAFI启动子介导的荧光素酶表达均比单独转染p73时的表达高,并且荧光素酶的表达和14-3-3σ的转染剂量之间存在着浓度依赖性(P<0.01).RT-PCR和Western blot结果也显示,转染p73可以使bax和p21WAF1的表达增加;共转染p73和14-3-3σ后,bax和p21WAF1的表达均比单独转染p73时的表达高(P<0.01).集落形成实验结果显示,在MDA-MB-436细胞系中,转染p73可以使细胞的克隆数减少;共转染p73和14-3-3σ后,细胞克隆数比单独转染p73时少(P<0.01).结论:14-3-3σ可以增加p73基因的转录活性,并且14-3-3σ对p73基因转录活性的调节存在剂量依赖性.

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

    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

  14. 14-3-3 family members act coordinately to regulate mitotic progression.

    Dalal, Sorab N; Yaffe, Michael B; DeCaprio, James A

    2004-05-01

    The mitosis promoting phosphatase, cdc25C, is a target of both the DNA replication and DNA damage checkpoint pathways. These pathways regulate cdc25C function, in part, by promoting the association of cdc25C with 14-3-3 proteins, which results in the retention of cdc25C in the cytoplasm. To determine which 14-3-3 proteins were required to regulate cdc25C function, we tested the ability of various 14-3-3 family members to form a complex with and negatively regulate cdc25C in human cells. Two 14-3-3 family members, 14-3-3epsilon and 14-3-3gamma specifically formed a complex with cdc25C but not with the 14-3-3 binding defective cdc25C mutant, S216A. In addition, 14-3-3epsilon and 14-3-3gamma inhibited the ability of cdc25C, but not the S216A mutant, to induce premature chromatin condensation (PCC) in U-2OS cells. These results suggested that the reduction in PCC by 14-3-3epsilon and 14-3-3gamma was due to inhibition of cdc25C function. In contrast, 14-3-3sigma was unable to form a complex with cdc25C, but was able to inhibit the ability of both wild type cdc25C and S216A to induce PCC. This suggests that 14-3-3sigma regulates entry into mitosis independently of cdc25C and 14-3-3epsilon and 14-3-3gamma. Thus, specific members of the 14-3-3 family of proteins may act coordinately to maintain the DNA replication checkpoint by regulating the activity of different cell cycle proteins. PMID:15107609

  15. Yeast 14-3-3 proteins participate in the regulation of cell cation homeostasis via interaction with Nha1 alkali-metal-cation/proton antiporter

    Zahrádka, Jaromír; Van Heusden, G.P.H.; Sychrová, Hana

    2012-01-01

    Roč. 1820, č. 7 (2012), s. 849-858. ISSN 0304-4165 R&D Projects: GA MŠk(CZ) LC531; GA MŠk(CZ) OC10012; GA AV ČR(CZ) IAA500110801 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : yeast * 14-3-3 proteins * ion homeostasis * Nha1 antiporter Subject RIV: CE - Biochemistry Impact factor: 3.848, year: 2012

  16. 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.

    Carla Rubio-Villena

    Full Text Available 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.

  17. Intracellular targeting of peroxiredoxin 6 to lysosomal organelles requires MAPK activity and binding to 14-3-3ε

    Sorokina, Elena M.; Feinstein, Sheldon I.; Zhou, Suiping; Fisher, Aron B.

    2011-01-01

    Peroxiredoxin 6 (Prdx6), a bifunctional protein with GSH peroxidase and lysosomal-type phospholipase A2 activities, has been localized to both cytosolic and acidic compartments (lamellar bodies and lysosomes) in lung alveolar epithelium. We postulate that Prdx6 subcellular localization affects the balance between the two activities. Immunostaining localized Prdx6 to lysosome-related organelles in the MLE12 and A549 alveolar epithelial cell lines. Inhibition of trafficking by brefeldin A indic...

  18. Inhibition of blue-light-dependent binding of 14-3-3 proteins to phototropins by hydrogen peroxide

    ZHANG Xiao; SHIMAZAKI Kenichiro

    2005-01-01

    @@ Phototropins, following the discovery of phytochromes[1,2] and cryptochromes[3,4], are the most recently characterized blue-light (BL) receptors in plants. The N- terminal regions of the proteins contain two light oxygen and voltage (LOV)――LOV1 and LOV2, which belong to PAS domain involved in protein-protein interaction and ligand binding, possessing non-covalent binding sites for the chromophore FMN[5]. The C-terminal regions contain Ser/Thr kinase domains[6].

  19. Phospho-specific recognition by 14-3-3 proteins and antibodies monitored by a high throughput label-free optical biosensor.

    Wu, Meng; Coblitz, Brian; Shikano, Sojin; Long, Shunyou; Spieker, Matt; Frutos, Anthony G; Mukhopadhyay, Sunil; Li, Min

    2006-10-16

    Label-free detection of molecular interactions has considerable potential in facilitating assay development. When combined with high throughput capability, it may be applied to small molecule screens for drug candidates. Phosphorylation is a key posttranslational process that confers diverse regulation in biological systems involving specific protein-protein interactions recognizing the phosphorylated motifs. Using a resonant waveguide grating biosensor, the Epic mark System, we have developed a generic assay to quantitatively measure phospho-specific interactions between a trafficking signal-phosphorylated SWTY peptide and 14-3-3 proteins or anti-phosphopeptide antibodies. Compared with a solution-based fluorescence anisotropy assay, our results support that the high throughput resonant waveguide grating biosensor system has favorable technical profiles in detecting protein-protein interactions that recognize phosphorylated motifs. Hence it provides a new generic HTS platform for phospho-detection. PMID:17011553

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

    Julie B. McLean; 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...

  1. The 14-3-3σ gene promoter is methylated in both human melanocytes and melanoma

    Recent evidence demonstrates that 14-3-3σ acts as a tumor suppressor gene inactivated by methylation of its 5' CpG islands in epithelial tumor cells, while remaining un-methylated in normal human epithelia. The methylation analysis of 14-3-3σ has been largely overlooked in melanoma. The methylation status of 14-3-3σ CpG island in melanocytes and melanoma cells was analyzed by methylation-specific sequencing (MSS) and quantitative methylation-specific PCR (Q-MSP). 14-3-3σ mRNA and protein expression in cell lines was detected by real-time RT-PCR and western blot. Melanoma cells were also treated by 5-aza-2'-deoxycytidine (DAC), a demethylating agent, and/or histone deacetylase inhibitor, Trichostatin A (TSA), to evaluate their effects on 14-3-3σ gene expression. 14-3-3σ is hypermethylated in both human melanocytes and most melanoma cells in a lineage-specific manner, resulting in the silencing of 14-3-3σ gene expression and the active induction of 14-3-3σ mRNA and protein expression following treatment with DAC. We also observed a synergistic effect upon gene expression when DAC was combined with TSA. The promoter methylation status of 14-3-3σ was analyzed utilizing Q-MSP in 20 melanoma tissue samples and 10 cell lines derived from these samples, showing that the majority of melanoma samples maintain their hypermethylation status of the 14-3-3σ gene. 14-3-3σ is hypermethylated in human melanoma in a cell-linage specific manner. Spontaneous demethylation and re-expression of 14-3-3σ is a rare event in melanoma, indicating 14-3-3σ might have a tentative role in the pathogenesis of melanoma

  2. P53 suppresses expression of the 14-3-3gamma oncogene

    Qi Wenqing

    2011-08-01

    Full Text Available Abstract Background 14-3-3 proteins are a family of highly conserved proteins that are involved in a wide range of cellular processes. Recent evidence indicates that some of these proteins have oncogenic activity and that they may promote tumorigenesis. We previously showed that one of the 14-3-3 family members, 14-3-3gamma, is over expressed in human lung cancers and that it can induce transformation of rodent cells in vitro. Methods qRTPCR and Western blot analysis were performed to examine 14-3-3gamma expression in non-small cell lung cancers (NSCLC. Gene copy number was analyzed by qPCR. P53 mutations were detected by direct sequencing and also by western blot. CHIP and yeast one hybrid assays were used to detect p53 binding to 14-3-3gamma promoter. Results Quantitative rtPCR results showed that the expression level of 14-3-3gamma was elevated in the majority of NSCLC that we examined which was also consistent with protein expression. Further analysis of the expression pattern of 14-3-3gamma in lung tumors showed a correlation with p53 mutations suggesting that p53 might suppress 14-3-3 gamma expression. Analysis of the gamma promoter sequence revealed the presence of a p53 consensus binding motif and in vitro assays demonstrated that wild-type p53 bound to this motif when activated by ionizing radiation. Deletion of the p53 binding motif eliminated p53's ability to suppress 14-3-3gamma expression. Conclusion Increased expression of 14-3-3gamma in lung cancer coincides with loss of functional p53. Hence, we propose that 14-3-3gamma's oncogenic activities cooperate with loss of p53 to promote lung tumorigenesis.

  3. P53 suppresses expression of the 14-3-3gamma oncogene

    14-3-3 proteins are a family of highly conserved proteins that are involved in a wide range of cellular processes. Recent evidence indicates that some of these proteins have oncogenic activity and that they may promote tumorigenesis. We previously showed that one of the 14-3-3 family members, 14-3-3gamma, is over expressed in human lung cancers and that it can induce transformation of rodent cells in vitro. qRTPCR and Western blot analysis were performed to examine 14-3-3gamma expression in non-small cell lung cancers (NSCLC). Gene copy number was analyzed by qPCR. P53 mutations were detected by direct sequencing and also by western blot. CHIP and yeast one hybrid assays were used to detect p53 binding to 14-3-3gamma promoter. Quantitative rtPCR results showed that the expression level of 14-3-3gamma was elevated in the majority of NSCLC that we examined which was also consistent with protein expression. Further analysis of the expression pattern of 14-3-3gamma in lung tumors showed a correlation with p53 mutations suggesting that p53 might suppress 14-3-3 gamma expression. Analysis of the gamma promoter sequence revealed the presence of a p53 consensus binding motif and in vitro assays demonstrated that wild-type p53 bound to this motif when activated by ionizing radiation. Deletion of the p53 binding motif eliminated p53's ability to suppress 14-3-3gamma expression. Increased expression of 14-3-3gamma in lung cancer coincides with loss of functional p53. Hence, we propose that 14-3-3gamma's oncogenic activities cooperate with loss of p53 to promote lung tumorigenesis

  4. Molecular characterization and expression analysis of three homoeologous Ta14S genes encoding 14-3-3 proteins in wheat (Triticum aestivum L.

    Xinguo Wang

    2016-06-01

    Full Text Available The purpose of this study was to characterize Ta14S homoeologs and assess their functions in wheat seed development. The genomic and cDNA sequences of three Ta14S homoeologous genes encoding 14-3-3 proteins were isolated. Sequence analysis revealed that the three homoeologs consisted of five exons and four introns and were very highly conserved in the coding regions and in exon/intron structure, whereas the cDNA sequences were variable in the 5′ and 3′-UTR. The three genes, designated as Ta14S-2A, Ta14S-2B and Ta14S-2D, were located in homoeologous group 2 chromosomes. The polypeptide chains of the three Ta14S genes were highly similar. These genes were most homologous to Hv14A from barley. Real-time quantitative PCR indicated that the three Ta14S genes were differentially expressed in different organs at different developmental stages and all exhibited greater expression in primary roots of 1-day-old germlings than in other tissues. Comparison of the expression patterns of the three homoeologous genes at different times after pollination also revealed that their expression was developmentally regulated. The transcription of Ta14S-2B was clearly higher during seed germination, whereas expressions of Ta14S-2A and Ta14S-2D were up-regulated at the beginning of seed imbibition (0–12 h, but declined thereafter. The results suggested that the three Ta14S homoeologous genes have regulatory roles in seed development and germination.

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

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

    2015-09-01

    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

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

    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

  7. The C-Terminal Segment of Yeast BMH Proteins Exhibits Different Structure Compared to Other 14-3-3 Protein Isoforms

    Veisová, Dana; Řežábková, L.; Štěpánek, M.; Novotná, P.; Herman, P.; Večeř, J.; Obšil, T.; Obšilová, Veronika

    2010-01-01

    Roč. 49, č. 18 (2010), s. 3853-3861. ISSN 0006-2960 R&D Projects: GA AV ČR(CZ) IAA501110801; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50110509 Keywords : yeast BMH proteins * sedimentation equilibrium and velocity measurements * dynamic light scattering Subject RIV: BO - Biophysics Impact factor: 3.226, year: 2010

  8. Structure of a 14-3-3σ–YAP phosphopeptide complex at 1.15 Å resolution

    The first structure of a 14-3-3 protein–phosphopeptide complex is reported at 1.15 Å resolution. The YAP 14-3-3-binding motif is revealed for the first time using crystallographic tools. The 14-3-3 proteins are a class of eukaryotic acidic adapter proteins, with seven isoforms in humans. 14-3-3 proteins mediate their biological function by binding to target proteins and influencing their activity. They are involved in pivotal pathways in the cell such as signal transduction, gene expression, enzyme activation, cell division and apoptosis. The Yes-associated protein (YAP) is a WW-domain protein that exists in two transcript variants of 48 and 54 kDa in humans. By transducing signals from the cytoplasm to the nucleus, YAP is important for transcriptional regulation. In both variants, interaction with 14-3-3 proteins after phosphorylation of Ser127 is important for nucleocytoplasmic trafficking, via which the localization of YAP is controlled. In this study, 14-3-3σ has been cloned, purified and crystallized in complex with a phosphopeptide from the YAP 14-3-3-binding domain, which led to a crystal that diffracted to 1.15 Å resolution. The crystals belonged to space group C2221, with unit-cell parameters a = 82.3, b = 112.1, c = 62.9 Å

  9. Nucleocytoplasmic Shuttling of the Golgi Phosphatidylinositol 4-Kinase Pik1 Is Regulated by 14-3-3 Proteins and Coordinates Golgi Function with Cell Growth

    Demmel, Lars; Beck, Mike; Klose, Christian; Schlaitz, Anne-Lore; Gloor, Yvonne; Hsu, Peggy P.; Havlis, Jan; Shevchenko, Andrej; Krause, Eberhard; Kalaidzidis, Yannis; Walch-Solimena, Christiane

    2008-01-01

    The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown...

  10. The interaction between ADAM22 and 14-3-3β

    ZHU; Pengcheng(朱鹏程); SANG; Yingying(桑瑛颖); XU; Rener(徐人尔); ZHAO; Jing(赵璟); LI; Changben(李昌本); ZHAO; Shouyuan(赵寿元)

    2002-01-01

    ADAM family consists of a number of transmembrane proteins that contain a disintegrin and metalloprotease domain. ADAMs are involved in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis and inflammatory response. The ADAM proteins have both cell adhesion and protease activities. Adam22 is highly expressed in human brain. The adam22-/- mice presented severe ataxia and died before weaning, but the function of ADAM22 is still unknown. 14-3-3β interacting with ADAM22 was detected by using yeast two-hybrid assay. The specificity of interaction between ADAM22 and 14-3-3β was proved by in vitro binding assay and immunoprecipitation. The major 14-3-3β binding site was located in the last 28 amino acid residues of ADAM22 cytoplasmic tail. Protein 14-3-3β is abundant and plays an important role in mediating cell diffusion, migration and cell cycle control. The interaction of ADAM22 and 14-3-3β suggests that the ADAM22 may play a crucial role in neural function and development.

  11. Differential Effects of Ethanol on c-Jun N-Terminal Kinase, 14-3-3 Proteins, and Bax in Postnatal Day 4 and Postnatal Day 7 Rat Cerebellum

    Heaton, Marieta Barrow; Paiva, Michael; Kubovic, Stacey; Kotler, Alexandra; Rogozinski, Jonathan; Swanson, Eric; Madorsky, Vladimir; Posados, Michelle

    2011-01-01

    These studies investigated ethanol effects on upstream cellular elements and interactions which contribute to Bax-related apoptosis in neonatal rat cerebellum at ages of peak ethanol sensitivity (postnatal day 4 [P4]), compared to later ages of relative resistance (P7). Analyses were made of basal levels of the pro-apoptotic c-jun N-termimal kinase (JNK), Bax, and the 14-3-3 anchoring proteins, as well as the responsiveness of these substances to ethanol at P4 versus P7. Dimerization of Bax w...

  12. 14-3-3σ controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

    14-3-3σ (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 14-3-3σ 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 14-3-3σ activity in corneal epithelial cells by overexpressing dominative negative 14-3-3σ 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 14-3-3σ mutant-expressing corneal epithelial cells. We conclude that 14-3-3σ is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

  13. 14-3-3{sigma} controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

    Xin, Ying [Stem Cell Institute, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Lu, Qingxian [Tumor Immunobiology Group, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Li, Qiutang, E-mail: q.li@louisville.edu [Stem Cell Institute, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States)

    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.

  14. 14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport

    Brunet, Anne; Kanai, Fumihiko; Stehn, Justine; Xu, Jian; Sarbassova, Dilara; Frangioni, John V.; Dalal, Sorab N.; DeCaprio, James A.; Greenberg, Michael E.; Yaffe, Michael B.

    2002-01-01

    14-3-3 proteins regulate the cell cycle and prevent apoptosis by controlling the nuclear and cytoplasmic distribution of signaling molecules with which they interact. Although the majority of 14-3-3 molecules are present in the cytoplasm, we show here that in the absence of bound ligands 14-3-3 homes to the nucleus. We demonstrate that phosphorylation of one important 14-3-3 binding molecule, the transcription factor FKHRL1, at the 14-3-3 binding site occurs within the nucleus immediately bef...

  15. 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

    Sakai Keiko

    2008-04-01

    Full Text Available Abstract Background Despite continuing advances in our understanding of AIDS pathogenesis, the mechanism of CD4+ T cell depletion in HIV-1-infected individuals remains unclear. The HIV-1 Vpr accessory protein causes cell death, likely through a mechanism related to its ability to arrest cells in the G2,M phase. Recent evidence implicated the scaffold protein, 14-3-3, in Vpr cell cycle blockade. Results We found that in human T cells, 14-3-3 plays an active role in mediating Vpr-induced cell cycle arrest and reveal a dramatic increase in the amount of Cdk1, Cdc25C, and CyclinB1 bound to 14-3-3 θ during Vprv-induced G2,M arrest. By contrast, a cell-cycle-arrest-dead Vpr mutant failed to augment 14-3-3 θ association with Cdk1 and CyclinB1. Moreover, G2,M arrest caused by HIV-1 infection strongly correlated with a disruption in 14-3-3 θ binding to centrosomal proteins, Plk1 and centrin. Finally, Vpr caused elevated levels of CyclinB1, Plk1, and Cdk1 in a complex with the nuclear transport and spindle assembly protein, importin β. Conclusion Thus, our data reveal a new facet of Vpr-induced cell cycle arrest involving previously unrecognized abnormal rearrangements of multiprotein assemblies containing key cell cycle regulatory proteins. Reviewers This article was reviewed by David Kaplan, Nathaniel R. Landau and Yan Zhou.

  16. The role of epigenetic inactivation of 14-3-3σin human cancer

    Dmitri LODYGIN; Heiko HERMEKING

    2005-01-01

    Cancer cells show characteristic alterations in DNA methylation patterns. Aberrant CpG methylation of specific promoters results in inactivation of tumor suppressor genes and therefore plays an important role in carcinogenesis. The p53-regulated gene 14-3-3σ undergoes frequent epigenetic silencing in several types of cancer, including carcinoma of the breast, prostate, and skin, suggesting that the loss of 14-3-3σ expression may be causally involved in tumor progression.Functional studies demonstrated that 14-3-3σ is involved in cell-cycle control and prevents the accumulation of chromosomal damage. The recent identification of novel 14-3-3σ-associated proteins by a targeted proteomics approach implies that 14-3-3σ regulates diverse cellular processes, which may become deregulated after silencing of 14-3-3σ expression in cancer cells.

  17. 14-3-3 Sigma And p53 Expression in Gastric Cancer and Its Clinical Applications

    Gilbert Mühlmann

    2010-01-01

    Full Text Available 14-3-3 sigma (σ induces G2 arrest enabling the repair of damaged DNA. The function of 14-3-3 σ is frequently lost in tumor cells, indicating a potential tumor suppressor function. The purpose of this study was to evaluate the prognostic value of 14-3-3 σ expression in human gastric cancer. 14-3-3 σ expression was analyzed by immunohistochemistry in 157 tumor samples of patients, who underwent resection for gastric cancer. Since 14-3-3 σ is involved in the p53 network, p53 expression was detected in parallel and correlated with 14-3-3 σ. 14-3-3 σ was found to be overexpressed in 75 (47.8% of 157 cases, the overexpression rate of p53 protein was 27.4%. 14-3-3 σ overexpression was statistically significantly associated with pT-stage (p=0.041 pN-stage (p=0.015 and UICC-stage (p=0.019 and showed a borderline significance with Lauren classification (p=0.057. Univariate survival calculations revealed a coexistent 14-3-3 σ and p53 overexpression as a significant predictor of disease-free survival. Multivariate analysis did not unfold 14-3-3 as an independent prognostic factor for disease-free survival and overall survival. Concomitant 14-3-3 σ and p53 overexpression in tumor cells of patients with gastric cancer identifies a population of patients with relatively unfavorable prognosis.

  18. Role of AKT/ PKB and 14-3-3 in the regulation of B cell receptor signaling and signalosome assembly

    Mohammad, Dara

    2015-01-01

    AKT/PKB is an oncogenic serine/threonine kinase regulated via the PI3K pathways. 14-3-3s represent a large group of adaptor proteins that are known to interact with a plethora of signaling proteins and regulate diverse signal transduction pathways. The B-cell antigen receptor (BCR) activation and signalosome assembly are dynamic processes controlled by protein phosphorylation. The signaling events share their functions in controlling cell proliferation, differentiation, and/or apoptosis. ...

  19. Over-expression of 14-3-3zeta is an early event in oral cancer

    The functional and clinical significance of 14-3-3 proteins in human cancers remain largely undetermined. Earlier, we have reported differential expression of 14-3-3ζ mRNA in oral squamous cell carcinoma (OSCC) by differential display. The clinical relevance of 14-3-3ζ protein in oral tumorigenesis was determined by immunohistochemistry in paraffin embedded sections of oral pre-malignant lesions (OPLs), OSCCs and histologically normal oral tissues and corroborated by Western Blotting. Co-immunoprecipitation assays were carried out to determine its association with NFκB, β-catenin and Bcl-2. Intense immunostaining of 14-3-3ζ protein was observed in 61/89 (69%) OPLs and 95/120 (79%) OSCCs. Immunohistochemistry showed significant increase in expression of 14-3-3ζ protein from normal mucosa to OPLs to OSCCs (ptrend < 0.001). Significant increase in expression of 14-3-3ζ protein was observed as early as in hyperplasia (p = 0.009), with further elevation in moderate and severe dysplasia, that was sustained in OSCCs. These findings were validated by Western blotting. Using Co-immunoprecipitation, we demonstrated that 14-3-3ζ protein binds to NFκB, β-catenin and Bcl-2, suggesting its involvement in cellular signaling, leading to proliferation of oral cancer cells. Our findings suggest that over-expression of 14-3-3ζ is an early event in oral tumorigenesis and may have an important role in its development and progression. Thus, 14-3-3ζ may serve as an important molecular target for designing novel therapy for oral cancer

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

    Mariana Ferreira Leal; Danielle Queiroz Calcagno; S(a)mia Demachki; Paulo Pimentel Assump(c)(a)o; Roger Chammas; Rommel Rodríguez Burbano; Marília de Arruda Cardoso Smith

    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.

  1. 14-3-3ζ Overexpression Defines High Risk for Breast Cancer Recurrence and Promotes Cancer Cell Survival

    Neal, Christopher L.; Yao, Jun; Yang, Wentao; ZHOU, XIAOYAN; Nguyen, Nina T.; LU, JING; Danes, Christopher G.; Guo, Hua; Lan, Keng-Hsueh; Ensor, Joe; Hittelman, Walter; Hung, Mien-Chie; Yu, Dihua

    2009-01-01

    The ubiquitously expressed 14-3-3 proteins are involved in numerous important cellular functions. The loss of 14-3-3σ is a common event in breast cancer; however, the role of other 14-3-3s in breast cancer is unclear. Recently, we found that 14-3-3ζ overexpression occurs in early stage breast diseases and contributes to transformation of human mammary epithelial cells. Here, we show that 14-3-3ζ overexpression also persisted in invasive ductal carcinoma and contributed to the further progress...

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

    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

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

    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

  4. Aberrant upregulation of 14-3-3ơ expression serves as an inferior prognostic biomarker for gastric cancer

    Li Hai-gang

    2011-09-01

    Full Text Available Abstract Background 14-3-3ơ is an intracellular, phosphoserine binding protein and proposed to be involved in tumorigenesis. However, the expression dynamics of 14-3-3ơ and its clinicopathological/prognostic significance in human tumors are still controversial. Methods The method of immunohistochemistry (IHC and Western blot were utilized to examine the protein expression of 14-3-3ơ in gastric cancer and paired normal adjacent gastric mucosal tissues. Receive operating characteristic (ROC curve analysis was employed to determine a cutoff score for 14-3-3ơ expression in a training set (n = 66. For validation, the ROC-derived cutoff score was subjected to analysis of the association of 14-3-3ơ expression with patient outcome and clinical characteristics in a testing set (n = 86 and overall patients (n = 152. Results The expression frequency and expression levels of 14-3-3ơ were significantly higher in gastric cancer than in normal gastric mucosal tissues. Correlation analysis demonstrated that high expression of 14-3-3ơ in gastric cancer was significantly correlated with clinical stage and tumor invasion. Furthermore, in the testing set and overall patients, Kaplan-Meier analysis showed that elevated 14-3-3ơ expression predicted poorer overall survival (OS and progression-free survival (PFS. Importantly, high 14-3-3ơ expression was also associated with shortened survival time in stage III and stage IV gastric cancer patients. Multivariate analyses revealed that 14-3-3ơ expression was an independent prognostic parameter in gastric cancer. Conclusions These findings provide evidence that high expression of 14-3-3ơ may be important in the tumor progression and servers as an independent molecular marker for poor prognosis of gastric cancer. Thus, overexpression of 14-3-3ơ identifies patients at high risk and is a novel therapeutic molecular target for this tumor.

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

    Cao, Lei; Lei, Hui; Chang, Ming-Ze; Liu, Zhi-Qin [Department of Neurological Disease, Xi' an Central Hospital, Xi' an Jiaotong University, Xi' an, Shaanxi 710000 (China); Bie, Xiao-Hua, E-mail: biexiaohua_xjtu@126.com [Department of Functional Neurosurgery, Xi' an Red Cross Hospital, Xi' an Jiaotong University, Xi' an, Shaanxi 710054 (China)

    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.

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

    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

  7. Effects of physical exercise on the P38MAPK/REDD1/14-3-3 pathways in the myocardium of diet-induced obesity rats.

    Pieri, B L S; Souza, D R; Luciano, T F; Marques, S O; Pauli, J R; Silva, A S R; Ropelle, E R; Pinho, R A; Lira, F S; De Souza, C T

    2014-08-01

    Obesity is associated with myocardial insulin resistance and impairment of the mammalian target of rapamycin (mTOR) signaling pathway. The activation of the mTOR cascade by exercise has been largely shown in skeletal muscle, but insufficiently analyzed in myocardial tissue. In addition, little is known regarding the mTOR upstream molecules in the hearts of obese animals and even less about the role of exercise in this process. Thus, the present study was aimed to evaluate the effects of physical exercise on P38 Mitogen-Activated Protein Kinase (P38MAPK) phosphorylation and the REDD1 (regulated in development and DNA damage responses 1) and 14-3-3 protein levels in the myocardium of diet-induced obesity (DIO) rats. After achievement of DIO and insulin resistance, Wistar rats were divided in 2 groups: sedentary obese rats and obese rats performed treadmill running (50-min/day, 5 days per week velocity of 1.0 km/h for 2 months). Forty-eight hours after the final physical exercise, the rats were killed, and the myocardial tissue was removed for Western blot analysis. DIO increased the REDD1 protein levels and reduced the 14-3-3 protein levels and P38MAPK, mTOR, P70S6k (p70 ribosomal S6 protein kinase), and 4EBP1 (4E-binding protein-1) phosphorylation. Interestingly, physical exercise reduced the REDD1 protein levels and increased the 14-3-3 protein levels and P38MAPK, mTOR, P70S6k, and 4EBP1 phosphorylation. Moreover, exercise increased the REDD1/14-3-3 association in the heart. Our results indicate that the phospho-P38MAPK, REDD1, and 14-3-3 protein levels were reduced in the myocardium of obese rats and that physical exercise increased the protein levels of these molecules. PMID:24691733

  8. Aberrant overexpression of an epithelial marker, 14-3-3σ, in a subset of hematological malignancies

    14-3-3σ is a p53-mediated cell-cycle inhibitor in epithelial cells. The expression of 14-3-3σ is frequently altered in cancers of epithelial origin associated with altered DNA methylation. Since its involvement in a non-epithelial tumor is unknown, we examined 14-3-3σ expression in patients with haematological malignancies. We analyzed 41 hematopoietic cell lines and 129 patients with a variety of hematological malignancies for 14-3-3σ expression with real-time RT-PCR. We also examined protein levels by Western blot analysis and DNA methylation status of the 14-3-3σ gene by methylation-specific PCR analysis of bisulfite-treated DNA. In addition, mutations of p53 gene were identified by RT-PCR-SSCP analysis and the expression levels of 14-3-3σ were compared with those of other cell-cycle inhibitor genes, CDKN2A and ARF. The expression levels of 14-3-3σ mRNA in almost all cell lines were low and comparable to those in normal hematopoietic cells except for 2 B-cell lines. On the contrary, 14-3-3σ mRNA was aberrantly overexpressed frequently in mature lymphoid malignancies (30 of 93, 32.3%) and rarely in acute leukemia (3 of 35, 8.6%). 14-3-3σ protein was readily detectable and roughly reflected the mRNA level. In contrast to epithelial tumors, methylation status of the 14-3-3σ gene was not associated with expression in hematological malignancies. Mutations of p53 were identified in 12 patients and associated with lower expression of 14-3-3σ. The expression levels of 14-3-3σ, CDKN2A and ARF were not correlated with but rather reciprocal to one another, suggesting that simultaneous overexpression of any two of them is incompatible with tumor growth. 14-3-3σ, an epithelial cell marker, was overexpressed significantly in a subset of mature lymphoid malignancies. This is the first report of aberrant 14-3-3σ expression in non-epithelial tumors in vivo. Since the significance of 14-3-3σ overexpression is unknown even in epithelial tumors such as pancreatic

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

    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.

  10. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells.

    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

  11. Pear 14-3-3a gene (Pp14-3-3a) is regulated during fruit ripening and senescense, and involved in response to salicylic acid and ethylene signalling

    Haiyan Shi; Yuxing Zhang

    2014-12-01

    14-3-3 proteins play important roles in regulating plant development and phytohormone (abscisic acid, gibberellin and brassinosteroids) signalling. However, their regulation in fruit ripening and senescense, and response to salicylic acid and ethylene signalling are yet to be illustrated. One cDNA encoding putative 14-3-3 protein was isolated from pear (Pyrus pyrifolia) and designated Pp14-3-3a. Phylogenetic analysis clearly demonstrated that Pp14-3-3a belonged to -like group of 14-3-3 super-families. Real-time quantitative PCR analysis indicated that the expression of Pp14-3-3a gene was developmentally regulated in the fruit. Further study demonstrated that Pp14-3-3a expression was inhibited by salicylic acid and induced by ethylene precursor 1-aminocyclopropane-1-carboxylic acid in pear fruit. These data suggested that Pp14-3-3a might be involved in response to salicylic acid and ethylene signalling during fruit ripening and senescence of pear.

  12. A role of TGFß1 dependent 14-3-3σ phosphorylation at Ser69 and Ser74 in the regulation of gene transcription, stemness and radioresistance.

    Olena Zakharchenko

    Full Text Available Transforming growth factor-β (TGFβ is a potent regulator of tumorigenesis, although mechanisms defining its tumor suppressing and tumor promoting activities are not understood. Here we describe phosphoproteome profiling of TGFβ signaling in mammary epithelial cells, and show that 60 identified TGFβ-regulated phosphoproteins form a network with scale-free characteristics. The network highlighted interactions, which may distribute signaling inputs to regulation of cell proliferation, metabolism, differentiation and cell organization. In this report, we identified two novel and TGFβ-dependent phosphorylation sites of 14-3-3σ, i.e. Ser69 and Ser74. We observed that 14-3-3σ phosphorylation is a feed-forward mechanism in TGFβ/Smad3-dependent transcription. TGFβ-dependent 14-3-3σ phosphorylation may provide a scaffold for the formation of the protein complexes which include Smad3 and p53 at the Smad3-specific CAGA element. Furthermore, breast tumor xenograft studies in mice and radiobiological assays showed that phosphorylation of 14-3-3σ at Ser69 and Ser74 is involved in regulation of cancer progenitor population and radioresistance in breast cancer MCF7 cells. Our data suggest that TGFβ-dependent phosphorylation of 14-3-3σ orchestrates a functional interaction of TGFβ/Smad3 with p53, plays a role in the maintenance of cancer stem cells and could provide a new potential target for intervention in breast cancer.

  13. 14-3-3 zeta is a molecular target in guggulsterone induced apoptosis in Head and Neck cancer cells

    The five-year survival rates for head and neck squamous cell carcinoma (HNSCC) patients are less than 50%, and the prognosis has not improved, despite advancements in standard multi-modality therapies. Hence major emphasis is being laid on identification of novel molecular targets and development of multi-targeted therapies. 14-3-3 zeta, a multifunctional phospho-serine/phospho-threonine binding protein, is emerging as an effector of pro-survival signaling by binding to several proteins involved in apoptosis (Bad, FKHRL1 and ASK1) and may serve as an appropriate target for head and neck cancer therapy. Herein, we determined effect of guggulsterone (GS), a farnesoid X receptor antagonist, on 14-3-3 zeta associated molecular pathways for abrogation of apoptosis in head and neck cancer cells. Head and neck cancer cells were treated with guggulsterone (GS). Effect of GS-treatment was evaluated using cell viability (MTT) assay and apoptosis was verified by annexin V, DNA fragmentation and M30 CytoDeath antibody assay. Mechanism of GS-induced apoptosis was determined by western blotting and co-IP assays using specific antibodies. Using in vitro models of head and neck cancer, we showed 14-3-3 zeta as a key player regulating apoptosis in GS treated SCC4 cells. Treatment with GS releases BAD from the inhibitory action of 14-3-3 zeta in proliferating HNSCC cells by activating protein phosphatase 2A (PP2A). These events initiate the intrinsic mitochondrial pathway of apoptosis, as revealed by increased levels of cytochrome c in cytoplasmic extracts of GS-treated SCC4 cells. In addition, GS treatment significantly reduced the expression of anti-apoptotic proteins, Bcl-2, xIAP, Mcl1, survivin, cyclin D1 and c-myc, thus committing cells to apoptosis. These events were followed by activation of caspase 9, caspase 8 and caspase 3 leading to cleavage of its downstream target, poly-ADP-ribose phosphate (PARP). GS targets 14-3-3 zeta associated cellular pathways for reducing

  14. Class-specific evolution and transcriptional differentiation of 14-3-3 family members in mesohexaploid Brassica rapa

    Ruby eChandna; Rehna eAugustine; Praveena eKanchupati; Roshan eKumar; Pawan eKumar; Arya, Gulab C.; Naveen Chandra Bisht

    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 cont...

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

    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 cont...

  16. 14-3-3σ regulates β-catenin-mediated mouse embryonic stem cell proliferation by sequestering GSK-3β.

    Tzu-Ching Chang

    Full Text Available BACKGROUND: Pluripotent embryonic stem cells are considered to be an unlimited cell source for tissue regeneration and cell-based therapy. Investigating the molecular mechanism underlying the regulation of embryonic stem cell expansion is thus important. 14-3-3 proteins are implicated in controlling cell division, signaling transduction and survival by interacting with various regulatory proteins. However, the function of 14-3-3 in embryonic stem cell proliferation remains unclear. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we show that all seven 14-3-3 isoforms were detected in mouse embryonic stem cells. Retinoid acid suppressed selectively the expression of 14-3-3σ isoform. Knockdown of 14-3-3σ with siRNA reduced embryonic stem cell proliferation, while only 14-3-3σ transfection increased cell growth and partially rescued retinoid acid-induced growth arrest. Since the growth-enhancing action of 14-3-3σ was abrogated by β-catenin knockdown, we investigated the influence of 14-3-3σ overexpression on β-catenin/GSK-3β. 14-3-3σ bound GSK-3β and increased GSK-3β phosphorylation in a PI-3K/Akt-dependent manner. It disrupted β-catenin binding by the multiprotein destruction complex. 14-3-3σ overexpression attenuated β-catenin phosphorylation and rescued the decline of β-catenin induced by retinoid acid. Furthermore, 14-3-3σ enhanced Wnt3a-induced β-catenin level and GSK-3β phosphorylation. DKK, an inhibitor of Wnt signaling, abolished Wnt3a-induced effect but did not interfere GSK-3β/14-3-3σ binding. SIGNIFICANCE: Our findings show for the first time that 14-3-3σ plays an important role in regulating mouse embryonic stem cell proliferation by binding and sequestering phosphorylated GSK-3β and enhancing Wnt-signaled GSK-3β inactivation. 14-3-3σ is a novel target for embryonic stem cell expansion.

  17. 14-3-3/HIP-55 complex increases the stability of HIP-55%14-3-3/HIP-55复合体增强HIP-55蛋白稳定性

    田爱炬; 李子健

    2015-01-01

    目的:用双分子荧光互补及免疫共沉淀技术验证HIP-55与14-3-3在HEK293细胞中存在相互作用,并进一步研究其生物学意义. 方法:利用GATEWAY系统构建PDEST-N-Venus-HIP-55WT(野生型),PDEST-N-Venus-HIP-55AA(突变体S269A/T291A),PDEST-GST-HIP-55WT及PDEST-C-Venus-14-3-3τ重组质粒,利用双分子荧光互补及免疫共沉淀技术检测两者的相互作用,同时应用14-3-3蛋白相互作用抑制肽R18和HIP-55蛋白突变体( HIP-55AA突变体S269A/T291A不能与14-3-3相互作用)作为工具研究两者结合后对嘌呤霉素诱导的HIP-55蛋白表达的影响. 结果:外源转入的Venus-HIP-55WT、Venus-HIP-55AA及Venus-14-3-3蛋白能够在HEK293细胞中表达;双分子荧光互补实验结果表明HIP-55与14-3-3存在相互作用,HIP-55蛋白的S269/T291位点介导HIP-55与14-3-3的相互作用;免疫共沉淀技术表明内源性HIP-55与14-3-3存在相互作用;进一步研究发现HIP-55与14-3-3复合体增强HIP-55蛋白的稳定性,保护HIP-55不被降解. 结论:14-3-3与HIP-55存在相互作用,14-3-3/HIP-55复合体可以促进HIP-55蛋白的稳定性.%Objective:To further demonstrate the interaction of a new 14-3-3 interaction protein hema-topoietic progenitor kinase 1 [ HPK1 ]-interacting protein ( HIP-55 ) and 14-3-3 proteins and its potential biological function in HEK293 cells. Methods:PDEST-N-Venus-HIP-55WT (wild type),PDEST-N-Ve-nus-HIP-55AA (mutants, S269A/T291A, abolishing the binding of HIP-55 to 14-3-3),PDEST-GST-HIP-55WT and PDEST-C-Venus-14-3-3τplasmids were constructed by gateway system. Their expressions were demonstrated by Western blotting method. Then we used Bimolecular Fluorescence Complementation ( BiFC) and co-immunoprecipitation ( co-IP) methods to demonstrate the interaction of HIP-55 and 14-3-3 in HEK293 cells. Moreover, the 14-3-3 antagonist peptide, R18 and HIP-55 protein mutant plasmid HIP-55 AA were used to detect the protein synthesis of HIP-55 at different time points

  18. 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

    Lixin Liu

    2015-07-01

    Full Text Available 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

  19. 14-3-3 regulates the nuclear import of class IIa histone deacetylases

    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.

  20. Interaction of hookworm 14-3-3 with the forkhead transcription factor DAF-16 requires intact Akt phosphorylation sites

    Hawdon John M

    2009-04-01

    Full Text Available Abstract Background Third-stage infective larvae (L3 of hookworms are in an obligatory state of developmental arrest that ends upon entering the definitive host, where they receive a signal that re-activates development. Recovery from the developmentally arrested dauer stage of Caenorhabditis elegans is analogous to the resumption of development during hookworm infection. Insulin-like signaling (ILS mediates recovery from arrest in C. elegans and activation of hookworm dauer L3. In C. elegans, phosphorylation of the forkhead transcription factor DAF-16 in response to ILS creates binding cites for the 14-3-3 protein Ce-FTT-2, which translocates DAF-16 out of the nucleus, resulting in resumption of reproductive development. Results To determine if hookworm 14-3-3 proteins play a similar role in L3 activation, hookworm FTT-2 was identified and tested for its ability to interact with A. caninum DAF-16 in vitro. The Ac-FTT-2 amino acid sequence was 91% identical to the Ce-FTT-2, and was most closely related to FTT-2 from other nematodes. Ac-FTT-2 was expressed in HEK 293T cells, and was recognized by an antibody against human 14-3-3β isoform. Reciprocal co-immunoprecipitations using anti-epitope tag antibodies indicated that Ac-FTT-2 interacts with Ac-DAF-16 when co-expressed in serum-stimulated HEK 293T cells. This interaction requires intact Akt consensus phosphorylation sites at serine107 and threonine312, but not serine381. Ac-FTT-2 was undetectable by Western blot in excretory/secretory products from serum-stimulated (activated L3 or adult A. caninum. Conclusion The results indicate that Ac-FTT-2 interacts with DAF-16 in a phosphorylation-site dependent manner, and suggests that Ac-FTT-2 mediates activation of L3 by binding Ac-DAF-16 during hookworm infection.

  1. 14-3-3σ is an independent prognostic biomarker for gastric cancer and is associated with apoptosis and proliferation in gastric cancer.

    Li, Yi-Liang; Liu, Lihua; Xiao, Yang; Zeng, Tao; Zeng, Chao

    2015-01-01

    14-3-3 proteins participate in various cellular processes, including apoptosis, proliferation and malignant transformation. 14-3-3σ, a member of the 14-3-3 protein family, is important in several types of cancer; however, little is known about the clinical significance and biological roles of 14-3-3σ in gastric cancer. The present study analyzed the expression pattern of 14-3-3σ in gastric cancer and investigated its correlation with the prognosis of gastric cancer patients. Furthermore, the association of 14-3-3σ with Ki-67, Bcl-2 and Bax was evaluated. 14-3-3σ was expressed at higher level in gastric cancer tissue compared with healthy gastric tissue, and 14-3-3σ expression was significantly correlated with tumor size and tumor node metastasis stage (Pknowledge, the present study data are the first to suggest that 14-3-3σ expression has been significantly associated with poor prognosis in gastric cancer. Additionally, 14-3-3σ overexpression was positively correlated with Ki-67 and Bcl-2 expression levels. Thus, 14-3-3σ is a potential prognostic marker for gastric cancer patients, and may be involved in regulating the apoptosis and proliferation of gastric cancer cells. PMID:25435977

  2. Rôle des protéines 14-3-3 dans la régulation de la longévité par la voie DAF-2/Insuline/IGF-1 chez Caenorhabditis elegans

    Araiz, Caroline

    2008-01-01

    14-3-3 proteins are ubiquitous proteins highly conserved among eukaryotes. They are involved in the regulation of several cellular processes by modifying the function and the localization of many targeted proteins. FTT-1 and FTT-2 are the two 14-3-3 orthologs identified in C. elegans. We showed that FTT proteins are involved in the main longevity pathway of C. elegans : the AF-2/Insulin/IGF-1-like pathway which also regulates stress resistance and metabolism through the activity of its final ...

  3. 14-3-3-zeta participates in TLR3-mediated TICAM-1 signal-platform formation.

    Funami, Kenji; Matsumoto, Misako; Obuse, Chikashi; Seya, Tsukasa

    2016-05-01

    Recognition of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) is important in innate immune signaling. Toll-like receptors (TLRs) are well-characterized PRRs and are pivotal in antiviral and antitumor host defense. TIR domain-containing adaptor molecule 1 (TICAM-1, also called TRIF) is an adapter molecule in TLR3- and TLR4-mediated IRF3 activation, late-phase NF-κB activation and MAPK-mediated AP-1 activation. When a TLR3 ligand is added to TLR3-positive cells, TICAM-1 transiently interacts with TLR3 and forms multimers in the cytosol. However, the precise mechanism of TICAM-1 multimer formation remains unknown. In this study, we identified 14-3-3-zeta as a molecule that functions in TLR3-mediated signaling. Knockdown of 14-3-3-zeta reduced production of type I interferon and inflammatory cytokines, nuclear translocation of IRF3 and phosphorylation of IκB via the TLR3-TICAM-1 pathway. Furthermore, TICAM-1 multimerization by ligand stimulation was prohibited by 14-3-3-zeta knockdown. These results suggest that 14-3-3-zeta is involved in the TLR3-TICAM-1 pathway in promoting multimerization of TICAM-1 for the formation of a TICAM-1 signalosome. PMID:27058640

  4. 14-3-3epsilon contributes to tumour suppression in laryngeal carcinoma by affecting apoptosis and invasion

    14-3-3epsilon regulates a wide range of biological processes, including cell cycle control, proliferation, and apoptosis, and plays a significant role in neurogenesis and the formation of malignant tumours. However, the exact function and regulatory mechanism of 14-3-3epsilon in carcinogenesis have not been elucidated. The expression of 14-3-3epsilon was assessed by RT-PCR and western blotting. The invasiveness and viability of Hep-2 cells were determined by the transwell migration assay and MTT assay, respectively. Cell cycle and apoptosis of Hep-2 cells were detected by flow cytometry. The mRNA and protein expression of 14-3-3epsilon in larynx squamous cell carcinoma (LSCC) tissues were significantly lower than those in clear surgical margin tissues. Statistical analysis showed that the 14-3-3epsilon protein level in metastatic lymph nodes was lower than that in paired tumour tissues. In addition, the protein level of 14-3-3epsilon in stage III or IV tumours was significantly lower than that in stage I or II tumours. Compared with control Hep-2 cells, the percentages of viable cells in the 14-3-3epsilon-GFP and negative control GFP groups were 36.68 ± 14.09% and 71.68 ± 12.10%, respectively. The proportions of S phase were 22.47 ± 3.36%, 28.17 ± 3.97% and 46.15 ± 6.82%, and the apoptotic sub-G1 populations were 1.23 ± 1.02%, 2.92 ± 1.59% and 13.72 ± 3.89% in the control, negative control GFP and 14-3-3epsilon-GFP groups, respectively. The percentages of the apoptotic cells were 0.84 ± 0.25%, 1.08 ± 0.24% and 2.93 ± 0.13% in the control, negative control GFP and 14-3-3epsilon-GFP groups, respectively. The numbers of cells that penetrated the filter membrane in the control, negative control GFP and 14-3-3epsilon-GFP groups were 20.65 ± 1.94, 17.63 ± 1.04 and 9.1 ± 0.24, respectively, indicating significant differences among the different groups. Decreased expression of 14-3-3epsilon in LSCC tissues contributes to the initiation and progression of LSCC

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

    Nefla, Meriam; Sudre, Laure; Denat, Guillaume; Priam, Sabrina; André-Leroux, Gwenaelle; 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 kno...

  6. Regulation of poly(A) polymerase by 14-3-3ε

    Kim, Hana; Lee, June Hyung; Lee, Younghoon

    2003-01-01

    Poly(A) polymerase (PAP) is a key enzyme responsible for the addition of the poly(A) at the 3′ end of pre-mRNA. The C-terminal region of mammalian PAP carries target sites for protein–protein interaction with the 25 kDa subunit of cleavage factor I and with splicing factors U1A and U2AF65. We used a yeast two-hybrid screen to identify 14-3-3ε as an additional protein binding to the C-terminal region of PAP. Interaction between PAP and 14-3-3ε was confirmed by both in vitro and in vivo binding...

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

    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 (pratio - 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

  8. Cloning, expression and identification of the 14-3-3 gene of Trichomonas vaginalis%阴道毛滴虫14-3-3基因的克隆、表达及鉴定

    刘畅; 张西臣; 丁鹤; 刘鑫; 宫鹏涛; 李建华; 李淑红; 李赫; 张国才; 杨举

    2012-01-01

    目的 克隆阴道毛滴虫14-3-3基因并进行原核表达.方法 根据阴道毛滴虫14-3-3基因序列设计特异性引物,以阴道毛滴虫cDNA为模板通过PCR扩增获得目的片段,与pMD-18-T连接,构建克隆载体pMD-Tv-14-3-3,经双酶切后回收目的片段,进行测序鉴定,然后与表达载体pGEX-T连接,构建原核表达载体pGEX-T-Tv-14-3-3,用异丙基-β-D硫代半乳糖苷(IPTG)诱导表达,通过SDS-PAGE及Western blot鉴定表达产物.结果 成功构建了阴道毛滴虫14-3-3基因原核表达载体pGEX-T-Tv-14-3-3;SDS-PAGE电泳检测显示,在IPTG诱导下,阳性菌高效表达分子质量单位为27 ku的蛋白质;Western blot显示表达产物可被抗阴道毛滴虫多克隆血清识别.结论 成功构建了阴道毛滴虫14-3-3基因原核表达载体,并在大肠埃希菌BL21(DE3)中高效表达.%Objective To clone and express 14-3-3 gene of Trichomonas vaginalis. Methods Special primers were designed on the basis of the reported T. vaginalis 14-3-3 gene. The 14-3-3 gene was amplified by PCR from the total DNA of T. vaginalis and was cloned into pMD-18-T to construct pMD-14-3-3. The plasmid pMD-14-3-3 was then digested with restriction ribozymes and subcloned into the prokaryotic expression plasmid pGEXT to construct pGEX-T-Tv-14-3-3. It was then expressed in E. coli BL21 (DE3) induced with IPTG. The fusion product was identified by SDS-PAGE and Western blot. Results A prokaryotic expression vector of the 14-3-3 gene was constructed and expressed in Esche-richia. coli. Induced with IPTG, the expressed recombinant protein was detected as a band of 27 ku by SDS-PAGE. A special reaction band to anti-14-3-3 sera was observed in Western blot. Conclusion The fusion protein of the 14-3-3 gene was successfully expressed in prokaryotic cells.

  9. The peripheral binding of 14-3-3γ to membranes involves isoform-specific histidine residues.

    Helene J Bustad

    Full Text Available 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.

  10. A quantitative 14-3-3 interaction screen connects the nuclear exosome targeting complex to the DNA damage response

    Blasius, Melanie; Wagner, Sebastian A; Choudhary, Chuna Ram;

    2014-01-01

    RNA metabolism is altered following DNA damage, but the underlying mechanisms are not well understood. Through a 14-3-3 interaction screen for DNA damage-induced protein interactions in human cells, we identified protein complexes connected to RNA biology. These include the nuclear exosome...

  11. Class-specific evolution and transcriptional differentiation of 14-3-3 family members in mesohexaploid Brassica rapa

    Ruby eChandna

    2016-01-01

    Full Text Available 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 (Ks0.48, 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.GRF14s indicated the occurrence of functional divergence of B. rapa 14-3-3 proteins during plant development and stress responses.

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

    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 0.48), 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

  13. Nuclear export regulation of COP1 by 14-3-3σ in response to DNA damage

    Chen Jian

    2010-09-01

    Full Text Available Abstract Mammalian constitutive photomorphogenic 1 (COP1 is a p53 E3 ubiquitin ligase involved in regulating p53 protein level. In plants, the dynamic cytoplasm/nucleus distribution of COP1 is important for its function in terms of catalyzing the degradation of target proteins. In mammalian cells, the biological consequence of cytoplasmic distribution of COP1 is not well characterized. Here, we show that DNA damage leads to the redistribution of COP1 to the cytoplasm and that 14-3-3σ, a p53 target gene product, controls COP1 subcellular localization. Investigation of the underlying mechanism suggests that COP1 S387 phosphorylation is required for COP1 to bind 14-3-3σ. Significantly, upon DNA damage, 14-3-3σ binds to phosphorylated COP1 at S387, resulting in COP1's accumulation in the cytoplasm. Cytoplasmic COP1 localization leads to its enhanced ubiquitination. We also show that N-terminal 14-3-3σ interacts with COP1 and promotes COP1 nuclear export through its NES sequence. Further, we show that COP1 is important in causing p53 nuclear exclusion. Finally, we demonstrate that 14-3-3σ targets COP1 for nuclear export, thereby preventing COP1-mediated p53 nuclear export. Together, these results define a novel, detailed mechanism for the subcellular localization and regulation of COP1 after DNA damage and provide a mechanistic explanation for the notion that 14-3-3σ's impact on the inhibition of p53 E3 ligases is an important step for p53 stabilization after DNA damage.

  14. Microphthalmia-associated Transcription Factor Interactions with 14-3-3 Modulate Differentiation of Committed Myeloid Precursors

    Bronisz, Agnieszka; Sharma, Sudarshana M.; Hu, Rong; Godlewski, Jakub; Tzivion, Guri; Mansky, Kim C.; Ostrowski, Michael C.

    2006-01-01

    The microphthalmia-associated transcription factor (MITF) is required for terminal osteoclast differentiation and is a target for signaling pathways engaged by colony stimulating factor (CSF)-1 and receptor-activator of nuclear factor-κB ligand (RANKL). Work presented here demonstrates that MITF can shuttle from cytoplasm to nucleus dependent upon RANKL/CSF-1 action. 14-3-3 was identified as a binding partner of MITF in osteoclast precursors, and overexpression of 14-3-3 in a transgenic model...

  15. Silencing neuroglobin enhances neuronal vulnerability to oxidative injury by down-regulating 14-3-3Y

    Shi-qiao YE; Xin-yu ZHOU; Xiao-jing LAI; Li ZHENG; Xiao-qian CHEN

    2009-01-01

    Aim:To explore the protective role and mechanism of endogenous neuroglobin (Ngb) in neuronal cells under oxidative stress.Methods:A stable N2a neuroblastoma cell line expressing the Ngb-siRNA plasmid (N2a/Ngb-siRNA) was established by neomycin screening.Reverse transcription (RT)-PCR and Western blot analysis were used to detect Ngb gene and protein levels.Hydrogen peroxide was used to induce oxidative stress in N2a cells.Cytotoxicity and cell viability were measured by lactate dehydrogenase (LDH) and WST-8 assays.Apoptotic cells were detected by Hoechst staining.Results:Cotransfection of Ngb-siRNA with Ngb-GFP plasmids suppressed the expression of Ngb-GFP in N2a cells.RT-PCR and Western blot analysis showed that the expression of endogenous Ngb was successfully knocked down to about 20% in N2a/Ngb-siRNA cells compared with control cells.A WST-8 assay demonstrated that viability was significantly decreased in N2a/Ngb-siRNA cells and N2a cells transiently transfected with Ngb-siRNA plasmids compared with controls following hydrogen peroxide treatment.An LDH assay demonstrated a time-dependent increase in the death of Ngb-siRNA-transfected N2a cells following hydrogen peroxide treatment.Hoechst staining demonstrated that the quantity of apoptotic cells among N2a/Ngb-siRNA cells following hydrogen peroxide treatment significantly increased compared with controls.In N2a/Ngb-siRNA cells,the expression level of activated caspase-3 significantly increased,whereas the expression of 14-3-3Y decreased compared with that of N2a/vec cells.Transfection of 14-3-3Y plasmids significantly enhanced the viability of N2a/Ngb-siRNA cells following hydrogen peroxide treatment compared with vector controls.Conclusion:Ngb contributes to neuronal defensive machinery against oxidative injuries by regulating 14-3-3Y expression.

  16. Progression of 14-3-3σ in nasopharyngeal carcinoma%14-3-3σ及其在鼻咽癌中的研究进展

    王堃; 易斌

    2011-01-01

    14-3-3σ,an vital tumor suppressor which is regulated by p53,plays a key role in cell cycle regulation, apoptosis, migration and proliferation, affecting tumor formation, invasion and metastasis. The methylation inactivation of 14-3-3σ is widely recognized as one of the mechanisms of tumorigenesis,and be associated with the metastasis of NPC.%14-3-3σ是p53调控的重要肿瘤抑制因子,在细胞周期调控、凋亡、迁移和扩散等过程中起关键作用,影响肿瘤的形成、浸润和转移.14-3-3σ甲基化失活是目前公认的肿瘤发生的重要机制之一,且与鼻咽癌的浸润转移等有关.

  17. Dysregulated 14-3-3 Family in Peripheral Blood Leukocytes of Patients with Schizophrenia

    Ying Qing; Liya Sun; Chao Yang; Jie Jiang; Xuhan Yang; Xiaowen Hu; Donghong Cui; Yifeng Xu; Lin He; Dongmei Han; Chunling Wan

    2016-01-01

    The 14-3-3 family, which is composed of seven distinct members in humans, plays important roles in the cell cycle, apoptosis, synaptic plasticity and neuronal differentiation and migration. Previous genetic and post-mortem gene expression studies have linked this family to schizophrenia. However, the direction of gene expression changes in these studies has been inconsistent, and reports of 14-3-3 gene expression in living schizophrenic patients are still lacking. Here, we assessed 14-3-3 gen...

  18. Characterization and subcellular localization of two 14-3-3 genes and their response to abiotic stress in wheat.

    Meng, Xiaodan; Chen, Xin; Wang, Yaying; Xiao, Ruixia; Liu, Hailun; Wang, Xinguo; Ren, Jiangping; Li, Yongchun; Niu, Hongbin; Wang, Xiang; Yin, Jun

    2014-02-01

    In order to investigate biological functions of the 14-3-3 genes and their response to abiotic stress, two cDNAs (designated as Ta14R1 and Ta14R2) encoding putative 14-3-3 proteins were isolated from wheat by PCR and rapid amplification of cDNA end (RACE) technique. The cDNA of Ta14R1 is 999bp and encodes a protein of 262 amino acids, while the cDNA of Ta14R2 is 897bp in length and encodes a protein of 261 amino acids. Transient expression assays using Ta14R1/Ta14R2-GFP fusion constructs indicated that Ta14R1 and Ta14R2 were located in cytoplasm and cell membrane but not in chloroplasts. Real-time quantitative (RT-PCR) analysis revealed that Ta14R1 and Ta14R2 were differentially expressed in wheat tissues and significantly up-regulated in roots and shoots 1d after germination, indicating they may play a role in process of seed germination. The expression of the two genes in roots and leaves were significantly induced by plant hormone ABA, as well as heat, cold and drought treatments, suggesting that the two 14-3-3 genes in wheat may be involved in ABA dependent stress-responding pathway and response to heat, cold and drought stress. PMID:24941745

  19. Functional relationship between CABIT, SAM and 14-3-3 binding domains of GAREM1 that play a role in its subcellular localization

    Nishino, Tasuku; Matsunaga, Ryota; Konishi, Hiroaki, E-mail: hkonishi@pu-hiroshima.ac.jp

    2015-08-21

    GAREM1 (Grb2-associated regulator of Erk/MAPK1) is an adaptor protein that is involved in the epidermal growth factor (EGF) pathway. The nuclear localization of GAREM1 depends on the nuclear localization sequence (NLS), which is located at the N-terminal CABIT (cysteine-containing, all in Themis) domain. Here, we identified 14-3-3ε as a GAREM-binding protein, and its binding site is closely located to the NLS. This 14-3-3 binding site was of the atypical type and independent of GAREM phosphorylation. Moreover, the binding of 14-3-3 had an effect on the nuclear localization of GAREM1. Unexpectedly, we observed that the CABIT domain had intramolecular association with the C-terminal SAM (sterile alpha motif) domain. This association might be inhibited by binding of 14-3-3 at the CABIT domain. Our results demonstrate that the mechanism underlying the nuclear localization of GAREM1 depends on its NLS in the CABIT domain, which is controlled by the binding of 14-3-3 and the C-terminal SAM domain. We suggest that the interplay between 14-3-3, SAM domain and CABIT domain might be responsible for the distribution of GAREM1 in mammalian cells. - Highlights: • 14-3-3ε regulated the nuclear localization of GAREM1 as its binding partner. • The atypical 14-3-3 binding site of GAREM1 is located near the NLS in CABIT domain. • The CABIT domain had intramolecular association with the SAM domain in GAREM1. • Subcellular localization of GAREM1 is affected with its CABIT-SAM interaction.

  20. Detection of PinX1 and 14-3-3 in the shrimp (Litopenaeus vannamei and study on gene expressions during viral infection and environmental stresses

    Potchanapond Graidist

    2010-12-01

    Full Text Available Two genes, PinX1 and 14-3-3, have been isolated and investigated for their expression in shrimp, Litopenaeusvannamei when infected with white spot syndrome virus (WSSV and subjected to environmental stresses. A putative PinX1protein of 180 amino acids showed a 65% similarity to the zebra fish PinX1 protein (Danio rerio and had a G-patch domainsimilar to human PinX1. The sequence of a full length cDNA of 14-3-3 has a very high similarity (96% to other shrimp 14-3-3-like protein (Feneropenaeus merguiensis and Penaeus monodon. Transcripts of PinX1 and 14-3-3 were up regulated in thehemolymph of viral infected shrimp with the highest expression level at 24 hrs p.i. Shrimp showing mortality characteristicshad very low expression of these two genes. In animals subjected to a combined low temperature (19-20°C and low oxygen(DO 1-1.5 mg/L for 24 hrs, an interesting result was that the transcript of PinX1 was drastically increased. In contrast, 14-3-3did not show any significant differences between the six treatments. The results of this work indicated that the PinX1 proteinmight play an important role in the shrimp response to viral infection and repose to certain stresses. In contrast the 14-3-3protein might play a particularly important role in the immune defended mechanisms of viral infections of shrimps.

  1. Factorial combinations of protein interactions generate a multiplicity of florigen activation complexes in wheat and barley.

    Li, Chengxia; Lin, Huiqiong; Dubcovsky, Jorge

    2015-10-01

    The FLOWERING LOCUS T (FT) protein is a central component of a mobile flowering signal (florigen) that is transported from leaves to the shoot apical meristem (SAM). Two FT monomers and two DNA-binding bZIP transcription factors interact with a dimeric 14-3-3 protein bridge to form a hexameric protein complex. This complex, designated as the 'florigen activation complex' (FAC), plays a critical role in flowering. The wheat homologue of FT, designated FT1 (= VRN3), activates expression of VRN1 in the leaves and the SAM, promoting flowering under inductive long days. In this study, we show that FT1, other FT-like proteins, and different FD-like proteins, can interact with multiple wheat and barley 14-3-3 proteins. We also identify the critical amino acid residues in FT1 and FD-like proteins required for their interactions, and demonstrate that 14-3-3 proteins are necessary bridges to mediate the FT1-TaFDL2 interaction. Using in vivo bimolecular fluorescent complementation (BiFC) assays, we demonstrate that the interaction between FT1 and 14-3-3 occurs in the cytoplasm, and that this complex is then translocated to the nucleus, where it interacts with TaFDL2 to form a FAC. We also demonstrate that a FAC including FT1, TaFDL2 and Ta14-3-3C can bind to the VRN1 promoter in vitro. Finally, we show that relative transcript levels of FD-like and 14-3-3 genes vary among tissues and developmental stages. Since FD-like proteins determine the DNA specificity of the FACs, variation in FD-like gene expression can result in spatial and temporal modulation of the effects of mobile FT-like signals. PMID:26252567

  2. Pim kinases phosphorylate multiple sites on Bad and promote 14-3-3 binding and dissociation from Bcl-XL

    Hastie C James

    2006-01-01

    Full Text Available Abstract Background Pim-1, 2 and 3 are a group of enzymes related to the calcium calmodulin family of protein kinases. Over-expression of Pim-1 and Pim-2 in mice promotes the development of lymphomas, and up-regulation of Pim expression has been observed in several human cancers. Results Here we show that the pim kinases are constitutively active when expressed in HEK-293 cells and are able to phosphorylate the Bcl-2 family member Bad on three residues, Ser112, Ser136 and Ser155 in vitro and in cells. In vitro mapping showed that Pim-2 predominantly phosphorylated Ser112, while Pim-1 phosphorylated Ser112, but also Ser136 and Ser155 at a reduced rate compared to Ser112. Pim-3 was found to be the least specific for Ser112, and the most effective at phosphorylating Ser136 and Ser155. Pim-3 was also able to phosphorylate other sites in Bad in vitro, including Ser170, another potential in vivo site. Mutation of Ser136 to alanine prevented the phosphorylation of Ser112 and Ser155 by Pim kinases in HEK-293 cells, suggesting that this site must be phosphorylated first in order to make the other sites accessible. Pim phosphorylation of Bad was also found to promote the 14-3-3 binding of Bad and block its association with Bcl-XL. Conclusion All three Pim kinase family members predominantly phosphorylate Bad on Ser112 and in addition are capable of phosphorylating Bad on multiple sites associated with the inhibition of the pro-apoptotic function of Bad in HEK-293 cells. This would be consistent with the proposed function of Pim kinases in promoting cell proliferation and preventing cell death.

  3. Superhelical DNA as a preferential binding target of 14-3-3 gamma protein

    Brázda, Václav; Čechová, J.; Coufal, Jan; Rumpel, S.; Jagelská, Eva

    2012-01-01

    Roč. 30, č. 4 (2012), s. 371-378. ISSN 0739-1102 R&D Projects: GA ČR(CZ) 301/10/1211; GA MŠk(CZ) LC 06035; GA AV ČR(CZ) M200040904 Institutional support: RVO:68081707 Keywords : TUMOR-SUPPRESSOR P53 * ASSOCIATES IN-VIVO * SUPERCOILED DNA Subject RIV: BO - Biophysics Impact factor: 4.986, year: 2010

  4. Expression of OsSPY and 14-3-3 genes involved in plant height variations of ion-beam-induced KDML 105 rice mutants

    The culm length of two semidwarf rice mutants (PKOS1, HyKOS1) obtained from low-energy N-ion beam bombardments of dehusked Thai jasmine rice (Oryza sativa L. cv. KDML 105) seeds showed 25.7% and 21.5% height reductions and one spindly rice mutant (TKOS4) showed 21.4% increase in comparison with that of the KDML 105 control. A cDNA-RAPD analysis identified differential gene expression in internode tissues of the rice mutants. Two genes identified from the cDNA-RAPD were OsSPY and 14-3-3, possibly associated with stem height variations of the semidwarf and spindly mutants, respectively. The OsSPY gene encoded the SPY protein which is considered to be a negative regulator of gibberellin (GA). On the other hand, the 14-3-3 encoded a signaling protein which can bind and prevent the RSG (repression of shoot growth) protein function as a transcriptional repressor of the kaurene oxidase (KO) gene in the GA biosynthetic pathway. Expression analysis of OsSPY, 14-3-3, RSG, KO, and SLR1 was confirmed in rice internode tissues during the reproductive stage of the plants by semi-quantitative RT-PCR technique. The expression analysis showed a clear increase of the levels of OsSPY transcripts in PKOS1 and HyKOS1 tissue samples compared to that of the KDML 105 and TKOS4 samples at the age of 50–60 days which were at the ages of internode elongation. The 14-3-3 expression had the highest increase in the TKOS4 samples compared to those in KDML 105, PKOS1 and HyKOS1 samples. The expression analysis of RSG and KO showed an increase in TKOS4 samples compared to that of the KDML 105 and that of the two semidwarf mutants. These results indicate that changes of OsSPY and 14-3-3 expression could affect internode elongation and cause the phenotypic changes of semidwarf and spindly rice mutants, respectively.

  5. Identification of the amino acids 300-600 of IRS-2 as 14-3-3 binding region with the importance of IGF-1/insulin-regulated phosphorylation of Ser-573.

    Sabine S Neukamm

    Full Text Available Phosphorylation of insulin receptor substrate (IRS-2 on tyrosine residues is a key event in IGF-1/insulin signaling and leads to activation of the PI 3-kinase and the Ras/MAPK pathway. Furthermore, phosphorylated serine/threonine residues on IRS-2 can induce 14-3-3 binding. In this study we searched IRS-2 for novel phosphorylation sites and investigated the interaction between IRS-2 and 14-3-3. Mass spectrometry identified a total of 24 serine/threonine residues on IRS-2 with 12 sites unique for IRS-2 while the other residues are conserved in IRS-1 and IRS-2. IGF-1 stimulation led to increased binding of 14-3-3 to IRS-2 in transfected HEK293 cells and this binding was prevented by inhibition of the PI 3-kinase pathway and an Akt/PKB inhibitor. Insulin-stimulated interaction between endogenous IRS-2 and 14-3-3 was observed in rat hepatoma cells and in mice liver after an acute insulin stimulus and refeeding. Using different IRS-2 fragments enabled localization of the IGF-1-dependent 14-3-3 binding region spanning amino acids 300-600. The 24 identified residues on IRS-2 included several 14-3-3 binding candidates in the region 300-600. Single alanine mutants of these candidates led to the identification of serine 573 as 14-3-3 binding site. A phospho-site specific antibody was generated to further characterize serine 573. IGF-1-dependent phosphorylation of serine 573 was reduced by inhibition of PI 3-kinase and Akt/PKB. A negative role of this phosphorylation site was implicated by the alanine mutant of serine 573 which led to enhanced phosphorylation of Akt/PKB in an IGF-1 time course experiment. To conclude, our data suggest a physiologically relevant role for IGF-1/insulin-dependent 14-3-3 binding to IRS-2 involving serine 573.

  6. Histone deacetylase turnover and recovery in sulforaphane-treated colon cancer cells: competing actions of 14-3-3 and Pin1 in HDAC3/SMRT corepressor complex dissociation/reassembly

    Williams David E

    2011-05-01

    Full Text Available Abstract Background Histone deacetylase (HDAC inhibitors are currently undergoing clinical evaluation as anti-cancer agents. Dietary constituents share certain properties of HDAC inhibitor drugs, including the ability to induce global histone acetylation, turn-on epigenetically-silenced genes, and trigger cell cycle arrest, apoptosis, or differentiation in cancer cells. One such example is sulforaphane (SFN, an isothiocyanate derived from the glucosinolate precursor glucoraphanin, which is abundant in broccoli. Here, we examined the time-course and reversibility of SFN-induced HDAC changes in human colon cancer cells. Results Cells underwent progressive G2/M arrest over the period 6-72 h after SFN treatment, during which time HDAC activity increased in the vehicle-treated controls but not in SFN-treated cells. There was a time-dependent loss of class I and selected class II HDAC proteins, with HDAC3 depletion detected ahead of other HDACs. Mechanism studies revealed no apparent effect of calpain, proteasome, protease or caspase inhibitors, but HDAC3 was rescued by cycloheximide or actinomycin D treatment. Among the protein partners implicated in the HDAC3 turnover mechanism, silencing mediator for retinoid and thyroid hormone receptors (SMRT was phosphorylated in the nucleus within 6 h of SFN treatment, as was HDAC3 itself. Co-immunoprecipitation assays revealed SFN-induced dissociation of HDAC3/SMRT complexes coinciding with increased binding of HDAC3 to 14-3-3 and peptidyl-prolyl cis/trans isomerase 1 (Pin1. Pin1 knockdown blocked the SFN-induced loss of HDAC3. Finally, SFN treatment for 6 or 24 h followed by SFN removal from the culture media led to complete recovery of HDAC activity and HDAC protein expression, during which time cells were released from G2/M arrest. Conclusion The current investigation supports a model in which protein kinase CK2 phosphorylates SMRT and HDAC3 in the nucleus, resulting in dissociation of the corepressor

  7. Identification of the Amino Acids 300–600 of IRS-2 as 14-3-3 Binding Region with the Importance of IGF-1/Insulin-Regulated Phosphorylation of Ser-573

    Neukamm, S.S.; Toth, R.; Morrice, N.; Campbell, D. G.; Mackintosh, C; Lehmann, R.; Haering, H; Schleicher, E. D.; Weigert, C.

    2012-01-01

    Phosphorylation of insulin receptor substrate (IRS)-2 on tyrosine residues is a key event in IGF-1/insulin signaling and leads to activation of the PI 3-kinase and the Ras/MAPK pathway. Furthermore, phosphorylated serine/threonine residues on IRS-2 can induce 14-3-3 binding. In this study we searched IRS-2 for novel phosphorylation sites and investigated the interaction between IRS-2 and 14-3-3. Mass spectrometry identified a total of 24 serine/threonine residues on IRS-2 with 12 sites unique...

  8. Orders induced by segments in floorplan partitions and (2-14-3,3-41-2)-avoiding permutations

    Asinowski, Andrei; Bousquet-Mélou, Mireille; Mansour, Toufik; Pinter, Ron

    2010-01-01

    Floorplan partitions are certain tilings of a rectangle by other rectangles. There are natural ways to order their elements (rectangles and segments). In particular, Ackerman, Barequet, and Pinter studied a pair of orders induced by neighborhood relations between rectangles of a floorplan partition, and obtained a natural bijection between these pairs and (2-41-3, 3-14-2)-avoiding permutations (also known as Baxter permutations). In the present paper, we study a pair of orders induced by neighborhood relations between segments of a floorplan partition. We obtain a natural bijection between these pairs and another family of permutations, namely (2-14-3,3-41-2)-avoiding permutations. We also enumerate these permutations, investigate relations between the two kinds of pairs of orders --- and correspondingly, between (2-14-3,3-41-2)-avoiding permutations and Baxter permutations --- and study the special case of "guillotine" partitions.

  9. Combinations of SPR and MS for characterization of native and recombinant proteins in cell lysates

    Borch, Jonas; Roepstorff, Peter

    2006-01-01

    14-3-3 proteins in order to test their activity. Specific binding of recombinant and native 14-3-3 proteins in complex mixtures to immobilized phosphopeptides and subsequent elution was also tested by SPR-MS. Ammonium sulfate precipitate fractions from lysates of E. coli expressing 14-3-3 protein and...... of cauliflower were investigated for specific binding to the phosphopeptide ligands immobilized on a sensorchip by SPR. Subsequently, the bound protein was eluted and analyzed by MS for characterization of intact mass and peptide mass mapping....

  10. Aqueous Extract from Hibiscus sabdariffa Linnaeus Ameliorate Diabetic Nephropathy via Regulating Oxidative Status and Akt/Bad/14-3-3γ in an Experimental Animal Model

    Shou-Chieh Wang

    2011-01-01

    Full Text Available Several studies point out that oxidative stress maybe a major culprit in diabetic nephropathy. Aqueous extract of Hibiscus sabdariffa L. (HSE has been demonstrated as having beneficial effects on anti-oxidation and lipid-lowering in experimental studies. This study aimed at investigating the effects of Hibiscus sabdariffa L. on diabetic nephropathy in streptozotocin induced type 1 diabetic rats. Our results show that HSE is capable of reducing lipid peroxidation, increasing catalase and glutathione activities significantly in diabetic kidney, and decreasing the plasma levels of triglyceride, low-density lipoprotein (LDL and increasing high-density lipoprotein (HDL value. In histological examination, HSE improves hyperglycemia-caused osmotic diuresis in renal proximal convoluted tubules (defined as hydropic change in diabetic rats. The study also reveals that up-regulation of Akt/Bad/14-3-3γ and NF-κB-mediated transcription might be involved. In conclusion, our results show that HSE possesses the potential effects to ameliorate diabetic nephropathy via improving oxidative status and regulating Akt/Bad/14-3-3γ signaling.

  11. Decreased expression of 14-3-3σ is predictive of poor prognosis for patients with human uterine papillary serous carcinoma.

    Suzuki, Fumihiko; Nagase, Satoru; Suzuki, Kichiya; Oba, Etsuko; Hiroki, Eri; Matsuda, Yukika; Akahira, Jun-Ichi; Nishigori, Hidekazu; Sugiyama, Takashi; Otsuki, Takeo; Yoshinaga, Kousuke; Takano, Tadao; Niikura, Hitoshi; Ito, Kiyoshi; Sasano, Hironobu; Yaegashi, Nobuo

    2013-01-01

    Uterine papillary serous carcinoma (UPSC) morphologically resembles ovarian serous carcinoma and is categorized as a type II endometrial cancer. UPSC comprises about 10% of all types of endometrial cancer and has an aggressive clinical course and a poor prognosis. The 14-3-3σ gene was originally discovered as a p53-inducible gene; its expression is induced by DNA damage in a p53-dependent manner, which leads to G2 arrest and repair of damaged DNA. Moreover, it has been reported that expression of 14-3-3σ is frequently lost in various types of human cancer, including ovarian cancer. We therefore examined the association between 14-3-3σ expression determined by immunohistochemistry and clinical outcomes of 51 patients with UPSC. UPSC was considered positive for 14-3-3σ when > 30% of tumor cells were stained with a specific antibody. Of these patients, 29 (58.7%) showed positive immunoreactivity for 14-3-3σ and 22 (41.3%) had decreased 14-3-3σ staining. Decreased immunoreactivity for 14-3-3σ was associated with stage (P = 0.001) and lymphovascular space involvement (P = 0.005). Moreover, decreased 14-3-3σ expression was an independent risk factor for reduced overall survival (P = 0.0416) in multivariate analysis. Direct bisulfite sequencing was performed to evaluate the methylation status of the 27 CpG islands in the promoter region and first exon of the 14-3-3σ gene. These CpG islands were hypermethylated in 30% of 14-3-3σ-positive UPSC and 80% of 14-3-3σ-negative UPSC, although the difference was not statistically significant. These findings suggest that decreased expression of immunoreactive 14-3-3σ may be a predictor of poor prognosis in patients with UPSC. PMID:24201220

  12. Combinatorial H3K9acS10ph Histone Modification in IgH Locus S Regions Targets 14-3-3 Adaptors and AID to Specify Antibody Class-Switch DNA Recombination

    Guideng Li

    2013-11-01

    Full Text Available Class-switch DNA recombination (CSR is central to the antibody response, in that it changes the immunoglobulin heavy chain (IgH constant region, thereby diversifying biological effector functions of antibodies. The activation-induced cytidine deaminase (AID-centered CSR machinery excises and rejoins DNA between an upstream (donor and a downstream (acceptor S region, which precede the respective constant region DNA. AID is stabilized on S regions by 14-3-3 adaptors. These adaptors display a high affinity for 5′-AGCT-3′ repeats, which recur in all S regions. However, how 14-3-3, AID, and the CSR machinery target exclusively the donor and acceptor S regions is poorly understood. Here, we show that histone methyltransferases and acetyltransferases are induced by CD40 or Toll-like receptor signaling and catalyze H3K4me3 and H3K9ac/K14ac histone modifications, which are enriched in S regions but do not specify the S region targets of CSR. By contrast, the combinatorial H3K9acS10ph modification specifically marks the S regions set to recombine and directly recruits 14-3-3 adaptors for AID stabilization there. Inhibition of the enzymatic activity of GCN5 and PCAF histone acetyltransferases reduces H3K9acS10ph in S regions, 14-3-3 and AID stabilization, and CSR. Thus, H3K9acS10ph is a histone code that is “written” specifically in S regions and is “read” by 14-3-3 adaptors to target AID for CSR as an important biological outcome.

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

    ... personality changes • Loss of muscle coordination • Vision problems • Insomnia (difficulty falling asleep or staying asleep) • Depression • Unusual sensations As the disease progresses, the person usually develops dementia. This leads to thinking problems that are severe enough to ...

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

    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...

  15. Crystal structure of the 14-3-3zeta:serotonin N-acetyltransferase complex. A role for scaffolding in enzyme regulation

    Obšil, Tomáš; Ghirlando, R.; Klein, D. C.; Ganguly, S.; Dyda, F.

    2001-01-01

    Roč. 105, č. 2 (2001), s. 257-267. ISSN 0092-8674 Institutional research plan: CEZ:AV0Z5011922 Keywords : 14-3-3 * AANAT * crystal structure Subject RIV: BO - Biophysics Impact factor: 29.210, year: 2001

  16. Amifostine alleviates radiation-induced lethal small bowel damage via promotion of 14-3-3σ-mediated nuclear p53 accumulation.

    Huang, Eng-Yen; Wang, Feng-Sheng; Chen, Yu-Min; Chen, Yi-Fan; Wang, Chung-Chi; Lin, I-Hui; Huang, Yu-Jie; Yang, Kuender D

    2014-10-30

    Amifostine (AM) is a radioprotector that scavenges free radicals and is used in patients undergoing radiotherapy. p53 has long been implicated in cell cycle arrest for cellular repair after radiation exposure. We therefore investigated the protective p53-dependent mechanism of AM on small bowel damage after lethal whole-abdominal irradiation (WAI). AM increased both the survival rate of rats and crypt survival following lethal 18 Gy WAI. The p53 inhibitor PFT-α compromised AM-mediated effects when administered prior to AM administration. AM significantly increased clonogenic survival in IEC-6 cells expressing wild type p53 but not in p53 knockdown cells. AM significantly increased p53 nuclear accumulation and p53 tetramer expression before irradiation through the inhibition of p53 degradation. AM inhibited p53 interactions with MDM2 but enhanced p53 interactions with 14-3-3σ. Knockdown of 14-3-3σ also compromised the effect of AM on clonogenic survival and p53 nuclear accumulation in IEC-6 cells. For the first time, our data reveal that AM alleviates lethal small bowel damage through the induction of 14-3-3σ and subsequent accumulation of p53. Enhancement of the p53/14-3-3σ interaction results in p53 tetramerization in the nucleus that rescues lethal small bowel damage. PMID:25230151

  17. BRF1 Protein Turnover and mRNA Decay Activity Are Regulated by Protein Kinase B at the Same Phosphorylation Sites▿

    Benjamin, Don; Schmidlin, Martin; Min, Lu; Gross, Brigitte; Moroni, Christoph

    2006-01-01

    BRF1 posttranscriptionally regulates mRNA levels by targeting ARE-bearing transcripts to the decay machinery. We previously showed that protein kinase B (PKB) phosphorylates BRF1 at Ser92, resulting in binding to 14-3-3 and impairment of mRNA decay activity. Here we identify an additional regulatory site at Ser203 that cooperates in vivo with Ser92. In vitro kinase labeling and wortmannin sensitivity indicate that Ser203 phosphorylation is also performed by PKB. Mutation of both serines to al...

  18. Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose

    Harthill, Jean E; Meek, Sarah E M; Morrice, Nick;

    2006-01-01

    Trehalose-6-phosphate is a 'sugar signal' that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP) enzymes. It also encodes class II proteins (TPS isoforms 5-11) that contain both TPS-like and TPP...

  19. Cell cycle checkpoints in Caenorhabditis elegans: the 14-3-3 gene par-5 is required for germline development and DNA damage response / Checkpoints del ciclo celular en Caenorhabditis elegans: el gen 14-3-3, par-5, es necesario para el desarrollo y respuesta al daño genómico de la línea germinal

    Aristizábal Corrales, David

    2012-01-01

    [spa] Las proteínas 14-3-3 han sido ampliamente estudiadas desde levadura hasta mamíferos y han sido asociadas con múltiples roles en procesos como ciclo celular, apoptosis y la respuesta al estrés. Así mismo estas proteínas se han visto involucradas en enfermedades neurodegenerativas y cáncer. De hecho, las proteínas 14-3-3 han sido propuestas como posibles agentes terapéuticos en el tratamiento contra el cáncer. En mamíferos existen 7 genes que codifican para proteínas 14-3-3, mientras en C...

  20. Cytoplasmic Localization of Human cdc25C during Interphase Requires an Intact 14-3-3 Binding Site

    Dalal, Sorab N.; Schweitzer, Colleen M.; Gan, Jianmin; DeCaprio, James A.

    1999-01-01

    cdc25C induces mitosis by activating the cdc2-cyclin B complex. The intracellular localization of cyclin B1 is regulated in a cell cycle-specific manner, and its entry into the nucleus may be required for the initiation of mitosis. To determine the cellular localization of cdc25C, monoclonal antibodies specific for cdc25C were developed and used to demonstrate that in human cells, cdc25C is retained in the cytoplasm during interphase. A deletion analysis identified a 58-amino-acid region (ami...

  1. The usefulness of S100P, mesothelin, fascin, prostate stem cell antigen, and 14-3-3 sigma in diagnosing pancreatic adenocarcinoma in cytological specimens obtained by endoscopic ultrasound guided fine-needle aspiration.

    Dim, Daniel C; Jiang, Feng; Qiu, Qi; Li, Ting; Darwin, Peter; Rodgers, William H; Peng, Hong Qi

    2014-03-01

    Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) of the pancreas is an efficient and minimally invasive procedure for the diagnosis and staging of pancreatic adenocarcinoma. Because of some limitations of EUS-FNA in diagnosis of well-differentiated or early stage cancers, the purpose of this study is to assess the added benefit of immunohistochemistry. We studied five proteins overexpressed in pancreatic adenocarcinoma, namely, prostate stem cell antigen, fascin, 14-3-3 sigma, mesothelin and S100P utilizing immunohistochemistry on paraffin sections from cellblocks obtained by EUS-FNA. Sixty-two cases of EUS-FNA of the pancreas that had follow-up histological and/or clinical diagnosis and sufficient material in cell blocks were included. Using histological diagnosis and/or clinical outcome as the reference standard, EUS-FNA shows the highest sensitivity (95%) and specificity (91%) and is superior to any marker in this study. Among five antibodies, S100P reveals the best diagnostic characters showing 90% of sensitivity and 67% of specificity. Fascin shows high specificity (92%) but low sensitivity (38%). Mesothelin has a moderate sensitivity (74%) and low specificity (33%), PSCA and 14-3-3 show high sensitivity but zero specificity. S100P and mesothelin were useful in nine indeterminate cases. S100P correctly predicted six of seven cancers and one of one without cancer and mesothelin correctly diagnosed five of seven cancers and one of two noncancers in this group. EUS-FNA cytomorphology is superior to any of the immunohistochemical markers used in this study. Use of S100P and mesothelin in cytologically borderline cases can increase the diagnostic accuracy in this group. PMID:21538952

  2. Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein

    Fuglsang, Anja Thoe; Guo, Yan; Cuin, Tracey A.;

    2007-01-01

    Regulation of the trans-plasma membrane pH gradient is an important part of plant responses to several hormonal and environmental cues, including auxin, blue light, and fungal elicitors. However, little is known about the signaling components that mediate this regulation. Here, we report that an ......Regulation of the trans-plasma membrane pH gradient is an important part of plant responses to several hormonal and environmental cues, including auxin, blue light, and fungal elicitors. However, little is known about the signaling components that mediate this regulation. Here, we report...

  3. p53突变型乳腺癌细胞系中14-3-3σ对p73基因稳定性的影响%Effect of 14-3-3σ on p73 stability in p53-mutant breast cancer cell line

    桑梅香; 杨瑞玲; 周军; 周岩; 耿翠芝; 单保恩

    2007-01-01

    目的 探讨在p53突变的乳腺癌细胞系MDA-MB-231中,14-3-3σ对p73基因稳定性的影响.方法 采用基因转染、反转录聚合酶链反应(RT-PCR)、蛋白质印迹检测(Western blot)和放线菌酮(CHX)蛋白抑制分析的方法研究14-3-3σ对p73基因稳定性的影响.结果 RT-PCR显示,单独转染1μg p73后,p73与GAPDH的灰度值比为0.635;转染1μg p73+1 μg 14-3-3σ和1 μg p73+2 μg 14-3-3σ后,p73与GAPDH的灰度值比分别为0.643和0.631.Western-blot显示,单独转染1μg p73后,p73与actin的灰度值比为0.333;转染1μg p73+1μg 14-3-3σ和1μg p73+2μg 14-3-3σ之后,p73与actin的灰度值比分别为0.797和0.826.放线菌酮蛋白抑制分析显示:单独转染p73的对照组,放线菌酮处理0、1、2、4、6 h后,p73与actin灰度比值分别为0.075、0.166、0.124、0.100和0.092;而共转染p73和14-3-3σ的实验组结果分别为0.963、0.244、0.244、0.234和0.185.结论 在转录水平上,14-3-3σ不影响p73基因的稳定性;而在蛋白表达水平上,14-3-3σ可以增加p73基因的稳定性.

  4. 核酸疫苗Sj14-3-3联合CpG和mIL-12免疫小鼠抗日本血吸虫攻击感染的研究%Immune protection of Sj14-3-3 nucleic acid vaccination with mIL-12 and CpG as adjuvants against Schistosoma japonicum in mice

    徐元宏; 胡元生; 沈继龙

    2006-01-01

    目的在证明核酸疫苗pcDNA3.1(+)-Sj14-3-3具有部分抗血吸虫感染的基础上,联合使用CpG和pcDNA3.1(+)-mIL-12,观察此二种佐剂在攻击感染小鼠的免疫效果及其抗血吸虫感染的保护机制.方法分别用pcDNA3.1(+)-Sj14-3-3+pcDNA3.1(+)-mIL-12、pcDNA3.1(+)-Sj14-3-3+CpG、pcDNA3.1(+)-mIL-12和CpG免疫小鼠.攻击感染后6 w计数成虫负荷和肝虫卵数;检测免疫后0 w、6 w和12 w小鼠血清总IgG、IgG1和IgG2a水平;检测小鼠脾细胞培养上清中IFN-γ和IL-4;流式细胞术检测免疫鼠脾细胞中CD4+和CD8+T细胞的比率.结果pcDNA3.1(+)-Sj14-3-3+pcDNA3.1(+)-mIL-12和pcDNA3.1(+)-Sj14-3-3+CpG免疫小鼠的减虫率分别为41.2%和28.7%;减卵率分别为52.6%和41.2%.单独使用pcDNA3.1(+)-mIL-12和CpG也有一定的免疫保护作用.保护性免疫主要通过诱导宿主产生CTL、TH1型和体液免疫应答.结论pcDNA3.1(+)-mIL-12和CpG具有较强的增强核酸疫苗pcDNA3.1(+)-Sj14-3-3抗血吸虫攻击感染作用.

  5. Cyclic nucleotide dependent dephosphorylation of regulator of G-protein signaling 18 in human platelets.

    Gegenbauer, Kristina

    2013-11-01

    Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets.

  6. Antiviral activities of whey proteins.

    Ng, Tzi Bun; Cheung, Randy Chi Fai; Wong, Jack Ho; Wang, Yan; Ip, Denis Tsz Ming; Wan, David Chi Cheong; Xia, Jiang

    2015-09-01

    Milk contains an array of proteins with useful bioactivities. Many milk proteins encompassing native or chemically modified casein, lactoferrin, alpha-lactalbumin, and beta-lactoglobulin demonstrated antiviral activities. Casein and alpha-lactalbumin gained anti-HIV activity after modification with 3-hydroxyphthalic anhydride. Many milk proteins inhibited HIV reverse transcriptase. Bovine glycolactin, angiogenin-1, lactogenin, casein, alpha-lactalbumin, beta-lactoglobulin, bovine lactoferrampin, and human lactoferrampin inhibited HIV-1 protease and integrase. Several mammalian lactoferrins prevented hepatitis C infection. Lactoferrin, methylated alpha-lactalbumin and methylated beta-lactoglobulin inhibited human cytomegalovirus. Chemically modified alpha-lactalbumin, beta-lactoglobulin and lysozyme, lactoferrin and lactoferricin, methylated alpha-lactalbumin, methylated and ethylated beta-lactoglobulins inhibited HSV. Chemically modified bovine beta-lactoglobulin had antihuman papillomavirus activity. Beta-lactoglobulin, lactoferrin, esterified beta-lactoglobulin, and esterified lactoferrindisplayed anti-avian influenza A (H5N1) activity. Lactoferrin inhibited respiratory syncytial virus, hepatitis B virus, adenovirus, poliovirus, hantavirus, sindbis virus, semliki forest virus, echovirus, and enterovirus. Milk mucin, apolactoferrin, Fe(3+)-lactoferrin, beta-lactoglobulin, human lactadherin, bovine IgG, and bovine kappa-casein demonstrated antihuman rotavirus activity. PMID:26198883

  7. 多房棘球绦虫重组质粒pGEX-EmⅡ/3-Em14-3-3在大肠埃希菌BL21(DE3)表达效率的研究%Study of expression efficiency of the recombinant plasmid pGEX-Em Ⅱ/3-Em14-3-3 of Echinococcus multilocularis in Escherichia coli BL21(DE3)

    杨梅; 李文桂; 朱佑明

    2007-01-01

    目的 研究多房棘球绦虫(Em)重组质粒pGEX-EmⅡ/3-Em14-3-3在大肠埃希菌BL21(DE3)中的表达效率.方法 通过PCR扩增EmⅡ/3和Em14-3-3抗原编码基因,然后采用基因拼接法(gene SOEing)剪接EmⅡ/3和Em14-3-3,得到EmⅡ/3-Em14-3-3融合基因;将该融合基因定向克隆于含有谷胱甘肽-S-转移酶(GST)基因的高效原核表达载体pGEX-1λT,经酶切鉴定后以IPTG诱导表达EmⅡ/3-Em14-3-3/GST融合蛋白;SDS-PAGE及Western blot对表达产物进行鉴定.结果 PCR成功扩增出2 554 bp的EmⅡ/3-Em14-3-3融合基因;双酶切证实EmⅡ/3-Em14-3-3融合基因插入pGEX-1λT中,成功构建了pGEX-EmⅡ/3-Em14-3-3重组质粒;SDS-PAGE及Western blot分析显示重组质粒转化宿主菌在IPTG诱导下高效表达了能被活动性泡球蚴病鼠血清识别的EmⅡ/3-Em14-3-3/GST融合蛋白,分子质量单位119 ku.结论 多房棘球绦虫EmⅡ/3-Em14-3-3融合基因在大肠埃希菌中获得了高效融合表达,表达出的EmⅡ/3-Em14-3-3重组蛋白具有特异的抗原性.

  8. Degradation of Activated Protein Kinases by Ubiquitination

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases.

  9. The polymorphism of YWHAE, a gene encoding 14-3-3epsilon, and brain morphology in schizophrenia: a voxel-based morphometric study.

    Mikio Kido

    Full Text Available BACKGROUND: YWHAE is a possible susceptibility gene for schizophrenia that encodes 14-3-3epsilon, a Disrupted-in-Schizophrenia 1 (DISC1-interacting molecule, but the effect of variation in its genotype on brain morphology remains largely unknown. METHODS: In this voxel-based morphometric magnetic resonance imaging study, we conducted whole-brain analyses regarding the effects of YWHAE single-nucleotide polymorphisms (SNPs (rs28365859, rs11655548, and rs9393 and DISC1 SNP (rs821616 on gray matter volume in a Japanese sample of 72 schizophrenia patients and 86 healthy controls. On the basis of a previous animal study, we also examined the effect of rs28365859 genotype specifically on hippocampal volume. RESULTS: Whole-brain analyses showed no significant genotype effect of these SNPs on gray matter volume in all subjects, but we found significant genotype-by-diagnosis interaction for rs28365859 in the left insula and right putamen. The protective C allele carriers of rs28365859 had a significantly larger left insula than the G homozygotes only for schizophrenia patients, while the controls with G allele homozygosity had a significantly larger right putamen than the C allele carriers. The C allele carriers had a larger right hippocampus than the G allele homozygotes in schizophrenia patients, but not in healthy controls. No significant interaction was found between rs28365859 and DISC1 SNP on gray matter volume. CONCLUSIONS: These different effects of the YWHAE (rs28365859 genotype on brain morphology in schizophrenia and healthy controls suggest that variation in its genotype might be, at least partly, related to the abnormal neurodevelopment, including in the limbic regions, reported in schizophrenia. Our results also suggest its specific role among YWHAE SNPs in the pathophysiology of schizophrenia.

  10. 14-3-3ζ Cooperates with ErbB2 to Promote Progression of Ductal Carcinoma in Situ to Invasive Breast Cancer by Inducing Epithelial-Mesenchymal Transition

    LU, JING; Guo, Hua; Treekitkarnmongkol, Warapen; Ping LI; Zhang, Jian; Shi, Bin; Ling, Chen; ZHOU, XIAOYAN; Chen, Tongzhen; Chiao, Paul J.; Feng, XinHua; Seewaldt, Victoria L.; Muller, William J; Sahin, Aysegul; Hung, Mien-Chie

    2009-01-01

    ErbB2, a metastasis-promoting oncoprotein, is overexpressed in ~25% of invasive/metastatic breast cancers, but in 50–60% of non-invasive ductal carcinomas in situ (DCIS). It has been puzzling how a subset of ErbB2-overexpressing DCIS develops into invasive breast cancer (IBC). We found that co-overexpression of 14-3-3ζ in ErbB2-overexpressing DCIS conferred a higher risk of progression to IBC. ErbB2 and 14-3-3ζ overexpression, respectively, increased cell migration and decreased cell adhesion...

  11. Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

    Sergiy; Kostenko; Gianina; Dumitriu; Kari; Jenssen; Lgreid; Ugo; Moens

    2011-01-01

    Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

  12. Protein-water dynamics in antifreeze protein III activity

    Xu, Yao; Bäumer, Alexander; Meister, Konrad; Bischak, Connor G.; DeVries, Arthur L.; Leitner, David M.; Havenith, Martina

    2016-03-01

    We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.

  13. Activity assay of membrane transport proteins

    Hao Xie

    2008-01-01

    Membrane transport proteins are integral membrane proteins and considered as potential drug targets. Activity assay of transport proteins is essential for developing drugs to target these proteins. Major issues related to activity assessment of transport proteins include availability of transporters,transport activity of transporters, and interactions between ligands and transporters. Researchers need to consider the physiological status of proteins (bound in lipid membranes or purified), availability and specificity of substrates, and the purpose of the activity assay (screening, identifying, or comparing substrates and inhibitors) before choosing appropriate assay strategies and techniques. Transport proteins bound in vesicular membranes can be assayed for transporting substrate across membranes by means of uptake assay or entrance counterflow assay. Alternatively, transport proteins can be assayed for interactions with ligands by using techniques such as isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, or surface plasmon resonance. Other methods and techniques such as fluorometry, scintillation proximity assay, electrophysiological assay, or stopped-flow assay could also be used for activity assay of transport proteins. In this paper the major strategies and techniques for activity assessment of membrane transport proteins are reviewed.

  14. Activity-Based Protein Profiling of Microbes

    Sadler, Natalie C.; Wright, Aaron T.

    2015-02-01

    Activity-Based Protein Profiling (ABPP) in conjunction with multimodal characterization techniques has yielded impactful findings in microbiology, particularly in pathogen, bioenergy, drug discovery, and environmental research. Using small molecule chemical probes that react irreversibly with specific proteins or protein families in complex systems has provided insights in enzyme functions in central metabolic pathways, drug-protein interactions, and regulatory protein redox, for systems ranging from photoautotrophic cyanobacteria to mycobacteria, and combining live cell or cell extract ABPP with proteomics, molecular biology, modeling, and other techniques has greatly expanded our understanding of these systems. New opportunities for application of ABPP to microbial systems include: enhancing protein annotation, characterizing protein activities in myriad environments, and reveal signal transduction and regulatory mechanisms in microbial systems.

  15. Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

    Ngo, HT; Pham, Long; Kim, JW;

    2013-01-01

    , approximately 100 cellular proteins were identified as HCV core-interacting partners. Of these candidates, mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) was selected for further characterization. MAPKAPK3 is a serine/threonine protein kinase that is activated by stress and growth...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA and......Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray...

  16. DNA-based control of protein activity.

    Engelen, W; Janssen, B M G; Merkx, M

    2016-03-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

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

    Kular, Jasreen; Scheer, Kaitlin G; Pyne, Natasha T;

    2015-01-01

    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 pr...

  18. Activated protein C modulates the proinflammatory activity of dendritic cells

    Matsumoto T

    2015-05-01

    Full Text Available Takahiro Matsumoto,1,2* Yuki Matsushima,1* Masaaki Toda,1 Ziaurahman Roeen,1 Corina N D'Alessandro-Gabazza,1,5 Josephine A Hinneh,1 Etsuko Harada,1,3 Taro Yasuma,4 Yutaka Yano,4 Masahito Urawa,1,5 Tetsu Kobayashi,5 Osamu Taguchi,5 Esteban C Gabazza1 1Department of Immunology, Mie University Graduate School of Medicine, Tsu, Mie Prefecture, 2BONAC Corporation, BIO Factory 4F, Fukuoka, 3Iwade Research Institute of Mycology, 4Department of Endocrinology, Diabetes and Metabolism, 5Department of Pulmonary and Critical Care Medicine, Mie University Graduate School of Medicine, Tsu, Mie Prefecture, Japan *These authors contributed equally to this work Background: Previous studies have demonstrated the beneficial activity of activated protein C in allergic diseases including bronchial asthma and rhinitis. However, the exact mechanism of action of activated protein C in allergies is unclear. In this study, we hypothesized that pharmacological doses of activated protein C can modulate allergic inflammation by inhibiting dendritic cells. Materials and methods: Dendritic cells were prepared using murine bone marrow progenitor cells and human peripheral monocytes. Bronchial asthma was induced in mice that received intratracheal instillation of ovalbumin-pulsed dendritic cells. Results: Activated protein C significantly increased the differentiation of tolerogenic plasmacytoid dendritic cells and the secretion of type I interferons, but it significantly reduced lipopolysaccharide-mediated maturation and the secretion of inflammatory cytokines in myeloid dendritic cells. Activated protein C also inhibited maturation and the secretion of inflammatory cytokines in monocyte-derived dendritic cells. Activated protein C-treated dendritic cells were less effective when differentiating naïve CD4 T-cells from Th1 or Th2 cells, and the cellular effect of activated protein C was mediated by its receptors. Mice that received adoptive transfer of activated protein C

  19. Chibby drives β catenin cytoplasmic accumulation leading to activation of the unfolded protein response in BCR-ABL1+ cells.

    Mancini, Manuela; Leo, Elisa; Takemaru, Ken-Ichi; Campi, Virginia; Borsi, Enrica; Castagnetti, Fausto; Gugliotta, Gabriele; Santucci, Maria Alessandra; Martinelli, Giovanni

    2013-09-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the constitutive tyrosine kinase (TK) activity of the BCR-ABL fusion protein. However, the phenotype of leukemic stem cells (LSC) is sustained by β catenin rather than by the BCR-ABL TK. β catenin activity in CML is contingent upon its stabilization proceeding from the BCR-ABL-induced phosphorylation at critical residues for interaction with the Adenomatous polyposis coli (APC)/Axin/glycogen synthase kinase 3 (GSK3) destruction complex or GSK3 inactivating mutations. Here we studied the impact of β catenin antagonist Chibby (CBY) on β catenin signaling in BCR-ABL1+ cells. CBY is a small conserved protein which interacts with β catenin and impairs β catenin-mediated transcriptional activation through two distinct molecular mechanisms: 1) competition with T cell factor (TCF) or lymphoid enhancer factor (LEF) for β catenin binding; and 2) nuclear export of β catenin via interaction with 14-3-3. We found that its enforced expression in K562 cell line promoted β catenin cytoplasmic translocation resulting in inhibition of target gene transcription. Moreover, cytoplasmic accumulation of β catenin activated the endoplasmic reticulum (ER) stress-associated pathway known as unfolded protein response (UPR). CBY-driven cytoplasmic accumulation of β catenin is also a component of BCR-ABL1+ cell response to the TK inhibitor Imatinib (IM). It evoked the UPR activation leading to the induction of BCL2-interacting mediator of cell death (BIM) by UPR sensors. BIM, in turn, contributed to the execution phase of apoptosis in the activation of ER resident caspase 12 and mobilization of Ca(2+) stores. PMID:23707389

  20. Activation and activities of the p53 tumour suppressor protein

    Bálint, É; Vousden, K H

    2001-01-01

    The p53 tumour suppressor protein inhibits malignant progression by mediating cell cycle arrest, apoptosis or repair following cellular stress. One of the major regulators of p53 function is the MDM2 protein, and multiple forms of cellular stress activate p53 by inhibiting the MDM2-mediated degradation of p53. Mutations in p53, or disruption of the pathways that allow activation of p53, seem to be a general feature of all cancers. Here we review recent advances in our understanding of the pat...

  1. Total Cellular RNA Modulates Protein Activity.

    Majumder, Subhabrata; DeMott, Christopher M; Reverdatto, Sergey; Burz, David S; Shekhtman, Alexander

    2016-08-16

    RNA constitutes up to 20% of a cell's dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase activity relative to that seen with RNA isolated from cells grown in the presence of dextrose and methanol. Because the total cellular RNA content changes with growth medium, protein-RNA quinary interactions can alter in-cell protein biochemistry and may play an important role in cell adaptation, critical to many physiological and pathological states. PMID:27456029

  2. Raman optical activity of proteins and glycoproteins

    Raman optical activity (ROA), measured in this project as a small difference in the intensity of Raman scattering from chiral molecules in right- and left-circularly polarised incident laser light, offers the potential to provide more information about the structure of biological molecules in aqueous solution than conventional spectroscopic techniques. Chapter one contains a general discussion of the relative merits of different spectroscopic techniques for structure determination of biomolecules, as well as a brief introduction to ROA. In Chapter two a theoretical analysis of ROA is developed, which extends the discussion in chapter one. The spectrometer setup and sample preparation is then discussed in chapter three. Instrument and sample conditions are monitored to ensure that the best results are obtained. As with any experimental project problems occur, which may result in a degradation of the spectra obtained. The cause of these problems was explored and remedied whenever possible. Chapter four introduces a brief account of protein, glycoprotein and carbohydrate structure and function, with a particular emphasis on the structure of proteins. In the remaining chapters experimental ROA results on proteins and glycoproteins, with some carbohydrate samples, from a wide range of sources are examined. For example, in chapter five some β-sheet proteins are examined. Structural features in these proteins are examined in the extended amide III region of their ROA spectra, revealing that ROA is sensitive to the rigidity or flexibility inherent in proteins. Chapter six concentrates on a group of proteins (usually glycoproteins) known as the serine proteinase inhibitors (serpins). Medically, the serpins are one of the most important groups of proteins of current interest, with wide-ranging implications in conditions such as Down's syndrome, Alzheimer's disease, and emphysema with associated cirrhosis of the liver. With favourable samples and conditions ROA may offer the

  3. [Mitogen-activated protein kinases in atherosclerosis].

    Bryk, Dorota; Olejarz, Wioletta; Zapolska-Downar, Danuta

    2014-01-01

    Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases) intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase), JNK (c-Jun N-terminal kinase) and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis. PMID:24491891

  4. Mitogen-activated protein kinases in atherosclerosis

    Dorota Bryk

    2014-01-01

    Full Text Available Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase, JNK (c-Jun N-terminal kinase and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis.

  5. Synaptic Vesicle Proteins and Active Zone Plasticity

    Kittel, Robert J.; Heckmann, Manfred

    2016-01-01

    Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone (AZ). The complex molecular architecture of AZs mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of AZs vary significantly, even for a given connection. Thus, there appear to be distinct AZ states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the AZ. The protein-rich cytomatrix at the active zone (CAZ) provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1) and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and AZ states, which has heretofore received little attention.

  6. [Antioxidant activity of cationic whey protein isolate].

    titova, M E; Komolov, S A; Tikhomirova, N A

    2012-01-01

    The process of lipid peroxidation (LPO) in biological membranes of cells is carried out by free radical mechanism, a feature of which is the interaction of radicals with other molecules. In this work we investigated the antioxidant activity of cationic whey protein isolate, obtained by the cation-exchange chromatography on KM-cellulose from raw cow's milk, in vitro and in vivo. In biological liquids, which are milk, blood serum, fetal fluids, contains a complex of biologically active substances with a unique multifunctional properties, and which are carrying out a protective, antimicrobial, regenerating, antioxidant, immunomodulatory, regulatory and others functions. Contents of the isolate were determined electrophoretically and by its biological activity. Cationic whey protein isolate included lactoperoxidase, lactoferrin, pancreatic RNase, lysozyme and angeogenin. The given isolate significantly has an antioxidant effect in model experimental systems in vitro and therefore may be considered as a factor that can adjust the intensity of lipid oxidation. In model solutions products of lipid oxidation were obtained by oxidation of phosphatidylcholine by hydrogen peroxide in the presence of a source of iron. The composition of the reaction mixture: 0,4 mM H2O2; 50 mcM of hemin; 2 mg/ml L-alpha-phosphatidylcholine from soybean (Sigma, German). Lipid peroxidation products were formed during the incubation of the reaction mixture for two hours at 37 degrees C. In our studies rats in the adaptation period immediately after isolation from the nest obtained from food given orally native cationic whey protein isolate at the concentration three times higher than in fresh cow's milk. On the manifestation of the antioxidant activity of cationic whey protein isolate in vivo evidence decrease of lipid peroxidation products concentration in the blood of rats from the experimental group receipt whey protein isolate in dos 0,6 mg/g for more than 20% (pwhey protein isolate has an

  7. Discovering Protein-Protein Interactions within the Programmed Cell Death Network Using a Protein-Fragment Complementation Screen

    Yuval Gilad

    2014-08-01

    Full Text Available Apoptosis and autophagy are distinct biological processes, each driven by a different set of protein-protein interactions, with significant crosstalk via direct interactions among apoptotic and autophagic proteins. To measure the global profile of these interactions, we adapted the Gaussia luciferase protein-fragment complementation assay (GLuc PCA, which monitors binding between proteins fused to complementary fragments of a luciferase reporter. A library encompassing 63 apoptotic and autophagic proteins was constructed for the analysis of ∼3,600 protein-pair combinations. This generated a detailed landscape of the apoptotic and autophagic modules and points of interface between them, identifying 46 previously unknown interactions. One of these interactions, between DAPK2, a Ser/Thr kinase that promotes autophagy, and 14-3-3τ, was further investigated. We mapped the region responsible for 14-3-3τ binding and proved that this interaction inhibits DAPK2 dimerization and activity. This proof of concept underscores the power of the GLuc PCA platform for the discovery of biochemical pathways within the cell death network.

  8. Protein C activators in snake venoms.

    Stocker, K; Fischer, H; Meier, J; Brogli, M; Svendsen, L

    1986-02-01

    Venoms of 32 snake species were tested for protein C (PC) activating potency. As measured with the chromogenic PC substrate D-Pro-L-Pro-L-Arg-pNA, eleven venoms were able to generate amidolytic activity from purified bovine PC. In five venom solutions (Bothrops moojeni, B. pradoi, Cerastes cerastes, Vipera lebetina and V. russellii) the PC activating potency was destroyed during 10 min heating at 70 degrees C at pH 3, whereas in six venom solutions (Agkistrodon contortrix contortrix, A. c. mokasen, A. c. pictigaster, Agkistrodon piscivorus, A. p. leucostoma and A. bilineatus) the PC activator was stable under these conditions. PC activator from A. c. contortrix (Protac) was purified to homogeneity and characterized as a single chain polypeptide with a molecular weight of approx. 39-42,000 Dalton. Protac does not exert proteinase activity and is not inhibited by proteinase inhibitors; PC activation with Protac seems to be a stoichiometric reaction. The use of Protac in quantitative PC determination bears significant advantages over the use of thrombin as an activator. In rabbits, i.v. injection of Protac caused a prolonged APTT and did not provoke acute toxic reactions. PMID:3755037

  9. Heat dissipation guides activation in signaling proteins.

    Weber, Jeffrey K; Shukla, Diwakar; Pande, Vijay S

    2015-08-18

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein-coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  10. Arabinogalactan proteins: focus on carbohydrate active enzymes

    Eva eKnoch

    2014-06-01

    Full Text Available Arabinogalactan proteins (AGPs are a highly diverse class of cell surface proteoglycans that are commonly found in most plant species. AGPs play important roles in many cellular processes during plant development, such as reproduction, cell proliferation, pattern formation and growth, and in plant-microbe interaction. However, little is known about the molecular mechanisms of their function. Numerous studies using monoclonal antibodies that recognize different AGP glycan epitopes have shown the appearance of a slightly altered AGP glycan in a specific stage of development in plant cells. Therefore, it is anticipated that the biosynthesis and degradation of AGP glycan is tightly regulated during development. Until recently, however, little was known about the enzymes involved in the metabolism of AGP glycans. In this review, we summarize recent discoveries of carbohydrate active enzymes (CAZy; http://www.cazy.org/ involved in the biosynthesis and degradation of AGP glycans, and we discuss the biological role of these enzymes in plant development.

  11. Reconstitution of Membrane Proteins into Model Membranes: Seeking Better Ways to Retain Protein Activities

    Trevor Lithgow; Lisa Martin; Hsin-Hui Shen

    2013-01-01

    The function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. The activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. The reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. However, the activity of mem...

  12. Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

    Sato, Shogo; Jung, Hunmin; Nakagawa, Tsutomu; Pawlosky, Robert; Takeshima, Tomomi; Lee, Wan-Ru; Sakiyama, Haruhiko; Laxman, Sunil; Wynn, R Max; Tu, Benjamin P; MacMillan, John B; De Brabander, Jef K; Veech, Richard L; Uyeda, Kosaku

    2016-05-13

    The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis. PMID:26984404

  13. Mitogen-activated protein kinase signaling in plants

    Rodriguez, Maria Cristina Suarez; Petersen, Morten; Mundy, John

    2010-01-01

    Eukaryotic mitogen-activated protein kinase (MAPK) cascades have evolved to transduce environmental and developmental signals into adaptive and programmed responses. MAPK cascades relay and amplify signals via three types of reversibly phosphorylated kinases leading to the phosphorylation of...... substrate proteins, whose altered activities mediate a wide array of responses, including changes in gene expression. Cascades may share kinase components, but their signaling specificity is maintained by spaciotemporal constraints and dynamic protein-protein interactions and by mechanisms that include...

  14. Human cytomegalovirus IE2 protein interacts with transcription activating factors

    徐进平; 叶林柏

    2002-01-01

    The human cytomegalovirus (HCMV) IE86 Cdna was cloned into Pgex-2T and fusion protein GST-IE86 was expressed in E. Coli. SDS-PAGE and Western blot assay indicated that fusion protein GST-IE86 with molecular weight of 92 ku is soluble in the supernatant of cell lysate. Protein GST and fusion protein GST-IE86 were purified by affinity chromatography. The technology of co-separation and specific affinity chromatography was used to study the interactions of HCMV IE86 protein with some transcriptional regulatory proteins and transcriptional factors. The results indicated that IE86 interacts separately with transcriptional factor TFIIB and promoter DNA binding transcription trans-activating factors SP1, AP1 and AP2 to form a heterogenous protein complex. These transcriptional trans-activating factors, transcriptional factor and IE86 protein were adsorbed and retained in the affinity chromatography simultaneously. But IE86 protein could not interact with NF-Кb, suggesting that the function of IE86 protein that can interact with transcriptional factor and transcriptional trans-activating factors has no relevance to protein glycosylation. IE86 protein probably has two domains responsible for binding transcriptional trans-activating regulatory proteins and transcriptional factors respectively, thus activating the transcription of many genes. The interactions accelerated the assembly of the transcriptional initiation complexes.

  15. 4-hydroxy-2, 3-nonenal activates activator protein-1 and mitogen-activated protein kinases in rat pancreatic stellate cells

    Kazuhiro Kikuta; Atsushi Masamune; Masahiro Satoh; Noriaki Suzuki; Tooru Shimosegawa

    2004-01-01

    AIM: Activated pancreatic stellate cells (PSCs) are implicated in the pathogenesis of pancreatic inflammation and fibrosis,where oxidative stress is thought to play a key role. 4-hydroxy2,3-nonenal (HNE) is generated endogenously during the process of lipid peroxidation, and has been accepted as a mediator of oxidative stress. The aim of this study was to clarify the effects of HNE on the activation of signal transduction pathways and cellular functions in PSCs.METHODS: PSCs were isolated from the pancreas of male Wistar rats after perfusion with collagenase P, and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. PSCs were treated with physiologically relevant and non-cytotoxic concentrations (up to 5 μmol/L)of HNE. Activation of transcription factors was examined by electrophoretic mobility shift assay and luciferase assay.Activation of mitogen-activated protein (MAP) kinases was assessed by Western blotting using anti-phosphospecific antibodies. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine. Production of type Ⅰ collagen and monocyte chemoattractant protein-1was determined by enzyme-linked immunosorbent assay.The effect of HNE on the transformation of freshly isolated PSCs in culture was also assessed.RESULTS: HNE activated activator protein-1, but not nuclear factor κB. In addition, HNE activated three classes of MAP kinases: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAP kinase. HNE increased type Ⅰ collagen production through the activation of p38 MAP kinase and c-Jun N-terminal kinase. HNE did not alter the proliferation,or monocyte chemoattractant protein-1 production. HNE did not initiate the transformation of freshly isolated PSCs to myofibroblast-like phenotype.CONCLUSION: Specific activation of these signal transduction pathways and altered cell functions such as collagen production by HNE may play a role in the pathogenesis of pancreatic

  16. Antioxidant activity of black bean (Phaseolus vulgaris L. protein hydrolysates

    Jarine Amaral do EVANGELHO

    2016-01-01

    Full Text Available Abstract The objective of this work was to study the effect of enzymatic hydrolysis of black bean protein concentrate using different enzymes. Bean proteins were extracted and hydrolyzed over a period of 120 min using the enzymes pepsin or alcalase. The protein hydrolysates’ molecular weight was assayed by electrophoresis and the antioxidant activity was evaluated by the capturing methods of free radicals ABTS●+ and DPPH. Electrophoretic results showed that the bands above 50 kDa disappeared, when the beans protein was subjected to hydrolysis with pepsin. The bean protein hydrolysate obtained by hydrolysis with alcalase enzyme, showed higher antioxidant activity for inhibition of the radical ABTS●+. However, the hydrolysates obtained by hydrolysis with pepsin had higher antioxidant activity for inhibition of the radical DPPH. The use of pepsin and alcalase enzymes, under the same reaction time, produced black bean protein hydrolysates with different molecular weight profiles and superior antioxidant activity than the native bean protein.

  17. Heat dissipation guides activation in signaling proteins

    Weber, Jeffrey K.; Shukla, Diwakar; Pande, Vijay S.

    2015-01-01

    As with their macroscopic counterparts, the moving parts of nanoscale protein machines grow hot while in operation. A portion of the energy biomolecules harness to perform meaningful work is always dissipated as heat into the surroundings. Here, we feature a methodology by which dominant dissipative trajectories can be extracted from detailed models of protein dynamics. In two important classes of signaling proteins [kinases and G-protein–coupled receptors (GPCRs)], we find that the regions o...

  18. Serum paraoxonase activity and protein thiols in patients with hyperlipidemia

    Mungli Prakash; Jeevan K Shetty; Sudeshna Tripathy; Pannuri Vikram; Manish Verma

    2009-01-01

    Objective: In the present study we evaluated the paraoxonase activity and protein thiols level in south Indian population with newly diagnosed hyperlipidemia. Methods: The study was conducted on 55 newly diagnosed hyperlipidemic pa-tients and 57 healthy controls. Serum paraoxonase activity and protein thiols were estimated by spectrophotometeric method and lipid profile by enzymatic kinetic assay method. Results: Serum paraoxonase activity, protein thiols and high density lipoprotein levels were low and total cholesterol, triglycerides and low density lipoprutein levels were high in patients with hyperlipidemia compared to healthy controls ( P < 0.01 ). Serum paranxonase activity correlated positively with protein thiols and high density lipoprotein (P<0.01). Conclusion: Decreased paraoxonase activity and protein thiols were found in patients with hyperlipi-demia. This may indicate the susceptibility of this population to accelerated atherogenesis and protein oxidation.

  19. Modeling Protein Folding and Applying It to a Relevant Activity

    Nelson, Allan; Goetze, Jim

    2004-01-01

    The different levels of protein structure that can be easily understood by creating a model that simulates protein folding, which can then be evaluated by applying it to a relevant activity, is presented. The materials required and the procedure for constructing a protein folding model are mentioned.

  20. Active Nuclear Import of Membrane Proteins Revisited

    Laba, Justyna K; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the m

  1. Antioxidant activity of whey protein hydrolysates in milk beverage system

    Mann, Bimlesh; Kumari, Anuradha; Kumar, Rajesh; Sharma, Rajan; Prajapati, Kishore; Mahboob, Shaik; Athira, S.

    2014-01-01

    The aim of the present study was to evaluate the antioxidant activity of flavoured milk enriched with antioxidative whey protein hydrolysates (WPHs) by radical scavenging method. Whey protein concentrate (WPC) was hydrolyzed by using three commercial proteases; flavouzyme, alcalase and corolase PP and these WPHs were analyzed for degree of hydrolysis and antioxidant activity. The antioxidant activities of these WPHs were evaluated using ABTS method. Trolox equivalent antioxidant activity of a...

  2. DIETARY PROTEIN AND LACTOSE INCREASE TRANSLATION INITIATION FACTOR ACTIVATION AND TISSUE PROTEIN SYNTHESIS IN NEONATAL PIGS

    Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in muscle and liver of pigs parenterally infused with amino acids and insulin. To examine the effects of enteral protein and carbohydrate on protein synthesis, pigs (n = 42, 1.7 kg body wt) were fed isocaloric milk die...

  3. Purification, renaturation, and reconstituted protein kinase activity of the Sendai virus large (L) protein: L protein phosphorylates the NP and P proteins in vitro.

    Einberger, H; Mertz, R; Hofschneider, P H; Neubert, W J

    1990-01-01

    Sodium dodecyl sulfate-solubilized Sendai virus large (L) protein was highly purified by a one-step procedure, using hydroxylapatite column chromatography. Monoclonal antibodies addressed to the carboxyl-terminal amino acid sequence of the L protein were used for monitoring L protein during purification. By removing sodium dodecyl sulfate from purified L protein, a protein kinase activity was successfully renatured. P and NP proteins served as its substrates. After immunoprecipitation with an...

  4. Protein determination in seeds by proton activation

    Morales, J. R.; Dinator, M. I.; Cerda, P.

    1989-04-01

    A proton beam of 6.6 MeV has been used to produce 11C and 13N in Araucaria Araucana seeds. Their positron decay allows determination of the N/C ratio. In seeds the nitrogen content is associated to proteins while carbon is spread in the organic material. Samples were irradiated for about 10 min with a beam intensity of 5 nA on areas of 1 mm 2. Slices of the seed were radially explored, showing a larger concentration of protein in the center.

  5. Protein determination in seeds by proton activation

    Morales, J.R.; Dinator, M.I. (Chile Univ., Santiago (Chile). Lab. de Fisica); Cerda, P. (Bio-Bio Univ., Chillan (Chile))

    1989-04-01

    A proton beam of 6.6 MeV has been used to produce /sup 11/C and /sup 13/N in Araucaria Araucana seeds. Their positron decay allows determination of the N/C ratio. In seeds the nitrogen content is associated to proteins while carbon is spread in the organic material. Samples were irradiated for about 10 min with a beam intensity of 5 nA on areas of 1 mm/sup 2/. Slices of the seed were radially explored, showing a larger concentration of protein in the center. (orig.).

  6. Monitoring G protein activation in cells with BRET

    Masuho, Ikuo; Martemyanov, Kirill A.; Lambert, Nevin A.

    2016-01-01

    Summary Live-cell assays based on fluorescence and luminescence are now indispensable tools for the study of G protein signaling. Assays based on fluorescence and bioluminescence resonance energy transfer (FRET and BRET) have been particularly valuable for monitoring changes in second messengers, protein-protein interactions, and protein conformation. Here we describe a BRET assay that monitors the release of free Gβγ dimers after activation of heterotrimers containing Gα subunits from all four G protein subfamilies. This assay provides useful kinetic and pharmacological information with reasonably high throughput using standard laboratory equipment. PMID:26260597

  7. New constitutive latex osmotin-like proteins lacking antifungal activity.

    Freitas, Cleverson D T; Silva, Maria Z R; Bruno-Moreno, Frederico; Monteiro-Moreira, Ana C O; Moreira, Renato A; Ramos, Márcio V

    2015-11-01

    Proteins that share similar primary sequences to the protein originally described in salt-stressed tobacco cells have been named osmotins. So far, only two osmotin-like proteins were purified and characterized of latex fluids. Osmotin from Carica papaya latex is an inducible protein lacking antifungal activity, whereas the Calotropis procera latex osmotin is a constitutive antifungal protein. To get additional insights into this subject, we investigated osmotins in latex fluids of five species. Two potential osmotin-like proteins in Cryptostegia grandiflora and Plumeria rubra latex were detected by immunological cross-reactivity with polyclonal antibodies produced against the C. procera latex osmotin (CpOsm) by ELISA, Dot Blot and Western Blot assays. Osmotin-like proteins were not detected in the latex of Thevetia peruviana, Himatanthus drasticus and healthy Carica papaya fruits. Later, the two new osmotin-like proteins were purified through immunoaffinity chromatography with anti-CpOsm immobilized antibodies. Worth noting the chromatographic efficiency allowed for the purification of the osmotin-like protein belonging to H. drasticus latex, which was not detectable by immunoassays. The identification of the purified proteins was confirmed after MS/MS analyses of their tryptic digests. It is concluded that the constitutive osmotin-like proteins reported here share structural similarities to CpOsm. However, unlike CpOsm, they did not exhibit antifungal activity against Fusarium solani and Colletotrichum gloeosporioides. These results suggest that osmotins of different latex sources may be involved in distinct physiological or defensive events. PMID:26231325

  8. Hydrodynamic collective effects of active proteins in biological membranes

    Koyano, Yuki; Mikhailov, Alexander S

    2016-01-01

    Lipid bilayers forming biological membranes are known to behave as viscous 2D fluids on submicrometer scales; usually they contain a large number of active protein inclusions. Recently, it has been shown [Proc. Nat. Acad. Sci. USA 112, E3639 (2015)] that such active proteins should in- duce non-thermal fluctuating lipid flows leading to diffusion enhancement and chemotaxis-like drift for passive inclusions in biomembranes. Here, a detailed analytical and numerical investigation of such effects is performed. The attention is focused on the situations when proteins are concentrated within lipid rafts. We demonstrate that passive particles tend to become attracted by active rafts and are accumulated inside them.

  9. Antioxidant activities of buttermilk proteins, whey proteins, and their enzymatic hydrolysates.

    Conway, Valérie; Gauthier, Sylvie F; Pouliot, Yves

    2013-01-16

    The oxygen radical absorbance capacities (ORAC) and metal chelating capacities (MCC) of protein concentrates prepared from buttermilk and cheese whey by ultrafiltration were compared with those of skim milk protein. Samples were also heat-denatured and hydrolyzed by pepsin for 2 h followed by trypsin for 3 h. The highest MCC was obtained for hydrolyzed skim milk protein. ORAC values ranged from 554.4 to 1319.6 μmol Trolox equivalents/g protein, with the highest value obtained for hydrolyzed buttermilk protein. Liquid-phase isoelectric focusing (IEF) of this hydrolysate yielded peptide fractions with lower ORAC values. LC-MS analysis of the hydrolyzed skim milk and buttermilk proteins and IEF fractions of the latter showed that peptides derived from milk fat globule membrane proteins, primarily butyrophilin, could be responsible for the superior antioxidant activity of buttermilk. These results suggest overall that hydrolyzed buttermilk protein could be used as a source of natural antioxidants. PMID:23244578

  10. Protein stability and enzyme activity at extreme biological temperatures

    Feller, Georges, E-mail: gfeller@ulg.ac.b [Laboratory of Biochemistry, Centre for Protein Engineering, Institute of Chemistry B6a, University of Liege, B-4000 Liege (Belgium)

    2010-08-18

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 {sup 0}C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins. (topical review)

  11. Anthocyanin antioxidant activity and partition behavior in whey protein emulsion.

    Viljanen, Kaarina; Kylli, Petri; Hubbermann, Eva-Maria; Schwarz, Karin; Heinonen, Marina

    2005-03-23

    The antioxidant activities of anthocyanins and anthocyanin fractions isolated from blackcurrants, raspberries, and lingonberries were investigated in whey protein-stabilized emulsion. The extent of protein oxidation was measured by determining the loss of tryptophan fluorescence and formation of protein carbonyl compounds and that of lipid oxidation by conjugated diene hydroperoxides and hexanal analyses. The antioxidant activity of berry anthocyanins increased with an increase in concentration. Blackcurrant anthocyanins were the most potent antioxidants toward both protein and lipid oxidation at all concentrations due to the beneficial combination of delphinidin and cyanidin glycosides. Most berry anthocyanins (69.4-72.8%) partitioned into the aqueous phase of the emulsion, thus being located favorably for antioxidant action toward protein oxidation. The presence of the lipid decreased the share of anthocyanin in the aqueous phase. Thus, the structure of food affects the antioxidant activity by influencing the partitioning of the antioxidant. PMID:15769130

  12. Antioxidant activity of black bean (Phaseolus vulgaris L.) protein hydrolysates

    Jarine Amaral do EVANGELHO; Jose de J. BERRIOS; Vânia Zanella PINTO; Mariana Dias ANTUNES; Nathan Levien VANIER; Elessandra da Rosa ZAVAREZE

    2016-01-01

    Abstract The objective of this work was to study the effect of enzymatic hydrolysis of black bean protein concentrate using different enzymes. Bean proteins were extracted and hydrolyzed over a period of 120 min using the enzymes pepsin or alcalase. The protein hydrolysates’ molecular weight was assayed by electrophoresis and the antioxidant activity was evaluated by the capturing methods of free radicals ABTS●+ and DPPH. Electrophoretic results showed that the bands above 50 kDa disappeared,...

  13. Controlled Activation of Protein Rotational Dynamics Using Smart Hydrogel Tethering

    Beech, Brenda M.; Xiong, Yijia; Boschek, Curt B.; Baird, Cheryl L.; Bigelow, Diana J.; Mcateer, Kathleen; Squier, Thomas C.

    2014-09-05

    Stimulus-responsive hydrogel materials that stabilize and control protein dynamics have the potential to enable a range of applications to take advantage of the inherent specificity and catalytic efficiencies of proteins. Here we describe the modular construction of a hydrogel using an engineered calmodulin (CaM) within a polyethylene glycol (PEG) matrix that involves the reversible tethering of proteins through an engineered CaM-binding sequence. For these measurements, maltose binding protein (MBP) was isotopically labeled with [13C] and [15N], permitting dynamic structural measurements using TROSY-HSQC NMR spectroscopy. Upon initial formation of hydrogels protein dynamics are suppressed, with concomitant increases in protein stability. Relaxation of the hydrogel matrix following transient heating results in the activation of protein dynamics and restoration of substrate-induced large-amplitude domain motions necessary for substrate binding.

  14. Mitogen activated protein kinases: a role in inflammatory bowel disease?

    Broom, O J; Widjaya, B; Troelsen, J;

    2009-01-01

    Since their discovery more than 15 years ago, the mitogen activated protein kinases (MAPK) have been implicated in an ever-increasingly diverse array of pathways, including inflammatory signalling cascades. Inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn's disease, are...... their related signalling proteins in influencing the progression of IBD....

  15. Modeling of Raman optical activity of globular proteins

    Kessler, Jiří; Kapitán, J.; Yamamoto, S.; Bouř, Petr

    Sapporo : Hokkaido University, 2015. s. 113. [International Conference on Chiroptical Spectroscopy /15./. 30.08.2015-03.09.2015, Sapporo] Institutional support: RVO:61388963 Keywords : vibrational optical activity * proteins * quantum chemistry Subject RIV: CF - Physical ; Theoretical Chemistry

  16. Dietary Protein Considerations to Support Active Aging

    Wall, Benjamin T.; Cermak, Naomi M.; van Loon, Luc J. C.

    2014-01-01

    Given our rapidly aging world-wide population, the loss of skeletal muscle mass with healthy aging (sarcopenia) represents an important societal and public health concern. Maintaining or adopting an active lifestyle alleviates age-related muscle loss to a certain extent. Over time, even small losses of muscle tissue can hinder the ability to maintain an active lifestyle and, as such, contribute to the development of frailty and metabolic disease. Considerable research focus has addressed the ...

  17. Acquired deficiencies of protein S. Protein S activity during oral anticoagulation, in liver disease, and in disseminated intravascular coagulation.

    D'Angelo, A.; Vigano-D'Angelo, S; Esmon, C T; Comp, P C

    1988-01-01

    Protein S is a vitamin K-dependent plasma protein which serves as the cofactor for activated protein C. Protein S circulates in both an active, free form and in an inactive complex with C4b-binding protein. To elucidate the role of protein S in disease states and during oral anticoagulation, we developed a functional assay for protein S that permits evaluation of the distribution of protein S between free and bound forms and permits determination of the specific activity of the free protein S...

  18. Protein kinase activity associated with the nuclear lamina.

    Dessev, G; Iovcheva, C; Tasheva, B; R. Goldman

    1988-01-01

    A nuclear lamina-enriched fraction from Ehrlich ascites tumor cells contains a tightly bound protein kinase activity, which phosphorylates in vitro the nuclear lamins, a 52-kilodalton protein, and several unknown minor components. The enzyme(s) is thermolabile, independent of Ca2+ and cAMP, and inhibited by quercetin. After treatment with 4 M urea it remains bound to the nuclear lamina in an active state, but it is irreversibly inactivated in 6 M urea. The lamin proteins are phosphorylated on...

  19. Stress-induced activation of protein kinase CK2 by direct interaction with p38 mitogen-activated protein kinase

    Sayed, M; Kim, S O; Salh, B S;

    2000-01-01

    Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2...

  20. C-Reactive Protein Activates Complement in Infarcted Human Myocardium

    Nijmeijer, Remco; Lagrand, Wim K.; Lubbers, Yvonne T. P.; Visser, Cees A.; Meijer, Chris J.L.M.; Niessen, Hans W. M.; Hack, C. Erik

    2003-01-01

    Circulating levels of C-reactive protein (CRP) constitute a cardiovascular risk marker. Immunohistochemical studies have revealed co-localization of CRP and activated complement in human infarcted myocardium suggesting CRP to enhance inflammation in ischemic myocardium by inducing local complement activation. The aim was to establish whether CRP activates complement in infarcted human myocardium and to assess the relationship between this activation and the duration of infarction. Myocardial ...

  1. Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms

    Massip, L; Garand, C; Labbé, A;

    2010-01-01

    show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1...... contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases.......Werner's syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we...

  2. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  3. Multistep Phosphorelay Proteins Transmit Oxidative Stress Signals to the Fission Yeast Stress-activated Protein Kinase

    Nguyen, Aaron Ngocky; Lee, Albert; Place, Warren; Shiozaki, Kazuhiro

    2000-01-01

    In response to oxidative stress, eukaryotic cells induce transcription of genes required for detoxification of oxidants. Here we present evidence that oxidative stress stimuli are transmitted by a multistep phosphorelay system to the Spc1/Sty1 stress-activated protein kinase in the fission yeast Schizosaccharomyces pombe. The fission yeast mpr1+ gene encodes a novel protein with a histidine-containing phosphotransfer domain homologous to the budding yeast Ypd1. Spc1 activation upon oxidative ...

  4. Dissociation of activated protein C functions by elimination of protein S cofactor enhancement.

    Harmon, Shona

    2008-11-07

    Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

  5. Cellular reprogramming through mitogen-activated protein kinases

    Justin eLee

    2015-10-01

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

  6. Mapping the interactome of overexpressed RAF kinase inhibitor protein in a gastric cancer cell line

    Gastric cancer (GC) is a threat to human health with increasing incidence and mortality worldwide. Down-regulation or absence of RAF kinase inhibitor protein (RKIP) was associated with the occurrence, differentiation, invasion, and metastasis of GC. This study aims to investigate the molecular mechanisms and biological functions of RKIP in the GC biology. The fusion expression plasmid pcDNA3.1-RKIP-3xFLAG was transfected into SGC7901 cells, the RKIP fusion proteins were purified with anti-flag M2 magnetic beads, and the RKIP-interacting proteins were identified with tandem mass spectrometry (MS/MS), and were analyzed with bioinformatics tools. Western blot and co-immunoprecipitation were used to confirm the interaction complex. A total of 72 RKIP-interacting proteins were identified by MS/MS. Those proteins play roles in enzyme metabolism, molecular chaperoning, biological oxidation, cytoskeleton organization, signal transduction, and enzymolysis. Three RKIP-interaction protein network diagrams were constructed with Michigan Molecular Interactions, functional linage network, and Predictome analysis to address the molecular pathways of the functional activity of RKIP. The MS/MS-characterized components of the existing interaction complex (RKIP, HSP90, 14-3-3ϵ, and keratin 8) were confirmed by Western blot analysis and co-immunoprecipitation. This study is the first discovery of the interaction of RKIP with HSP90, 14-3-3, and keratin. The present data would provide insight into the molecular mechanisms of how RKIP inhibits the occurrence and development of GC

  7. Constitutive Photomorphogensis Protein1 (COP1 mediated p53 pathway and its oncogenic role

    Md. Golam Rabbani

    2014-05-01

    Full Text Available We have reviewed the COP1 mediated tumor suppressor protein p53 pathway and its oncogenic role. COP1 is a negative regulator of p53 and acts as a pivotal controller of p53-Akt death-live switch (Protein kinase B. In presence of p53, COP1 is overexpressed in breast, ovarian, gastric cancers, even without MDM2 (Mouse double minute-2 amplification. Following DNA damage, COP1 is phosphorylated instantly by ATM (Ataxia telangiectasia mutated and degraded by 14-3-3 and #963; following nuclear export and enhancing ubiquitination. In ATM lacking cell, other kinases, i.e. ATR (ataxia telangiectasia and Rad3-related protein, Jun kinases and DNA-PK (DNA-dependent protein kinase cause COP1 and CSN3 (COP9 signalosome complex subunit-3 phosphorylation and initiate COP1's down regulation. Although, it has been previously found that co-knockout of MDM2 and COP1 enhance p53's half life by eight fold, the reason is still unknown. Additionally, while interacting with p53, COP1 upregulate MDM2's E3 ubiquitin ligase, Akt, CSN6 (COP9 signalosome 6 activity and inhibit 14-3-3 and #963;'s negative regulation on MDM2 and COP1 itself. Conclusively, there persists an amplification loop among COP1, MDM2, Akt and 14-3-3 and #963; to regulate p53's stability and activity. However, the role of another tumor suppressor PTEN (phosphatase and tensin homologue is yet to be discovered. This study provides insight on the molecular genetic pathways related to cancer and might be helpful for therapeutic inventions. [Biomed Res Ther 2014; 1(5.000: 142-151

  8. Conserved interaction of the papillomavirus E2 transcriptional activator proteins with human and yeast TFIIB proteins.

    Benson, J D; Lawande, R; Howley, P M

    1997-01-01

    Papillomavirus early gene expression is regulated by the virus gene-encoded E2 proteins. The best-characterized E2 protein, encoded by bovine papillomavirus type 1 (BPV-1), has been shown to interact with basal transcription factor IIB (TFIIB) and the TATA binding protein basal transcription factor (N. M. Rank and P. F. Lambert, J. Virol. 69:6323-6334, 1995). We demonstrate that the potent E2 transcriptional activator protein encoded by a gene of human PV type 16 also interacts with TFIIB in ...

  9. Contractions activate hormone-sensitive lipase in rat muscle by protein kinase C and mitogen-activated protein kinase

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia;

    2003-01-01

    contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction......-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50% by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None of the...

  10. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins. PMID:27080133

  11. Anthelmintic activity of Leucaena leucocephala protein extracts on Haemonchus contortus.

    Soares, Alexandra Martins dos Santos; de Araújo, Sandra Alves; Lopes, Suzana Gomes; Costa Junior, Livio Martins

    2015-01-01

    The objective of this study was to evaluate the effects of protein extracts obtained from the plant Leucaena leucocephala on the nematode parasite Haemonchus contortus. The seeds, shell and cotyledon of L. leucocephala were separated and their proteins extracted using a sodium phosphate buffer, and named as TE (total seed extract), SE (shell extract) and CE (cotyledon extract). Soluble protein content, protease, protease inhibitory and chitinase activity assays were performed. Exsheathment inhibition of H. contortus larvae were performed at concentrations of 0.6 mg mL-1, and egg hatch assays were conducted at protein concentrations of 0.8, 0.4, 0.2, 0.1 and 0.05 mg mL-1. The effective concentration for 50% hatching inhibition (EC50) was estimated by probit. Different proportions of soluble proteins, protease and chitinase were found in TE and CE. Protease inhibitory activity was detected in all extracts. The EC50 of the CE and TE extracts were 0.48 and 0.33 mg mL-1, respectively. No ovicidal effects on H. contortus were detected in SE extracts, and none of the protein extracts demonstrated larvicidal effects on H. contortus. We therefore conclude that protein extracts of L. leucocephala had a detrimental effect on nematode eggs, which can be correlated with the high protease and chitinase activity of these extracts. PMID:26689178

  12. Organization, Structure and Activity of Proteins in Monolayers

    Boucher,J.; Trudel, E.; Methot, M.; Desmeules, P.; Salesse, C.

    2007-01-01

    Many different processes take place at the cell membrane interface. Indeed, for instance, ligands bind membrane proteins which in turn activate peripheral membrane proteins, some of which are enzymes whose action is also located at the membrane interface. Native cell membranes are difficult to use to gain information on the activity of individual proteins at the membrane interface because of the large number of different proteins involved in membranous processes. Model membrane systems, such as monolayers at the air-water interface, have thus been extensively used during the last 50 years to reconstitute proteins and to gain information on their organization, structure and activity in membranes. In the present paper, we review the recent work we have performed with membrane and peripheral proteins as well as enzymes in monolayers at the air-water interface. We show that the structure and orientation of gramicidin has been determined by combining different methods. Furthermore, we demonstrate that the secondary structure of rhodopsin and bacteriorhodopsin is indistinguishable from that in native membranes when appropriate conditions are used. We also show that the kinetics and extent of monolayer binding of myristoylated recoverin is much faster than that of the nonmyristoylated form and that this binding is highly favored by the presence polyunsaturated phospholipids. Moreover, we show that the use of fragments of RPE65 allow determine which region of this protein is most likely involved in membrane binding. Monomolecular films were also used to further understand the hydrolysis of organized phospholipids by phospholipases A2 and C.

  13. G protein activation stimulates phospholipase D signaling in plants

    Munnik, T.; Arisz, S.A.; Vrije, de T.; Musgrave, A.

    1995-01-01

    We provide direct evidence for phospholipase D (PLD) signaling in plants by showing that this enzyme is stimulated by the G protein activators mastoparan, ethanol, and cholera toxin. An in vivo assay for PLD activity in plant cells was developed based on the use of a "reporter alcohol" rather than w

  14. Activation of repair and checkpoints by double-strand breaks of DNA. Activational cascade of protein phosphorylation

    Molecular mechanisms of double-strand breaks repair and checkpoints include phosphorylations of repair and checkpoint-proteins by protein kinases. Chemical modification of proteins has different consequences including activation, changing of affinity to proteins and localization

  15. Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

    Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

    2005-01-01

    The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research. PMID:16302727

  16. Heated Proteins are Still Active in a Functionalized Nanoporous Support

    Chen, Baowei; Qi, Wen N.; Li, Xiaolin; Lei, Chenghong; Liu, Jun

    2013-07-08

    We report that even under the heated condition, the conformation and activity of a protein can be hoarded in a functionalized nanoporous support via non-covalent interaction, although the hoarded protein was not exhibiting the full protein activity, the protein released subsequently still maintained its native conformation and activity. Glucose oxidase (GOX) was spontaneously and largely entrapped in aminopropyl-functionalized mesoporous silica (NH2-FMS) at 20 oC via a dominant electrostatic interaction. Although FMS-GOX displayed 45% activity of the free enzyme in solution, the GOX released from FMS exhibited its 100% activity prior to the entrapment. Surprisingly, the released GOX from FMS still maintained 89% of its initial activity prior to the entrapment after FMS-GOX was incubated at 60 oC for 1 h prior to release, while the free GOX in solution lost nearly all activity under the same incubation. Intrinsic fluorescence emission of GOX and native electrophoresis demonstrated that the heating resulted in significant conformational changes and oligomeric structures of the free GOX, but FMS efficiently maintained the thermal stability of GOX therein and resisted the thermal denaturation and oligomeric aggregation.

  17. Redox Regulation of the AMP-Activated Protein Kinase

    Yingying Han; Qilong Wang; Ping Song; Yi Zhu; Ming-Hui Zou

    2010-01-01

    Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death. Objectives The aim of this study is to determine if AMP-activated protein kinase (AMPK), a cellular energy sensor, is activated by oxidants generated by Berberine in endothelial cells (EC). Methods Bovine aortic endothelial cells (BAEC) were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation. Results In BAEC, Berberine caused a dos...

  18. Redox Regulation of Protein Tyrosine Phosphatase Activity by Hydroxyl Radical

    Meng, Fan-Guo; Zhang, Zhong-Yin

    2012-01-01

    Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H2O2 include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H2O2 abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. ...

  19. Nanoencapsulation of Biologically Active Peptides from Whey Proteins

    Sebnem Tellioglu Harsa

    2014-06-01

    Full Text Available "Now a days consumers, in order to feed with balanced diet, prefer healthy and reliable foods. In this respect food manufacturers are trying to respond the demands of consumers by developing new types of foods such as diet foods ( low calorie foods, modified foods (organic foods and functional foods (probiotic and prebiotics. Thus, production of nutritious, functional and beneficial foods has become a growing sector in the United States and European countries. Proteins are major source of many bioactive peptides. Bioactive peptides have been defined as specific protein fragments that have a positive impact on body functions and may ultimately influence human health. These peptides stay inactive within the main protein structure and activated by the enzymatic hydrolysis. These bioactive peptides, derived from proteins, are able to influence basic body systems (cardiovascular, nervous, gastrointestinal and immune systems and show multi-functional character. Due to these properties, studies have recently been focused on milk proteins and their bioactive peptides. Such peptides are inactive within the sequence of the milk protein. Whey contains a multitude of biologically active proteins and peptides. Physiologically active serum proteins are serum albumin, immunoglobulins, proteose-peptone, lactoferrin, lactoperoxidase and growth factors. In addition to these, enzymatic degradation of serum proteins releases a number of bioactive peptides such as alfa-lactophorin, beta-lactophorin, beta- lactotensin, lactokinin, albutensin, serophorin and lactoferricin. One of the common qualities of bioactive substances is their sensitivity to the physical and chemical properties of the environment. For this reason, the usefulness of bioactive components in food is limited by the structure. In order to sustain bioavailibility of these peptides, limiting its relationship with the media by encapsulation technology is one of them osthotly debated issues on in recent

  20. Protein determination in soya bean by fast neutron activation analysis

    For a non-destructive determination of the protein content in soya bean samples, 14-MeV neutron activation analysis was applied. To check the method, the results obtained by X-ray fluorescence analysis and the Kjeldahl procedure were compared. For pressed pellet samples of about 1 g with 15 min irradiation and 10 min measuring times the accuracy of the protein determination was found to be 15%. (author) 7 refs.; 4 figs.; 3 tabs

  1. Emerging Roles of AMP-Activated Protein Kinase

    Fritzen, Andreas Mæchel

    The cellular energy sensor AMP-activated protein kinase (AMPK) is activated, when the energy balance of the cell decreases. AMPK has been proposed to regulate multiple metabolic processes. However, much of the evidence for these general effects of AMPK relies on investigations in cell systems or...... exercise appears to inhibit pyruvate dehydrogenase (PDH) activity by an immediate up-regulation of pyruvate dehydrogenase kinase 4 (PDK4) protein content. Consequently, this may inhibit glucose oxidation and thereby generate conditions for increased FA oxidation and glycogen resynthesis in skeletal muscle...... importance for prioritising energy dissipation, inhibition of lipid storage pathways and regulation of mitochondrial and metabolic proteins, but this needs further investigations. In addition, we provide evidence that AMPK is regulating autophagic signalling in skeletal muscle. Thus, in skeletal muscle AMPK...

  2. Antioxidant activity of whey protein hydrolysates in milk beverage system.

    Mann, Bimlesh; Kumari, Anuradha; Kumar, Rajesh; Sharma, Rajan; Prajapati, Kishore; Mahboob, Shaik; Athira, S

    2015-06-01

    The aim of the present study was to evaluate the antioxidant activity of flavoured milk enriched with antioxidative whey protein hydrolysates (WPHs) by radical scavenging method. Whey protein concentrate (WPC) was hydrolyzed by using three commercial proteases; flavouzyme, alcalase and corolase PP and these WPHs were analyzed for degree of hydrolysis and antioxidant activity. The antioxidant activities of these WPHs were evaluated using ABTS method. Trolox equivalent antioxidant activity of all the hydrolysates i.e. flavourzyme (0.81 ± 0.04), alcalase (1.16 ± 0.05) and corolase (1.42 ± 0.12) was higher than the WPC (0.19 ± 0.01). Among these, whey protein hydrolysates prepared using corolase showed maximum antioxidant activity. Total 15 β-lactoglobulin, 1 α-lactoalbumin, and 6 β-casein derived peptide fragments were identified in the WPHs by LC-MS/MS. Due to their size and characteristic amino acid composition, all the identified peptides may contribute for the antioxidant activity. The strawberry and chocolate flavoured milk was supplemented with WPC and WPHs and 2 % addition has shown increase in antioxidant activity upto 42 %. The result suggests that WPH could be used as natural biofunctional ingredients in enhancing antioxidant properties of food products. PMID:26028704

  3. Antioxidant activity of Fish Protein Hydrolysates from Sardinella longiceps

    JEEVITHA K

    2014-12-01

    Full Text Available Sardinella longiceps were hydrolysed with the different concentration of proteolytic enzymes trypsin to obtain peptides with antioxidant activity. The degree of hydrolysis and yield of hydrolysates were found increasing with increasing the concentration of enzyme. The antioxidant activities of hydrolysates were investigated through various assays. The hydrolysate exhibited the higher reducing power capability. The hydrolysates has shown a higher scavenging activity against DPPH, Superoxide, hydroxyl radical and metal chelating activity at the maximum concentration of 5mg/ml. The finding of this study reveals that, protein hydrolysates produced has potent antioxidant properties and it could be used as a food supplement in nutraceutical and pharmaceutical industry.

  4. Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis.

    Kohansal-Nodehi, Mahdokht; Chua, John Je; Urlaub, Henning; Jahn, Reinhard; Czernik, Dominika

    2016-01-01

    Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity. PMID:27115346

  5. Methods to distinguish various types of protein phosphatase activity

    To distinguish the action of protein Tyr(P) and protein Ser(P)/Thr(P) phosphatases on 32P-labeled phosphoproteins in subcellular fractions different inhibitors and activators are utilized. Comparison of the effects of added compounds provides a convenient, indirect method to characterize dephosphorylation reactions. Protein Tyr(P) phosphatases are specifically inhibited by micromolar Zn2+ or vanadate, and show maximal activity in the presence of EDTA. The other class of cellular phosphatases, specific for protein Ser(P) and Thr(P) residues, are inhibited by fluoride and EDTA. In this class of enzymes two major functional types can be distinguished: those sensitive to inhibition by the heat-stable protein inhibitor-2 and not stimulated by polycations, and those not sensitive to inhibition and stimulated by polycations. Preparation of 32P-labeled Tyr(P) and Ser(P) phosphoproteins also is presented for the direct measurement of phosphatase activities in preparations by the release of acid-soluble [32P]phosphate

  6. Detergent activation of the binding protein in the folate radioassay

    A minor cow's whey protein associated with β-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to β-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants

  7. Tau protein

    Frederiksen, Jette Lautrup Battistini; Kristensen, Kim; Bahl, Jmc;

    2011-01-01

    Background: Tau protein has been proposed as biomarker of axonal damage leading to irreversible neurological impairment in MS. CSF concentrations may be useful when determining risk of progression from ON to MS. Objective: To investigate the association between tau protein concentration and 14...... the Department of Neurology of Glostrup Hospital, University of Copenhagen, Denmark, were included. CSF samples were analysed for tau protein and 14-3-3 protein, and clinical and paraclinical information was obtained from medical records. Results: The study shows a significantly increased...... concentration of tau protein in CSF from patients with relapsing-remitting MS and patients monosymptomatic at onset who progressed to MS, but interestingly no increased tau protein concentration in monosymptomatic ON. The concentration of tau protein was significantly correlated to Expanded Disability Status...

  8. Quantifying Agonist Activity at G Protein-coupled Receptors

    Ehlert, Frederick J.; Suga, Hinako; Griffin, Michael T.

    2011-01-01

    When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.

  9. Activity-Based Protein Profiling of Rhomboid Proteases in Liposomes

    Wolf, E. V.; Seybold, M.; Hadravová, Romana; Stříšovský, Kvido; Verhelst, S. H. L.

    2015-01-01

    Roč. 16, č. 11 (2015), s. 1616-1621. ISSN 1439-4227 R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : activity -based protein profiling * chemical probes * inhibitors * intramembrane proteases * liposomes Subject RIV: CE - Biochemistry Impact factor: 3.088, year: 2014

  10. Reassessing the Potential Activities of Plant CGI-58 Protein.

    Khatib, Abdallah; Arhab, Yani; Bentebibel, Assia; Abousalham, Abdelkarim; Noiriel, Alexandre

    2016-01-01

    Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed. PMID:26745266