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Sample records for 14-3-3 protein family

  1. Structural basis for protein–protein interactions in the 14-3-3 protein family

    PubMed Central

    Yang, Xiaowen; Lee, Wen Hwa; Sobott, Frank; Papagrigoriou, Evangelos; Robinson, Carol V.; Grossmann, J. Günter; Sundström, Michael; Doyle, Declan A.; Elkins, Jonathan M.

    2006-01-01

    The seven members of the human 14-3-3 protein family regulate a diverse range of cell signaling pathways by formation of protein–protein complexes with signaling proteins that contain phosphorylated Ser/Thr residues within specific sequence motifs. Previously, crystal structures of three 14-3-3 isoforms (zeta, sigma, and tau) have been reported, with structural data for two isoforms deposited in the Protein Data Bank (zeta and sigma). In this study, we provide structural detail for five 14-3-3 isoforms bound to ligands, providing structural coverage for all isoforms of a human protein family. A comparative structural analysis of the seven 14-3-3 proteins revealed specificity determinants for binding of phosphopeptides in a specific orientation, target domain interaction surfaces and flexible adaptation of 14-3-3 proteins through domain movements. Specifically, the structures of the beta isoform in its apo and peptide bound forms showed that its binding site can exhibit structural flexibility to facilitate binding of its protein and peptide partners. In addition, the complex of 14-3-3 beta with the exoenzyme S peptide displayed a secondary structural element in the 14-3-3 peptide binding groove. These results show that the 14-3-3 proteins are adaptable structures in which internal flexibility is likely to facilitate recognition and binding of their interaction partners. PMID:17085597

  2. A fusicoccin binding protein belongs to the family of 14-3-3 brain protein homologs.

    PubMed Central

    Korthout, H A; de Boer, A H

    1994-01-01

    The fusicoccin binding protein (FCBP) is a highly conserved plasma membrane protein present in all higher plants tested thus far. It exhibits high- and low-affinity binding for the fungal toxin fusicoccin (FC). We purified the active FCBP from a fraction highly enriched in plasma membrane by selective precipitation and anion exchange chromatography. After SDS-PAGE, the two FCBP subunits of 30 and 31 kD were detected as major bands. Amino acid sequence analysis of the 31-kD polypeptide displayed a high degree of identity with so-called 14-3-3 proteins, a class of mammalian brain proteins initially described as regulators of neurotransmitter synthesis and protein kinase C inhibitors. Thereafter, we affinity purified the 30- and 31-kD FCBP subunits, using biotinylated FC in combination with a monomeric avidin column. Immunodecoration of these 30- and 31-kD FCBP subunits with polyclonal antibodies raised against a 14-3-3 homolog from yeast confirmed the identity of the FCBP as a 14-3-3 homolog. Similar to all 14-3-3 protein homologs, the FCBP seems to exist as a dimer in native form. Thus far, the FCBP is the only 14-3-3 homolog with a receptor-like function. The conserved structure of the 14-3-3 protein family is a further indication that the FCBP plays an important role in the physiology of higher plants. PMID:7827499

  3. Yeast 14-3-3 proteins.

    PubMed

    van Heusden, G Paul H; Steensma, H Yde

    2006-02-01

    14-3-3 proteins form a family of highly conserved proteins which are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. They interact with more than 200 different, mostly phosphorylated proteins. The molecular consequences of 14-3-3 binding are diverse: this binding may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization, to the interaction with other proteins or to shielding of binding sites. The binding partners, and hence the 14-3-3 proteins, are involved in almost every cellular process and 14-3-3 proteins have been linked to several diseases, such as cancer, Alzheimer's disease, the neurological Miller-Dieker and spinocerebellar ataxia type 1 diseases and bovine spongiform encephalopathy (BSE). The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe both have two genes encoding 14-3-3 proteins, BMH1 and BMH2 and rad24 and rad25, respectively. In these yeasts, 14-3-3 proteins are essential in most laboratory strains. As in higher eukaryotes, yeast 14-3-3 proteins bind to numerous proteins involved in a variety of cellular processes. Recent genome-wide studies on yeast strains with impaired 14-3-3 function support the participation of 14-3-3 proteins in numerous yeast cellular processes. Given the high evolutionary conservation of the 14-3-3 proteins, the experimental accessibility and relative simplicity of yeasts make them excellent model organisms for elucidating the function of the 14-3-3 protein family.

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

    PubMed Central

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

    2012-01-01

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

  5. Association of GABA(B) receptors and members of the 14-3-3 family of signaling proteins.

    PubMed

    Couve, A; Kittler, J T; Uren, J M; Calver, A R; Pangalos, M N; Walsh, F S; Moss, S J

    2001-02-01

    Two GABA(B) receptors, GABA(B)R1 and GABA(B)R2, have been cloned recently. Unlike other G protein-coupled receptors, the formation of a heterodimer between GABA(B)R1 and GABA(B)R2 is required for functional expression. We have used the yeast two hybrid system to identify proteins that interact with the C-terminus of GABA(B)R1. We report a direct association between GABA(B) receptors and two members of the 14-3-3 protein family, 14-3-3eta and 14-3-3zeta. We demonstrate that the C-terminus of GABA(B)R1 associates with 14-3-3zeta in rat brain preparations and tissue cultured cells, that they codistribute after rat brain fractionation, colocalize in neurons, and that the binding site overlaps partially with the coiled-coil domain of GABA(B)R1. Furthermore we show a reduced interaction between the C-terminal domains of GABA(B)R1 and GABA(B)R2 in the presence of 14-3-3. The results strongly suggest that GABA(B)R1 and 14-3-3 associate in the nervous system and begin to reveal the signaling complexities of the GABA(B)R1/GABA(B)R2 receptor heterodimer.

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

    PubMed

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

    2015-01-01

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

  7. 14-3-3 proteins: regulators of numerous eukaryotic proteins.

    PubMed

    van Heusden, G Paul H

    2005-09-01

    14-3-3 proteins form a family of highly conserved proteins capable of binding to more than 200 different mostly phosphorylated proteins. They are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. 14-3-3 binding partners are involved in almost every cellular process and 14-3-3 proteins play a key role in these processes. 14-3-3 proteins interact with products encoded by oncogenes, with filament forming proteins involved in Alzheimer'ss disease and many other proteins related to human diseases. Disturbance of the interactions with 14-3-3 proteins may lead to diseases like cancer and the neurological Miller-Dieker disease. The molecular consequences of 14-3-3 binding are diverse and only partly understood. Binding of a protein to a 14-3-3 protein may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization or to the interaction with other proteins. Currently genome- and proteome-wide studies are contributing to a wider knowledge of this important family of proteins.

  8. Controllability of protein-protein interaction phosphorylation-based networks: Participation of the hub 14-3-3 protein family

    PubMed Central

    Uhart, Marina; Flores, Gabriel; Bustos, Diego M.

    2016-01-01

    Posttranslational regulation of protein function is an ubiquitous mechanism in eukaryotic cells. Here, we analyzed biological properties of nodes and edges of a human protein-protein interaction phosphorylation-based network, especially of those nodes critical for the network controllability. We found that the minimal number of critical nodes needed to control the whole network is 29%, which is considerably lower compared to other real networks. These critical nodes are more regulated by posttranslational modifications and contain more binding domains to these modifications than other kinds of nodes in the network, suggesting an intra-group fast regulation. Also, when we analyzed the edges characteristics that connect critical and non-critical nodes, we found that the former are enriched in domain-to-eukaryotic linear motif interactions, whereas the later are enriched in domain-domain interactions. Our findings suggest a possible structure for protein-protein interaction networks with a densely interconnected and self-regulated central core, composed of critical nodes with a high participation in the controllability of the full network, and less regulated peripheral nodes. Our study offers a deeper understanding of complex network control and bridges the controllability theorems for complex networks and biological protein-protein interaction phosphorylation-based networked systems. PMID:27195976

  9. Controllability of protein-protein interaction phosphorylation-based networks: Participation of the hub 14-3-3 protein family.

    PubMed

    Uhart, Marina; Flores, Gabriel; Bustos, Diego M

    2016-05-19

    Posttranslational regulation of protein function is an ubiquitous mechanism in eukaryotic cells. Here, we analyzed biological properties of nodes and edges of a human protein-protein interaction phosphorylation-based network, especially of those nodes critical for the network controllability. We found that the minimal number of critical nodes needed to control the whole network is 29%, which is considerably lower compared to other real networks. These critical nodes are more regulated by posttranslational modifications and contain more binding domains to these modifications than other kinds of nodes in the network, suggesting an intra-group fast regulation. Also, when we analyzed the edges characteristics that connect critical and non-critical nodes, we found that the former are enriched in domain-to-eukaryotic linear motif interactions, whereas the later are enriched in domain-domain interactions. Our findings suggest a possible structure for protein-protein interaction networks with a densely interconnected and self-regulated central core, composed of critical nodes with a high participation in the controllability of the full network, and less regulated peripheral nodes. Our study offers a deeper understanding of complex network control and bridges the controllability theorems for complex networks and biological protein-protein interaction phosphorylation-based networked systems.

  10. 14-3-3 proteins are promising LRRK2 interactors.

    PubMed

    Rudenko, Iakov N; Cookson, Mark R

    2010-09-15

    Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most common cause of familial PD (Parkinson's disease). Mutations that cause PD are found in either the GTPase or kinase domains of LRRK2 or an intervening sequence called the COR [C-terminus of ROC (Ras of complex proteins)] domain. As well as the two catalytic domains, LRRK2 possesses several protein-protein interaction domains, but their function and the proteins with which they interact are poorly understood. In this issue of the Biochemical Journal, Nichols et al. study the interaction of the N-terminal region of LRRK2 with 14-3-3 proteins, regulatory proteins that often bind to phosphorylated regions of components of cell signalling pathways. Using a combination of techniques, Nichols et al. have identified two residues (Ser910 and Ser935) that are critically responsible for 14-3-3 binding. The interaction of LRRK2 with 14-3-3 proteins can prevent dephosphorylation of Ser910/Ser935 and stabilize LRRK2 structure, perhaps by influencing the dimerization of LRRK2. The ability to interact with 14-3-3 correlates with the pattern of intracellular LRRK2 distribution. Collectively, these new results identify a potentially important regulatory mechanism of this complex protein and might provide ways to think about therapeutic opportunities for PD.

  11. 14-3-3 Proteins: insights from genome-wide studies in yeast.

    PubMed

    van Heusden, G Paul H

    2009-11-01

    14-3-3 proteins form a family of highly conserved, acidic, dimeric proteins. These proteins have been identified in all eukaryotic species investigated, often in multiple isoforms, up to 13 in the plant Arabidopsis thaliana. Hundreds of proteins, from diverse eukaryotic organisms, implicated in numerous cellular processes, have been identified as binding partners of 14-3-3 proteins. Therefore, the major activity of 14-3-3 proteins seems to be its ability to bind other intracellular proteins. Binding to 14-3-3 proteins may result in a conformational change of the protein required for its full activity or for inhibition of its activity, in interaction between two binding partners or in a different subcellular localization. Most of these interactions take place after phosphorylation of the binding partners. These observations suggest a major role of 14-3-3 proteins in regulatory networks. Here, the information on 14-3-3 proteins gathered from several genome- and proteome-wide studies in the yeast Saccharomyces cerevisiae is reviewed. In particular, the protein kinases responsible for the phosphorylation of 14-3-3 binding partners, phosphorylation of 14-3-3 proteins themselves, the transcriptional regulation of the 14-3-3 genes, and the role of 14-3-3 proteins in transcription are addressed. These large scale studies may help understand the function of 14-3-3 proteins at a cellular level rather than at the level of a single process.

  12. An account of fungal 14-3-3 proteins.

    PubMed

    Kumar, Ravinder

    2017-02-24

    14-3-3s are a group of relatively low molecular weight, acidic, dimeric, protein(s) conserved from single-celled yeast to multicellular vertebrates including humans. Despite lacking catalytic activity, these proteins have been shown to be involved in multiple cellular processes. Apart from their role in normal cellular physiology, recently these proteins have been implicated in various medical consequences. In this present review, fungal 14-3-3 protein localization, interactions, transcription, regulation, their role in the diverse cellular process including DNA duplication, cell cycle, protein trafficking or secretion, apoptosis, autophagy, cell viability under stress, gene expression, spindle positioning, role in carbon metabolism have been discussed. In the end, I also highlighted various roles of yeasts 14-3-3 proteins in tabular form. Thus this review with primary emphasis on yeast will help in appreciating the significance of 14-3-3 proteins in cell physiology.

  13. 14-3-3 proteins regulate Tctp–Rheb interaction for organ growth in Drosophila

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    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

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

    PubMed

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

    2014-10-01

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

  16. Arabidopsis 14-3-3 Proteins: Fascinating and Less Fascinating Aspects

    PubMed Central

    Jaspert, Nina; Throm, Christian; Oecking, Claudia

    2011-01-01

    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. Such fascinating discoveries, however, 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 recent surveys of the Arabidopsis 14-3-3 interactome. PMID:22639620

  17. Fusicoccin, 14-3-3 Proteins, and Defense Responses in Tomato Plants1

    PubMed Central

    Roberts, Michael R.; Bowles, Dianna J.

    1999-01-01

    Fusicoccin (FC) is a fungal toxin that activates the plant plasma membrane H+-ATPase by binding with 14-3-3 proteins, causing membrane hyperpolarization. Here we report on the effect of FC on a gene-for-gene pathogen-resistance response and show that FC application induces the expression of several genes involved in plant responses to pathogens. Ten members of the FC-binding 14-3-3 protein gene family were isolated from tomato (Lycopersicon esculentum) to characterize their role in defense responses. Sequence analysis is suggestive of common biochemical functions for these tomato 14-3-3 proteins, but their genes showed different expression patterns in leaves after challenges. Different specific subsets of 14-3-3 genes were induced after treatment with FC and during a gene-for-gene resistance response. Possible roles for the H+-ATPase and 14-3-3 proteins in responses to pathogens are discussed. PMID:10198082

  18. Mechanism of inhibition of protein kinase C by 14-3-3 isoforms. 14-3-3 isoforms do not have phospholipase A2 activity.

    PubMed Central

    Robinson, K; Jones, D; Patel, Y; Martin, H; Madrazo, J; Martin, S; Howell, S; Elmore, M; Finnen, M J; Aitken, A

    1994-01-01

    The ability of individual members of the 14-3-3 protein family to inhibit protein kinase C (PKC) has been studied by using a synthetic peptide based on the specific 80 kDa substrate for PKC (MARCKS protein) in two different assay systems. Recombinant 14-3-3 and isoforms renatured by a novel method after separation by reverse-phase h.p.l.c. were studied. The detailed effects of diacylglycerol and the phorbol ester phorbol 12-myristate 13-acetate on the inhibition were also investigated. This suggests that one of the sites of interaction of 14-3-3 may be the cysteine-rich (C1) domain in PKC. Since a region in secreted phospholipase A2 (PLA2) shares similarity with this domain, the ability of 14-3-3 to interact with mammalian PLA2 was studied. Cytosolic PLA2 has some similarity to the C2 region of PKC, and the effect of 14-3-3 on this class of PLA2 was also analysed. In contrast with a previous report, no PLA2 activity was found in brain 14-3-3, nor in any of the recombinant proteins tested. These include zeta 14-3-3 isoform, on which the original observation was made. Images Figure 2 PMID:8192676

  19. 14-3-3 proteins: key regulators of cell division, signalling and apoptosis.

    PubMed

    van Hemert, M J; Steensma, H Y; van Heusden, G P

    2001-10-01

    The 14-3-3 proteins constitute a family of conserved proteins present in all eukaryotic organisms so far investigated. These proteins have attracted interest because they are involved in important cellular processes such as signal transduction, cell-cycle control, apoptosis, stress response and malignant transformation and because at least 100 different binding partners for the 14-3-3 proteins have been reported. Although the exact function of 14-3-3 proteins is still unknown, they are known to (1) act as adaptor molecules stimulating protein-protein interactions, (2) regulate the subcellular localisation of proteins and (3) activate or inhibit enzymes. In this review, we discuss the role of the 14-3-3 proteins in three cellular processes: cell cycle control, signal transduction and apoptosis. These processes are regulated by the 14-3-3 proteins at multiple steps. The 14-3-3 proteins have an overall inhibitory effect on cell cycle progression and apoptosis, whereas in signal transduction they may act as stimulatory or inhibitory factors. This article contains supplementary material which may be viewed at the BioEssays website at http://www.interscience.wiley.com/jpages/0265-9247/Suppmat/23/v23_10.936.

  20. Analysis of 14-3-3 Family Member Function in Xenopus Embryos by Microinjection of Antisense Morpholino Oligos

    NASA Astrophysics Data System (ADS)

    Lau, Jeffrey M. C.; Muslin, Anthony J.

    The 14-3-3 intracellular phosphoserine/threonine-binding proteins are adapter molecules that regulate signal transduction, cell cycle, nutrient sensing, apoptotic, and cytoskeletal pathways. There are seven 14-3-3 family members, encoded by separate genes, in vertebrate organisms. To evaluate the role of individual 14-3-3 proteins in vertebrate embryonic development, we utilized an antisense morpholino oligo microinjection technique in Xenopus laevis embryos. By use of this method, we showed that embryos lacking specific 14-3-3 proteins displayed unique phenotypic abnormalities. Specifically, embryos lacking 14-3-3 τ exhibited gastrulation and axial patterning defects, but embryos lacking 14-3-3 γ exhibited eye defects without other abnormalities, and embryos lacking 14-3-3 ζ appeared completely normal. These and other results demonstrate the power and specificity of the morpholino antisense oligo microinjection technique.

  1. Identification and characterization of protein 14-3-3 in carcinogenic liver fluke Opisthorchis viverrini.

    PubMed

    Kafle, Alok; Puchadapirom, Pranom; Plumworasawat, Sirikanya; Dontumprai, Rieofarng; Chan-On, Waraporn; Buates, Sureemas; Laha, Thewarach; Sripa, Banchob; Suttiprapa, Sutas

    2016-10-27

    Protein 14-3-3s are abundant phospho-serine/threonine binding proteins, which are highly conserved among eukaryotes. Members of this protein family mediate metabolism and signal transduction networks through binding to hundreds of other protein partners. Protein 14-3-3s have been studied in other species of parasitic helminthes, but little is known about this protein in the carcinogenic liver fluke Opisthorchis viverrini. In this study, we identified and characterized protein 14-3-3s of O. viverrini. Seven protein 14-3-3 encoded sequences were retrieved from the O. viverrini genome database. Multiple alignment and phylogenetic analysis were performed. Two isoforms (protein 14-3-3 zeta and protein 14-3-3 epsilon) that have been previously found in the excretory-secretory (ES) products of O. viverrini were produced as recombinant protein in E. coli and the proteins were then used to immunize mice to obtain specific antibodies. Western blot analysis showed that both proteins were detected in all obtainable developmental stages of O. viverrini and the ES products. Immunolocalization revealed that both isoforms were expressed throughout tissues and organs except the gut epithelium. The highest expression was observed in testes especially in developing spermatocytes, suggesting their role in spermatogenesis. Prominent expression was also detected on tegumental surface of the parasite and on epical surface of bile duct epithelium indicates their additional role in host-parasite interaction. These findings indicate that protein 14-3-3s play important role in the life cycle of the parasite and might be involved in the pathogenesis of O. viverrini infection.

  2. The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

    PubMed

    Satoh, Jun-ichi; Onoue, Hiroyuki; Arima, Kunimasa; Yamamura, Takashi

    2005-10-01

    The 14-3-3 protein family consists of acidic 30-kDa proteins composed of 7 isoforms expressed abundantly in neurons and glial cells of the central nervous system (CNS). The 14-3-3 protein identified in the cerebrospinal fluid provides a surrogate marker for premortem diagnosis of Creutzfeldt-Jakob disease, although an active involvement of 14-3-3 in the pathogenesis of prion diseases remains unknown. By protein overlay and mass spectrometric analysis of protein extract of NTera2-derived differentiated neurons, we identified heat shock protein Hsp60 as a 14-3-3-interacting protein. The 14-3-3zeta and gamma isoforms interacted with Hsp60, suggesting that the interaction is not isoform-specific. Furthermore, the interaction was identified in SK-N-SH neuroblastoma, U-373MG astrocytoma, and HeLa cervical carcinoma cells. The cellular prion protein (PrPC) along with Hsp60 was coimmunoprecipitated with 14-3-3 in the human brain protein extract. By protein overlay, 14-3-3 interacted with both recombinant human Hsp60 and PrPC produced by Escherichia coli, indicating that the molecular interaction is phosphorylation-independent. The 14-3-3-binding domain was located in the N-terminal half (NTF) of Hsp60 spanning amino acid residues 27-287 and the NTF of PrPC spanning amino acid residues 23-137. By immunostaining, the 14-3-3 protein Hsp60 and PrPC were colocalized chiefly in the mitochondria of human neuronal progenitor cells in culture, and were coexpressed most prominently in neurons and reactive astrocytes in the human brain. These observations indicate that the 14-3-3 protein forms a molecular complex with Hsp60 and PrPC in the human CNS under physiological conditions and suggest that this complex might become disintegrated in the pathologic process of prion diseases.

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

    PubMed Central

    Dhaubhadel, Sangeeta; Bian, Shaomin; Li, Xuyan

    2015-01-01

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

  4. alpha-Synuclein shares physical and functional homology with 14-3-3 proteins.

    PubMed

    Ostrerova, N; Petrucelli, L; Farrer, M; Mehta, N; Choi, P; Hardy, J; Wolozin, B

    1999-07-15

    alpha-Synuclein has been implicated in the pathophysiology of many neurodegenerative diseases, including Parkinson's disease (PD) and Alzheimer's disease. Mutations in alpha-synuclein cause some cases of familial PD (Polymeropoulos et al., 1997; Kruger et al., 1998). In addition, many neurodegenerative diseases show accumulation of alpha-synuclein in dystrophic neurites and in Lewy bodies (Spillantini et al., 1998). Here, we show that alpha-synuclein shares physical and functional homology with 14-3-3 proteins, which are a family of ubiquitous cytoplasmic chaperones. Regions of alpha-synuclein and 14-3-3 proteins share over 40% homology. In addition, alpha-synuclein binds to 14-3-3 proteins, as well as some proteins known to associate with 14-3-3, including protein kinase C, BAD, and extracellular regulated kinase, but not Raf-1. We also show that overexpression of alpha-synuclein inhibits protein kinase C activity. The association of alpha-synuclein with BAD and inhibition of protein kinase C suggests that increased expression of alpha-synuclein could be harmful. Consistent with this hypothesis, we observed that overexpression of wild-type alpha-synuclein is toxic, and overexpression of alpha-synuclein containing the A53T or A30P mutations exhibits even greater toxicity. The activity and binding profile of alpha-synuclein suggests that it might act as a protein chaperone and that accumulation of alpha-synuclein could contribute to cell death in neurodegenerative diseases.

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

    PubMed

    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.

  6. Multisite phosphorylation of 14-3-3 proteins by calcium-dependent protein kinases

    PubMed Central

    Swatek, Kirby N.; Wilson, Rashaun S.; Ahsan, Nagib; Tritz, Rebecca L.; Thelen, Jay J.

    2014-01-01

    Plant 14-3-3 proteins are phosphorylated at multiple sites in vivo; however, the protein kinase(s) responsible are unknown. Of the 34 CPK (calcium-dependent protein kinase) paralogues in Arabidopsis thaliana, three (CPK1, CPK24 and CPK28) contain a canonical 14-3-3-binding motif. These three, in addition to CPK3, CPK6 and CPK8, were tested for activity against recombinant 14-3-3 proteins χ and ε. Using an MS-based quantitative assay we demonstrate phosphorylation of 14-3-3 χ and ε at a total of seven sites, one of which is an in vivo site discovered in Arabidopsis. CPK autophosphorylation was also comprehensively monitored by MS and revealed a total of 45 sites among the six CPKs analysed, most of which were located within the N-terminal variable and catalytic domains. Among these CPK autophosphorylation sites was Tyr463 within the calcium-binding EF-hand domain of CPK28. Of all CPKs assayed, CPK28, which contained an autophosphorylation site (Ser43) within a canonical 14-3-3-binding motif, showed the highest activity against 14-3-3 proteins. Phosphomimetic mutagenesis of Ser72 to aspartate on 14-3-3χ, which is adjacent to the 14-3-3-binding cleft and conserved among all 14-3-3 isoforms, prevented 14-3-3-mediated inhibition of phosphorylated nitrate reductase. PMID:24438037

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

    SciTech Connect

    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

    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.

  8. Membrane proteins as 14-3-3 clients in functional regulation and intracellular transport.

    PubMed

    Smith, Andrew J; Daut, Jürgen; Schwappach, Blanche

    2011-06-01

    14-3-3 proteins regulate the function and subcellular sorting of membrane proteins. Often, 14-3-3 binding to client proteins requires phosphorylation of the client, but the relevant kinase is unknown in most cases. We summarize current progress in identifying kinases that target membrane proteins with 14-3-3 binding sites and discuss the molecular mechanisms of 14-3-3 action. One of the kinases involved is Akt/PKB, which has recently been shown to activate the 14-3-3-dependent switch in a number of client membrane proteins.

  9. 14-3-3 proteins, FHA domains and BRCT domains in the DNA damage response.

    PubMed

    Mohammad, Duaa H; Yaffe, Michael B

    2009-09-02

    The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phosphorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G(2)/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.

  10. IDENTIFICATION AND EXPRESSION ANALYSIS OF TWO 14-3-3 PROTEINS IN THE MOSQUITO Aedes aegypti, AN IMPORTANT ARBOVIRUSES VECTOR.

    PubMed

    Trujillo-Ocampo, Abel; Cázares-Raga, Febe Elena; Celestino-Montes, Antonio; Cortés-Martínez, Leticia; Rodríguez, Mario H; Hernández-Hernández, Fidel de la Cruz

    2016-11-01

    The 14-3-3 proteins are evolutionarily conserved acidic proteins that form a family with several isoforms in many cell types of plants and animals. In invertebrates, including dipteran and lepidopteran insects, only two isoforms have been reported. 14-3-3 proteins are scaffold molecules that form homo- or heterodimeric complexes, acting as molecular adaptors mediating phosphorylation-dependent interactions with signaling molecules involved in immunity, cell differentiation, cell cycle, proliferation, apoptosis, and cancer. Here, we describe the presence of two isoforms of 14-3-3 in the mosquito Aedes aegypti, the main vector of dengue, yellow fever, chikungunya, and zika viruses. Both isoforms have the conserved characteristics of the family: two protein signatures (PS1 and PS2), an annexin domain, three serine residues, targets for phosphorylation (positions 58, 184, and 233), necessary for their function, and nine alpha helix-forming segments. By sequence alignment and phylogenetic analysis, we found that the molecules correspond to Ɛ and ζ isoforms (Aeae14-3-3ε and Aeae14-3-3ζ). The messengers and protein products were present in all stages of the mosquito life cycle and all the tissues analyzed, with a small predominance of Aeae14-3-3ζ except in the midgut and ovaries of adult females. The 14-3-3 proteins in female midgut epithelial cells were located in the cytoplasm. Our results may provide insights to further investigate the functions of these proteins in mosquitoes.

  11. Identification of novel 14-3-3ζ interacting proteins by quantitative immunoprecipitation combined with knockdown (QUICK).

    PubMed

    Ge, Feng; Li, Wen-Liang; Bi, Li-Jun; Tao, Sheng-Ce; Zhang, Zhi-Ping; Zhang, Xian-En

    2010-11-05

    The family of 14-3-3 proteins has emerged as critical regulators of diverse cellular responses under both physiological and pathological conditions. To gain insight into the molecular action of 14-3-3ζ in multiple myeloma (MM), we performed a systematic proteomic analysis of 14-3-3ζ-associated proteins. This analysis, recently developed by Matthias Mann, termed quantitative immunoprecipitation combined with knockdown (QUICK), integrates RNAi, SILAC, immunoprecipitation, and quantitative MS technologies. Quantitative mass spectrometry analysis allowed us to distinguish 14-3-3ζ-interacting proteins from background proteins, resulting in the identification of 292 proteins in total with 95 novel interactions. Three 14-3-3ζ-interacting proteins-BAX, HSP70, and BAG3-were further confirmed by reciprocal coimmunoprecipitations and colocalization analysis. Our results therefore not only uncover a large number of novel 14-3-3ζ-associated proteins that possess a variety of cellular functions, but also provide new research directions for the study of the functions of 14-3-3ζ. This study also demonstrated that QUICK is a useful approach to detect specific protein-protein interactions with very high confidence and may have a wide range of applications in the investigation of protein complex interaction networks.

  12. Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes

    PubMed Central

    2004-01-01

    14-3-3 proteins exert an extraordinarily widespread influence on cellular processes in all eukaryotes. They operate by binding to specific phosphorylated sites on diverse target proteins, thereby forcing conformational changes or influencing interactions between their targets and other molecules. In these ways, 14-3-3s ‘finish the job’ when phosphorylation alone lacks the power to drive changes in the activities of intracellular proteins. By interacting dynamically with phosphorylated proteins, 14-3-3s often trigger events that promote cell survival – in situations from preventing metabolic imbalances caused by sudden darkness in leaves to mammalian cell-survival responses to growth factors. Recent work linking specific 14-3-3 isoforms to genetic disorders and cancers, and the cellular effects of 14-3-3 agonists and antagonists, indicate that the cellular complement of 14-3-3 proteins may integrate the specificity and strength of signalling through to different cellular responses. PMID:15167810

  13. Regulation of transcription by Saccharomyces cerevisiae 14-3-3 proteins

    PubMed Central

    Bruckmann, Astrid; Steensma, H. Yde; Teixeira de Mattos, M. Joost; van Heusden, G. Paul H.

    2004-01-01

    14-3-3 proteins form a family of highly conserved eukaryotic proteins involved in a wide variety of cellular processes, including signalling, apoptosis, cell-cycle control and transcriptional regulation. More than 150 binding partners have been found for these proteins. The yeast Saccharomyces cerevisiae has two genes encoding 14-3-3 proteins, BMH1 and BMH2. A bmh1 bmh2 double mutant is unviable in most laboratory strains. Previously, we constructed a temperature-sensitive bmh2 mutant and showed that mutations in RTG3 and SIN4, both encoding transcriptional regulators, can suppress the temperature-sensitive phenotype of this mutant, suggesting an inhibitory role of the 14-3-3 proteins in Rtg3-dependent transcription [van Heusden and Steensma (2001) Yeast 18, 1479–1491]. In the present paper, we report a genome-wide transcription analysis of a temperature-sensitive bmh2 mutant. Steady-state mRNA levels of 60 open reading frames were increased more than 2.0-fold in the bmh2 mutant, whereas those of 78 open reading frames were decreased more than 2.0-fold. In agreement with our genetic experiments, six genes known to be regulated by Rtg3 showed elevated mRNA levels in the mutant. In addition, several genes with other cellular functions, including those involved in gluconeogenesis, ergosterol biosynthesis and stress response, had altered mRNA levels in the mutant. Our data show that the yeast 14-3-3 proteins negatively regulate Rtg3-dependent transcription, stimulate the transcription of genes involved in ergosterol metabolism and in stress response and are involved in transcription regulation of multiple other genes. PMID:15142031

  14. Regulation of transcription by Saccharomyces cerevisiae 14-3-3 proteins.

    PubMed

    Bruckmann, Astrid; Steensma, H Yde; Teixeira De Mattos, M Joost; Van Heusden, G Paul H

    2004-09-15

    14-3-3 proteins form a family of highly conserved eukaryotic proteins involved in a wide variety of cellular processes, including signalling, apoptosis, cell-cycle control and transcriptional regulation. More than 150 binding partners have been found for these proteins. The yeast Saccharomyces cerevisiae has two genes encoding 14-3-3 proteins, BMH1 and BMH2. A bmh1 bmh2 double mutant is unviable in most laboratory strains. Previously, we constructed a temperature-sensitive bmh2 mutant and showed that mutations in RTG3 and SIN4, both encoding transcriptional regulators, can suppress the temperature-sensitive phenotype of this mutant, suggesting an inhibitory role of the 14-3-3 proteins in Rtg3-dependent transcription [van Heusden and Steensma (2001) Yeast 18, 1479-1491]. In the present paper, we report a genome-wide transcription analysis of a temperature-sensitive bmh2 mutant. Steady-state mRNA levels of 60 open reading frames were increased more than 2.0-fold in the bmh2 mutant, whereas those of 78 open reading frames were decreased more than 2.0-fold. In agreement with our genetic experiments, six genes known to be regulated by Rtg3 showed elevated mRNA levels in the mutant. In addition, several genes with other cellular functions, including those involved in gluconeogenesis, ergosterol biosynthesis and stress response, had altered mRNA levels in the mutant. Our data show that the yeast 14-3-3 proteins negatively regulate Rtg3-dependent transcription, stimulate the transcription of genes involved in ergosterol metabolism and in stress response and are involved in transcription regulation of multiple other genes.

  15. Human 14-3-3 gamma protein results in abnormal cell proliferation in the developing eye of Drosophila melanogaster

    PubMed Central

    Hong, Sophia W; Qi, Wenqing; Brabant, Marc; Bosco, Giovanni; Martinez, Jesse D

    2008-01-01

    Background 14-3-3 proteins are a family of adaptor proteins that participate in a wide variety of cellular processes. Recent evidence indicates that the expression levels of these proteins are elevated in some human tumors providing circumstantial evidence for their involvement in human cancers. However, the mechanism through which these proteins act in tumorigenesis is uncertain. Results To determine whether elevated levels of 14-3-3 proteins may perturb cell growth we overexpressed human 14-3-3 gamma (h14-3-3 gamma) in Drosophila larvae using the heat shock promoter or the GMR-Gal4 driver and then examined the effect that this had on cell proliferation in the eye imaginal discs of third instar larvae. We found that induction of h14-3-3 gamma resulted in the abnormal appearance of replicating cells in the differentiating proneural photoreceptor cells of eye imaginal discs where h14-3-3 gamma was driven by the heat shock promoter. Similarly, we found that driving h14-3-3 gamma expression specifically in developing eye discs with the GMR-Gal4 driver resulted in increased numbers of replicative cells following the morphogenetic furrow. Interestingly, we found that the effects of overexpressing h1433 gamma on eye development were increased in a genetic background where String (cdc25) function was compromised. Conclusion Taken together our results indicate that h14-3-3 gamma can promote abnormal cell proliferation and may act through Cdc25. This has important implications for 14-3-3 gamma as an oncogene as it suggests that elevated levels of 14-3-3 may confer a growth advantage to cells that overexpress it. PMID:18194556

  16. Small-Molecule Stabilization of 14-3-3 Protein-Protein Interactions Stimulates Axon Regeneration.

    PubMed

    Kaplan, Andrew; Morquette, Barbara; Kroner, Antje; Leong, SooYuen; Madwar, Carolin; Sanz, Ricardo; Banerjee, Sara L; Antel, Jack; Bisson, Nicolas; David, Samuel; Fournier, Alyson E

    2017-03-08

    Damaged central nervous system (CNS) neurons have a poor ability to spontaneously regenerate, causing persistent functional deficits after injury. Therapies that stimulate axon growth are needed to repair CNS damage. 14-3-3 adaptors are hub proteins that are attractive targets to manipulate cell signaling. We identify a positive role for 14-3-3s in axon growth and uncover a developmental regulation of the phosphorylation and function of 14-3-3s. We show that fusicoccin-A (FC-A), a small-molecule stabilizer of 14-3-3 protein-protein interactions, stimulates axon growth in vitro and regeneration in vivo. We show that FC-A stabilizes a complex between 14-3-3 and the stress response regulator GCN1, inducing GCN1 turnover and neurite outgrowth. These findings show that 14-3-3 adaptor protein complexes are druggable targets and identify a new class of small molecules that may be further optimized for the repair of CNS damage.

  17. Efficient nuclear export of p65-IkappaBalpha complexes requires 14-3-3 proteins.

    PubMed

    Aguilera, Cristina; Fernández-Majada, Vanessa; Inglés-Esteve, Julia; Rodilla, Verónica; Bigas, Anna; Espinosa, Lluís

    2006-09-01

    IkappaB are responsible for maintaining p65 in the cytoplasm under non-stimulating conditions and promoting the active export of p65 from the nucleus following NFkappaB activation to terminate the signal. We now show that 14-3-3 proteins regulate the NFkappaB signaling pathway by physically interacting with p65 and IkappaBalpha proteins. We identify two functional 14-3-3 binding domains in the p65 protein involving residues 38-44 and 278-283, and map the interaction region of IkappaBalpha in residues 60-65. Mutation of these 14-3-3 binding domains in p65 or IkappaBalpha results in a predominantly nuclear distribution of both proteins. TNFalpha treatment promotes recruitment of 14-3-3 and IkappaBalpha to NFkappaB-dependent promoters and enhances the binding of 14-3-3 to p65. Disrupting 14-3-3 activity by transfection with a dominant-negative 14-3-3 leads to the accumulation of nuclear p65-IkappaBalpha complexes and the constitutive association of p65 with the chromatin. In this situation, NFkappaB-dependent genes become unresponsive to TNFalpha stimulation. Together our results indicate that 14-3-3 proteins facilitate the nuclear export of IkappaBalpha-p65 complexes and are required for the appropriate regulation of NFkappaB signaling.

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

    PubMed

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

    2012-05-04

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

  19. 14-3-3β protein expression in eosinophilic meningitis caused by Angiostrongylus cantonensis infection

    PubMed Central

    2014-01-01

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

  20. Regulation of the wheat MAP kinase phosphatase 1 by 14-3-3 proteins.

    PubMed

    Ghorbel, Mouna; Cotelle, Valérie; Ebel, Chantal; Zaidi, Ikram; Ormancey, Mélanie; Galaud, Jean-Philippe; Hanin, Moez

    2017-04-01

    Plant MAP kinase phosphatases (MKPs) are major regulators of MAPK signaling pathways and play crucial roles in controlling growth, development and stress responses. The presence of several functional domains in plant MKPs such as a dual specificity phosphatase catalytic domain, gelsolin, calmodulin-binding and serine-rich domains, suggests that MKPs can interact with distinct cellular partners, others than MAPKs. In this report, we identified a canonical mode I 14-3-3-binding motif (574KLPSLP579) located at the carboxy-terminal region of the wheat MKP, TMKP1. We found that this motif is well-conserved among other MKPs from monocots including Hordeum vulgare, Brachypodium distachyon and Aegilops taushii. Using co-immunoprecipitation assays, we provide evidence for interaction between TMKP1 and 14-3-3 proteins in wheat. Moreover, the phosphatase activity of TMKP1 is increased in a phospho-dependent manner by either Arabidopsis or yeast 14-3-3 isoforms. TMKP1 activation by 14-3-3 proteins is enhanced by Mn(2+), whereas in the presence of Ca(2+) ions, TMKP1 activation was limited to Arabidopsis 14-3-3φ (phi), an isoform harboring an EF-hand motif. Such findings strongly suggest that 14-3-3 proteins, in conjunction with specific divalent cations, may stimulate TMKP1 activity and point-out that 14-3-3 proteins bind and regulate the activity of a MKP in eukaryotes.

  1. 14-3-3 Proteins are essential for regulation of RTG3-dependent transcription in Saccharomyces cerevisiae.

    PubMed

    van Heusden, G P; Steensma, H Y

    2001-12-01

    14-3-3 proteins comprise a family of highly conserved proteins that bind more than 60 different, mostly phosphorylated, proteins. The yeast Saccharomyces cerevisiae has two genes, BMH1 and BMH2, encoding 14-3-3 proteins. Disruption of both genes together is lethal. In this study we constructed a mutant with a single, temperature-sensitive bmh allele. Recessive mutations in SIN4 and RTG3 can suppress the temperature-sensitive phenotype of this mutant. These genes encode a global transcriptional regulator and a basic helix-loop-helix transcription factor, respectively. The yeast 14-3-3 proteins were shown to bind to the Rtg3 protein. Overexpression of RTG3 is lethal even in wild-type cells. These genetic and biochemical data are consistent with a model in which the 14-3-3 proteins are required to keep the Rtg3 protein in an inactive state, which is (one of) the essential function(s) of the 14-3-3 proteins.

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

    PubMed Central

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

    2016-01-01

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

  3. Light modulated activity of root alkaline/neutral invertase involves the interaction with 14-3-3 proteins.

    PubMed

    Gao, Jing; van Kleeff, Paula J M; Oecking, Claudia; Li, Ka Wan; Erban, Alexander; Kopka, Joachim; Hincha, Dirk K; de Boer, Albertus H

    2014-12-01

    Alkaline/neutral invertases (A/N-Invs) are now recognized as essential proteins in plant life. They catalyze the irreversible breakdown of sucrose into glucose and fructose and thus supply the cells with energy as well as signaling molecules. In this study we report on a mechanism that affects the activity of the cytosolic invertase AtCINV1 (At-A/N-InvG or AT1G35580). We demonstrate that Ser547 at the extreme C-terminus of the AtCINV1 protein is a substrate of calcium-dependent kinases (CPK3 and 21) and that phosphorylation creates a high-affinity binding site for 14-3-3 proteins. The invertase as such has basal activity, but we provide evidence that interaction with 14-3-3 proteins enhances its activity. The analysis of three quadruple 14-3-3 mutants generated from six T-DNA insertion mutants of the non-epsilon family shows both specificity as well as redundancy for this function of 14-3-3 proteins. The strong reduction in hexose levels in the roots of one 14-3-3 quadruple mutant plant is in line with the activating function of 14-3-3 proteins. The physiological relevance of this mechanism that affects A/N-invertase activity is underscored by the light-induced activation and is another example of the central role of 14-3-3 proteins in mediating dark/light signaling. The nature of the light-induced signal that travels from the shoot to root and the question whether this signal is transmitted via cytosolic Ca(++) changes that activate calcium-dependent kinases, await further study.

  4. CDPKs and 14-3-3 Proteins: Emerging Duo in Signaling.

    PubMed

    Ormancey, Mélanie; Thuleau, Patrice; Mazars, Christian; Cotelle, Valérie

    2017-03-01

    Calcium-dependent protein kinases (CDPKs) are Ca(2+)-sensors that play pivotal roles in plant development and stress responses. They have the unique ability to directly translate intracellular Ca(2+) signals into reversible phosphorylation events of diverse substrates which can mediate interactions with 14-3-3 proteins to modulate protein functions. Recent studies have revealed roles for the coordinated action of CDPKs and 14-3-3s in regulating diverse aspects of plant biology including metabolism, development, and stress responses. We review here the underlying interaction and cross-regulation of the two signaling proteins, and we discuss how this insight has led to the emerging concept of CDPK/14-3-3 signaling modules that could contribute to response specificity.

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

    MedlinePlus

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

  6. Loss of Par3 promotes lung adenocarcinoma metastasis through 14-3-3ζ protein.

    PubMed

    Song, Tong; Tian, Xia; Kai, Fan; Ke, Jiang; Wei, Zhai; Jing-Song, Li; Si-Hua, Wang; Jian-Jun, Wang

    2016-09-27

    Partitioning defective protein 3 (Par3) can activate the Tiam1/Rac pathway to inhibit invasion and metastasis in many cancers; however, the role of Par3 in lung adenocarcinoma remains unknown. Here we show that Par3 is downregulated in lung adenocarcinoma tissues and is associated with higher rates of lymph node metastasis and recurrence. Our functional study demonstrated that knock-down of Par3 promoted lung adenocarcinoma cell growth, cell migration, tumor formation, and metastasis, all of which were effectively inhibited when 14-3-3ζ was silenced. We found that Par3 binded with 14-3-3ζ protein and also showed that Par3 abrogated the binding of 14-3-3ζ to Tiam1, which was responsible for Rac1 activation. Knock-down of 14-3-3ζ inhibited Tiam1/Rac-GTP activation and blocked the invasive behavior of cells lacking Par3. These data suggest that loss of Par3 promotes metastatic behavior in lung adenocarcinoma cells through 14-3-3ζ protein.

  7. Loss of Par3 promotes lung adenocarcinoma metastasis through 14-3-3ζ protein

    PubMed Central

    Tong, Song; Xia, Tian; Fan, Kai; Jiang, Ke; Zhai, Wei; Li, Jing-Song; Wang, Si-Hua; Wang, Jian-Jun

    2016-01-01

    Partitioning defective protein 3 (Par3) can activate the Tiam1/Rac pathway to inhibit invasion and metastasis in many cancers; however, the role of Par3 in lung adenocarcinoma remains unknown. Here we show that Par3 is downregulated in lung adenocarcinoma tissues and is associated with higher rates of lymph node metastasis and recurrence. Our functional study demonstrated that knock-down of Par3 promoted lung adenocarcinoma cell growth, cell migration, tumor formation, and metastasis, all of which were effectively inhibited when 14-3-3ζ was silenced. We found that Par3 binded with 14-3-3ζ protein and also showed that Par3 abrogated the binding of 14-3-3ζ to Tiam1, which was responsible for Rac1 activation. Knock-down of 14-3-3ζ inhibited Tiam1/Rac-GTP activation and blocked the invasive behavior of cells lacking Par3. These data suggest that loss of Par3 promotes metastatic behavior in lung adenocarcinoma cells through 14-3-3ζ protein. PMID:27588399

  8. Neuroprotective Effect of TAT-14-3-3ε Fusion Protein against Cerebral Ischemia/Reperfusion Injury in Rats

    PubMed Central

    Liu, Xiaoyan; Hu, Wenhui; Wang, Yinye

    2014-01-01

    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 activation. PMID

  9. 14-3-3 proteins associate with phosphorylated simple epithelial keratins during cell cycle progression and act as a solubility cofactor

    PubMed Central

    1996-01-01

    14-3-3 is a ubiquitous protein family that interacts with several signal transduction kinases. We show that 14-3-3 proteins associate with keratin intermediate filament polypeptides 8 and 18 (K8/18) that are expressed in simple-type epithelia. The association is stoichiometrically significant (> or = one 14-3-3 molecule/keratin tetramer), occurs preferentially with K18, and is phosphorylation- and cell cycle-dependent in that it occurs during S/G2/M phases of the cell cycle when keratins become hyperphosphorylated. Binding of phospho- K8/18 to 14-3-3 can be reconstituted in vitro using recombinant 14-3-3 or using total cellular cytosol. Phosphatase treatment results in dissociation of 14-3-3, and dephosphorylation of phospho-K8/18 prevents reconstitution of the binding. Three cellular keratin subpopulations were analyzed that showed parallel gradients of keratin phosphorylation and 14-3-3 binding. Incubation of 14-3-3 with keratins during or after in vitro filament assembly results in sequestering of additional soluble keratin, only in cases when the keratins were hyperphosphorylated. Our results demonstrate a stoichiometrically significant cell cycle- and phosphorylation-regulated binding of 14-3-3 proteins to K18 and in vitro evidence of a simple epithelial keratin sequestering role for 14-3-3 proteins. PMID:8609167

  10. Abscisic acid and 14-3-3 proteins control K channel activity in barley embryonic root.

    PubMed

    van den Wijngaard, Paul W J; Sinnige, Mark P; Roobeek, Ilja; Reumer, Annet; Schoonheim, Peter J; Mol, Jos N M; Wang, Mei; De Boer, Albertus H

    2005-01-01

    Germination of seeds proceeds in general in two phases, an initial imbibition phase and a subsequent growth phase. In grasses like barley, the latter phase is evident as the emergence of the embryonic root (radicle). The hormone abscisic acid (ABA) inhibits germination because it prevents the embryo from entering and completing the growth phase. Genetic and physiological studies have identified many steps in the ABA signal transduction cascade, but how it prevents radicle elongation is still not clear. For elongation growth to proceed, uptake of osmotically active substances (mainly K(+)) is essential. Therefore, we have addressed the question of how the activity of K(+) permeable ion channels in the plasma membrane of radicle cells is regulated under conditions of slow (+ABA) and rapid germination (+fusicoccin). We found that ABA arrests radicle growth, inhibits net K(+) uptake and reduces the activity of K(+) (in) channels as measured with the patch-clamp technique. In contrast, fusicoccin (FC), a well-known stimulator of germination, stimulates radicle growth, net K(+) uptake and reduces the activity of K(+) (out) channels. Both types of channels are under the control of 14-3-3 proteins, known as integral components of signal transduction pathways and instrumental in FC action. Intriguingly, 14-3-3 affected both channels in an opposite fashion: whereas K(+) (in) channel activity was fully dependent upon 14-3-3 proteins, K(+) (out) channel activity was reduced by 14-3-3 proteins by 60%. Together with previous data showing that 14-3-3 proteins control the activity of the plasma membrane H(+)-ATPase, this makes 14-3-3 a prime candidate for molecular master regulator of the cellular osmo-pump. Regulation of the osmo-pump activity by ABA and FC is an important mechanism in controlling the growth of the embryonic root during seed germination.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-01-01

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

  13. Identification of cofilin and LIM-domain-containing protein kinase 1 as novel interaction partners of 14-3-3 zeta.

    PubMed Central

    Birkenfeld, Jörg; Betz, Heinrich; Roth, Dagmar

    2003-01-01

    Proteins of the 14-3-3 family have been implicated in various physiological processes, and are thought to function as adaptors in various signal transduction pathways. In addition, 14-3-3 proteins may contribute to the reorganization of the actin cytoskeleton by interacting with as yet unidentified actin-binding proteins. Here we show that the 14-3-3 zeta isoform interacts with both the actin-depolymerizing factor cofilin and its regulatory kinase, LIM (Lin-11/Isl-1/Mec-3)-domain-containing protein kinase 1 (LIMK1). In both yeast two-hybrid assays and glutathione S-transferase pull-down experiments, these proteins bound efficiently to 14-3-3 zeta. Deletion analysis revealed consensus 14-3-3 binding sites on both cofilin and LIMK1. Furthermore, the C-terminal region of 14-3-3 zeta inhibited the binding of cofilin to actin in co-sedimentation experiments. Upon co-transfection into COS-7 cells, 14-3-3 zeta-specific immunoreactivity was redistributed into characteristic LIMK1-induced actin aggregations. Our data are consistent with 14-3-3-protein-induced changes to the actin cytoskeleton resulting from interactions with cofilin and/or LIMK1. PMID:12323073

  14. Destabilisation of dimeric 14-3-3 proteins as a novel approach to anti-cancer therapeutics.

    PubMed

    Woodcock, Joanna M; Coolen, Carl; Goodwin, Katy L; Baek, Dong Jae; Bittman, Robert; Samuel, Michael S; Pitson, Stuart M; Lopez, Angel F

    2015-06-10

    14-3-3 proteins play a pivotal role in controlling cell proliferation and survival, two commonly dysregulated hallmarks of cancers. 14-3-3 protein expression is enhanced in many human cancers and correlates with more aggressive tumors and poor prognosis, suggesting a role for 14-3-3 proteins in tumorigenesis and/or progression. We showed previously that the dimeric state of 14-3-3 proteins is regulated by the lipid sphingosine, a physiological inducer of apoptosis. As the functions of 14-3-3 proteins are dependent on their dimeric state, this sphingosine-mediated 14-3-3 regulation provides a possible means to target dimeric 14-3-3 for therapeutic effect. However, sphingosine mimics are needed that are not susceptible to sphingolipid metabolism. We show here the identification and optimization of sphingosine mimetics that render dimeric 14-3-3 susceptible to phosphorylation at a site buried in the dimer interface and induce mitochondrial-mediated apoptosis. Two such compounds, RB-011 and RB-012, disrupt 14-3-3 dimers at low micromolar concentrations and induce rapid down-regulation of Raf-MAPK and PI3K-Akt signaling in Jurkat cells. Importantly, both RB-011 and RB-012 induce apoptosis of human A549 lung cancer cells and RB-012, through disruption of MAPK signaling, reduces xenograft growth in mice. Thus, these compounds provide proof-of-principle for this novel 14-3-3-targeting approach for anti-cancer drug discovery.

  15. 14-3-3 and its binding partners are regulators of protein–protein interactions during spermatogenesis

    PubMed Central

    Sun, Shengyi; Wong, Elissa W P; Li, Michelle W M; Lee, Will M; Cheng, C Yan

    2009-01-01

    During spermatogenesis, spermiation takes place at the adluminal edge of the seminiferous epithelium at stage VIII of the epithelial cycle during which fully developed spermatids (i.e. spermatozoa) detach from the epithelium in adult rat testes. This event coincides with the migration of preleptotene/leptotene spermatocytes across the blood–testis barrier from the basal to the apical (or adluminal) compartment. At stage XIV of the epithelial cycle, Pachytene spermatocytes (diploid, 2n) differentiate into diplotene spermatocytes (tetraploid, 4n) in the apical compartment of the epithelium, which begin meiosis I to be followed by meiosis II to form spermatids (haploid, 1n) at stage XIVof the epithelial cycle. These spermatids, in turn, undergo extensive morphological changes and traverse the seminiferous epithelium until they differentiate into elongated spermatids. Thus, there are extensive changes at the Sertoli–Sertoli and Sertoli–germ cell interface via protein ‘coupling’ and ‘uncoupling’ between cell adhesion protein complexes, as well as changes in interactions between integral membrane proteins and their peripheral adaptors, regulatory protein kinases and phosphatases, and the cytoskeletal proteins. These precisely coordinated protein–protein interactions affect cell adhesion and cell movement. In this review, we focus on the 14-3-3 protein family, whose members have different binding partners in the seminiferous epithelium. Recent studies have illustrated that 14-3-3 affects protein–protein interactions in the seminiferous epithelium, and regulates cell adhesion possibly via its effects on intracellular protein trafficking and cell-polarity proteins. This review provides a summary on the latest findings regarding the role of 14-3-3 family of proteins and their potential implications on spermatogenesis. We also highlight research areas that deserve attentions by investigators. PMID:19366886

  16. Quantifying protein interaction dynamics by SWATH mass spectrometry: application to the 14-3-3 system.

    PubMed

    Collins, Ben C; Gillet, Ludovic C; Rosenberger, George; Röst, Hannes L; Vichalkovski, Anton; Gstaiger, Matthias; Aebersold, Ruedi

    2013-12-01

    Protein complexes and protein interaction networks are essential mediators of most biological functions. Complexes supporting transient functions such as signal transduction processes are frequently subject to dynamic remodeling. Currently, the majority of studies on the composition of protein complexes are carried out by affinity purification and mass spectrometry (AP-MS) and present a static view of the system. For a better understanding of inherently dynamic biological processes, methods to reliably quantify temporal changes of protein interaction networks are essential. Here we used affinity purification combined with sequential window acquisition of all theoretical spectra (AP-SWATH) mass spectrometry to study the dynamics of the 14-3-3β scaffold protein interactome after stimulation of the insulin-PI3K-AKT pathway. The consistent and reproducible quantification of 1,967 proteins across all stimulation time points provided insights into the 14-3-3β interactome and its dynamic changes following IGF1 stimulation. We therefore establish AP-SWATH as a tool to quantify dynamic changes in protein-complex interaction networks.

  17. Stimulation of 14-3-3 protein and its isoform on histamine secretion from permeabilized rat peritoneal mast cells.

    PubMed

    Fujii, Toshihiro; Ueeda, Takayuki

    2002-12-01

    The effect of the 14-3-3 protein, an adaptor protein of intracellular signal pathways, on histamine release from rat peritoneal mast cells was investigated. The exogenous 14-3-3 protein from bovine brain increased the Ca(2+)-dependent histamine release from permeabilized mast cells, but only slightly affected the non-permeabilized cells. Partial amino acid sequences showed that the bovine brain 14-3-3 protein contained 14-3-3beta, gamma and zeta isoforms, and that these recombinant isoforms were prepared. Among them, 14-3-3zeta was an active species while the 14-3-3beta and gamma were inactive for histamine release from the permeabilized mast cells. Approximately 15% of the histamine release was stimulated by 14-3-3zeta at 2.5 microM, and half-maximal stimulation occurred at 1 microM. Treatment of the mast cells with wortmannin or staurosporine completely inhibited the stimulatory effect on histamine release caused by Ca(2+) or Ca(2+)/14-3-3zeta, and genistein partially inhibited both stimulatory effects. PD 98059, however, had little effect on the histamine release. These results suggest the possibility that 14-3-3zeta is associated with signal transduction for degranulation of the mast cells.

  18. RGS3 interacts with 14-3-3 via the N-terminal region distinct from the RGS (regulator of G-protein signalling) domain.

    PubMed Central

    Niu, Jiaxin; Scheschonka, Astrid; Druey, Kirk M; Davis, Amanda; Reed, Eleanor; Kolenko, Vladimir; Bodnar, Richard; Voyno-Yasenetskaya, Tatyana; Du, Xiaoping; Kehrl, John; Dulin, Nickolai O

    2002-01-01

    RGS3 belongs to a family of the regulators of G-protein signalling (RGS), which bind and inhibit the G alpha subunits of heterotrimeric G-proteins via a homologous RGS domain. Increasing evidence suggests that RGS proteins can also interact with targets other than G-proteins. Employing yeast two-hybrid screening of a cDNA library, we identified an interaction between RGS3 and the phosphoserine-binding protein 14-3-3. This interaction was confirmed by in vitro binding and co-immunoprecipitation experiments. RGS3-deletion analysis revealed the presence of a single 14-3-3-binding site located outside of the RGS domain. Ser(264) was then identified as the 14-3-3-binding site of RGS3. The S(264)A mutation resulted in the loss of RGS3 binding to 14-3-3, without affecting its ability to bind G alpha(q). Signalling studies showed that the S(264)A mutant was more potent than the wild-type RGS3 in inhibition of G-protein-mediated signalling. Binding experiments revealed that RGS3 exists in two separate pools, either 14-3-3-bound or G-protein-bound, and that the 14-3-3-bound RGS3 is unable to interact with G-proteins. These data are consistent with the model wherein 14-3-3 serves as a scavenger of RGS3, regulating the amounts of RGS3 available for binding G-proteins. This study describes a new level in the regulation of G-protein signalling, in which the inhibitors of G-proteins, RGS proteins, can themselves be regulated by phosphorylation and binding 14-3-3. PMID:11985497

  19. Protein Phosphatase 2A Reactivates FOXO3a through a Dynamic Interplay with 14-3-3 and AKT

    PubMed Central

    Singh, Amrik; Ye, Min; Bucur, Octavian; Zhu, Shudong; Tanya Santos, Maria; Rabinovitz, Isaac; Wei, Wenyi; Gao, Daming; Hahn, William C.

    2010-01-01

    Forkhead box transcription factor FOXO3a, a key regulator of cell survival, is regulated by reversible phosphorylation and subcellular localization. Although the kinases regulating FOXO3a activity have been characterized, the role of protein phosphatases (PP) in the control of FOXO3a subcellular localization and function is unknown. In this study, we detected a robust interaction between FOXO3a and PP2A. We further demonstrate that 14-3-3, while not impeding the interaction between PP2A and FOXO3a, restrains its activity toward AKT phosphorylation sites T32/S253. Disruption of PP2A function revealed that after AKT inhibition, PP2A-mediated dephosphorylation of T32/S253 is required for dissociation of 14-3-3, nuclear translocation, and transcriptional activation of FOXO3a. Our findings reveal that distinct phosphatases dephosphorylate conserved AKT motifs within the FOXO family and that PP2A is entwined in a dynamic interplay with AKT and 14-3-3 to directly regulate FOXO3a subcellular localization and transcriptional activation. PMID:20110348

  20. Toxoplasma gondii: Effect of infection on expression of 14-3-3 proteins in human epithelial cells

    PubMed Central

    Monroy, Fernando P.

    2008-01-01

    14-3-3 proteins are expressed in most eukaryotes organisms and play varied and crucial roles in a wide range of regulatory processes. In mammalian cells, seven 14-3-3 isoforms have been identified. However; it is not known what effect infection has on 14-3-3 isoform expression. In this study human colonic carcinoma cell lines were infected with Toxoplasma gondii for 24 h and expression of 14-3-3 proteins was determined by RT-PCR. HT-29 cells only expressed 3 out of the 7 isoforms while 5 and all 7 isoforms were found in HCT-116 and Caco-2 cells respectively. Infection had little or no effect in the expression of 14-3-3γ, ε, σ, and ξ; but in HCT-116 cells induced expression of 14-3-3η and σ, while 14-3-3β, η, and ξ were induced in HT-29 cells. If 14-3-3 proteins are involved in cell survival and/or prevention of parasite replication, longer incubation times may be required as no differences in percentage of infection were found among the cell lines at 24 h post-infection. PMID:17825295

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

    PubMed

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

    2016-05-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  3. Quantitative proteomic dissection of a native 14-3-3ε interacting protein complex associated with hepatocellular carcinoma.

    PubMed

    Bai, Chen; Tang, Siwei; Bai, Chen; Chen, Xian

    2014-04-01

    The 14-3-3 proteins regulate diverse biological processes that are implicated in cancer development, and seven 14-3-3 isoforms were identified with isoform-specific roles in different human tumors. In our previous work, we dissected the interactome of 14-3-3ε formed during the DNA damage response in a hepatocellular carcinoma (HCC) cell using an AACT/SILAC-based quantitative proteomic approach. In this study, we used a similar proteomic approach to profile/identify the 14-3-3ε interactome formed in native HCC cells. Functional categorization and data-dependent network analysis of the native HCC-specific 14-3-3ε interactome revealed that 14-3-3ε is involved in the regulation of multiple biological processes (BPs)/pathways, including cell cycle control, apoptosis, signal transduction, transport, cell adhesion, carbohydrate metabolism, and nucleic acid metabolism. Biological validation further supports that 14-3-3ε, via association with multiple BP/pathway-specific proteins, coordinates the regulation of proliferation, survival, and metastasis of HCC. The findings in this study, together with those of our previous study, provide an extensive profile of the 14-3-3ε interaction network in HCC cells, which should be valuable for understanding the pathology of HCC and HCC therapy.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    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.

  6. Inhibition of the Arabidopsis Salt Overly Sensitive Pathway by 14-3-3 Proteins[C][W

    PubMed Central

    Zhou, Huapeng; Lin, Huixin; Chen, She; Becker, Katia; Yang, Yongqing; Zhao, Jinfeng; Kudla, Jörg; Schumaker, Karen S.; Guo, Yan

    2014-01-01

    The Salt Overly Sensitive (SOS) pathway regulates intracellular sodium ion (Na+) homeostasis and salt tolerance in plants. Until recently, little was known about the mechanisms that inhibit the SOS pathway when plants are grown in the absence of salt stress. In this study, we report that the Arabidopsis thaliana 14-3-3 proteins λ and κ interact with SOS2 and repress its kinase activity. Growth in the presence of salt decreases the interaction between SOS2 and the 14-3-3 proteins, leading to kinase activation in planta. 14-3-3 λ interacts with the SOS2 junction domain, which is important for its kinase activity. A phosphorylation site (Ser-294) is identified within this domain by mass spectrometry. Mutation of Ser-294 to Ala or Asp does not affect SOS2 kinase activity in the absence of the 14-3-3 proteins. However, in the presence of 14-3-3 proteins, the inhibition of SOS2 activity is decreased by the Ser-to-Ala mutation and enhanced by the Ser-to-Asp exchange. These results identify 14-3-3 λ and κ as important regulators of salt tolerance. The inhibition of SOS2 mediated by the binding of 14-3-3 proteins represents a novel mechanism that confers basal repression of the SOS pathway in the absence of salt stress. PMID:24659330

  7. Discovery of 14-3-3 protein-protein interaction inhibitors that sensitize multidrug-resistant cancer cells to doxorubicin and the Akt inhibitor GSK690693.

    PubMed

    Mori, Mattia; Vignaroli, Giulia; Cau, Ylenia; Dinić, Jelena; Hill, Richard; Rossi, Matteo; Colecchia, David; Pešić, Milica; Link, Wolfgang; Chiariello, Mario; Ottmann, Christian; Botta, Maurizio

    2014-05-01

    14-3-3 is a family of highly conserved adapter proteins that is attracting much interest among medicinal chemists. Small-molecule inhibitors of 14-3-3 protein-protein interactions (PPIs) are in high demand, both as tools to increase our understanding of 14-3-3 actions in human diseases and as leads to develop innovative therapeutic agents. Herein we present the discovery of novel 14-3-3 PPI inhibitors through a multidisciplinary strategy combining molecular modeling, organic synthesis, image-based high-content analysis of reporter cells, and in vitro assays using cancer cells. Notably, the two most active compounds promoted the translocation of c-Abl and FOXO pro-apoptotic factors into the nucleus and sensitized multidrug-resistant cancer cells to apoptotic inducers such as doxorubicin and the pan-Akt inhibitor GSK690693, thus becoming valuable lead candidates for further optimization. Our results emphasize the possible role of 14-3-3 PPI inhibitors in anticancer combination therapies.

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

    PubMed

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

    2015-02-01

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

  9. 14-3-3 Protein isoforms and atypical patterns of the 14-3-3 assay in the diagnosis of Creutzfeldt-Jakob disease.

    PubMed

    Sánchez-Valle, Raquel; Saiz, Albert; Graus, Francesc

    2002-03-01

    A positive 14-3-3 assay is a criterion for probable Creutzfeldt-Jakob disease (CJD). Cerebrospinal fluid (CSF) 14-3-3 is usually detected by immunoblot using an antibody that recognizes all of the 14-3-3 isoforms. In a few cases, the antibody recognizes an inferior band and this pattern is associated with false positive results. We analyzed 43 CSF (26 CJD, 17 controls) samples using antibodies against specific isoforms (beta, epsilon, gamma, tau, xi) and compared the results with those obtained with the standard antibody. The anti-gamma and anti-beta antibody achieved similar results but the presence of atypical patterns made the standard antibody more accurate for the CJD diagnosis. To study the nature of the inferior band, CSF samples were probed with antibodies against light chain immunoglobulins, and immunoblots of human IgG with the standard antibody. The experiments suggested a cross-reaction of the anti-14-3-3 antibody with light chain immunoglobulins.

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

    PubMed Central

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

    2007-01-01

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

  11. Molecular cloning of cDNA for the zeta isoform of the 14-3-3 protein: homologous sequences in the 3'-untranslated region of frog and human zeta isoforms.

    PubMed

    Miura, I; Nakajima, T; Ohtani, H; Kashiwagi, A; Nakamura, M

    1997-10-01

    14-3-3 proteins constitute a family of well-conserved eukaryotic proteins that possess diverse biochemical activities such as regulation of gene transcription, cell proliferation and activation of protein kinase C. At least 7 subtypes (alpha to theta) of 14-3-3 protein are known, but the zeta subtype of this protein has been cloned only in mammals. We cloned the zeta subtype of 14-3-3 protein (14-3-3 zeta) from the frog, Rana rugosa. The sequence encoded 245 amino acids that share 92% identity with rat and bovine 14-3-3 zeta s, and 92% with human phospholipase A2 (PLA2; 14-3-3 zeta). Northern blot analysis revealed a single band of about 1.8 kb in tadpoles at stage 25. The 14-3-3 zeta mRNA level was high in the brain, lung, spleen and kidney, and low in the heart and testis, as opposed to the mRNA level, which was only faintly detected in the liver, pancreas, ovary and muscle. Furthermore, high similarity in the 3'-untranslated region (3'-UTR) was observed between frog and human 14-3-3 zeta cDNA. The results suggest that 14-3-3 zeta is highly conserved throughout eukaryotic evolution, and that the homologous sequence in the 3'-UTR of 14-3-3 zeta cDNA may be conserved in frogs and humans.

  12. Organellar proteomics of human platelet dense granules reveals that 14-3-3zeta is a granule protein related to atherosclerosis.

    PubMed

    Hernandez-Ruiz, Laura; Valverde, Federico; Jimenez-Nuñez, Maria D; Ocaña, Esther; Sáez-Benito, Ana; Rodríguez-Martorell, Javier; Bohórquez, Juan-Carlos; Serrano, Aurelio; Ruiz, Felix A

    2007-11-01

    Dense granules, a type of platelet secretory organelle, are known to accumulate high concentrations of small molecules such as calcium, adenine nucleotides, serotonin, pyrophosphate, and polyphosphate. Protein composition of these granules has been obscure, however. In this paper, we use proteomics techniques to describe, for the first time, the soluble protein composition of platelet dense granules. We have isolated highly enriched human platelet dense granule fractions that have been analyzed using two proteomics methods. Using this approach, we have identified 40 proteins, and most of them, such as actin-associated proteins, glycolytic enzymes, and regulatory proteins, have not previously been related to the organelle. We have focused our efforts on studying 14-3-3zeta, a member of a conserved family of proteins that interact with hundreds of different proteins. We have demonstrated that 14-3-3zeta is localized mostly on dense granules and that it is secreted after platelet activation. As some proteins secreted from activated platelets could promote the development of atherosclerosis and thrombosis, we have studied the expression of 14-3-3zeta in sections of human abdominal aorta of patients with aneurysm, identifying it at the atherosclerotic plaques. Together, our results reveal new details of the composition of the platelet dense granule and suggest an extracellular function for 14-3-3zeta associated with atherosclerosis.

  13. The 14-3-3s

    PubMed Central

    Ferl, Robert J; Manak, Michael S; Reyes, Matthew F

    2002-01-01

    Multiple members of the 14-3-3 protein family have been found in all eukaryotes so far investigated, yet they are apparently absent from prokaryotes. The major native forms of 14-3-3s are homo- and hetero-dimers, the biological functions of which are to interact physically with specific client proteins and thereby effect a change in the client. As a result, 14-3-3s are involved in a vast array of processes such as the response to stress, cell-cycle control, and apoptosis, serving as adapters, activators, and repressors. There are currently 133 full-length sequences available in GenBank for this highly conserved protein family. A phylogenetic tree based on the conserved middle core region of the protein sequences shows that, in plants, the 14-3-3 family can be divided into two clearly defined groups. The core region encodes an amphipathic groove that binds the multitude of client proteins that have conserved 14-3-3-recognition sequences. The amino and carboxyl termini of 14-3-3 proteins are much more divergent than the core region and may interact with isoform-specific client proteins and/or confer specialized subcellular and tissue localization. PMID:12184815

  14. Interaction of 14-3-3 proteins with the estrogen receptor alpha F domain provides a drug target interface.

    PubMed

    De Vries-van Leeuwen, Ingrid J; da Costa Pereira, Daniel; Flach, Koen D; Piersma, Sander R; Haase, Christian; Bier, David; Yalcin, Zeliha; Michalides, Rob; Feenstra, K Anton; Jiménez, Connie R; de Greef, Tom F A; Brunsveld, Luc; Ottmann, Christian; Zwart, Wilbert; de Boer, Albertus H

    2013-05-28

    Estrogen receptor alpha (ERα) is involved in numerous physiological and pathological processes, including breast cancer. Breast cancer therapy is therefore currently directed at inhibiting the transcriptional potency of ERα, either by blocking estrogen production through aromatase inhibitors or antiestrogens that compete for hormone binding. Due to resistance, new treatment modalities are needed and as ERα dimerization is essential for its activity, interference with receptor dimerization offers a new opportunity to exploit in drug design. Here we describe a unique mechanism of how ERα dimerization is negatively controlled by interaction with 14-3-3 proteins at the extreme C terminus of the receptor. Moreover, the small-molecule fusicoccin (FC) stabilizes this ERα/14-3-3 interaction. Cocrystallization of the trimeric ERα/14-3-3/FC complex provides the structural basis for this stabilization and shows the importance of phosphorylation of the penultimate Threonine (ERα-T(594)) for high-affinity interaction. We confirm that T(594) is a distinct ERα phosphorylation site in the breast cancer cell line MCF-7 using a phospho-T(594)-specific antibody and by mass spectrometry. In line with its ERα/14-3-3 interaction stabilizing effect, fusicoccin reduces the estradiol-stimulated ERα dimerization, inhibits ERα/chromatin interactions and downstream gene expression, resulting in decreased cell proliferation. Herewith, a unique functional phosphosite and an alternative regulation mechanism of ERα are provided, together with a small molecule that selectively targets this ERα/14-3-3 interface.

  15. Engagement of beta2 integrins recruits 14-3-3 proteins to c-Cbl in human neutrophils.

    PubMed

    Melander, Fredrik; Andersson, Tommy; Dib, Karim

    2004-05-14

    We found that engagement of beta2 integrins on human neutrophils triggered both tyrosine and serine phosphorylation of c-Cbl. Pretreatment of the neutrophils with the broad range protein kinase C (PKC) inhibitor GF-109203X blocked the serine but not the tyrosine phosphorylation of c-Cbl. Moreover, the Src kinase inhibitor PP1 prevented the beta2 integrin-induced tyrosine phosphorylation of c-Cbl but not the simultaneous serine phosphorylation. These results indicate that Src family kinases and PKC can separately modulate the properties of c-Cbl. Indeed, tyrosine kinase-dependent phosphorylation of c-Cbl regulated the ubiquitin ligase activity of that protein, whereas PKC-dependent phosphorylation of c-Cbl had no such effect. Instead, c-Cbl that underwent PKC-induced serine phosphorylation associated with the scaffolding and anti-apoptotic 14-3-3 proteins. Consequently, c-Cbl can independently target proteins for degradation or intracellular localization and may initiate an anti-apoptotic signal in neutrophils.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-10-28

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

  18. The Saccharomyces cerevisiae 14-3-3 protein Bmh2 is required for regulation of the phosphorylation status of Fin1, a novel intermediate filament protein.

    PubMed Central

    Mayordomo, Isabel; Sanz, Pascual

    2002-01-01

    In order to identify proteins that interact with Bmh2, a yeast member of the 14-3-3 protein family, we performed a two-hybrid screening using LexA-Bmh2 as bait. We identified Fin1, a novel intermediate filament protein, as the protein that showed the highest degree of interaction. We also identified components of the vesicular transport machinery such as Gic2 and Msb3, proteins involved in transcriptional regulation such as Mbf1, Gcr2 and Reg2, and a variety of other different proteins (Ppt1, Lre1, Rps0A and Ylr177w). We studied the interaction between Bmh2 and Fin1 in more detail and found that Bmh2 only interacted with phosphorylated forms of Fin1. In addition, we showed that Glc7, the catalytic subunit of the protein phosphatase 1 complex, was also able to interact with Fin1. PMID:11931638

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

    PubMed

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

    2014-03-01

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

  20. Identification of a 14-3-3 protein from Lentinus edodes that interacts with CAP (adenylyl cyclase-associated protein), and conservation of this interaction in fission yeast.

    PubMed

    Zhou, G L; Yamamoto, T; Ozoe, F; Yano, D; Tanaka, K; Matsuda, H; Kawamukai, M

    2000-01-01

    We previously identified a gene encoding a CAP (adenylyl cyclase-associated protein) homologue from the edible Basidiomycete Lentinus edodes. To further discover the cellular functions of the CAP protein, we searched for CAP-interacting proteins using a yeast two-hybrid system. Among the candidates thus obtained, many clones encoded the C-terminal half of an L. edodes 14-3-3 homologue (designated cip3). Southern blot analysis indicated that L. edodes contains only one 14-3-3 gene. Overexpression of the L. edodes 14-3-3 protein in the fission yeast Schizosaccharomyces pombe rad24 null cells complemented the loss of endogenous 14-3-3 protein functions in cell morphology and UV sensitivity, suggesting functional conservation of 14-3-3 proteins between L. edodes and S. pombe. The interaction between L. edodes CAP and 14-3-3 protein was restricted to the N-terminal domain of CAP and was confirmed by in vitro co-precipitation. Results from both the two-hybrid system and in vivo co-precipitation experiments showed the conservation of this interaction in S. pombe. The observation that a 14-3-3 protein interacts with the N-terminal portion of CAP but not with full-length CAP in L. edodes and S. pombe suggests that the C-terminal region of CAP may have a negative effect on the interaction between CAP and 14-3-3 proteins, and 14-3-3 proteins may play a role in regulation of CAP function.

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

    PubMed Central

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

    2015-01-01

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

  2. 14-3-3 Proteins SGF14c and SGF14l Play Critical Roles during Soybean Nodulation1[W][OA

    PubMed Central

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

    2012-01-01

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

  3. Two genes encoding GF14 (14-3-3) proteins in Zea mays. Structure, expression, and potential regulation by the G-box binding complex.

    PubMed Central

    de Vetten, N C; Ferl, R J

    1994-01-01

    Two maize (Zea mays) genes, designated GRF1 and GRF2, have been isolated and characterized. The proteins encoded by these genes, called GF14 proteins, participate in protein/DNA complexes and show more than 60% identity with a highly conserved, widely distributed protein family, collectively referred to as 14-3-3 proteins. Members of the 14-3-3 protein family have been reported to activate Tyr and Trp hydroxylases, modulate protein kinase C activity, and activate ADP-ribosyltransferase. The mRNAs of the GRF genes are encoded by six exons interrupted by five introns. The transcriptional units of the GRF genes were found to be very similar, with complete conservation of the intron positions. In addition, the length and nucleotide sequences of the two genes' introns were highly conserved. The 5' flanking sequences of the two GRF genes were compared and regions of homology and divergence identified. This comparison revealed the presence of a conserved G-box element in the 5' flanking region of both genes. Electrophoretic mobility shift assays of maize protein extract with the GRF G-box indicates that GBF binds to this G-box site in the 5' up stream region of GRF. Antibody supershifts indicate that GF14 protein is associated with the G-box-binding complex that interacts with the GRF upstream region. PMID:7846163

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

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

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

    2009-11-20

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

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

    PubMed

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

    2016-09-01

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

  7. Deleting the 14-3-3 protein Bmh1 extends life span in Saccharomyces cerevisiae by increasing stress response.

    PubMed

    Wang, Chen; Skinner, Craig; Easlon, Erin; Lin, Su-Ju

    2009-12-01

    Enhanced stress response has been suggested to promote longevity in many species. Calorie restriction (CR) and conserved nutrient-sensing target of rapamycin (TOR) and protein kinase A (PKA) pathways have also been suggested to extend life span by increasing stress response, which protects cells from age-dependent accumulation of oxidative damages. Here we show that deleting the yeast 14-3-3 protein, Bmh1, extends chronological life span (CLS) by activating the stress response. 14-3-3 proteins are highly conserved chaperone-like proteins that play important roles in many cellular processes. bmh1Delta-induced heat resistance and CLS extension require the general stress-response transcription factors Msn2, Msn4, and Rim15. The bmh1Delta mutant also displays a decreased reactive oxygen species level and increased heat-shock-element-driven transcription activity. We also show that BMH1 genetically interacts with CR and conserved nutrient-sensing TOR- and PKA-signaling pathways to regulate life span. Interestingly, the level of phosphorylated Ser238 on Bmh1 increases during chronological aging, which is delayed by CR or by reduced TOR activities. In addition, we demonstrate that PKA can directly phosphorylate Ser238 on Bmh1. The status of Bmh1 phosphorylation is therefore likely to play important roles in life-span regulation. Together, our studies suggest that phosphorylated Bmh1 may cause inhibitory effects on downstream longevity factors, including stress-response proteins. Deleting Bmh1 may eliminate the inhibitory effects of Bmh1 on these longevity factors and therefore extends life span.

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

    PubMed

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

    2016-06-01

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

  9. Plasma Membrane CRPK1-Mediated Phosphorylation of 14-3-3 Proteins Induces Their Nuclear Import to Fine-Tune CBF Signaling during Cold Response.

    PubMed

    Liu, Ziyan; Jia, Yuxin; Ding, Yanglin; Shi, Yiting; Li, Zhen; Guo, Yan; Gong, Zhizhong; Yang, Shuhua

    2017-04-06

    In plant cells, changes in fluidity of the plasma membrane may serve as the primary sensor of cold stress; however, the precise mechanism and how the cell transduces and fine-tunes cold signals remain elusive. Here we show that the cold-activated plasma membrane protein cold-responsive protein kinase 1 (CRPK1) phosphorylates 14-3-3 proteins. The phosphorylated 14-3-3 proteins shuttle from the cytosol to the nucleus, where they interact with and destabilize the key cold-responsive C-repeat-binding factor (CBF) proteins. Consistent with this, the crpk1 and 14-3-3κλ mutants show enhanced freezing tolerance, and transgenic plants overexpressing 14-3-3λ show reduced freezing tolerance. Further study shows that CRPK1 is essential for the nuclear translocation of 14-3-3 proteins and for 14-3-3 function in freezing tolerance. Thus, our study reveals that the CRPK1-14-3-3 module transduces the cold signal from the plasma membrane to the nucleus to modulate CBF stability, which ensures a faithfully adjusted response to cold stress of plants.

  10. Structural characterization of a unique interface between carbohydrate response element-binding protein (ChREBP) and 14-3-3β protein.

    PubMed

    Ge, Qiang; Huang, Nian; Wynn, R Max; Li, Yang; Du, Xinlin; Miller, Bonnie; Zhang, Hong; Uyeda, Kosaku

    2012-12-07

    Carbohydrate response element-binding protein (ChREBP) is an insulin-independent, glucose-responsive transcription factor that is expressed at high levels in liver hepatocytes where it plays a critical role in converting excess carbohydrates to fat for storage. In response to fluctuating glucose levels, hepatic ChREBP activity is regulated in large part by nucleocytoplasmic shuttling of ChREBP protein via interactions with 14-3-3 proteins. The N-terminal ChREBP regulatory region is necessary and sufficient for glucose-responsive ChREBP nuclear import and export. Here, we report the crystal structure of a complex of 14-3-3β bound to the N-terminal regulatory region of ChREBP at 2.4 Å resolution. The crystal structure revealed that the α2 helix of ChREBP (residues 117-137) adopts a well defined α-helical conformation and binds 14-3-3 in a phosphorylation-independent manner that is different from all previously characterized 14-3-3 and target protein-binding modes. ChREBP α2 interacts with 14-3-3 through both electrostatic and van der Waals interactions, and the binding is partially mediated by a free sulfate or phosphate. Structure-based mutagenesis and binding assays indicated that disrupting the observed 14-3-3 and ChREBP α2 interface resulted in a loss of complex formation, thus validating the novel protein interaction mode in the 14-3-3β·ChREBP α2 complex.

  11. Protein kinase C-alpha regulates insulin action and degradation by interacting with insulin receptor substrate-1 and 14-3-3 epsilon.

    PubMed

    Oriente, Francesco; Andreozzi, Francesco; Romano, Chiara; Perruolo, Giuseppe; Perfetti, Anna; Fiory, Francesca; Miele, Claudia; Beguinot, Francesco; Formisano, Pietro

    2005-12-09

    Protein kinase C (PKC)-alpha exerts a regulatory function on insulin action. We showed by overlay blot that PKCalpha directly binds a 180-kDa protein, corresponding to IRS-1, and a 30-kDa molecular species, identified as 14-3-3epsilon. In intact NIH-3T3 cells overexpressing insulin receptors (3T3-hIR), insulin selectively increased PKCalpha co-precipitation with IRS-1, but not with IRS-2, and with 14-3-3epsilon, but not with other 14-3-3 isoforms. Overexpression of 14-3-3epsilon in 3T3-hIR cells significantly reduced IRS-1-bound PKCalpha activity, without altering IRS-1/PKCalpha co-precipitation. 14-3-3epsilon overexpression also increased insulin-stimulated insulin receptor and IRS-1 tyrosine phosphorylation, followed by increased activation of Raf1, ERK1/2, and Akt/protein kinase B. Insulin-induced glycogen synthase activity and thymidine incorporation were also augmented. Consistently, selective depletion of 14-3-3epsilon by antisense oligonucleotides caused a 3-fold increase of IRS-1-bound PKCalpha activity and a similarly sized reduction of insulin receptor and IRS-1 tyrosine phosphorylation and signaling. In turn, selective inhibition of PKCalpha expression by antisense oligonucleotides reverted the negative effect of 14-3-3epsilon depletion on insulin signaling. Moreover, PKCalpha inhibition was accompanied by a >2-fold decrease of insulin degradation. Similar results were also obtained by overexpressing 14-3-3epsilon. Thus, in NIH-3T3 cells, insulin induces the formation of multimolecular complexes, including IRS-1, PKCalpha, and 14-3-3epsilon. The presence of 14-3-3epsilon in the complex is not necessary for IRS-1/PKCalpha interaction but modulates PKCalpha activity, thereby regulating insulin signaling and degradation.

  12. Biophysical Characterization of Essential Phosphorylation at the Flexible C-Terminal Region of C-Raf with 14-3-3ζ Protein

    PubMed Central

    Gayen, Nilanjan; Mroue, Kamal H.; Kar, Rajiv K.; Mandal, Atin K.; Bhunia, Anirban

    2015-01-01

    Phosphorylation at the C-terminal flexible region of the C-Raf protein plays an important role in regulating its biological activity. Auto-phosphorylation at serine 621 (S621) in this region maintains C-Raf stability and activity. This phosphorylation mediates the interaction between C-Raf and scaffold protein 14-3-3ζ to activate the downstream MEK kinase pathway. In this study, we have defined the interaction of C-terminal peptide sequence of C-Raf with 14-3-3ζ protein and determined the possible structural adaptation of this region. Biophysical elucidation of the interaction was carried out using phosphopeptide (residue number 615–630) in the presence of 14-3-3ζ protein. Using isothermal titration calorimetry (ITC), a high binding affinity with micro-molar range was found to exist between the peptide and 14-3-3ζ protein, whereas the non-phosphorylated peptide did not show any appreciable binding affinity. Further interaction details were investigated using several biophysical techniques such as circular dichroism (CD), fluorescence, and nuclear magnetic resonance (NMR) spectroscopy, in addition to molecular modeling. This study provides the molecular basis for C-Raf C-terminal-derived phosphopeptide interaction with 14-3-3ζ protein as well as structural insights responsible for phosphorylated S621-mediated 14-3-3ζ binding at an atomic resolution. PMID:26295714

  13. The Saccharomyces cerevisiae 14-3-3 proteins Bmh1 and Bmh2 directly influence the DNA damage-dependent functions of Rad53

    PubMed Central

    Usui, Takehiko; Petrini, John H. J.

    2007-01-01

    In this study, we mutated autophosphorylation sites in Rad53 based on their conservation with previously identified autophosphorylation sites in the mammalian Rad53 ortholog, Chk2. As with wild-type Rad53, the autophosphorylation mutant, rad53-TA, undergoes Mec1/Tel1-dependent interactions with Rad9 and Dun1 in response to genotoxic stress. Whereas rad53-TA in vitro kinase activity is severely impaired, the rad53-TA strains are not completely deficient for cell-cycle checkpoint functions, indicating that the mutant kinase retains a basal level of function. We describe a genetic interaction among Rad53, Dun1, and the 14-3-3 proteins Bmh1 and Bmh2 and present evidence that 14-3-3 proteins directly facilitate Rad53 function in vivo. The data presented account for the previously observed checkpoint defects associated with 14-3-3 mutants in Saccharomyces pombe and Saccharomyces cerevisiae. The 14-3-3 functional interaction appears to modulate Rad53 activity, reminiscent of 14-3-3's effect on human Raf1 kinase and distinct from the indirect mode of regulation by 14-3-3 observed for Chk1 or Cdc25. PMID:17299042

  14. Proteomic identification of 14-3-3ϵ as a linker protein between pERK1/2 inhibition and BIM upregulation in human osteosarcoma cells.

    PubMed

    Kim, Kyung Ok; Hsu, Anny C; Lee, Heon Goo; Patel, Neel; Chandhanayingyong, Chandhanarat; Hickernell, Thomas; Lee, Francis Young-In

    2014-06-01

    Despite advancements in multimodality chemotherapy, conventional cytotoxic treatments still remain ineffective for a subset of patients with aggressive metastatic or multifocal osteosarcoma. It has been shown that pERK1/2 inhibition enhances chemosensitivity to doxorubicin and promotes osteosarcoma cell death in vivo and in vitro. One of the pro-apoptotic mechanisms is upregulation of Bim by pERK1/2 inhibitors. To this end, we examined proteomic changes of 143B human osteosarcoma cells with and without treatment of PD98059, pERK1/2 inhibitor. Specifically, we identified 14-3-3ϵ protein as a potential mediator of Bim expression in response to inhibition of pERK1/2. We hypothesized that 14-3-3ϵ mediates upregulation of Bim expression after pERK1/2 inhibition. We examined the expression of Bim after silencing 14-3-3ϵ using siRNA. The 14-3-3ϵ gene silencing resulted in downregulation of Bim expression after PD98059 treatment. These data indicate that 14-3-3ϵ is required for Bim expression and that it has an anti-cancer effect under pERK1/2 inhibition in 143B cells. By playing an essential role upstream of Bim, 14-3-3ϵ may potentially be a coadjuvant factor synergizing the effect of pERK1/2 inhibitors in addition to conventional cytotoxic agents for more effective osteosarcoma treatments.

  15. Structural Basis for the Interaction of a Human Small Heat Shock Protein with the 14-3-3 Universal Signaling Regulator.

    PubMed

    Sluchanko, Nikolai N; Beelen, Steven; Kulikova, Alexandra A; Weeks, Stephen D; Antson, Alfred A; Gusev, Nikolai B; Strelkov, Sergei V

    2017-02-07

    By interacting with hundreds of protein partners, 14-3-3 proteins coordinate vital cellular processes. Phosphorylation of the small heat shock protein, HSPB6, within its intrinsically disordered N-terminal domain activates its interaction with 14-3-3, ultimately triggering smooth muscle relaxation. After analyzing the binding of an HSPB6-derived phosphopeptide to 14-3-3 using isothermal calorimetry and X-ray crystallography, we have determined the crystal structure of the complete assembly consisting of the 14-3-3 dimer and full-length HSPB6 dimer and further characterized this complex in solution using fluorescence spectroscopy, small-angle X-ray scattering, and limited proteolysis. We show that selected intrinsically disordered regions of HSPB6 are transformed into well-defined conformations upon the interaction, whereby an unexpectedly asymmetric structure is formed. This structure provides the first atomic resolution snapshot of a human small HSP in functional state, explains how 14-3-3 proteins sequester their regulatory partners, and can inform the design of small-molecule interaction modifiers to be used as myorelaxants.

  16. Structural basis for the interaction of a human small heat shock protein with the 14-3-3 universal signaling regulator

    PubMed Central

    Sluchanko, Nikolai N.; Beelen, Steven; Kulikova, Alexandra A.; Weeks, Stephen D.; Antson, Alfred A.; Gusev, Nikolai B.; Strelkov, Sergei V.

    2017-01-01

    Summary By interacting with hundreds of protein partners, 14-3-3 proteins coordinate vital cellular processes. Phosphorylation of the small heat shock protein HSPB6 within its intrinsically disordered N-terminal domain activates its interaction with 14-3-3, ultimately triggering smooth muscle relaxation. After analyzing the binding of an HSPB6-derived phosphopeptide to 14-3-3 using isothermal calorimetry and X-ray crystallography, we have determined the crystal structure of the complete assembly consisting of the 14-3-3 dimer and full-length HSPB6 dimer and further characterized this complex in solution using fluorescence spectroscopy, small-angle X-ray scattering and limited proteolysis. We show that selected intrinsically disordered regions of HSPB6 are transformed into well-defined conformations upon the interaction, whereby an unexpectedly asymmetric structure is formed. This structure provides the first-ever atomic resolution snapshot of a human small HSP in functional state, explains how 14-3-3 proteins sequester their regulatory partners, and can inform the design of small-molecule interaction modifiers to be used as myorelaxants. PMID:28089448

  17. Differences in Spatial Expression between 14-3-3 Isoforms in Germinating Barley Embryos1

    PubMed Central

    Testerink, Christa; van der Meulen, René M.; Oppedijk, Berry J.; de Boer, Albertus H.; Heimovaara-Dijkstra, Sjoukje; Kijne, Jan W.; Wang, Mei

    1999-01-01

    The family of 14-3-3 proteins is ubiquitous in eukaryotes and has been shown to exert an array of functions. We were interested in the possible role of 14-3-3 proteins in seed germination. Therefore, we studied the expression of 14-3-3 mRNA and protein in barley (Hordeum distichum L.) embryos during germination. With the use of specific cDNA probes and antibodies, we could detect individual expression of three 14-3-3 isoforms, 14-3-3A, 14-3-3B, and 14-3-3C. Each homolog was found to be expressed in barley embryos. Whereas protein levels of all three isoforms were constant during germination, mRNA expression was found to be induced upon imbibition of the grains. The induction of 14-3-3A gene expression during germination was different from that of 14-3-3B and 14-3-3C. In situ immunolocalization analysis showed similar spatial expression for 14-3-3A and 14-3-3B, while 14-3-3C expression was markedly different. Whereas 14-3-3A and 14-3-3B were expressed throughout the embryo, 14-3-3C expression was tissue specific, with the strongest expression observed in the scutellum and the L2 layer of the shoot apical meristem. These results show that 14-3-3 homologs are differently regulated in barley embryos, and provide a first step in acquiring more knowledge about the role of 14-3-3 proteins in the germination process. PMID:10482663

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

    PubMed Central

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

    2016-01-01

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

  19. Extract from mistletoe, Viscum album L., reduces Hsp27 and 14-3-3 protein expression and induces apoptosis in C6 rat glioma cells.

    PubMed

    Uçar, E Ö; Arda, N; Aitken, A

    2012-08-24

    Extracts of mistletoe (Viscum album) are intensively used in complementary medicine, but their mechanisms are not fully understood in most cases, and the effects on metabolism have not been investigated in detail. However, some biologically active natural products are well known to provoke unexpected cellular responses. They reduce overexpression of heat shock proteins (Hsps) in cancer cells. The aim of the current study was to determine whether methanolic extract of V. album, which possesses antioxidant activity, has an effect on expression levels of Hsp27 and 14-3-3 proteins in a C6 glioma cell line. For the first time, the apoptosis-inducing effect of this extract was also determined via caspase-3 activation in the cells. Overexpression of Hsps was induced by heat shock at 42°C for 1 h. Expression levels of Hsp27 and 14-3-3 proteins were determined using Western blot analysis. The apoptosis-inducing effect was also evaluated via caspase-3 activation in C6 glioma cells. Pretreatment of the cells with a nontoxic dose (100 μg/mL) of V. album extract before heat shock significantly reduced expression levels of Hsp27 (73%) and 14-3-3β (124%), 14-3-3γ (23%), and 14-3-3ζ (84%) proteins. Pretreatment with the extract before heat shock increased apoptosis via caspase-3 activation (60%) in C6 glioma cells. This result suggested that the methanolic extract of V. album downregulates expression of Hsp27 and 14-3-3 chaperone proteins and induces apoptosis, which warrants further exploration as a potential bioactive compound for cancer therapy.

  20. A Member of the 14-3-3 Gene Family in Brachypodium distachyon, BdGF14d, Confers Salt Tolerance in Transgenic Tobacco Plants

    PubMed Central

    He, Yuan; Zhang, Yang; Chen, Lihong; Wu, Chunlai; Luo, Qingchen; Zhang, Fan; Wei, Qiuhui; Li, Kexiu; Chang, Junli; Yang, Guangxiao; He, Guangyuan

    2017-01-01

    Plant 14-3-3 proteins are involved in diverse biological processes, but for the model monocotyledonous species, Brachypodium distachyon, their roles in abiotic stress tolerance are not well understood. In this study, a total of eight Bd14-3-3 genes were identified from B. distachyon and these were designated respectively as BdGF14a–BdGF14g. The qRT-PCR analyses of 3-month-old plants of B. distachyon showed that these genes were all expressed in the stems, leaves, and spikelets. By contrast, most of the plants had relatively lower transcriptional levels in their roots, except for the BdGF14g gene. The different expression profiles of the Bd14-3-3s under various stress treatments, and the diverse interaction patterns between Bd14-3-3s and BdAREB/ABFs, suggested that these gene products probably had a range of functions in the stress responses. The NaCl-induced Bd14-3-3 gene, BdGF14d, was selected for overexpression in tobacco. BdGF14d was found to be localized throughout the cell and it conferred enhanced tolerance to salt in the transgenic plants. Lowered contents of malondialdehyde, H2O2, and Na+, and lower relative electronic conductance (Rec%), yet greater activities of catalase and peroxidase, were observed in the overexpressing plants. Higher photosynthetic rate, transpiration rate, stomatal conductance, and water use efficiency were measured in the transgenic lines. Following abscisic acid (ABA) or NaCl treatment, stomatal aperture in leaves of the BdGF14d-overexpression plants was significantly lower than in leaves of the wild type (WT) controls. The stress-related marker genes involved in the ABA signaling pathway, the reactive oxygen species (ROS)-scavenging system, and the ion transporters were all up-regulated in the BdGF14d-overexpressing plants as compared with WT. Taken together, these results demonstrate that the Bd14-3-3 genes play important roles in abiotic stress tolerance. The ABA signaling pathway, the ROS-scavenging system, and ion

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

    PubMed

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

    2014-10-01

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

  2. Meiotic failure in cyclin A1-deficient mouse spermatocytes triggers apoptosis through intrinsic and extrinsic signaling pathways and 14-3-3 proteins

    PubMed Central

    Panigrahi, Sunil K.; Manterola, Marcia; Wolgemuth, Debra J.

    2017-01-01

    Cyclin A1 (Ccna1), a member of the mammalian A type cyclins, is most abundantly expressed in spermatocytes and is essential for spermatogenesis in the mouse. Ccna1- deficient spermatocytes arrest at late meiotic prophase and undergo apoptosis. To further delineate the mechanisms and key factors involved in this process, we have examined changes in expression of genes involved in both intrinsic and extrinsic signaling pathways that trigger apoptosis in the mutant spermatocytes. Our results show that both pathways are involved, and that the factors involved in the intrinsic pathway were expressed earlier than those involved in the extrinsic pathway. We have also begun to identify in vivo Ccna1-interacting proteins, using an unbiased biochemical approach, and identified 14-3-3, a key regulator of apoptosis, as a Ccna1-interacting protein. Expression levels of 14-3-3 proteins remain unchanged between wild type and mutant testes but there were differences in the subcellular distribution. In wild type control, 14-3-3 is detected in both cytosolic and nuclear fractions whereas it is restricted to the cytoplasm in mutant testes. This differential distribution of 14-3-3 may contribute to the induction of apoptosis in Ccna1-deficient spermatocytes. These results provide insight into the apoptotic mechanisms and pathways that are triggered when progression through the meiotic cell cycle is defective in male gametogenesis. PMID:28301569

  3. Induction of Androgen Formation in the Male by a TAT-VDAC1 Fusion Peptide Blocking 14-3-3ɛ Protein Adaptor and Mitochondrial VDAC1 Interactions

    PubMed Central

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

    2014-01-01

    Low testosterone (T), a major cause of male hypogonadism and infertility, is linked to mood changes, fatigue, osteoporosis, reduced bone-mass index, and aging. The treatment of choice, T replacement therapy, has been linked with increased risk for prostate cancer and luteinizing hormone (LH) suppression, and shown to lead to infertility, cardiovascular diseases, and obesity. Alternate methods to induce T with lower side effects are desirable. In search of the mechanisms regulating T synthesis in the testes, we identified the 14-3-3ɛ protein adaptor as a negative regulator of steroidogenesis. Steroidogenesis begins in mitochondria. 14-3-3ɛ interacts with the outer mitochondrial membrane voltage-dependent anion channel (VDAC1) protein, forming a scaffold that limits the availability of cholesterol for steroidogenesis. We report the development of a tool able to induce endogenous T formation. Peptides able to penetrate testes conjugated to 14-3-3ɛ site of interaction with VDAC1 blocked 14-3-3ɛ-VDAC1 interactions while at the same time increased VDAC1-translocator protein (18 kDa) interactions that induced steroid formation in rat testes, leading to increased serum T levels. These peptides rescued intratesticular and serum T formation in adult male rats treated with gonadotropin-releasing hormone antagonist, which dampened LH and T production. PMID:24947306

  4. Two cytosolic puromycin-sensitive aminopeptidase isozymes in chicken brain: molecular homology to brain-specific 14-3-3 protein.

    PubMed

    Hui, K S; Saito, M; Hui, M; Saito, M; Lajtha, A; Yamamoto, K; Osawa, T

    1993-05-01

    Two puromycin-sensitive aminopeptidase isozymes (PSA-I and PSA-II) were isolated from chicken brain cytosol by ammonium sulfate fractionation followed by column chromatography on Cellex D and AH-Sepharose 4B and separated on Bio-Gel HTP. Each was purified to homogeneity on Sephadex G-200, Arg-Tyr-AH-Sepharose, Bio-Gel HTP, and preparative gel electrophoresis. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, PSA-I appeared to be a monomer with a molecular mass of 105 kDa, and PSA-II to be composed of two subunits of 25 kDa and 100 kDa. The tryptic maps of 100 kDa and 105 kDa protein in HPLC are different in peak frequency, height, and composition. The internal peptide sequence of PSA-I has a considerable homology to PSA-II. Both isozymes have repeated copies of common peptide segments and have no significant sequence homology to other peptidases and proteinases. These thio and Co(2+)-activated isozymes have a neutral pH optimum and are inhibited by puromycin and bestatin. PSA-II is more sensitive to trypsin and heat treatment, has a lower Km to Met-enkephalin, and is more active on Arg BNA and Pro BNA. Our results suggest that PSA-I and PSA-II derive from translation of two RNAs of a new gene family related to the brain-specific 14-3-3 protein.

  5. Protein kinase CK2 interacts at the neuromuscular synapse with Rapsyn, Rac1, 14-3-3γ, and Dok-7 proteins and phosphorylates the latter two.

    PubMed

    Herrmann, Dustin; Straubinger, Marion; Hashemolhosseini, Said

    2015-09-11

    Previously, we demonstrated that the protein kinase CK2 associates with and phosphorylates the receptor tyrosine kinase MuSK (muscle specific receptor tyrosine kinase) at the neuromuscular junction (NMJ), thereby preventing fragmentation of the NMJs (Cheusova, T., Khan, M. A., Schubert, S. W., Gavin, A. C., Buchou, T., Jacob, G., Sticht, H., Allende, J., Boldyreff, B., Brenner, H. R., and Hashemolhosseini, S. (2006) Genes Dev. 20, 1800-1816). Here, we asked whether CK2 interacts with other proteins involved in processes at the NMJ, which would be consistent with the previous observation that CK2 appears enriched at the NMJ. We identified the following proteins to interact with protein kinase CK2: (a) the α and β subunits of the nicotinic acetylcholine receptors with weak interaction, (b) dishevelled (Dsh), and (c) another four proteins, Rapsyn, Rac1, 14-3-3γ, and Dok-7, with strong interaction. CK2 phosphorylated 14-3-3γ at serine residue 235 and Dok-7 at several serine residues but does not phosphorylate Rapsyn or Rac1. Furthermore, phosphomimetic Dok-7 mutants aggregated nicotinic acetylcholine receptors in C2C12 myotubes with significantly higher frequency than wild type Dok-7. Additionally, we mapped the interacting epitopes of all four binding partners to CK2 and thereby gained insights into the potential role of the CK2/Rapsyn interaction.

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

    SciTech Connect

    Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.; E-mail: andy.blakely@vanderbilt.edu

    2005-08-05

    The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH{sub 2}-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking.

  7. The 14-3-3 protein Bmh1 functions in the spindle position checkpoint by breaking Bfa1 asymmetry at yeast centrosomes.

    PubMed

    Caydasi, Ayse Koca; Micoogullari, Yagmur; Kurtulmus, Bahtiyar; Palani, Saravanan; Pereira, Gislene

    2014-07-15

    In addition to their well-known role in microtubule organization, centrosomes function as signaling platforms and regulate cell cycle events. An important example of such a function is the spindle position checkpoint (SPOC) of budding yeast. SPOC is a surveillance mechanism that ensures alignment of the mitotic spindle along the cell polarity axis. Upon spindle misalignment, phosphorylation of the SPOC component Bfa1 by Kin4 kinase engages the SPOC by changing the centrosome localization of Bfa1 from asymmetric (one centrosome) to symmetric (both centrosomes). Here we show that, unexpectedly, Kin4 alone is unable to break Bfa1 asymmetry at yeast centrosomes. Instead, phosphorylation of Bfa1 by Kin4 creates a docking site on Bfa1 for the 14-3-3 family protein Bmh1, which in turn weakens Bfa1-centrosome association and promotes symmetric Bfa1 localization. Consistently, BMH1-null cells are SPOC deficient. Our work thus identifies Bmh1 as a new SPOC component and refines the molecular mechanism that breaks Bfa1 centrosome asymmetry upon SPOC activation.

  8. The 14-3-3 protein Bmh1 functions in the spindle position checkpoint by breaking Bfa1 asymmetry at yeast centrosomes

    PubMed Central

    Caydasi, Ayse Koca; Micoogullari, Yagmur; Kurtulmus, Bahtiyar; Palani, Saravanan; Pereira, Gislene

    2014-01-01

    In addition to their well-known role in microtubule organization, centrosomes function as signaling platforms and regulate cell cycle events. An important example of such a function is the spindle position checkpoint (SPOC) of budding yeast. SPOC is a surveillance mechanism that ensures alignment of the mitotic spindle along the cell polarity axis. Upon spindle misalignment, phosphorylation of the SPOC component Bfa1 by Kin4 kinase engages the SPOC by changing the centrosome localization of Bfa1 from asymmetric (one centrosome) to symmetric (both centrosomes). Here we show that, unexpectedly, Kin4 alone is unable to break Bfa1 asymmetry at yeast centrosomes. Instead, phosphorylation of Bfa1 by Kin4 creates a docking site on Bfa1 for the 14-3-3 family protein Bmh1, which in turn weakens Bfa1–centrosome association and promotes symmetric Bfa1 localization. Consistently, BMH1-null cells are SPOC deficient. Our work thus identifies Bmh1 as a new SPOC component and refines the molecular mechanism that breaks Bfa1 centrosome asymmetry upon SPOC activation. PMID:24850890

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

    PubMed

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

    2016-01-21

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

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

    PubMed Central

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

    2016-01-01

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

  11. ­­Presence and distribution of 14-3-3 proteins in human ocular surface tissues

    PubMed Central

    Shankardas, Jwalitha; Senchyna, Michelle

    2008-01-01

    Purpose 14–3-3 is a highly conserved, ubiquitously expressed family of proteins. At least seven mammalian isoforms (β, ε, γ, η, θ, σ, and ζ) are known. These proteins associate with over 200 different target molecules and activate several downstream signaling cascades involved in the regulation of metabolism, cell cycle, apoptosis, protein trafficking, transcription, stress responses, and malignant transformations. We are interested in the role of these proteins in the mechanisms regulating homeostasis and the pathologies of the human ocular surface. Therefore, our purpose is to determine the expression of the 14–3-3 proteins in the human cornea, the conjunctiva, and the primary cells comprising these tissues. Methods Using immunofluorescence, we determined the expression of 14–3-3 β, ε, γ, η, θ, σ, and ζ in paraffin sections of the human cornea and conjunctiva. Using indirect immunofluorescence and western blot analysis, we also determined the expression of these isoforms in primary corneal epithelial cells, keratocytes, endothelial cells, and primary conjunctival epithelial cells. The expressions of these isoforms in primary epithelial and endothelial cells were compared with the same expressions in several corneal cell lines. Western blot analysis was used to determine the presence of 14–3-3 isoforms in the culture medium from corneal epithelial cells, cell lines, and the tear fluid. Results All the 14–3-3 isoforms were expressed in the corneal and conjunctival epithelia as well as primary epithelial cells and cell lines. Expression of 14–3-3 σ was confined to epithelial cells and was secreted into the culture medium of primary cells and cell lines. We also report for the first time that two of the secreted isoforms, 14–3-3 γ and ζ, are also present in the human tear fluid. Conclusions We have determined that all the mammalian 14–3-3 isoforms are expressed in the human cornea, conjunctiva, and the component cells and that the 14

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

    PubMed Central

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

    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. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p–14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling. PMID:18172025

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

    PubMed Central

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

    2016-01-01

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

  14. The Double-Edged Sword of Prostate Cancer: 14-3-3(sigma)

    DTIC Science & Technology

    2005-12-01

    are highly conserved (1,2). The 14-3-3 proteins play important roles in many biological activities by directly binding to and altering the...apoptotic cell death. 15. SUBJECT TERMS No subject terms provided. 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18 . NUMBER OF...as Adriamycin and mitoxantrone (unpublished observations). 14-3-3σ is a member of a highly conserved family of 14-3-3 proteins that are present in

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

    PubMed

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

    2014-01-01

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

  16. The Pseudomonas syringae effector HopQ1 promotes bacterial virulence and interacts with tomato 14-3-3 proteins in a phosphorylation-dependent manner.

    PubMed

    Li, Wei; Yadeta, Koste A; Elmore, James Mitch; Coaker, Gitta

    2013-04-01

    A key virulence strategy of bacterial pathogens is the delivery of multiple pathogen effector proteins into host cells during infection. The Hrp outer protein Q (HopQ1) effector from Pseudomonas syringae pv tomato (Pto) strain DC3000 is conserved across multiple bacterial plant pathogens. Here, we investigated the virulence function and host targets of HopQ1 in tomato (Solanum lycopersicum). Transgenic tomato lines expressing dexamethasone-inducible HopQ1 exhibited enhanced disease susceptibility to virulent Pto DC3000, the Pto ΔhrcC mutant, and decreased expression of a pathogen-associated molecular pattern-triggered marker gene after bacterial inoculation. HopQ1-interacting proteins were coimmunoprecipitated and identified by mass spectrometry. HopQ1 can associate with multiple tomato 14-3-3 proteins, including TFT1 and TFT5. HopQ1 is phosphorylated in tomato, and four phosphorylated peptides were identified by mass spectrometry. HopQ1 possesses a conserved mode I 14-3-3 binding motif whose serine-51 residue is phosphorylated in tomato and regulates its association with TFT1 and TFT5. Confocal microscopy and fractionation reveal that HopQ1 exhibits nucleocytoplasmic localization, while HopQ1 dephosphorylation mimics exhibit more pronounced nuclear localization. HopQ1 delivered from Pto DC3000 was found to promote bacterial virulence in the tomato genotype Rio Grande 76R. However, the HopQ1(S51A) mutant delivered from Pto DC3000 was unable to promote pathogen virulence. Taken together, our data demonstrate that HopQ1 enhances bacterial virulence and associates with tomato 14-3-3 proteins in a phosphorylation-dependent manner that influences HopQ1's subcellular localization and virulence-promoting activities in planta.

  17. Involvement of 14-3-3 protein GRF9 in root growth and response under polyethylene glycol-induced water stress.

    PubMed

    He, Yuchi; Wu, Jingjing; Lv, Bing; Li, Jia; Gao, Zhiping; Xu, Weifeng; Baluška, František; Shi, Weiming; Shaw, Pang Chui; Zhang, Jianhua

    2015-04-01

    Plant 14-3-3 proteins are phosphoserine-binding proteins that regulate a wide array of targets via direct protein-protein interactions. In this study, the role of a 14-3-3 protein, GRF9, in plant response to water stress was investigated. Arabidopsis wild-type, GRF9-deficient mutant (grf9), and GRF9-overexpressing (OE) plants were treated with polyethylene glycol (PEG) to induce mild water stress. OE plant showed better whole-plant growth and root growth than the wild type under normal or water stress conditions while the grf9 mutant showed worse growth. In OE plants, GRF9 favours the allocation of shoot carbon to roots. In addition, GRF9 enhanced proton extrusion, mainly in the root elongation zone and root hair zone, and maintained root growth under mild water stress. Grafting among the wild type, OE, and grf9 plants showed that when OE plants were used as the scion and GRF9 was overexpressed in the shoot, it enhanced sucrose transport into the root, and when OE plants were used as rootstock and GRF9 was overexpressed in the root, it caused more release of protons into the root surface under water stress. Taken together, the results suggest that under PEG-induced water stress, GRF9 is involved in allocating more carbon from the shoot to the root and enhancing proton secretion in the root growing zone, and this process is important for root response to mild water stress.

  18. Antioxidant effects of carnitine supplementation on 14-3-3 protein isoforms in the aged rat hippocampus detected using fully automated two-dimensional chip gel electrophoresis.

    PubMed

    Iwamoto, M; Miura, Y; Tsumoto, H; Tanaka, Y; Morisawa, H; Endo, T; Toda, T

    2014-12-01

    We here described the antioxidant effects of carnitine supplementation on 14-3-3 protein isoforms in the aged rat hippocampus detected using the fully automated two-dimensional chip gel electrophoresis system (Auto2D). This system was easy and convenient to use, and the resolution obtained was more sensitive and higher than that of conventional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). We separated and identified five isoforms of the 14-3-3 protein (beta/alpha, gamma, epsilon, zeta/delta, and eta) using the Auto2D system. We then examined the antioxidant effects of carnitine supplementation on the protein profiles of the cytosolic fraction in the aged rat hippocampus, demonstrating that carnitine supplementation suppressed the oxidation of methionine residues in these isoforms. Since methionine residues are easily oxidized to methionine sulfoxide, the convenient and high-resolution 2-D PAGE system can be available to analyze methionine oxidation avoiding artifactual oxidation. We showed here that the Auto2D system was a very useful tool for studying antioxidant effects through proteomic analysis of protein oxidation.

  19. 14-3-3 phosphoprotein interaction networks – does isoform diversity present functional interaction specification?

    PubMed Central

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

    2012-01-01

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

  20. Unveiling equal importance of two 14-3-3 proteins for morphogenesis, conidiation, stress tolerance and virulence of an insect pathogen.

    PubMed

    Liu, Qian; Li, Jin-Gen; Ying, Sheng-Hua; Wang, Juan-Juan; Sun, Wen-Liang; Tian, Chao-Guang; Feng, Ming-Guang

    2015-04-01

    Two conserved 14-3-3 proteins orthologous to Saccharomyces cerevisiae Bmh1/2 are poorly understood in filamentous fungi. Here we show that Bmh1 and Bmh2 contribute equally to the fundamental biology and physiology of Beauveria bassiana by targeting many sets of proteins/enzymes. Single Bmh deletion caused similar upregulation of another. Excellent knockdown (∼91%) expressions of Bmh1 in ΔBmh2 and Bmh2 in ΔBmh1 resulted in equally more severe multiphenotypic defects than the single deletions, including G2 /M transition, blastospore size, carbon/nitrogen utilization, conidiation, germination and conidial tolerances to high osmolarity, oxidation, cell wall stress, high temperature and UV-B irradiation. All the deletion and deletion/knockdown mutants showed similar defects in blastospore yield and density, hyphal septation and cell size, hyphal responses to most chemical stresses and virulence. All the defects were evident with altered transcripts of phenotype-related genes and well restored by each Bmh complementation. Our Bmh1- and Bmh2-specific transcriptomes generated under osmotic and oxidative stresses revealed up to 6% genes differentially expressed by at least twofold in the fungal genome. Many of those were greatly depressed or co-depressed in ΔBmh1 and ΔBmh2. Our findings provide a thorough insight into the functions and complementary effects of the two 14-3-3 proteins in the filamentous entomopathogen.

  1. Crystal structures of a yeast 14-3-3 protein from Lachancea thermotolerans in the unliganded form and bound to a human lipid kinase PI4KB-derived peptide reveal high evolutionary conservation.

    PubMed

    Eisenreichova, Andrea; Klima, Martin; Boura, Evzen

    2016-11-01

    14-3-3 proteins bind phosphorylated binding partners to regulate several of their properties, including enzymatic activity, stability and subcellular localization. Here, two crystal structures are presented: the crystal structures of the 14-3-3 protein (also known as Bmh1) from the yeast Lachancea thermotolerans in the unliganded form and bound to a phosphopeptide derived from human PI4KB (phosphatidylinositol 4-kinase B). The structures demonstrate the high evolutionary conservation of ligand recognition by 14-3-3 proteins. The structural analysis suggests that ligand recognition by 14-3-3 proteins evolved very early in the evolution of eukaryotes and remained conserved, underlying the importance of 14-3-3 proteins in physiology.

  2. 14-3-3 inhibition promotes dopaminergic neuron loss and 14-3-3θ overexpression promotes recovery in the MPTP mouse model of Parkinson's disease

    PubMed Central

    Ding, Huiping; Underwood, Rachel; Lavalley, Nicholas; Yacoubian, Talene A.

    2015-01-01

    14-3-3s are a highly conserved protein family that plays important roles in cell survival and interact with several proteins implicated in Parkinson's disease (PD). Disruption of 14-3-3 expression and function has been implicated in the pathogenesis of PD. We have previously shown that increasing the expression level of 14-3-3θ is protective against rotenone and 1-methyl-4-phenylpyridinium (MPP+) in cultured cells. Here, we extend our studies to examine the effects of 14-3-3s in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. We first investigated whether targeted nigral 14-3-3θ overexpression mediated by adeno-associated virus offers neuroprotection against MPTP-induced toxicity. 14-3-3θ overexpression using this approach did not reduce MPTP-induced dopaminergic cell loss in the substantia nigra nor the depletion of dopamine and its metabolites in the striatum at three weeks after MPTP administration. However, 14-3-3θ-overexpressing mice showed a later partial recovery in striatal dopamine metabolites at eight weeks after MPTP administration compared to controls, suggesting that 14-3-3θ overexpression may help in the functional recovery of those dopaminergic neurons that survive. Conversely, we investigated whether disrupting 14-3-3 function in transgenic mice expressing the pan 14-3-3 inhibitor difopein exacerbates MPTP-induced toxicity. We found that difopein expression promoted dopaminergic cell loss in response to MPTP treatment. Together, these findings suggest that 14-3-3θ overexpression promotes recovery of dopamine metabolites whereas 14-3-3 inhibition exacerbates neuron loss in the MPTP mouse model of PD. PMID:26314634

  3. Osmoregulation in Lilium pollen grains occurs via modulation of the plasma membrane H+ ATPase activity by 14-3-3 proteins.

    PubMed

    Pertl, Heidi; Pöckl, Magdalena; Blaschke, Christian; Obermeyer, Gerhard

    2010-12-01

    To allow successful germination and growth of a pollen tube, mature and dehydrated pollen grains (PGs) take up water and have to adjust their turgor pressure according to the water potential of the surrounding stigma surface. The turgor pressure of PGs of lily (Lilium longiflorum) was measured with a modified pressure probe for simultaneous recordings of turgor pressure and membrane potential to investigate the relation between water and electrogenic ion transport in osmoregulation. Upon hyperosmolar shock, the turgor pressure decreased, and the plasma membrane (PM) hyperpolarizes in parallel, whereas depolarization of the PM was observed with hypoosmolar treatment. An acidification and alkalinization of the external medium was monitored after hyper- and hypoosmotic treatments, respectively, and pH changes were blocked by vanadate, indicating a putative role of the PM H(+) ATPase. Indeed, an increase in PM-associated 14-3-3 proteins and an increase in PM H(+) ATPase activity were detected in PGs challenged by hyperosmolar medium. We therefore suggest that in PGs the PM H(+) ATPase via modulation of its activity by 14-3-3 proteins is involved in the regulation of turgor pressure.

  4. Osmoregulation in Lilium Pollen Grains Occurs via Modulation of the Plasma Membrane H+ ATPase Activity by 14-3-3 Proteins1[C][W][OA

    PubMed Central

    Pertl, Heidi; Pöckl, Magdalena; Blaschke, Christian; Obermeyer, Gerhard

    2010-01-01

    To allow successful germination and growth of a pollen tube, mature and dehydrated pollen grains (PGs) take up water and have to adjust their turgor pressure according to the water potential of the surrounding stigma surface. The turgor pressure of PGs of lily (Lilium longiflorum) was measured with a modified pressure probe for simultaneous recordings of turgor pressure and membrane potential to investigate the relation between water and electrogenic ion transport in osmoregulation. Upon hyperosmolar shock, the turgor pressure decreased, and the plasma membrane (PM) hyperpolarizes in parallel, whereas depolarization of the PM was observed with hypoosmolar treatment. An acidification and alkalinization of the external medium was monitored after hyper- and hypoosmotic treatments, respectively, and pH changes were blocked by vanadate, indicating a putative role of the PM H+ ATPase. Indeed, an increase in PM-associated 14-3-3 proteins and an increase in PM H+ ATPase activity were detected in PGs challenged by hyperosmolar medium. We therefore suggest that in PGs the PM H+ ATPase via modulation of its activity by 14-3-3 proteins is involved in the regulation of turgor pressure. PMID:20974894

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

    PubMed Central

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

    2014-01-01

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

  6. The Arabidopsis SERK1 protein interacts with the AAA-ATPase AtCDC48, the 14-3-3 protein GF14lambda and the PP2C phosphatase KAPP.

    PubMed

    Rienties, Ingrid M; Vink, Josefien; Borst, Jan Willem; Russinova, Eugenia; de Vries, Sacco C

    2005-06-01

    Leucine-rich repeat (LRR)-containing transmembrane receptor-like kinases (RLKs) are important components of plant signal transduction. The Arabidopsis thaliana somatic embryogenesis receptor-like kinase 1 (AtSERK1) is an LRR-RLK proposed to participate in a signal transduction cascade involved in embryo development. By yeast two-hybrid screening we identified AtCDC48, a homologue of the mammalian AAA-ATPase p97 and GF14lambda, a member of the Arabidopsis family of 14-3-3 proteins as AtSERK1 interactors. In vitro, the AtSERK1 kinase domain is able to transphosphorylate and bind both AtCDC48 and GF14lambda. In yeast, AtCDC48 interacts with GF14lambda and with the PP2C phosphatase KAPP. In plant protoplasts AtSERK1 interacts with GF14lambda.

  7. 14-3-3ζ Interacts with Stat3 and Regulates Its Constitutive Activation in Multiple Myeloma Cells

    PubMed Central

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

    2012-01-01

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

  8. Self-association of the spindle pole body-related intermediate filament protein Fin1p and its phosphorylation-dependent interaction with 14-3-3 proteins in yeast.

    PubMed

    van Hemert, Martijn J; Deelder, André M; Molenaar, Chris; Steensma, H Yde; van Heusden, G Paul H

    2003-04-25

    The Fin1 protein of the yeast Saccharomyces cerevisiae forms filaments between the spindle pole bodies of dividing cells. In the two-hybrid system it binds to 14-3-3 proteins, which are highly conserved proteins involved in many cellular processes and which are capable of binding to more than 120 different proteins. Here, we describe the interaction of the Fin1 protein with the 14-3-3 proteins Bmh1p and Bmh2p in more detail. Purified Fin1p interacts with recombinant yeast 14-3-3 proteins. This interaction is strongly reduced after dephosphorylation of Fin1p. Surface plasmon resonance analysis showed that Fin1p has a higher affinity for Bmh2p than for Bmh1p (K(D) 289 versus 585 nm). Sequences in both the central and C-terminal part of Fin1p are required for the interaction with Bmh2p in the two-hybrid system. In yeast strains lacking 14-3-3 proteins Fin1 filament formation was observed, indicating that the 14-3-3 proteins are not required for this process. Fin1 also interacts with itself in the two-hybrid system. For this interaction sequences at the C terminus, containing one of two putative coiled-coil regions, are sufficient. Fin1p-Fin1p interactions were demonstrated in vivo by fluorescent resonance energy transfer between cyan fluorescent protein-labeled Fin1p and yellow fluorescent protein-labeled Fin1p.

  9. Development of a dot blot assay with antibodies to recombinant “core” 14-3-3 protein: Evaluation of its usefulness in diagnosis of Creutzfeldt–Jakob disease

    PubMed Central

    Subramanian, Sarada; Mahadevan, Anita; Satishchandra, Parthasarathy; Shankar, Susarla Krishna

    2016-01-01

    Background and Purpose: Definitive diagnosis of Creutzfeldt–Jakob disease (CJD) requires demonstration of infective prion protein (PrPSc) in brain tissues by immunohistochemistry or immunoblot, making antemortem diagnosis of CJD difficult. The World Health Organization (WHO) recommends detection of 14-3-3 protein in cerebrospinal fluid (CSF) in cases of dementia, with clinical correlation, as a useful diagnostic marker for CJD, obviating the need for brain biopsy. This facility is currently available in only a few specialized centers in the West and no commercial kit is available for clinical diagnostic use in India. Hence the objective of this study was to develop an in-house sensitive assay for quantitation of 14-3-3 protein and to evaluate its diagnostic potential to detect 14-3-3 proteins in CSF as a biomarker in suspected cases of CJD. Materials and Methods: A minigene expressing the “core” 14-3-3 protein was synthesized by overlapping polymerase chain reaction (PCR) and the recombinant protein was produced by employing a bacterial expression system. Polyclonal antibodies raised in rabbit against the purified recombinant protein were used for developing a dot blot assay with avidin-biotin technology for signal amplification and quantitation of 14-3-3 protein in CSF. Results: The results in the present study suggest the diagnostic potential of the dot blot method with about 10-fold difference (P< 0.001) in the CSF levels of 14-3-3 protein between the CJD cases (N= 50) and disease controls (N= 70). The receiver operating characteristic (ROC) analysis of the results suggested an optimal cutoff value of 2 ng/mL. Conclusions: We have developed an indigenous, economical, and sensitive dot blot method for the quantitation of 14-3-3 protein in CSF. PMID:27293331

  10. A20 zinc finger protein inhibits TNF-induced apoptosis and stress response early in the signaling cascades and independently of binding to TRAF2 or 14-3-3 proteins.

    PubMed

    Lademann, U; Kallunki, T; Jäättelä, M

    2001-03-01

    A20 zinc finger protein is a negative regulator of tumor necrosis factor (TNF)-induced signaling pathways leading to apoptosis, stress response and inflammation. A20 has been shown to bind to TNF-receptor-associated factor 2 (TRAF2) and 14-3-3 chaperone proteins. Our data indicate that the zinc finger domain of A20 is sufficient and that neither TRAF2 nor 14-3-3 binding is necessary for the inhibitory effects of A20. Mutations in the 14-3-3 binding site of A20 did, however, result in a partial cleavage of A20 protein suggesting that 14-3-3 chaperone proteins may stabilize A20. Furthermore, we show that A20 acts early in TNF-induced signaling cascades blocking both TNF-induced rapid activation of c-Jun N-terminal kinase and processing of the receptor-associated caspase-8. Taken together our data indicate that the zinc finger domain of A20 contains all necessary functional domains required for the inhibition of TNF signaling and that A20 may function at the level of the receptor signaling complex.

  11. Up-regulation and interaction of the plasma membrane H(+)-ATPase and the 14-3-3 protein are involved in the regulation of citrate exudation from the broad bean (Vicia faba L.) under Al stress.

    PubMed

    Chen, Qi; Guo, Chuan-Long; Wang, Ping; Chen, Xuan-Qin; Wu, Kong-Huan; Li, Kui-Zhi; Yu, Yong-Xiong; Chen, Li-Mei

    2013-09-01

    Our previous study showed that citrate excretion coupled with a concomitant release of protons was involved in aluminum (Al) resistance in the broad bean. Furthermore, genes encoding plasma membrane (PM) H(+)-ATPase (vha2) and the 14-3-3 protein (vf14-3-3b) were up-regulated by Al in Al-resistant (YD) broad bean roots. In this study, the roles of PM H(+)-ATPase (E.C. 3.6.3.6) and the 14-3-3 protein in the regulation of citrate secretion were further investigated in Al-resistant (YD) and Al-sensitive (AD) broad bean cultivars under Al stress. The results showed that greater citrate exudation was positively correlated with higher activities of PM H(+)-ATPase in roots of YD than AD. Real-time RT-PCR analysis revealed that vha2 was clearly up-regulated by Al in YD but not in AD roots, whereas the transcription levels of vf14-3-3b were elevated in a time-dependent manner in both YD and AD roots. Immunoprecipitation and Western analysis suggested that phosphorylation and interaction with the vf14-3-3b protein of the VHA2 were enhanced in YD roots but not in AD roots with increasing Al treatment time. Fusicoccin or adenosine 5'-monophosphate increased or decreased the interaction between the phosphorylated VHA2 and the vf14-3-3b protein, followed by an enhancement or reduction of the PM H(+)-ATPase activity and citrate exudation in both cultivars under Al stress conditions, respectively. Taken together, these results suggested that Al enhanced the expression and interaction of the PM H(+)-ATPase and the 14-3-3 protein, which thereby led to higher activity of the PM H(+)-ATPase and more citrate exudation from YD plants.

  12. Plant 14-3-3s: omnipotent metabolic phosphopartners?

    PubMed

    Sehnke, P C; Ferl, R J

    2000-10-31

    The accurate regulation of metabolism is crucial to the existence all organisms. The inappropriate activation of metabolic enzymes can waste precious energy; likewise, the failure to activate metabolic enzymes can disrupt homeostasis and lead to suboptimal cellular (and organismic) responses. Plants use several means to control their metabolic proteins, including a two-step process of protein phosphorylation and subsequent binding by phosphospecific binding proteins termed 14-3-3 proteins. Sehnke and Ferl discuss how 14-3-3 proteins regulate the activity of nitrate reductase and the H(+)-ATPase pump in plants, and compare the functions of 14-3-3 proteins in plants and animals.

  13. Phosphorylation of HopQ1, a Type III Effector from Pseudomonas syringae, Creates a Binding Site for Host 14-3-3 Proteins1[C][W][OA

    PubMed Central

    Giska, Fabian; Lichocka, Małgorzata; Piechocki, Marcin; Dadlez, Michał; Schmelzer, Elmon; Hennig, Jacek; Krzymowska, Magdalena

    2013-01-01

    HopQ1 (for Hrp outer protein Q), a type III effector secreted by Pseudomonas syringae pv phaseolicola, is widely conserved among diverse genera of plant bacteria. It promotes the development of halo blight in common bean (Phaseolus vulgaris). However, when this same effector is injected into Nicotiana benthamiana cells, it is recognized by the immune system and prevents infection. Although the ability to synthesize HopQ1 determines host specificity, the role it plays inside plant cells remains unexplored. Following transient expression in planta, HopQ1 was shown to copurify with host 14-3-3 proteins. The physical interaction between HopQ1 and 14-3-3a was confirmed in planta using the fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique. Moreover, mass spectrometric analyses detected specific phosphorylation of the canonical 14-3-3 binding site (RSXpSXP, where pS denotes phosphoserine) located in the amino-terminal region of HopQ1. Amino acid substitution within this motif abrogated the association and led to altered subcellular localization of HopQ1. In addition, the mutated HopQ1 protein showed reduced stability in planta. These data suggest that the association between host 14-3-3 proteins and HopQ1 is important for modulating the properties of this bacterial effector. PMID:23396834

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

    PubMed Central

    Marcos, Caroline Maria; da Silva, Julhiany de Fátima; de Oliveira, Haroldo Cesar; 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

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

    PubMed

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

    2016-07-15

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

  16. GARNL1, a major RalGAP α subunit in skeletal muscle, regulates insulin-stimulated RalA activation and GLUT4 trafficking via interaction with 14-3-3 proteins.

    PubMed

    Chen, Qiaoli; Quan, Chao; Xie, Bingxian; Chen, Liang; Zhou, Shuilian; Toth, Rachel; Campbell, David G; Lu, Shuangshuang; Shirakawa, Ryutaro; Horiuchi, Hisanori; Li, Chaojun; Yang, Zhongzhou; MacKintosh, Carol; Wang, Hong Yu; Chen, Shuai

    2014-08-01

    Insulin and muscle contraction each stimulate translocation of the glucose transporter GLUT4 to the plasma membrane in skeletal muscle, an important process regulating whole-body glucose homeostasis. RalA mediates insulin-stimulated GLUT4 translocation; however, it is unclear how this small GTPase is regulated in skeletal muscle in response to insulin. Here, we identified GARNL1/RalGAPα1, a major α subunit of the Ral-GTPase activating protein in skeletal muscle, as a protein whose phosphorylation and binding to the regulatory 14-3-3 proteins is stimulated by insulin and also by muscle contraction. The insulin-stimulated interaction with 14-3-3 involved PKB/Akt-mediated phosphorylation of Thr(735) on GARNL1/RalGAPα1. Knockdown of GARNL1/RalGAPα1 increased, while overexpression of GARNL1/RalGAPα1(Thr735Ala) mutant protein decreased, the RalA activation and the RalA-dependent GLUT4 translocation in response to insulin in muscle cells. These findings show that GARNL1/RalGAPα1 is the missing link that connects the insulin-PKB/Akt signaling pathway with the activation of the RalA small GTPase in muscle cells. GARNL1/RalGAPα1 and its phosphorylation and/or binding to 14-3-3s are critical for GLUT4 trafficking through RalA in muscle cells.

  17. Characteristics of Korean patients with suspected Creutzfeldt-Jakob disease with 14-3-3 protein in cerebrospinal fluid: Preliminary study of the Korean Creutzfeldt-Jakob disease active surveillance program

    PubMed Central

    Lim, Jae-Sung; Kwon, Hyung-Min; Jang, Jae-Won; Ju, Young-Ran; Kim, SuYeon; Park, Young Ho; Park, So Young; Kim, SangYun

    2015-01-01

    Abstract Although Korea had a national surveillance system for Creutzfeldt-Jakob disease (CJD), it was mainly dependent on attending physician's reports. Thus, little prospective data about the epidemiology, characteristics, and final diagnoses of suspected patients were available. We have established a nationwide network for the active surveillance of patients with suspected CJD. When the requested cerebrospinal fluid (CSF) samples tested positive for 14-3-3 protein, we investigated the clinical characteristics of the corresponding patients and followed them until their final diagnoses were confirmed. A total of 218 samples were requested for CSF assays from May 2010 to August 2012, and 106 (48.6%) were positive for 14-3-3 protein. In 89 patients with complete clinical data, 38 (42.7%) were diagnosed with probable CJD and the estimated annual occurrence of CJD was 16.3 persons-per-year. The most common diagnoses of the remainder were central nervous system infection and any-cause encephalopathy. Non-CJD subjects showed worse initial consciousness levels than CJD patients. This preliminary study showed that the number of reported cases of CJD and the true positivity rates of CSF 14-3-3 protein assays were both low in Korea. An active surveillance system is urgently needed to provide the latest nationwide epidemiological data of CJD. PMID:25996401

  18. Characteristics of Korean patients with suspected Creutzfeldt-Jakob disease with 14-3-3 protein in cerebrospinal fluid: Preliminary study of the Korean Creutzfeldt-Jakob disease active surveillance program.

    PubMed

    Lim, Jae-Sung; Kwon, Hyung-Min; Jang, Jae-Won; Ju, Young-Ran; Kim, SuYeon; Park, Young Ho; Park, So Young; Kim, SangYun

    2015-01-01

    Although Korea had a national surveillance system for Creutzfeldt-Jakob disease (CJD), it was mainly dependent on attending physician's reports. Thus, little prospective data about the epidemiology, characteristics, and final diagnoses of suspected patients were available. We have established a nationwide network for the active surveillance of patients with suspected CJD. When the requested cerebrospinal fluid (CSF) samples tested positive for 14-3-3 protein, we investigated the clinical characteristics of the corresponding patients and followed them until their final diagnoses were confirmed. A total of 218 samples were requested for CSF assays from May 2010 to August 2012, and 106 (48.6%) were positive for 14-3-3 protein. In 89 patients with complete clinical data, 38 (42.7%) were diagnosed with probable CJD and the estimated annual occurrence of CJD was 16.3 persons-per-year. The most common diagnoses of the remainder were central nervous system infection and any-cause encephalopathy. Non-CJD subjects showed worse initial consciousness levels than CJD patients. This preliminary study showed that the number of reported cases of CJD and the true positivity rates of CSF 14-3-3 protein assays were both low in Korea. An active surveillance system is urgently needed to provide the latest nationwide epidemiological data of CJD.

  19. The Acid-sensitive, Anesthetic-activated Potassium Leak Channel, KCNK3, Is Regulated by 14-3-3β-dependent, Protein Kinase C (PKC)-mediated Endocytic Trafficking*

    PubMed Central

    Gabriel, Luke; Lvov, Anatoli; Orthodoxou, Demetra; Rittenhouse, Ann R.; Kobertz, William R.; Melikian, Haley E.

    2012-01-01

    The acid-sensitive neuronal potassium leak channel, KCNK3, is vital for setting the resting membrane potential and is the primary target for volatile anesthetics. Recent reports demonstrate that KCNK3 activity is down-regulated by PKC; however, the mechanisms responsible for PKC-induced KCNK3 down-regulation are undefined. Here, we report that endocytic trafficking dynamically regulates KCNK3 activity. Phorbol esters and Group I metabotropic glutamate receptor (mGluR) activation acutely decreased both native and recombinant KCNK3 currents with concomitant KCNK3 surface losses in cerebellar granule neurons and cell lines. PKC-mediated KCNK3 internalization required the presence of both 14-3-3β and a novel potassium channel endocytic motif, because depleting either 14-3-3β protein levels or ablating the endocytic motif completely abrogated PKC-regulated KCNK3 trafficking. These results demonstrate that neuronal potassium leak channels are not static membrane residents but are subject to 14-3-3β-dependent regulated trafficking, providing a straightforward mechanism to modulate neuronal excitability and synaptic plasticity by Group I mGluRs. PMID:22846993

  20. 14-3-3 Protects against stress-induced apoptosis

    PubMed Central

    Clapp, C; Portt, L; Khoury, C; Sheibani, S; Norman, G; Ebner, P; Eid, R; Vali, H; Mandato, C A; Madeo, F; Greenwood, M T

    2012-01-01

    Expression of human Bax, a cardinal regulator of mitochondrial membrane permeabilization, causes death in yeast. We screened a human cDNA library for suppressors of Bax-mediated yeast death and identified human 14-3-3β/α, a protein whose paralogs have numerous chaperone-like functions. Here, we show that, yeast cells expressing human 14-3-3β/α are able to complement deletion of the endogenous yeast 14-3-3 and confer resistance to a variety of different stresses including cadmium and cycloheximide. The expression of 14-3-3β/α also conferred resistance to death induced by the target of rapamycin inhibitor rapamycin and by starvation for the amino acid leucine, conditions that induce autophagy. Cell death in response to these autophagic stimuli was also observed in the macroautophagic-deficient atg1Δ and atg7Δ mutants. Furthermore, 14-3-3β/α retained its ability to protect against the autophagic stimuli in these autophagic-deficient mutants arguing against so called ‘autophagic death'. In line, analysis of cell death markers including the accumulation of reactive oxygen species, membrane integrity and cell surface exposure of phosphatidylserine indicated that 14-3-3β/α serves as a specific inhibitor of apoptosis. Finally, we demonstrate functional conservation of these phenotypes using the yeast homolog of 14-3-3: Bmh1. In sum, cell death in response to multiple stresses can be counteracted by 14-3-3 proteins. PMID:22785534

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

    PubMed Central

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

    2016-01-01

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

  2. Phosphorylation-dependent inhibition of Cdc42 GEF Gef1 by 14-3-3 protein Rad24 spatially regulates Cdc42 GTPase activity and oscillatory dynamics during cell morphogenesis.

    PubMed

    Das, Maitreyi; Nuñez, Illyce; Rodriguez, Marbelys; Wiley, David J; Rodriguez, Juan; Sarkeshik, Ali; Yates, John R; Buchwald, Peter; Verde, Fulvia

    2015-10-01

    Active Cdc42 GTPase, a key regulator of cell polarity, displays oscillatory dynamics that are anticorrelated at the two cell tips in fission yeast. Anticorrelation suggests competition for active Cdc42 or for its effectors. Here we show how 14-3-3 protein Rad24 associates with Cdc42 guanine exchange factor (GEF) Gef1, limiting Gef1 availability to promote Cdc42 activation. Phosphorylation of Gef1 by conserved NDR kinase Orb6 promotes Gef1 binding to Rad24. Loss of Rad24-Gef1 interaction increases Gef1 protein localization and Cdc42 activation at the cell tips and reduces the anticorrelation of active Cdc42 oscillations. Increased Cdc42 activation promotes precocious bipolar growth activation, bypassing the normal requirement for an intact microtubule cytoskeleton and for microtubule-dependent polarity landmark Tea4-PP1. Further, increased Cdc42 activation by Gef1 widens cell diameter and alters tip curvature, countering the effects of Cdc42 GTPase-activating protein Rga4. The respective levels of Gef1 and Rga4 proteins at the membrane define dynamically the growing area at each cell tip. Our findings show how the 14-3-3 protein Rad24 modulates the availability of Cdc42 GEF Gef1, a homologue of mammalian Cdc42 GEF DNMBP/TUBA, to spatially control Cdc42 GTPase activity and promote cell polarization and cell shape emergence.

  3. Phosphorylation-dependent inhibition of Cdc42 GEF Gef1 by 14-3-3 protein Rad24 spatially regulates Cdc42 GTPase activity and oscillatory dynamics during cell morphogenesis

    PubMed Central

    Das, Maitreyi; Nuñez, Illyce; Rodriguez, Marbelys; Wiley, David J.; Rodriguez, Juan; Sarkeshik, Ali; Yates, John R.; Buchwald, Peter; Verde, Fulvia

    2015-01-01

    Active Cdc42 GTPase, a key regulator of cell polarity, displays oscillatory dynamics that are anticorrelated at the two cell tips in fission yeast. Anticorrelation suggests competition for active Cdc42 or for its effectors. Here we show how 14-3-3 protein Rad24 associates with Cdc42 guanine exchange factor (GEF) Gef1, limiting Gef1 availability to promote Cdc42 activation. Phosphorylation of Gef1 by conserved NDR kinase Orb6 promotes Gef1 binding to Rad24. Loss of Rad24–Gef1 interaction increases Gef1 protein localization and Cdc42 activation at the cell tips and reduces the anticorrelation of active Cdc42 oscillations. Increased Cdc42 activation promotes precocious bipolar growth activation, bypassing the normal requirement for an intact microtubule cytoskeleton and for microtubule-dependent polarity landmark Tea4-PP1. Further, increased Cdc42 activation by Gef1 widens cell diameter and alters tip curvature, countering the effects of Cdc42 GTPase-activating protein Rga4. The respective levels of Gef1 and Rga4 proteins at the membrane define dynamically the growing area at each cell tip. Our findings show how the 14-3-3 protein Rad24 modulates the availability of Cdc42 GEF Gef1, a homologue of mammalian Cdc42 GEF DNMBP/TUBA, to spatially control Cdc42 GTPase activity and promote cell polarization and cell shape emergence. PMID:26246599

  4. Tomato 14-3-3 protein 7 (TFT7) positively regulates immunity-associated programmed cell death by enhancing accumulation and signaling ability of MAPKKKalpha

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Programmed cell death (PCD) is triggered when Pto, a serine-threonine protein kinase recognizes either the AvrPto or AvrPtoB effector from Pseudomonas syringae pv. tomato. This PCD requires MAPKKKalpha as a positive regulator in tomato and Nicotiana benthamiana. To examine how PCD-eliciting activi...

  5. 14-3-3 sigma and 14-3-3 zeta plays an opposite role in cell growth inhibition mediated by transforming growth factor-beta 1.

    PubMed

    Hong, Hye-Young; Jeon, Woo-Kwang; Bae, Eun-Jin; Kim, Shin-Tae; Lee, Ho-Jae; Kim, Seong-Jin; Kim, Byung-Chul

    2010-03-01

    The expression of 14-3-3 proteins is dysregulated in various types of cancer. This study was undertaken to investigate the effects of 14-3-3 zeta and 14-3-3 sigma on cell growth inhibition mediated by transforming growth factor-beta 1 (TGF-beta1). Mouse mammary epithelial cells (Eph4) that are transformed with oncogenic c-H-Ras (EpRas) and no longer sensitive to TGF-beta1-mediated growth inhibition displayed increased expression of 14-3-3 zeta and decreased expression of 14-3-3 sigma compared with parental Eph4 cells. Using small interfering RNA-mediated knockdown and overexpression of 14-3-3 sigma or 14-3-3 zeta, we showed that 14-3-3 sigma is required for TGF-beta1-mediated growth inhibition whereas 14-3-3 zeta negatively modulates this growth inhibitory response. Notably, overexpression of 14-3-3 zeta increased the level of Smad3 protein that is phosphorylated at linker regions and cannot mediate the TGF-beta1 growth inhibitory response. Consistent with this finding, mutation of the 14-3-3 zeta phosphorylation sites in Smad3 markedly reduced the 14-3-3 zeta-mediated inhibition of TGF-beta1-induced p15 promoter-reporter activity and cell cycle arrest, suggesting that these residues are critical targets of 14-3-3 zeta in the suppression of TGF-beta1-mediated growth. Taken together, our findings indicate that dysregulation of 14-3-3 sigma or 14-3-3 zeta contributes to TGF-beta1 resistance in cancer cells.

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

    PubMed

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

    2015-07-01

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

  7. 14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-06-01

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

  9. Rac1 activation driven by 14-3-3ζ dimerization promotes prostate cancer cell-matrix interactions, motility and transendothelial migration.

    PubMed

    Goc, Anna; Abdalla, Maha; Al-Azayzih, Ahmad; Somanath, Payaningal R

    2012-01-01

    14-3-3 proteins are ubiquitously expressed dimeric adaptor proteins that have emerged as key mediators of many cell signaling pathways in multiple cell types. Its effects are mainly mediated by binding to selective phosphoserine/threonine proteins. The importance of 14-3-3 proteins in cancer have only started to become apparent and its exact role in cancer progression as well as the mechanisms by which 14-3-3 proteins mediate cancer cell function remain unknown. While protein 14-3-3σ is widely accepted as a tumor suppressor, 14-3-3ζ, β and γ isoforms have been shown to have tumor promoting effects. Despite the importance of 14-3-3 family in mediating various cell processes, the exact role and mechanism of 14-3-3ζ remain unexplored. In the current study, we investigated the role of protein 14-3-3ζ in prostate cancer cell motility and transendothelial migration using biochemical, molecular biology and electric cell-substrate impedance sensing approaches as well as cell based functional assays. Our study indicated that expression with wild-type protein 14-3-3ζ significantly enhanced Rac activity in PC3 cells. In contrast, expression of dimer-resistant mutant of protein 14-3-3ζ (DM-14-3-3) inhibited Rac activity and associated phosphorylation of p21 activated kinase-1 and 2. Expression with wild-type 14-3-3ζ or constitutively active Rac1 enhanced extracellular matrix recognition, lamellipodia formation, cell migration and trans-endothelial migration by PC3 cells. In contrast, expression with DM 14-3-3ζ or DN-Rac1 in PC3 cells significantly inhibited these cell functions. Our results demonstrate for the first time that 14-3-3ζ enhances prostate cancer cell-matrix interactions, motility and transendothelial migration in vitro via activation of Rac1-GTPase and is an important target for therapeutic interventions for prostate cancer.

  10. A novel interaction partner for the C-terminus of Arabidopsis thaliana plasma membrane H+-ATPase (AHA1 isoform): site and mechanism of action on H+-ATPase activity differ from those of 14-3-3 proteins.

    PubMed

    Morandini, Piero; Valera, Marco; Albumi, Cristina; Bonza, Maria Cristina; Giacometti, Sonia; Ravera, Giuseppe; Murgia, Irene; Soave, Carlo; De Michelis, Maria Ida

    2002-08-01

    Using the two-hybrid technique we identified a novel protein whose N-terminal 88 amino acids (aa) interact with the C-terminal regulatory domain of the plasma membrane (PM) H+-ATPase from Arabidopsis thaliana (aa 847-949 of isoform AHA1). The corresponding gene has been named Ppi1 for Proton pump interactor 1. The encoded protein is 612 aa long and rich in charged and polar residues, except for the extreme C-terminus, where it presents a hydrophobic stretch of 24 aa. Several genes in the A. thaliana genome and many ESTs from different plant species share significant similarity (50-70% at the aa level over stretches of 200-600 aa) to Ppi1. The PPI1 N-terminus, expressed in bacteria as a fusion protein with either GST or a His-tag, binds the PM H+-ATPase in overlay experiments. The same fusion proteins and the entire coding region fused to GST stimulate H+-ATPase activity. The effect of the His-tagged peptide is synergistic with that of fusicoccin (FC) and of tryptic removal of a C-terminal 10 kDa fragment. The His-tagged peptide binds also the trypsinised H+-ATPase. Altogether these results indicate that PPI1 N-terminus is able to modulate the PM H+-ATPase activity by binding to a site different from the 14-3-3 binding site and is located upstream of the trypsin cleavage site.

  11. Human 14-3-3 Paralogs Differences Uncovered by Cross-Talk of Phosphorylation and Lysine Acetylation

    PubMed Central

    Uhart, Marina; Bustos, Diego M.

    2013-01-01

    The 14-3-3 protein family interacts with more than 700 different proteins in mammals, in part as a result of its specific phospho-serine/phospho-threonine binding activity. Upon binding to 14-3-3, the stability, subcellular localization and/or catalytic activity of the ligands are modified. Seven paralogs are strictly conserved in mammalian species. Although initially thought as redundant, the number of studies showing specialization is growing. We created a protein-protein interaction network for 14-3-3, kinases and their substrates signaling in human cells. We included information of phosphorylation, acetylation and other PTM sites, obtaining a complete representation of the 14-3-3 binding partners and their modifications. Using a computational system approach we found that networks of each 14-3-3 isoform are statistically different. It was remarkable to find that Tyr was the most phosphorylatable amino acid in domains of 14-3-3 epsilon partners. This, together with the over-representation of SH3 and Tyr_Kinase domains, suggest that epsilon could be involved in growth factors receptors signaling pathways particularly. We also found that within zeta’s network, the number of acetylated partners (and the number of modify lysines) is significantly higher compared with each of the other isoforms. Our results imply previously unreported hidden differences of the 14-3-3 isoforms interaction networks. The phosphoproteome and lysine acetylome within each network revealed post-transcriptional regulation intertwining phosphorylation and lysine acetylation. A global understanding of these networks will contribute to predict what could occur when regulatory circuits become dysfunctional or are modified in response to external stimuli. PMID:23418452

  12. Identification of 14-3-3epsilon substrates from embryonic murine brain.

    PubMed

    Ballif, Bryan A; Cao, Zhongwei; Schwartz, Daniel; Carraway, Kermit L; Gygi, Steven P

    2006-09-01

    Mice deficient in 14-3-3epsilon exhibit abnormal neuronal migration and die perinatally. We report here the first large-scale analysis of 14-3-3 interacting partners from primary animal tissue, identifying from embryonic murine brain 163 14-3-3epsilon interacting proteins and 85 phosphorylation sites on these proteins. Phosphorylation of the deubiquitinating enzyme USP8 at serine 680 was found essential for its interaction with 14-3-3epsilon and for maintaining USP8 in the cytosol.

  13. Overexpression of 14-3-3σ counteracts tumorigenicity by positively regulating p73 in vivo

    PubMed Central

    GENG, CUIZHI; SANG, MEIXIANG; YANG, RUILING; GAO, WEI; ZHOU, TAO; WANG, SHIJIE

    2011-01-01

    14-3-3σ, one of the 14-3-3 family members, was initially identified as a human mammary epithelium-specific marker 1. The expression of 14-3-3σ is directly regulated by p53. It has been demonstrated that 14-3-3σ stabilizes p53 and enhances its transcriptional activity through the interaction with p53, suggesting that 14-3-3σ has a positive feedback effect on p53. Our previous study showed that 14-3-3σ is a direct transcriptional target of p73 and enhances the p73-mediated transcriptional as well as pro-apoptotic activity in vitro. In the present study, we explored the tumor-suppressive effect of 14-3-3σ by establishing a breast cancer xenograft nude mouse model with an inducible expression of 14-3-3σ or with an inducible expression of p53/p73 plus 14-3-3σ with ADR treatment. Tumor formation was then assayed. Moreover, 66 primary breast cancer specimens and paired tumor-free breast specimens obtained from the female patients were examined. Results showed that the expression of p73 and 14-3-3σ in breast cancer specimens was significantly lower than the tumor-free breast specimens and that 14-3-3σ expression was positively correlated with the expression of p73. Furthermore, overexpression of 14-3-3σ counteracts tumorigenicity by positively regulating p73 in p53-mutated or -deficient cancers in vivo. Therefore, our results may lead to the use of 14-3-3σ in the therapeutic application for the p53-mutated and p73-expressed breast cancer patients. PMID:22848285

  14. Deletion of the cruciform binding domain in CBP/14-3-3 displays reduced origin binding and initiation of DNA replication in budding yeast

    PubMed Central

    Yahyaoui, Wafaa; Callejo, Mario; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2007-01-01

    Background Initiation of eukaryotic DNA replication involves many protein-protein and protein-DNA interactions. We have previously shown that 14-3-3 proteins bind cruciform DNA and associate with mammalian and yeast replication origins in a cell cycle dependent manner. Results By expressing the human 14-3-3ε, as the sole member of 14-3-3 proteins family in Saccharomyces cerevisiae, we show that 14-3-3ε complements the S. cerevisiae Bmh1/Bmh2 double knockout, conserves its cruciform binding activity, and associates in vivo with the yeast replication origins ARS307. Deletion of the α5-helix, the potential cruciform binding domain of 14-3-3, decreased the cruciform binding activity of the protein as well as its association with the yeast replication origins ARS307 and ARS1. Furthermore, the mutant cells had a reduced ability to stably maintain plasmids bearing one or multiple origins. Conclusion 14-3-3, a cruciform DNA binding protein, associates with yeast origins of replication and functions as an initiator of DNA replication, presumably through binding to cruciform DNA forming at yeast replicators. PMID:17430600

  15. On the role of residue phosphorylation in 14-3-3 partners: AANAT as a case study.

    PubMed

    Masone, Diego; Uhart, Marina; Bustos, Diego M

    2017-04-07

    Twenty years ago, a novel concept in protein structural biology was discovered: the intrinsically disordered regions (IDRs). These regions remain largely unstructured under native conditions and the more are studied, more properties are attributed to them. Possibly, one of the most important is their ability to conform a new type of protein-protein interaction. Besides the classical domain-to-domain interactions, IDRs follow a 'fly-casting' model including 'induced folding'. Unfortunately, it is only possible to experimentally explore initial and final states. However, the complete movie of conformational changes of protein regions and their characterization can be addressed by in silico experiments. Here, we simulate the binding of two proteins to describe how the phosphorylation of a single residue modulates the entire process. 14-3-3 protein family is considered a master regulator of phosphorylated proteins and from a modern point-of-view, protein phosphorylation is a three component system, with writers (kinases), erasers (phosphatases) and readers. This later biological role is attributed to the 14-3-3 protein family. Our molecular dynamics results show that phosphorylation of the key residue Thr31 in a partner of 14-3-3, the aralkylamine N-acetyltransferase, releases the fly-casting mechanism during binding. On the other hand, the non-phosphorylation of the same residue traps the proteins, systematically and repeatedly driving the simulations into wrong protein-protein conformations.

  16. On the role of residue phosphorylation in 14-3-3 partners: AANAT as a case study

    PubMed Central

    Masone, Diego; Uhart, Marina; Bustos, Diego M.

    2017-01-01

    Twenty years ago, a novel concept in protein structural biology was discovered: the intrinsically disordered regions (IDRs). These regions remain largely unstructured under native conditions and the more are studied, more properties are attributed to them. Possibly, one of the most important is their ability to conform a new type of protein-protein interaction. Besides the classical domain-to-domain interactions, IDRs follow a ‘fly-casting’ model including ‘induced folding’. Unfortunately, it is only possible to experimentally explore initial and final states. However, the complete movie of conformational changes of protein regions and their characterization can be addressed by in silico experiments. Here, we simulate the binding of two proteins to describe how the phosphorylation of a single residue modulates the entire process. 14-3-3 protein family is considered a master regulator of phosphorylated proteins and from a modern point-of-view, protein phosphorylation is a three component system, with writers (kinases), erasers (phosphatases) and readers. This later biological role is attributed to the 14-3-3 protein family. Our molecular dynamics results show that phosphorylation of the key residue Thr31 in a partner of 14-3-3, the aralkylamine N-acetyltransferase, releases the fly-casting mechanism during binding. On the other hand, the non-phosphorylation of the same residue traps the proteins, systematically and repeatedly driving the simulations into wrong protein-protein conformations. PMID:28387381

  17. Modulation of 14-3-3/phosphotarget interaction by physiological concentrations of phosphate and glycerophosphates.

    PubMed

    Sluchanko, Nikolai N; Chebotareva, Natalia A; Gusev, Nikolai B

    2013-01-01

    Molecular mechanisms governing selective binding of a huge number of various phosphorylated protein partners to 14-3-3 remain obscure. Phosphate can bind to 14-3-3 and therefore being present at high intracellular concentration, which undergoes significant changes under physiological conditions, phosphate can theoretically regulate interaction of 14-3-3 with phosphorylated targets. In order to check this hypothesis we analyzed effect of phosphate and other natural abundant anions on interaction of 14-3-3 with phosphorylated human small heat shock protein HspB6 (Hsp20) participating in regulation of different intracellular processes. Inorganic phosphate, glycerol-1-phosphate and glycerol-2-phosphate at physiologically relevant concentrations (5-15 mM) significantly destabilized complexes formed by 14-3-3ζ and phosphorylated HspB6 (pHspB6), presumably, via direct interaction with the substrate-binding site of 14-3-3. Phosphate also destabilized complexes between pHspB6 and 14-3-3γ or the monomeric mutant form of 14-3-3ζ. Inorganic sulfate and pyrophosphate were less effective in modulation of 14-3-3 interaction with its target protein. The inhibitory effect of all anions on pHspB6/14-3-3 interaction was concentration-dependent. It is hypothesized that physiological changes in phosphate anions concentration can modulate affinity and specificity of interaction of 14-3-3 with its multiple targets and therefore the actual phosphointeractome of 14-3-3.

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

    PubMed

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

    2013-01-01

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

  19. Molecular characterization of cotton 14-3-3L gene preferentially expressed during fiber elongation.

    PubMed

    Shi, Haiyan; Wang, Xiulan; Li, Dengdi; Tang, Wenkai; Wang, Hong; Xu, Wenliang; Li, Xuebao

    2007-02-01

    The 14-3-3 protein, highly conserved in all eukaryotic cells, is an important regulatory protein. It plays an important role in the growth, amplification, apoptosis, signal transduction, and other crucial life activities of cells. A cDNA encoding a putative 14-3-3 protein was isolated from cotton fiber cDNA library. The cDNA, designated as Gh14-3-3L (Gossypium hirsutum 14-3-3-like), is 1,029 bp in length (including a 762 bp long open reading frame and 5'-/3'-untranslated regions) and deduced a protein with 253 amino acids. The Gh14-3-3L shares higher homology with the known plant 14-3-3 proteins, and possesses the basic structure of 14-3-3 proteins: one dimeric domain, one phosphoralated-serine rich motif, four CC domains, and one EF Hand motif. Northern blotting analysis showed that Gh14-3-3L was predominantly expressed during early fiber development, and reached to the peak of expression in 10 days post anthers (DPA) fiber cells, suggesting that the gene may be involved in regulating fiber elongation. The gene is also expressed at higher level in both ovule and petal, but displays lower or undetectable level of activity in other tissues of cotton.

  20. A dual phosphorylation switch controls 14-3-3-dependent cell surface expression of TASK-1

    PubMed Central

    Kilisch, Markus; Lytovchenko, Olga; Arakel, Eric C.; Bertinetti, Daniela; Schwappach, Blanche

    2016-01-01

    ABSTRACT The transport of the K+ channels TASK-1 and TASK-3 (also known as KCNK3 and KCNK9, respectively) to the cell surface is controlled by the binding of 14-3-3 proteins to a trafficking control region at the extreme C-terminus of the channels. The current model proposes that phosphorylation-dependent binding of 14-3-3 sterically masks a COPI-binding motif. However, the direct effects of phosphorylation on COPI binding and on the binding parameters of 14-3-3 isoforms are still unknown. We find that phosphorylation of the trafficking control region prevents COPI binding even in the absence of 14-3-3, and we present a quantitative analysis of the binding of all human 14-3-3 isoforms to the trafficking control regions of TASK-1 and TASK-3. Surprisingly, the affinities of 14-3-3 proteins for TASK-1 are two orders of magnitude lower than for TASK-3. Furthermore, we find that phosphorylation of a second serine residue in the C-terminus of TASK-1 inhibits 14-3-3 binding. Thus, phosphorylation of the trafficking control region can stimulate or inhibit transport of TASK-1 to the cell surface depending on the target serine residue. Our findings indicate that control of TASK-1 trafficking by COPI, kinases, phosphatases and 14-3-3 proteins is highly dynamic. PMID:26743085

  1. 14-3-3 Mediates Histone Cross-Talk during Transcription Elongation in Drosophila

    PubMed Central

    Karam, Caline S.; Kellner, Wendy A.; Takenaka, Naomi; Clemmons, Alexa W.; Corces, Victor G.

    2010-01-01

    Post-translational modifications of histone proteins modulate the binding of transcription regulators to chromatin. Studies in Drosophila have shown that the phosphorylation of histone H3 at Ser10 (H3S10ph) by JIL-1 is required specifically during early transcription elongation. 14-3-3 proteins bind H3 only when phosphorylated, providing mechanistic insights into the role of H3S10ph in transcription. Findings presented here show that 14-3-3 functions downstream of H3S10ph during transcription elongation. 14-3-3 proteins localize to active genes in a JIL-1–dependent manner. In the absence of 14-3-3, levels of actively elongating RNA polymerase II are severely diminished. 14-3-3 proteins interact with Elongator protein 3 (Elp3), an acetyltransferase that functions during transcription elongation. JIL-1 and 14-3-3 are required for Elp3 binding to chromatin, and in the absence of either protein, levels of H3K9 acetylation are significantly reduced. These results suggest that 14-3-3 proteins mediate cross-talk between histone phosphorylation and acetylation at a critical step in transcription elongation. PMID:20532201

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

    SciTech Connect

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

    2010-05-28

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

  3. Nucleotide sequence and expression of the 14-3-3 from the halotolerant alga Dunaliella salina.

    PubMed

    Wang, Tian-yun; Jing, Chang-Qin; Dong, Wei-Hua; Zhang, Jun-He; Zhang, Yu

    2010-02-01

    Previously we reported the nucleotide sequence of a 14-3-3 cDNA cloned from the unicellular green alga Dunaliella salina, however, the nucleotide sequence of this gene have not been reported so far. In the present study, the cloning and characterization of the nucleotide sequence, the gene copy and expression were undertaken. The coding sequence of the gene was found to be interrupted by five introns of 132, 266, 153, 152 and 625 bp, respectively. Introns 3-5 were found in conserved positions as compared to the Chlamydomonas reinhardtii 14-3-3 gene. D. salina 14-3-3 cDNA was inserted into the prokaryotic expression plasmid pET-28 and transformed into E. coli BL21, and the recombinant expressed 14-3-3 protein was purified from E. coli and immunized the rabbit. Indirect ELISA coated with 14-3-3 illustrated that the rabbit antisera titration was 1:1.00E + 06. Western blotting assays confirmed that prepared rabbit antibodies could recognize the recombinant 14-3-3 protein. Southern blotting results showed that there was only one copy of the 14-3-3 present in the genome of D. salina and 14-3-3 expression did not change throughout the Dnualiella cell cycle.

  4. Locomotor hyperactivity in 14-3-3ζ KO mice is associated with dopamine transporter dysfunction

    PubMed Central

    Ramshaw, H; Xu, X; Jaehne, E J; McCarthy, P; Greenberg, Z; Saleh, E; McClure, B; Woodcock, J; Kabbara, S; Wiszniak, S; Wang, Ting-Yi; Parish, C; van den Buuse, M; Baune, B T; Lopez, A; Schwarz, Q

    2013-01-01

    Dopamine (DA) neurotransmission requires a complex series of enzymatic reactions that are tightly linked to catecholamine exocytosis and receptor interactions on pre- and postsynaptic neurons. Regulation of dopaminergic signalling is primarily achieved through reuptake of extracellular DA by the DA transporter (DAT) on presynaptic neurons. Aberrant regulation of DA signalling, and in particular hyperactivation, has been proposed as a key insult in the presentation of schizophrenia and related neuropsychiatric disorders. We recently identified 14-3-3ζ as an essential component of neurodevelopment and a central risk factor in the schizophrenia protein interaction network. Our analysis of 14-3-3ζ-deficient mice now shows that baseline hyperactivity of knockout (KO) mice is rescued by the antipsychotic drug clozapine. 14-3-3ζ KO mice displayed enhanced locomotor hyperactivity induced by the DA releaser amphetamine. Consistent with 14-3-3ζ having a role in DA signalling, we found increased levels of DA in the striatum of 14-3-3ζ KO mice. Although 14-3-3ζ is proposed to modulate activity of the rate-limiting DA biosynthesis enzyme, tyrosine hydroxylase (TH), we were unable to identify any differences in total TH levels, TH localization or TH activation in 14-3-3ζ KO mice. Rather, our analysis identified significantly reduced levels of DAT in the absence of notable differences in RNA or protein levels of DA receptors D1–D5. Providing insight into the mechanisms by which 14-3-3ζ controls DAT stability, we found a physical association between 14-3-3ζ and DAT by co-immunoprecipitation. Taken together, our results identify a novel role for 14-3-3ζ in DA neurotransmission and provide support to the hyperdopaminergic basis of pathologies associated with schizophrenia and related disorders. PMID:24301645

  5. Drosophila 14-3-3/PAR-5 is an essential mediator of PAR-1 function in axis formation.

    PubMed

    Benton, Richard; Palacios, Isabel M; St Johnston, Daniel

    2002-11-01

    PAR-1 kinases are required to determine the anterior-posterior (A-P) axis in C. elegans and Drosophila, but little is known about their molecular function. We identified 14-3-3 proteins as Drosophila PAR-1 interactors and show that PAR-1 binds a domain of 14-3-3 distinct from the phosphoserine binding pocket. PAR-1 kinases phosphorylate proteins to generate 14-3-3 binding sites and may therefore directly deliver 14-3-3 to these targets. 14-3-3 mutants display identical phenotypes to par-1 mutants in oocyte determination and the polarization of the A-P axis. Together, these results indicate that PAR-1's function is mediated by the binding of 14-3-3 to its substrates. The C. elegans 14-3-3 protein, PAR-5, is also required for A-P polarization, suggesting that this is a conserved mechanism by which PAR-1 establishes cellular asymmetries.

  6. 14-3-3ε Is Required for Germ Cell Migration in Drosophila

    PubMed Central

    Tsigkari, K. Kirki; Acevedo, Summer F.; Skoulakis, Efthimios M. C.

    2012-01-01

    Although 14-3-3 proteins participate in multiple biological processes, isoform-specific specialized functions, as well as functional redundancy are emerging with tissue and developmental stage-specificity. Accordingly, the two 14-3-3ε proteins in Drosophila exhibit functional specificity and redundancy. Homozygotes for loss of function alleles of D14-3-3ε contain significantly fewer germ line cells (pole cells) in their gonads, a phenotype not shared by mutants in the other 14-3-3 gene leo. We show that although D14-3-3ε is enriched within pole cells it is required in mesodermal somatic gonad precursor cells which guide pole cells in their migration through the mesoderm and coalesce with them to form the embryonic gonad. Loss of D14-3-3ε results in defective pole cell migration, reduced pole cell number. We present evidence that D14-3-3ε loss results in reduction or loss of the transcription factor Zfh-1, one of the main regulatory molecules of the pole cell migration, from the somatic gonad precursor cells. PMID:22666326

  7. Subcellular Targeting of p33ING1b by Phosphorylation-Dependent 14-3-3 Binding Regulates p21WAF1 Expression

    PubMed Central

    Gong, Wei; Russell, Michael; Suzuki, Keiko; Riabowol, Karl

    2006-01-01

    ING1 is a type II tumor suppressor that affects cell growth, stress signaling, apoptosis, and DNA repair by altering chromatin structure and regulating transcription. Decreased ING1 expression is seen in several human cancers, and mislocalization has been noted in diverse types of cancer cells. Aberrant targeting may, therefore, functionally inactivate ING1. Bioinformatics analysis identified a sequence between the nuclear localization sequence and plant homeodomain domains of ING1 that closely matched the binding motif of 14-3-3 proteins that target cargo proteins to specific subcellular locales. We find that the widely expressed p33ING1b splicing isoform of ING1 interacts with members of the 14-3-3 family of proteins and that this interaction is regulated by the phosphorylation status of ING1. 14-3-3 binding resulted in significant amounts of p33ING1b protein being tethered in the cytoplasm. As shown previously, ectopic expression of p33ING1b increased levels of the p21Waf1 cyclin-dependent kinase inhibitor upon UV-induced DNA damage. Overexpression of 14-3-3 inhibited the up-regulation of p21Waf1 by p33ING1b, consistent with the idea that mislocalization blocks at least one of ING1's biological activities. These data support the idea that the 14-3-3 proteins play a crucial role in regulating the activity of p33ING1b by directing its subcellular localization. PMID:16581770

  8. Transgenic Overexpression of 14-3-3 Zeta Protects Hippocampus against Endoplasmic Reticulum Stress and Status Epilepticus In Vivo

    PubMed Central

    Brennan, Gary P.; Jimenez-Mateos, Eva M.; McKiernan, Ross C.; Engel, Tobias; Tzivion, Guri; Henshall, David C.

    2013-01-01

    14-3-3 proteins are ubiquitous molecular chaperones that are abundantly expressed in the brain where they regulate cell functions including metabolism, the cell cycle and apoptosis. Brain levels of several 14-3-3 isoforms are altered in diseases of the nervous system, including epilepsy. The 14-3-3 zeta (ζ) isoform has been linked to endoplasmic reticulum (ER) function in neurons, with reduced levels provoking ER stress and increasing vulnerability to excitotoxic injury. Here we report that transgenic overexpression of 14-3-3ζ in mice results in selective changes to the unfolded protein response pathway in the hippocampus, including down-regulation of glucose-regulated proteins 78 and 94, activating transcription factors 4 and 6, and Xbp1 splicing. No differences were found between wild-type mice and transgenic mice for levels of other 14-3-3 isoforms or various other 14-3-3 binding proteins. 14-3-3ζ overexpressing mice were potently protected against cell death caused by intracerebroventricular injection of the ER stressor tunicamycin. 14-3-3ζ overexpressing mice were also potently protected against neuronal death caused by prolonged seizures. These studies demonstrate that increased 14-3-3ζ levels protect against ER stress and seizure-damage despite down-regulation of the unfolded protein response. Delivery of 14-3-3ζ may protect against pathologic changes resulting from prolonged or repeated seizures or where injuries provoke ER stress. PMID:23359526

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

    PubMed

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

    2012-02-01

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

  10. PRMT5 C-terminal Phosphorylation Modulates a 14-3-3/PDZ Interaction Switch.

    PubMed

    Espejo, Alexsandra B; Gao, Guozhen; Black, Karynne; Gayatri, Sitaram; Veland, Nicolas; Kim, Jeesun; Chen, Taiping; Sudol, Marius; Walker, Cheryl; Bedford, Mark T

    2017-02-10

    PRMT5 is the primary enzyme responsible for the deposition of the symmetric dimethylarginine in mammalian cells. In an effort to understand how PRMT5 is regulated, we identified a threonine phosphorylation site within a C-terminal tail motif, which is targeted by the Akt/serum- and glucocorticoid-inducible kinases. While investigating the function of this posttranslational modification, we serendipitously discovered that its free C-terminal tail binds PDZ domains (when unphosphorylated) and 14-3-3 proteins (when phosphorylated). In essence, a phosphorylation event within the last few residues of the C-terminal tail generates a posttranslational modification-dependent PDZ/14-3-3 interaction "switch." The C-terminal motif of PRMT5 is required for plasma membrane association, and loss of this switching capacity is not compatible with life. This signaling phenomenon was recently reported for the HPV E6 oncoprotein but has not yet been observed for mammalian proteins. To investigate the prevalence of PDZ/14-3-3 switching in signal transduction, we built a protein domain microarray that harbors PDZ domains and 14-3-3 proteins. We have used this microarray to interrogate the C-terminal tails of a small group of candidate proteins and identified ERBB4, PGHS2, and IRK1 (as well as E6 and PRMT5) as conforming to this signaling mode, suggesting that PDZ/14-3-3 switching may be a broad biological paradigm.

  11. 14-3-3ζ Orchestrates Mammary Tumor Onset and Progression via miR221-Mediated Cell Proliferation

    PubMed Central

    Wyszomierski, Shannon L.; Wang, Qingfei; Li, Ping; Sahin, Ozgur; Xiao, Yi; Zhang, Siyuan; Xiong, Yan; Yang, Jun; Wang, Hai; Guo, Hua; Zhang, Jitao D.; Medina, Daniel; Muller, William J.; Yu, Dihua

    2013-01-01

    14-3-3ζ is overexpressed in over 40% of breast cancers but its pathophysiological relevance to tumorigenesis has not been established. Here we show that 14-3-3ζ overexpression is sufficient to induce tumorigenesis in a transgenic mouse model of breast cancer. MMTV-LTR promoter driven HA-14-3-3ζ transgenic mice (MMTV-HA-14-3-3ζ) developed mammary tumors whereas control mice did not. Whey acidic protein promoter driven HA-14-3-3ζ transgenic mice (WAP-HA-14-3-3ζ) developed hyperplastic lesions and showed increased susceptibility to carcinogen-induced tumorigenesis. When crossed with MMTV-neu transgenic mice, 14-3-3ζ.neu transgenic mice exhibited accelerated mammary tumorigenesis and metastasis compared to MMTV-neu mice. Mechanistically, 14-3-3ζ overexpression enhanced MAPK/c-Jun signaling leading to increased miR-221 transcription, which inhibited p27 CDKI translation, and consequently, promoted cell proliferation. Importantly, this 14-3-3ζ/miR-221/p27/proliferation axis is also functioning in patients' breast tumors and associates with high grade cancers. Taken together, our findings show that 14-3-3ζ overexpression has a causal role in mammary tumorigenesis and progression, acting through miR-221 in cooperation with known oncogenic events to drive neoplastic cell proliferation. PMID:24197133

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

    PubMed Central

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

    2015-01-01

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

  13. 14-3-3σ regulates keratinocyte proliferation and differentiation by modulating Yap1 cellular localization

    PubMed Central

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

    2015-01-01

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

  14. Neurodevelopmental and neuropsychiatric behaviour defects arise from 14-3-3ζ deficiency.

    PubMed

    Cheah, P S; Ramshaw, H S; Thomas, P Q; Toyo-Oka, K; Xu, X; Martin, S; Coyle, P; Guthridge, M A; Stomski, F; van den Buuse, M; Wynshaw-Boris, A; Lopez, A F; Schwarz, Q P

    2012-04-01

    Complex neuropsychiatric disorders are believed to arise from multiple synergistic deficiencies within connected biological networks controlling neuronal migration, axonal pathfinding and synapse formation. Here, we show that deletion of 14-3-3ζ causes neurodevelopmental anomalies similar to those seen in neuropsychiatric disorders such as schizophrenia, autism spectrum disorder and bipolar disorder. 14-3-3ζ-deficient mice displayed striking behavioural and cognitive deficiencies including a reduced capacity to learn and remember, hyperactivity and disrupted sensorimotor gating. These deficits are accompanied by subtle developmental abnormalities of the hippocampus that are underpinned by aberrant neuronal migration. Significantly, 14-3-3ζ-deficient mice exhibited abnormal mossy fibre navigation and glutamatergic synapse formation. The molecular basis of these defects involves the schizophrenia risk factor, DISC1, which interacts isoform specifically with 14-3-3ζ. Our data provide the first evidence of a direct role for 14-3-3ζ deficiency in the aetiology of neurodevelopmental disorders and identifies 14-3-3ζ as a central risk factor in the schizophrenia protein interaction network.

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

    PubMed Central

    Motokura, Toru; Nakamura, Yukari; Sato, Hiroyuki

    2007-01-01

    Background 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. Methods 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. Results 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. Conclusion 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

  16. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells.

    PubMed

    Li, Tong; Paudel, Hemant K

    2016-01-01

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

  17. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells

    PubMed Central

    Li, Tong; Paudel, Hemant K.

    2016-01-01

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

  18. 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.

    PubMed

    Shi, Haiyan; Zhang, Yuxing

    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 ε-like group of 14-3-3 superfamilies. 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.

  19. 14-3-3 antagonizes Ras-mediated Raf-1 recruitment to the plasma membrane to maintain signaling fidelity.

    PubMed

    Light, Yvonne; Paterson, Hugh; Marais, Richard

    2002-07-01

    We have investigated the role that S259 phosphorylation, S621 phosphorylation, and 14-3-3 binding play in regulating Raf-1 activity. We show that 14-3-3 binding, rather than Raf-1 phosphorylation, is required for the correct regulation of kinase activity. Phosphorylation of S621 is not required for activity, but 14-3-3 binding is essential. When 14-3-3 binding to conserved region 2 (CR2) was disrupted, Raf-1 basal kinase activity was elevated and it could be further activated by (V12,G37)Ras, (V23)TC21, and (V38)R-Ras. Disruption of 14-3-3 binding at CR2 did not recover binding of Raf-1 to (V12,G37)Ras but allowed more efficient recruitment of Raf-1 to the plasma membrane and stimulated its phosphorylation on S338. Finally, (V12)Ras, but not (V12,G37)Ras, displaced 14-3-3 from full-length Raf-1 and the Raf-1 bound to Ras. GTP was still phosphorylated on S259. Our data suggest that stable association of Raf-1 with the plasma membrane requires Ras-mediated displacement of 14-3-3 from CR2. Small G proteins that cannot displace 14-3-3 fail to recruit Raf-1 to the membrane efficiently and so fail to stimulate kinase activity.

  20. 14-3-3-dependent inhibition of the deubiquitinating activity of UBPY and its cancellation in the M phase.

    PubMed

    Mizuno, Emi; Kitamura, Naomi; Komada, Masayuki

    2007-10-01

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

  1. Impairment of GABAB receptor dimer by endogenous 14-3-3ζ in chronic pain conditions

    PubMed Central

    Laffray, Sophie; Bouali-Benazzouz, Rabia; Papon, Marie-Amélie; Favereaux, Alexandre; Jiang, Yang; Holm, Tina; Spriet, Corentin; Desbarats, Pascal; Fossat, Pascal; Le Feuvre, Yves; Decossas, Marion; Héliot, Laurent; Langel, Ulo; Nagy, Frédéric; Landry, Marc

    2012-01-01

    In the central nervous system, the inhibitory GABAB receptor is the archetype of heterodimeric G protein-coupled receptors (GPCRs). However, the regulation of GABAB dimerization, and more generally of GPCR oligomerization, remains largely unknown. We propose a novel mechanism for inhibition of GPCR activity through de-dimerization in pathological conditions. We show here that 14-3-3ζ, a GABAB1-binding protein, dissociates the GABAB heterodimer, resulting in the impairment of GABAB signalling in spinal neurons. In the dorsal spinal cord of neuropathic rats, 14-3-3ζ is overexpressed and weakens GABAB inhibition. Using anti-14-3-3ζ siRNA or competing peptides disrupts 14-3-3ζ/GABAB1 interaction and restores functional GABAB heterodimers in the dorsal horn. Importantly, both strategies greatly enhance the anti-nociceptive effect of intrathecal Baclofen in neuropathic rats. Taken together, our data provide the first example of endogenous regulation of a GPCR oligomeric state and demonstrate its functional impact on the pathophysiological process of neuropathic pain sensitization. PMID:22692127

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

    PubMed

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

    2014-01-01

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

  3. Isolation and expression analysis of a homolog of the 14-3-3 epsilon gene in the diamondback moth, Plutella xylostella.

    PubMed

    Yoo, Ji Yeon; Hwang, Se Hui; Han, Yeon Soo; Cho, Saeyoull

    2011-02-01

    A full-length 14-3-3 gene homolog (also referred to as the Px14-3-3 epsilon "ε" or Px14-3-3ε gene) was cloned from cDNA of the diamondback moth, Plutella xylostella. The Px14-3-3 transcript is 789 nucleotides in length, and the predicted polypeptide is 263 amino acids in length, with a calculated molecular mass of 29.6 kDa. The Px14-3-3 gene contains the typical and predicted 14-3-3 domains and motifs. The amino acid sequence of the diamondback moth 14-3-3 gene is very similar to that of other insect epsilons (ε) but not to other insect zetas (ζ). In particular, the protein sequence of the Px14-3-3 gene shows high identity to the Bombyx mori epsilon (96.2%). Western blot analysis using an antibody against Px14-3-3ε verified the expression of 14-3-3ε in the larval, pupal, and adult stages. The Px14-3-3ε expression patterns in all the different tissue types were examined in the fourth instar larvae. Px14-3-3ε was detected in every tissue examined, including the body fat, hemocytes, brain, gut, and cuticle.

  4. [Inhibitory effect of 14-3-3ζ on the proliferation of HL-60 cells and HL-60/VCR cells].

    PubMed

    Liang, Rong; Chen, Xie-Qun; Wang, Zhe; Xiong, Hua; Bai, Qing-Xian; Gao, Guang-Xun; Dong, Bao-Xia; Zhu, Hua-Feng

    2013-08-01

    This study was aimed to investigate the expression and role of 14-3-3ζ in the AML cell lines: sensitive HL-60 and drug-resistant HL-60/VCR cells. Semi-quantitative RT-PCR and Western blot were respectively used to examine the expression of mdr1 mRNA and Pgp in AML cell lines to validate the results of microarray. Western blot was performed to investigate the expression of Pgp, 14-3-3ζ, and anti-apoptosis protein BCL-2, MCL-1 proteins. Immunofluorescence assay was used to detect the subcellular location of 14-3-3ζ protein in HL-60 and HL-60/VCR cells by laser scanning confocal microscopy. Transduction with siRNA was used to silence 14-3-3ζ in AML cell lines. Cell count method and flow cytometry of cell cycle were used to analyze the changes of growth of AML cells. The results found that mdr1 mRNA and Pgp did not expressed in HL-60 cells, but significantly overexpressed in HL-60/VCR cells. Except 14-3-3σ, the expression of other subtypes of 14-3-3 was higher in HL-60/VCR cells than that in HL-60 cells, especially 14-3-3ζ. The higher expression of 14-3-3ζ, BCL-2, MCL-1 protein was observed in HL-60/VCR cells than that in HL-60 cells. These results were same results from gene chip. It was also noticed that 14-3-3ζ was located in the cytoplasma and nuclei of AML cell lines, especially over-expressed in HL-60/VCR cells. Furthermore, suppression of 14-3-3ζ by RNA interference resulted in inhibition of the proliferation of AML cells with decreased protein expression of BCL-2 and MCL-1, especially in HL-60/VCR cells. It is concluded that 14-3-3ζ plays an important role in proliferation of AML cells and associates with BCL-2 and MCL-1 expression. These results suggested that development of therapy targeting 14-3-3ζ may provide novel, effective strategies for refractory and relapsed AML.

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

    PubMed

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

    2016-05-13

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

  6. Cannabinoid receptor activation inhibits cell cycle progression by modulating 14-3-3β.

    PubMed

    Jung, Hye-Won; Park, Inae; Ghil, Sungho

    2014-09-01

    Cannabinoids display various pharmacological activities, including tumor regression, anti-inflammatory and neuroprotective effects. To investigate the molecular mechanisms underlying the pharmacological effects of cannabinoids, we used a yeast two-hybrid system to screen a mouse brain cDNA library for proteins interacting with type 1 cannabinoid receptor (CB1R). Using the intracellular loop 3 of CB1R as bait, we identified 14-3-3β as an interacting partner of CB1R and confirmed their interaction using affinity-binding assays. 14-3-3β has been reported to induce a cell cycle delay at the G2/M phase. We tested the effects of cannabinoids on cell cycle progression in HeLa cells synchronized using a double-thymidine block-and-release protocol and found an increase in the population of G2/M phase cells. We further found that CB1R activation augmented the interaction of 14-3-3β with Wee1 and Cdc25B, and promoted phosphorylation of Cdc2 at Tyr-15. These results suggest that cannabinoids induce cell cycle delay at the G2/M phase by activating 14-3-3β.

  7. 14-3-3ζ up-regulates hypoxia-inducible factor-1α in hepatocellular carcinoma via activation of PI3K/Akt/NF-кB signal transduction pathway

    PubMed Central

    Tang, Yufu; Lv, Pengfei; Sun, Zhongyi; Han, Lei; Luo, Bichao; Zhou, Wenping

    2015-01-01

    14-3-3ζ protein, a member of 14-3-3 family, plays important roles in multiple cellular processes. Our previous study showed that 14-3-3ζ could bind to regulate the expression of hypoxia-inducible factor-1α (HIF-1α), which is induced by hypoxia and a crucial factor for induction of tumor metastasis. Moreover, we also have confirmed the response of 14-3-3ζ to hypoxia in our unpublished data as well. Thus, in the present study, we attempted to reveal that whether the regulation effect of 14-3-3ζ on HIF-1α functioned in a similar pattern as hypoxia. Stable regulation of 14-3-3ζ in human HCC cell line SMMC-772 and HCC-LM3 was achieved. The regulation of 14-3-3ζ on HIF-1α mRNA transcription was evaluated by luciferase activity assay and quantitative real-time PCR (qPCR). The effect of 14-3-3ζ on the production of HIF-1α and pathways determining HIF-1α’s response to hypoxia was assessed using western blotting assay. Our results showed that regulation of 14-3-3ζ expression influenced the activity of HIF-1α, phosphatidyl inositol 3-kinase (PI3K), Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), and nuclear factor kappa B (NF-кB). Blocking of these pathways using indicated inhibitors revealed that 14-3-3ζ enhanced the production of HIF-1α via the activation of PI3K/Akt/NF-кB pathway, which was identical to hypoxia induced HIF-1α expression. For the first time, our study described the key role of 14-3-3ζ in the HIF-1α production in HCC cells. And the molecule exerted its function on HIF-1α both by directly binding to it and via PI3K/Akt/NF-кB signal transduction pathway. PMID:26884855

  8. 14-3-3{sigma} controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

    SciTech Connect

    Xin, Ying; Lu, Qingxian; Li, Qiutang

    2010-02-19

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

  9. 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

    PubMed Central

    Bolton, Diane L; Barnitz, Robert A; Sakai, Keiko; Lenardo, Michael J

    2008-01-01

    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. PMID:18445273

  10. 14-3-3-dependent inhibition of the deubiquitinating activity of UBPY and its cancellation in the M phase

    SciTech Connect

    Mizuno, Emi; Kitamura, Naomi; Komada, Masayuki

    2007-10-01

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

  11. miR-137 modulates coelomocyte apoptosis by targeting 14-3-3ζ in the sea cucumber Apostichopus japonicus.

    PubMed

    Lv, Miao; Chen, Huahui; Shao, Yina; Li, Chenghua; Xu, Wei; Zhang, Weiwei; Zhao, Xuelin; Duan, Xuemei

    2017-02-01

    MicroRNAs (miRNAs) have emerged as key regulators in the host immune response and play a pivotal role in host-pathogen interactions by suppressing the transcriptional and post-transcriptional expression of target genes. miR-137, a well-documented tumor repressor, was previously found by high-throughput sequencing to be differentially expressed in diseased specimens of the sea cucumber Apostichopus japonicus. In this study, we identified 14-3-3ζ protein (Aj14-3-3ζ) as a novel target of miR-137 using isobaric tags for relative and absolute quantification (iTRAQ) and transcriptome screening. Expression analysis indicated that consistently depressed expression profiles of miR-137 and Aj14-3-3ζ were detected in both LPS-exposed primary coelomocytes and Vibrio splendidus-challenged sea cucumbers, suggesting a positive regulatory interaction. Consistently, miR-137 overexpression or inhibition in vitro and in vivo showed no effect on Aj14-3-3ζ mRNA levels, but the concentration of Aj14-3-3ζ protein was induced or repressed, respectively. Moreover, siRNA-mediated Aj14-3-3ζ knockdown in vivo decreased both mRNA and protein expression levels of Aj14-3-3ζ and significantly promoted coelomocyte apoptosis as assessed by flow cytometry, consistent with miR-137 inhibition. Overall, these results enhance our understanding of miR-137 regulatory roles in sea cucumber pathogenesis.

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

    PubMed Central

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

    2012-01-01

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

  13. Rethinking the role of phosducin: Light-regulated binding of phosducin to 14-3-3 in rod inner segments

    PubMed Central

    Nakano, Koichi; Chen, Jing; Tarr, George E.; Yoshida, Tatsuro; Flynn, Julia M.; Bitensky, Mark W.

    2001-01-01

    Phosducin (Pd), a small protein found abundantly in photoreceptors, is widely assumed to regulate light sensitivity in the rod outer segment through interaction with the heterotrimeric G protein transducin. But, based on histochemistry and Western blot analysis, Pd is found almost entirely in the inner segment in both light and dark, most abundantly near the rod synapse. We report a second small protein, 14-3-3, in the rod with a similar distribution. By immunoprecipitation, phospho-Pd is found to interact with 14-3-3 in material from dark-adapted retina, and this interaction is markedly diminished by light, which dephosphorylates Pd. Conversely, unphosphorylated Pd binds to inner segment G protein(s) in the light. From these results and reported functions of 14-3-3, we have constructed a hypothesis for the regulation of light sensitivity at the level of rod synapse. By dissociating the Pd/14-3-3 complex, light enables both proteins to function in this role. PMID:11287646

  14. Transcriptional regulation of YWHAZ, the gene encoding 14-3-3ζ.

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2016-03-01

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

  16. 14-3-3ε Plays a Role in Cardiac Ventricular Compaction by Regulating the Cardiomyocyte Cell Cycle

    PubMed Central

    Kosaka, Yasuhiro; Cieslik, Katarzyna A.; Li, Ling; Lezin, George; Maguire, Colin T.; Saijoh, Yukio; Toyo-oka, Kazuhito; Gambello, Michael J.; Vatta, Matteo; Wynshaw-Boris, Anthony; Baldini, Antonio; Yost, H. Joseph

    2012-01-01

    Trabecular myocardium accounts for the majority of the ventricles during early cardiogenesis, but compact myocardium is the primary component at later developmental stages. Elucidation of the genes regulating compact myocardium development is essential to increase our understanding of left ventricular noncompaction (LVNC), a cardiomyopathy characterized by increased ratios of trabecular to compact myocardium. 14-3-3ε is an adapter protein expressed in the lateral plate mesoderm, but its in vivo cardiac functions remain to be defined. Here we show that 14-3-3ε is expressed in the developing mouse heart as well as in cardiomyocytes. 14-3-3ε deletion did not appear to induce compensation by other 14-3-3 isoforms but led to ventricular noncompaction, with features similar to LVNC, resulting from a selective reduction in compact myocardium thickness. Abnormal compaction derived from a 50% decrease in cardiac proliferation as a result of a reduced number of cardiomyocytes in G2/M and the accumulation of cardiomyocytes in the G0/G1 phase of the cell cycle. These defects originated from downregulation of cyclin E1 and upregulation of p27Kip1, possibly through both transcriptional and posttranslational mechanisms. Our work shows that 14-3-3ε regulates cardiogenesis and growth of the compact ventricular myocardium by modulating the cardiomyocyte cell cycle via both cyclin E1 and p27Kip1. These data are consistent with the long-held view that human LVNC may result from compaction arrest, and they implicate 14-3-3ε as a new candidate gene in congenital human cardiomyopathies. PMID:23071090

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

    PubMed Central

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

    2014-01-01

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

  18. SAP family proteins.

    PubMed

    Fujita, A; Kurachi, Y

    2000-03-05

    Thus far, five members including Dlg, SAP97/hDlg, SAP90/PSD-95, SAP102, and PSD-93/chapsyn110 which belong to SAP family have been identified. Recent studies have revealed that these proteins play important roles in the localization and function of glutamate receptors and K(+) channels. Although most of them have been reported to be localized to the synapse, only one member, SAP97, is expressed also in the epithelial cells. In this review, we have summarized structural characters of SAP family proteins and discuss their functions in neurons and epithelial cells.

  19. The RGK family: a regulatory tail of small GTP-binding proteins.

    PubMed

    Kelly, Kathleen

    2005-12-01

    RGK proteins are small Ras-related GTP-binding proteins that function as potent inhibitors of voltage-dependent calcium channels, and two members of the family, Gem and Rad, modulate Rho-dependent remodeling of the cytoskeleton. Within the Ras superfamily, RGK proteins have distinct structural and regulatory characteristics. It is an open question as to whether RGK proteins catalyze GTP hydrolysis in vivo. Binding of calmodulin and the 14-3-3 protein to RGK proteins controls downstream pathways. Here, we discuss the structural and functional properties of RGK proteins and highlight recent work by Beguin and colleagues addressing the mechanism of Gem regulation by calmodulin and 14-3-3.

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

    PubMed Central

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

    2015-01-01

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

  1. 14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression

    PubMed Central

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

    2016-01-01

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

  2. Anchoring of both PKA and 14-3-3 inhibits the Rho-GEF activity of the AKAP-Lbc signaling complex.

    PubMed

    Diviani, Dario; Abuin, Liliane; Cotecchia, Susanna; Pansier, Laetitia

    2004-07-21

    A-kinase anchoring proteins (AKAPs) target the cAMP-regulated protein kinase (PKA) to its physiological substrates. We recently identified a novel anchoring protein, called AKAP-Lbc, which functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha subunit of the heterotrimeric G protein G12. Here, we identified 14-3-3 as a novel regulatory protein interacting with AKAP-Lbc. Elevation of the cellular concentration of cAMP activates the PKA holoenzyme anchored to AKAP-Lbc, which phosphorylates the anchoring protein on the serine 1565. This phosphorylation event induces the recruitment of 14-3-3, which inhibits the Rho-GEF activity of AKAP-Lbc. AKAP-Lbc mutants that fail to interact with PKA or with 14-3-3 show a higher basal Rho-GEF activity as compared to the wild-type protein. This suggests that, under basal conditions, 14-3-3 maintains AKAP-Lbc in an inactive state. Therefore, while it is known that AKAP-Lbc activity can be stimulated by Galpha12, in this study we demonstrated that it is inhibited by the anchoring of both PKA and 14-3-3.

  3. Origin and evolution of a new exon of 14-3-3ξ in bees and phylogenetic analysis.

    PubMed

    Zhan, Leilei; Jiang, Chao

    2013-04-01

    Mutually exclusive splicing, one type of alternative splicing, involves selection of alternatively spliced exons arranged in tandem and creates protein products with substitution of one segment of the amino acid sequence for another. Previous studies revealed that exon 5 of 14-3-3ξ from Apis mellifera (western honeybee) had three mutually exclusive exons, while orthologous exon of Nasonia vitripennis (parasitic wasp) had only two, suggesting that cases of exon gain or loss might have happened during the evolution of hymenopteran species. In the current study, we annotated and analyzed the 14-3-3ξ genes from 20 hymenopteran species successfully, and the results of phylogenetic analysis revealed the presence of a new mutually exclusive exon in corbiculate bees. In addition, we found that duplication via staggered homologous recombination was responsible for the origin of the new exon.

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

    SciTech Connect

    Nishino, Tasuku; Matsunaga, Ryota; Konishi, Hiroaki

    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.

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

    PubMed Central

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

    2016-01-01

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

  6. Induction of expression of a 14-3-3 gene in response to copper exposure in the marine alga, Fucus vesiculosus.

    PubMed

    Owen, Jennifer R; Morris, Ceri A; Nicolaus, Beate; Harwood, John L; Kille, Peter

    2012-01-01

    The macro-alga Fucus vesiculosus has a broad global and estuarine distribution and exhibits exceptional resistance to toxic metals, the molecular basis of which is poorly understood. To address this issue a cDNA library was constructed from an environmental isolate of F. vesiculosus growing in an area with chronic copper pollution. Characterisation of this library led to the identification of a cDNA encoding a protein known to be synthesised in response to toxicity, a full length 14-3-3 exhibiting a 71% identity to human/mouse epsilon isoform, 70-71% identity to yeast BMH1/2 and 95 and 71% identity to the Ectocarpus siliculosus 14-3-3 isoforms 1 and 2 respectively. Preliminary characterisation of the expression profile of the 14-3-3 indicated concentration- and time-dependent inductions on acute exposure of F. vesiculosus of copper (3-30 μg/l). Higher concentrations of copper (≥150 μg/l) did not elicit significant induction of the 14-3-3 gene compared with the control even though levels of both intracellular copper and the expression of a cytosolic metal chaperone, metallothionein, continued to rise. Analysis of gene expression within environmental isolates demonstrated up-regulation of the 14-3-3 gene associated with the known copper pollution gradient. Here we report for the first time, identification of a gene encoding a putative 14-3-3 protein in a multicellular alga and provide preliminary evidence to link the induction of this 14-3-3 gene to copper exposure in this alga. Interestingly, the threshold exposure profile may be associated with a decrease in the organism's ability to control copper influx so that it perceives copper as a toxic response.

  7. Loss of ypk1 function causes rapamycin sensitivity, inhibition of translation initiation and synthetic lethality in 14-3-3-deficient yeast.

    PubMed Central

    Gelperin, Daniel; Horton, Lynn; DeChant, Anne; Hensold, Jack; Lemmon, Sandra K

    2002-01-01

    14-3-3 proteins bind to phosphorylated proteins and regulate a variety of cellular activities as effectors of serine/threonine phosphorylation. To define processes requiring 14-3-3 function in yeast, mutants with increased sensitivity to reduced 14-3-3 protein levels were identified by synthetic lethal screening. One mutation was found to be allelic to YPK1, which encodes a Ser/Thr protein kinase. Loss of Ypk function causes hypersensitivity to rapamycin, similar to 14-3-3 mutations and other mutations affecting the TOR signaling pathway in yeast. Similar to treatment with rapamycin, loss of Ypk function disrupted translation, at least in part by causing depletion of eIF4G, a central adaptor protein required for cap-dependent mRNA translation initiation. In addition, Ypk1 as well as eIF4G protein levels were rapidly depleted upon nitrogen starvation, but not during glucose starvation, even though both conditions inhibit translation initiation. These results suggest that Ypk regulates translation initiation in response to nutrient signals, either through the TOR pathway or in a functionally related pathway parallel to TOR. PMID:12196392

  8. Chemical Genetics of 14-3-3 Regulation and Role in Tumor Development

    DTIC Science & Technology

    2005-11-01

    from proteolysis (e.g. plant nitrate reductase) or from dephosphorylation (e.g. Raf and BAD). However, to date the most common mode of 14-3-3 function is...the general CRMI-mediated nuclear export I further studied the effects of haloprogin. This compound is a topical antifungal agent whose target is

  9. 14-3-3ζ and aPKC-ι synergistically facilitate epithelial-mesenchymal transition of cholangiocarcinoma via GSK-3β/snail signaling pathway

    PubMed Central

    He, Jun-chuang; Wang, Jian-ming; Schemmer, Peter; Ma, Chao-qun; Qian, Ya-wei; Yao, Wei; Zhang, Jian; Qi, Wei-peng; Fu, Yang; Feng, Wei; Yang, Tao

    2016-01-01

    Cholangiocarcinoma (CCA) invasion and metastasis are the primary causes of poor survival rates in patients. The epithelial-mesenchymal transition (EMT) is a crucial step in cancer invasion and metastasis. However, it is still unclear of the molecular mechanism. In this study, the expression of 14-3-3ζ and atypical protein kinase C-ι (aPKC-ι) was further detected in CCA tissues and cell lines. Meanwhile, we established the EMT model of CCA cells and investigated 14-3-3ζ and aPKC-ι co-regulatory effect on the EMT in vitro and in vivo. Further, we identified the downstream molecular glycogen synthase kinase 3 beta (GSK-3β)/Snail signalling pathway that contribute to regulating the EMT. Our data showed that the expression of 14-3-3ζ and aPKC-ι was synergistically increased in CCA tissues compared with adjacent noncancerous tissues and was intimately associated with differentiation and the tumour-node-metastasis (TNM) stage. Multivariate Cox regression analysis indicated that high 14-3-3ζ and aPKC-ι expression separately predicted a poor prognosis and were independent prognostic indicators in patients with CCA. The CO-IP experiment confirmed that the mutual binding relationship between 14-3-3ζ and aPKC-ι. Small interfering RNAs and siRNA rescue experiment demonstrated that 14-3-3ζ and aPKC-ι regulated each other. In addition, 14-3-3ζ and aPKC-ι pretreatment by si-RNA inhibit the phosphorylated GSK-3β and Snail expression during EMT. Meanwhile, silence of 14-3-3ζ or aPKC-ι suppressed CCA cells migration, metastasis and proliferation in vitro and in vivo. Our study demonstrates that 14-3-3ζ and aPKC-ι synergistically facilitate EMT of CCA via GSK-3β/Snail signalling pathway, and may be potential therapeutic target for CCA. PMID:27409422

  10. Chemical Genetics of 14-3-3 Regulation and Role in Tumor Development

    DTIC Science & Technology

    2006-11-01

    antifungal chemical typically used in the treatment of fungal (Tinea) skin such as athlete’s foot, jock itch, ringworm and tinea versicolor...DeCaprio JA: Cytoplasmic localization of human cdc25C during interphase requires an intact 14-3-3 binding site. Mol Cell Biol 1999, 19(6):4465- 4479. 12...epidermal growth factor receptor signaling pathways of the human prostate epithelial cell line M12. Oncogene 2004, 23(41):6881-6889. 15. Lopez-Girona

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

    SciTech Connect

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

    2015-07-10

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

  12. Internal Amino Acids Promote Gap1 Permease Ubiquitylation via TORC1/Npr1/14-3-3-Dependent Control of the Bul Arrestin-Like Adaptors

    PubMed Central

    Merhi, Ahmad

    2012-01-01

    Ubiquitylation of many plasma membrane proteins promotes their endocytosis followed by degradation in the lysosome. The yeast general amino acid permease, Gap1, is ubiquitylated and downregulated when a good nitrogen source like ammonium is provided to cells growing on a poor nitrogen source. This ubiquitylation requires the Rsp5 ubiquitin ligase and the redundant arrestin-like Bul1 and Bul2 adaptors. Previous studies have shown that Gap1 ubiquitylation involves the TORC1 kinase complex, which inhibits the Sit4 phosphatase. This causes inactivation of the protein kinase Npr1, which protects Gap1 against ubiquitylation. However, the mechanisms inducing Gap1 ubiquitylation after Npr1 inactivation remain unknown. We here show that on a poor nitrogen source, the Bul adaptors are phosphorylated in an Npr1-dependent manner and bound to 14-3-3 proteins that protect Gap1 against downregulation. After ammonium is added and converted to amino acids, the Bul proteins are dephosphorylated, dissociate from the 14-3-3 proteins, and undergo ubiquitylation. Furthermore, dephosphorylation of Bul requires the Sit4 phosphatase, which is essential to Gap1 downregulation. The data support the emerging concept that permease ubiquitylation results from activation of the arrestin-like adaptors of the Rsp5 ubiquitin ligase, this coinciding with their dephosphorylation, dissociation from the inhibitory 14-3-3 proteins, and ubiquitylation. PMID:22966204

  13. The Pfam protein families database.

    PubMed

    Finn, Robert D; Tate, John; Mistry, Jaina; Coggill, Penny C; Sammut, Stephen John; Hotz, Hans-Rudolf; Ceric, Goran; Forslund, Kristoffer; Eddy, Sean R; Sonnhammer, Erik L L; Bateman, Alex

    2008-01-01

    Pfam is a comprehensive collection of protein domains and families, represented as multiple sequence alignments and as profile hidden Markov models. The current release of Pfam (22.0) contains 9318 protein families. Pfam is now based not only on the UniProtKB sequence database, but also on NCBI GenPept and on sequences from selected metagenomics projects. Pfam is available on the web from the consortium members using a new, consistent and improved website design in the UK (http://pfam.sanger.ac.uk/), the USA (http://pfam.janelia.org/) and Sweden (http://pfam.sbc.su.se/), as well as from mirror sites in France (http://pfam.jouy.inra.fr/) and South Korea (http://pfam.ccbb.re.kr/).

  14. Costunolide and dehydrocostuslactone combination treatment inhibit breast cancer by inducing cell cycle arrest and apoptosis through c-Myc/p53 and AKT/14-3-3 pathway.

    PubMed

    Peng, Zhangxiao; Wang, Yan; Fan, Jianhui; Lin, Xuejing; Liu, Chunying; Xu, Yang; Ji, Weidan; Yan, Chao; Su, Changqing

    2017-01-24

    Our previous studies demonstrated that volatile oil from saussurea lappa root (VOSL), rich in two natural sesquiterpene lactones, costunolide (Cos) and dehydrocostuslactone (Dehy), exerts better anti-breast cancer efficacy and lower side effects than Cos or Dehy alone in vivo, however, their anti-cancer molecular mechanisms were still unknown. In this study, we investigated the underlying mechanisms of Cos and Dehy combination treatment (CD) on breast cancer cells through proteomics technology coupled with Western blot validation. Ingenuity Pathways Analysis (IPA) results based on the differentially expressed proteins revealed that both VOSL and CD affect the 14-3-3-mediated signaling, c-Myc mediated apoptosis signaling and protein kinase A (PKA) signaling. Western blot coupled with cell cycle and apoptosis analysis validated the results of proteomics analysis. Cell cycle arrest and apoptosis were induced in a dose-dependent manner, and the expressions of p53 and p-14-3-3 were significantly up-regulated, whereas the expressions of c-Myc, p-AKT, p-BID were significantly down-regulated, furthermore, the ratio of BAX/BCL-2 were significantly increased in breast cancer cells after CD and VOSL treatment. The findings indicated that VOSL and CD could induce breast cancer cell cycle arrest and apoptosis through c-Myc/p53 and AKT/14-3-3 signaling pathways and may be novel effective candidates for breast cancer treatment.

  15. Accumulation and tolerance to cadmium heavy metal ions and induction of 14-3-3 gene expression in response to cadmium exposure in Coprinus atramentarius.

    PubMed

    Xie, Chengjian; Hu, Liujie; Yang, Yongzhu; Liao, Dunxiu; Yang, Xingyong

    2017-03-01

    Cadmium (Cd), one of the most toxic heavy-metal pollutants, has a strong and irreversible tendency to accumulate. Bioremediation is a promising technology to remedy and control heavy metal pollutants because of its low cost and ability to recycle heavy metals. Coprinus atramentarius is recognized as being able to accumulate heavy metal ions. In this work, C. atramentarius is cultivated on a solid medium containing Cd(2+) ions to analyze its ability to tolerate different concentrations of the heavy metal ion. It is found that the growth of C. atramentarius is not significantly inhibited when the concentration of Cd(2+) is less than 0.6mgL(-1). The accumulation capacity of C. atramentarius at different Cd(2+) concentrations also was determined. The results show that 76% of the Cd(2+) present can be accumulated even when the concentration of the Cd(2+) is 1mgL(-1). The different proteins of C. atramentarius exposed to Cd(2+) were further analyzed using gel electrophoresis. A 14-3-3 protein was identified and shown to be significantly up-regulated. In a further study, a full-length 14-3-3 gene was cloned containing a 759bp open reading frame encoding a polypeptide consisting of 252 amino acids and 3 introns. The gene expression work also showed that the 14-3-3 was significantly induced, and showed coordinated patterns of expression, with Cd(2+) exposure.

  16. Costunolide and dehydrocostuslactone combination treatment inhibit breast cancer by inducing cell cycle arrest and apoptosis through c-Myc/p53 and AKT/14-3-3 pathway

    PubMed Central

    Peng, Zhangxiao; Wang, Yan; Fan, Jianhui; Lin, Xuejing; Liu, Chunying; Xu, Yang; Ji, Weidan; Yan, Chao; Su, Changqing

    2017-01-01

    Our previous studies demonstrated that volatile oil from saussurea lappa root (VOSL), rich in two natural sesquiterpene lactones, costunolide (Cos) and dehydrocostuslactone (Dehy), exerts better anti-breast cancer efficacy and lower side effects than Cos or Dehy alone in vivo, however, their anti-cancer molecular mechanisms were still unknown. In this study, we investigated the underlying mechanisms of Cos and Dehy combination treatment (CD) on breast cancer cells through proteomics technology coupled with Western blot validation. Ingenuity Pathways Analysis (IPA) results based on the differentially expressed proteins revealed that both VOSL and CD affect the 14-3-3-mediated signaling, c-Myc mediated apoptosis signaling and protein kinase A (PKA) signaling. Western blot coupled with cell cycle and apoptosis analysis validated the results of proteomics analysis. Cell cycle arrest and apoptosis were induced in a dose-dependent manner, and the expressions of p53 and p-14-3-3 were significantly up-regulated, whereas the expressions of c-Myc, p-AKT, p-BID were significantly down-regulated, furthermore, the ratio of BAX/BCL-2 were significantly increased in breast cancer cells after CD and VOSL treatment. The findings indicated that VOSL and CD could induce breast cancer cell cycle arrest and apoptosis through c-Myc/p53 and AKT/14-3-3 signaling pathways and may be novel effective candidates for breast cancer treatment. PMID:28117370

  17. In-vivo administration of clozapine affects behaviour but does not reverse dendritic spine deficits in the 14-3-3ζ KO mouse model of schizophrenia-like disorders.

    PubMed

    Jaehne, Emily J; Ramshaw, Hayley; Xu, Xiangjun; Saleh, Eiman; Clark, Scott R; Schubert, Klaus Oliver; Lopez, Angel; Schwarz, Quenten; Baune, Bernhard T

    2015-11-01

    Clozapine is an atypical antipsychotic drug used in the treatment of schizophrenia, which has been shown to reverse behavioural and dendritic spine deficits in mice. It has recently been shown that deficiency of 14-3-3ζ has an association with schizophrenia, and that a mouse model lacking this protein displays several schizophrenia-like behavioural deficits. To test the effect of clozapine in this mouse model, 14-3-3ζ KO mice were administered clozapine (5mg/kg) for two weeks prior to being analysed in a test battery of cognition, anxiety, and despair (depression-like) behaviours. Following behavioural testing brain samples were collected for analysis of specific anatomical defects and dendritic spine formation. We found that clozapine reduced despair behaviour of 14-3-3ζ KO mice in the forced swim test (FST) and altered the behaviour of wild types and 14-3-3ζ KO mice in the Y-maze task. In contrast, clozapine had no effects on hippocampal laminar defects or decreased dendritic spine density observed in 14-3-3ζ KO mice. Our results suggest that clozapine may have beneficial effects on clinical behaviours associated with deficiencies in the 14-3-3ζ molecular pathway, despite having no effects on morphological defects. These findings may provide mechanistic insight to the action of this drug.

  18. BIS targeting induces cellular senescence through the regulation of 14-3-3 zeta/STAT3/SKP2/p27 in glioblastoma cells.

    PubMed

    Lee, J-J; Lee, J-S; Cui, M N; Yun, H H; Kim, H Y; Lee, S H; Lee, J-H

    2014-11-20

    Cellular senescence is an important mechanism for preventing tumor progression. The elevated expression of Bcl-2-interacting cell death suppressor (BIS), an anti-apoptotic and anti-stress protein, often correlates with poor prognosis in several cancers including glioblastoma; however, the role of BIS in the regulation of senescence has not been well defined. Here, we describe for the first time that the depletion of BIS induces G1 arrest and cellular senescence through the accumulation of p27 that is independent of p53, p21 or p16. The increase in p27 expression in BIS-depleted cells was attributable to an impairment of the ubiquitin-mediated degradation of p27, which was caused by a decrease in S-phase kinase-associated protein 2 (SKP2) at the transcriptional level. As an underlying molecular mechanism, we demonstrate that the loss of activity of signal transducer and activator of transcription 3 (STAT3) was specifically linked to the suppression of SKP2 expression. Despite a reduction in phospho-STAT3 levels, total STAT3 levels were unexpectedly increased by BIS depletion, specifically in the insoluble fraction. Our results show that 14-3-3ζ expression is decreased by BIS knockdown and that 14-3-3ζ depletion per se significantly induced senescence phenotypes. In addition, the ectopic expression of 14-3-3ζ blocked senescence caused by BIS depletion, which was paralleled with a decrease in insoluble STAT3 in A172 glioblastoma cells. These findings indicate that the impairment of the protein quality control conferred by BIS and/or 14-3-3ζ is critical for BIS depletion-induced senescence. Moreover, BIS knockdown also induced senescence along with an accumulation of total STAT3 and p27 in several different cell types as well as embryonic fibroblasts derived from Bis-knock out mice with/without variations in 14-3-3ζ levels. Therefore, our findings suggest that a downregulation of BIS expression could serve as a potential strategy for restricting tumor progression

  19. Supervised Protein Family Classification and New Family Construction

    PubMed Central

    Yi, Gangman; Thon, Michael R.

    2012-01-01

    Abstract The goal of protein family classification is to group proteins into families so that proteins within the same family have common function or are related by ancestry. While supervised classification algorithms are available for this purpose, most of these approaches focus on assigning unclassified proteins to known families but do not allow for progressive construction of new families from proteins that cannot be assigned. Although unsupervised clustering algorithms are also available, they do not make use of information from known families. By computing similarities between proteins based on pairwise sequence comparisons, we develop supervised classification algorithms that achieve improved accuracy over previous approaches while allowing for construction of new families. We show that our algorithm has higher accuracy rate and lower mis-classification rate when compared to algorithms that are based on the use of multiple sequence alignments and hidden Markov models, and our algorithm performs well even on families with very few proteins and on families with low sequence similarity. A software program implementing the algorithm (SClassify) is available online (http://faculty.cse.tamu.edu/shsze/sclassify). PMID:22876787

  20. Defective erythroid differentiation in miR-451 mutant mice mediated by 14-3-3ζ

    PubMed Central

    Patrick, David M.; Zhang, Cheng C.; Tao, Ye; Yao, Huiyu; Qi, Xiaoxia; Schwartz, Robert J.; Jun-Shen Huang, Lily; Olson, Eric N.

    2010-01-01

    Erythrocyte formation occurs throughout life in response to cytokine signaling. We show that microRNA-451 (miR-451) regulates erythropoiesis in vivo. Mice lacking miR-451 display a reduction in hematrocrit, an erythroid differentiation defect, and ineffective erythropoiesis in response to oxidative stress. 14-3-3ζ, an intracellular regulator of cytokine signaling that is repressed by miR-451, is up-regulated in miR-451−/− erythroblasts, and inhibition of 14-3-3ζ rescues their differentiation defect. These findings reveal an essential role of 14-3-3ζ as a mediator of the proerythroid differentiation actions of miR-451, and highlight the therapeutic potential of miR-451 inhibitors. PMID:20679397

  1. Metabolic-Stress-Induced Rearrangement of the 14-3-3ζ Interactome Promotes Autophagy via a ULK1- and AMPK-Regulated 14-3-3ζ Interaction with Phosphorylated Atg9

    PubMed Central

    Weerasekara, Vajira K.; Panek, David J.; Broadbent, David G.; Mortenson, Jeffrey B.; Mathis, Andrew D.; Logan, Gideon N.; Prince, John T.; Thomson, David M.; Thompson, J. Will

    2014-01-01

    14-3-3ζ promotes cell survival via dynamic interactions with a vast network of binding partners, many of which are involved in stress regulation. We show here that hypoxia (low glucose and oxygen) triggers a rearrangement of the 14-3-3ζ interactome to favor an interaction with the core autophagy regulator Atg9A. Our data suggest that the localization of mammalian Atg9A to autophagosomes requires phosphorylation on the C terminus of Atg9A at S761, which creates a 14-3-3ζ docking site. Under basal conditions, this phosphorylation is maintained at a low level and is dependent on both ULK1 and AMPK. However, upon induction of hypoxic stress, activated AMPK bypasses the requirement for ULK1 and mediates S761 phosphorylation directly, resulting in an increase in 14-3-3ζ interactions, recruitment of Atg9A to LC3-positive autophagosomes, and enhanced autophagosome production. These data suggest a novel mechanism whereby the level of autophagy induction can be modulated by AMPK/ULK1-mediated phosphorylation of mammalian Atg9A. PMID:25266655

  2. Protein family classification using sparse Markov transducers.

    PubMed

    Eskin, E; Grundy, W N; Singer, Y

    2000-01-01

    In this paper we present a method for classifying proteins into families using sparse Markov transducers (SMTs). Sparse Markov transducers, similar to probabilistic suffix trees, estimate a probability distribution conditioned on an input sequence. SMTs generalize probabilistic suffix trees by allowing for wild-cards in the conditioning sequences. Because substitutions of amino acids are common in protein families, incorporating wildcards into the model significantly improves classification performance. We present two models for building protein family classifiers using SMTs. We also present efficient data structures to improve the memory usage of the models. We evaluate SMTs by building protein family classifiers using the Pfam database and compare our results to previously published results.

  3. 14-3-3theta Protects against Neurotoxicity in a Cellular Parkinson's Disease Model through Inhibition of the Apoptotic Factor Bax

    PubMed Central

    Slone, Sunny R.; Lesort, Mathieu; Yacoubian, Talene A.

    2011-01-01

    Disruption of 14-3-3 function by alpha-synuclein has been implicated in Parkinson's disease. As 14-3-3s are important regulators of cell death pathways, disruption of 14-3-3s could result in the release of pro-apoptotic factors, such as Bax. We have previously shown that overexpression of 14-3-3θ reduces cell loss in response to rotenone and MPP+ in dopaminergic cell culture and reduces cell loss in transgenic C. elegans that overexpress alpha-synuclein. In this study, we investigate the mechanism for 14-3-3θ's neuroprotection against rotenone toxicity. While 14-3-3s can inhibit many pro-apoptotic factors, we demonstrate that inhibition of one factor in particular, Bax, is important to 14-3-3s' protection against rotenone toxicity in dopaminergic cells. We found that 14-3-3θ overexpression reduced Bax activation and downstream signaling events, including cytochrome C release and caspase 3 activation. Pharmacological inhibition or shRNA knockdown of Bax provided protection against rotenone, comparable to 14-3-3θ's neuroprotective effects. A 14-3-3θ mutant incapable of binding Bax failed to protect against rotenone. These data suggest that 14-3-3θ's neuroprotective effects against rotenone are at least partially mediated by Bax inhibition and point to a potential therapeutic role of 14-3-3s in Parkinson's disease. PMID:21799745

  4. Regulation of IRSp53-Dependent Filopodial Dynamics by Antagonism between 14-3-3 Binding and SH3-Mediated Localization ▿ †

    PubMed Central

    Robens , Jeffrey M.; Yeow-Fong, Lee; Ng, Elsa; Hall, Christine; Manser, Ed

    2010-01-01

    Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia. PMID:19933840

  5. Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14-3-3 isoforms to suppress effector-triggered immunity.

    PubMed

    Teper, Doron; Salomon, Dor; Sunitha, Sukumaran; Kim, Jung-Gun; Mudgett, Mary Beth; Sessa, Guido

    2014-01-01

    Effector-triggered immunity (ETI) to host-adapted pathogens is associated with rapid cell death at the infection site. The plant-pathogenic bacterium Xanthomonas euvesicatoria (Xcv) interferes with plant cellular processes by injecting effector proteins into host cells through the type III secretion system. Here, we show that the Xcv effector XopQ suppresses cell death induced by components of the ETI-associated MAP kinase cascade MAPKKKα MEK2/SIPK and by several R/avr gene pairs. Inactivation of xopQ by insertional mutagenesis revealed that this effector inhibits ETI-associated cell death induced by avirulent Xcv in resistant pepper (Capsicum annuum), and enhances bacterial growth in resistant pepper and tomato (Solanum lycopersicum). Using protein-protein interaction studies in yeast (Saccharomyces cerevisiae) and in planta, we identified the tomato 14-3-3 isoform SlTFT4 and homologs from other plant species as XopQ interactors. A mutation in the putative 14-3-3 binding site of XopQ impaired interaction of the effector with CaTFT4 in yeast and its virulence function in planta. Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv. Silencing of NbTFT4 in Nicotiana benthamiana significantly reduced cell death induced by MAPKKKα. In addition, silencing of CaTFT4 in pepper delayed the appearance of ETI-associated cell death and enhanced growth of virulent and avirulent Xcv, demonstrating the requirement of TFT4 for plant immunity to Xcv. Our results suggest that the XopQ virulence function is to suppress ETI and immunity-associated cell death by interacting with TFT4, which is an important component of ETI and a bona fide target of XopQ.

  6. Protein family classification using sparse markov transducers.

    PubMed

    Eskin, Eleazar; Noble, William Stafford; Singer, Yoram

    2003-01-01

    We present a method for classifying proteins into families based on short subsequences of amino acids using a new probabilistic model called sparse Markov transducers (SMT). We classify a protein by estimating probability distributions over subsequences of amino acids from the protein. Sparse Markov transducers, similar to probabilistic suffix trees, estimate a probability distribution conditioned on an input sequence. SMTs generalize probabilistic suffix trees by allowing for wild-cards in the conditioning sequences. Since substitutions of amino acids are common in protein families, incorporating wild-cards into the model significantly improves classification performance. We present two models for building protein family classifiers using SMTs. As protein databases become larger, data driven learning algorithms for probabilistic models such as SMTs will require vast amounts of memory. We therefore describe and use efficient data structures to improve the memory usage of SMTs. We evaluate SMTs by building protein family classifiers using the Pfam and SCOP databases and compare our results to previously published results and state-of-the-art protein homology detection methods. SMTs outperform previous probabilistic suffix tree methods and under certain conditions perform comparably to state-of-the-art protein homology methods.

  7. Protein function annotation using protein domain family resources.

    PubMed

    Das, Sayoni; Orengo, Christine A

    2016-01-15

    As a result of the genome sequencing and structural genomics initiatives, we have a wealth of protein sequence and structural data. However, only about 1% of these proteins have experimental functional annotations. As a result, computational approaches that can predict protein functions are essential in bridging this widening annotation gap. This article reviews the current approaches of protein function prediction using structure and sequence based classification of protein domain family resources with a special focus on functional families in the CATH-Gene3D resource.

  8. The lipocalin protein family: structure and function.

    PubMed Central

    Flower, D R

    1996-01-01

    The lipocalin protein family is a large group of small extracellular proteins. The family demonstrates great diversity at the sequence level; however, most lipocalins share three characteristic conserved sequence motifs, the kernel lipocalins, while a group of more divergent family members, the outlier lipocalins, share only one. Belying this sequence dissimilarity, lipocalin crystal structures are highly conserved and comprise a single eight-stranded continuously hydrogen-bonded antiparallel beta-barrel, which encloses an internal ligand-binding site. Together with two other families of ligand-binding proteins, the fatty-acid-binding proteins (FABPs) and the avidins, the lipocalins form part of an overall structural superfamily: the calycins. Members of the lipocalin family are characterized by several common molecular-recognition properties: the ability to bind a range of small hydrophobic molecules, binding to specific cell-surface receptors and the formation of complexes with soluble macromolecules. The varied biological functions of the lipocalins are mediated by one or more of these properties. In the past, the lipocalins have been classified as transport proteins; however, it is now clear that the lipocalins exhibit great functional diversity, with roles in retinol transport, invertebrate cryptic coloration, olfaction and pheromone transport, and prostaglandin synthesis. The lipocalins have also been implicated in the regulation of cell homoeostasis and the modulation of the immune response, and, as carrier proteins, to act in the general clearance of endogenous and exogenous compounds. PMID:8761444

  9. Characteristic motifs for families of allergenic proteins

    PubMed Central

    Ivanciuc, Ovidiu; Garcia, Tzintzuni; Torres, Miguel; Schein, Catherine H.; Braun, Werner

    2008-01-01

    The identification of potential allergenic proteins is usually done by scanning a database of allergenic proteins and locating known allergens with a high sequence similarity. However, there is no universally accepted cut-off value for sequence similarity to indicate potential IgE cross-reactivity. Further, overall sequence similarity may be less important than discrete areas of similarity in proteins with homologous structure. To identify such areas, we first classified all allergens and their subdomains in the Structural Database of Allergenic Proteins (SDAP, http://fermi.utmb.edu/SDAP/) to their closest protein families as defined in Pfam, and identified conserved physicochemical property motifs characteristic of each group of sequences. Allergens populate only a small subset of all known Pfam families, as all allergenic proteins in SDAP could be grouped to only 130 (of 9318 total) Pfams, and 31 families contain more than four allergens. Conserved physicochemical property motifs for the aligned sequences of the most populated Pfam families were identified with the PCPMer program suite and catalogued in the webserver Motif-Mate (http://born.utmb.edu/motifmate/summary.php). We also determined specific motifs for allergenic members of a family that could distinguish them from non-allergenic ones. These allergen specific motifs should be most useful in database searches for potential allergens. We found that sequence motifs unique to the allergens in three families (seed storage proteins, Bet v 1, and tropomyosin) overlap with known IgE epitopes, thus providing evidence that our motif based approach can be used to assess the potential allergenicity of novel proteins. PMID:18951633

  10. Protein function prediction using domain families

    PubMed Central

    2013-01-01

    Here we assessed the use of domain families for predicting the functions of whole proteins. These 'functional families' (FunFams) were derived using a protocol that combines sequence clustering with supervised cluster evaluation, relying on available high-quality Gene Ontology (GO) annotation data in the latter step. In essence, the protocol groups domain sequences belonging to the same superfamily into families based on the GO annotations of their parent proteins. An initial test based on enzyme sequences confirmed that the FunFams resemble enzyme (domain) families much better than do families produced by sequence clustering alone. For the CAFA 2011 experiment, we further associated the FunFams with GO terms probabilistically. All target proteins were first submitted to domain superfamily assignment, followed by FunFam assignment and, eventually, function assignment. The latter included an integration step for multi-domain target proteins. The CAFA results put our domain-based approach among the top ten of 31 competing groups and 56 prediction methods, confirming that it outperforms simple pairwise whole-protein sequence comparisons. PMID:23514456

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

    PubMed Central

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

    2014-01-01

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

  12. Differential 14-3-3 sigma DNA methylation and expression in c-myc- and activated H-ras-transformed cells under r- and K-selection.

    PubMed

    Sato, Hiroyuki; Nakamura, Yukari; Motokura, Toru

    2006-05-08

    We cloned rat 14-3-3 sigma, a mediator of p53 tumor suppressor, as a target of K-selection. 14-3-3 sigma expression is suppressed with DNA methylation in breast cancers while its overexpression with hypomethylation is frequent in pancreatic cancers. These opposite findings were recapitulated through r- and K-selection of transformed rat embryo fibroblasts. 14-3-3 sigma expression was suppressed with DNA methylation after r-selection and the gene was overexpressed and demethylated in K-selected cells. 5-aza-2'-deoxycytidine recovered 14-3-3 sigma expression in r-selected cells. The presence of heterogeneous methylation patterns and expression levels before selection suggests that different 14-3-3 sigma expression levels play a role as a prerequisite for selection and clonal evolution.

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

    PubMed

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

    2015-12-21

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

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

    PubMed

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

    2015-07-24

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

  15. FIGfams : yet another set of protein families.

    SciTech Connect

    Meyer, F.; Overbeek, R.; Rodriguez, A.; Mathematics and Computer Science; Univ. of Chicago; Fellowship for the Interpretation of Genomes

    2009-11-01

    We present FIGfams, a new collection of over 100,000 protein families that are the product of manual curation and close strain comparison. Using the Subsystem approach the manual curation is carried out, ensuring a previously unattained degree of throughput and consistency. FIGfams are based on over 950,000 manually annotated proteins and across many hundred Bacteria and Archaea. Associated with each FIGfam is a two-tiered, rapid, accurate decision procedure to determine family membership for new proteins. FIGfams are freely available under an open source license. These can be downloaded at ftp://ftp.theseed.org/FIGfams/. The web site for FIGfams is http://www.theseed.org/wiki/FIGfams/.

  16. Fos family protein degradation by the proteasome.

    PubMed

    Gomard, Tiphanie; Jariel-Encontre, Isabelle; Basbous, Jihane; Bossis, Guillaume; Moquet-Torcy, Gabriel; Mocquet-Torcy, Gabriel; Piechaczyk, Marc

    2008-10-01

    c-Fos proto-oncoprotein defines a family of closely related transcription factors (Fos proteins) also comprising Fra-1, Fra-2, FosB and DeltaFosB, the latter two proteins being generated by alternative splicing. Through the regulation of many genes, most of them still unidentified, they regulate major functions from the cell level up to the whole organism. Thus they are involved in the control of proliferation, differentiation and apoptosis, as well as in the control of responses to stresses, and they play important roles in organogenesis, immune responses and control of cognitive functions, among others. Fos proteins are intrinsically unstable. We have studied how two of them, c-Fos and Fra-1, are degraded. Departing from the classical scenario where unstable key cell regulators are hydrolysed by the proteasome after polyubiquitination, we showed that the bulk of c-Fos and Fra-1 can be broken down independently of any prior ubiquitination. Certain conserved structural domains suggest that similar mechanisms may also apply to Fra-2 and FosB. Computer search indicates that certain motifs shared by the Fos proteins and putatively responsible for instability are found in no other protein, suggesting the existence of degradation mechanisms specific for this protein family. Under particular signalling conditions, others have shown that a part of cytoplasmic c-Fos requires ubiquitination for fast turnover. This poses the question of the multiplicity of degradation pathways that apply to proteins depending on their intracellular localization.

  17. Novel protein families in archaean genomes.

    PubMed Central

    Ouzonis, C; Kyrpides, N; Sander, C

    1995-01-01

    In a quest for novel functions in archaea, all archaean hypothetical open reading frames (ORFs), as annotated in the Swiss-Prot protein sequence database, were used to search the latest databases for the identification of characterized homologues. Of the 95 hypothetical archaean ORFs, 25 were found to be homologous to another hypothetical archaean ORF, while 36 were homologous to non-archaean proteins, of which as many as 30 were homologous to a characterized protein family. Thus the level of sequence similarity in this set reaches 64%, while the level of function assignment is only 32%. Of the ORFs with predicted functions, 12 homologies are reported here for the first time and represent nine new functions and one gene duplication at an acetyl-coA synthetase locus. The novel functions include components of the transcriptional and translational apparatus, such as ribosomal proteins, modification enzymes and a translation initiation factor. In addition, new enzymes are identified in archaea, such as cobyric acid synthase, dCTP deaminase and the first archaean homologues of a new subclass of ATP binding proteins found in fungi. Finally, it is shown that the putative laminin receptor family of eukaryotes and an archaean homologue belong to the previously characterized ribosomal protein family S2 from eubacteria. From the present and previous work, the major implication is that archaea seem to have a mode of expression of genetic information rather similar to eukaryotes, while eubacteria may have proceeded into unique ways of transcription and translation. In addition, with the detection of proteins in various metabolic and genetic processes in archaea, we can further predict the presence of additional proteins involved in these processes. PMID:7899076

  18. PI 3-kinase-dependent phosphorylation of Plk1–Ser99 promotes association with 14-3-3γ and is required for metaphase–anaphase transition

    PubMed Central

    Kasahara, Kousuke; Goto, Hidemasa; Izawa, Ichiro; Kiyono, Tohru; Watanabe, Nobumoto; Elowe, Sabine; Nigg, Erich A; Inagaki, Masaki

    2013-01-01

    Polo-like kinase 1 (Plk1) controls multiple aspects of mitosis and is activated through its phosphorylation at Thr210. Here we identify Ser99 on Plk1 as a novel mitosis-specific phosphorylation site, which operates independently of Plk1–Thr210 phosphorylation. Plk1–Ser99 phosphorylation creates a docking site for 14-3-3γ, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3γ or replacement of wild-type (WT) Plk1 by a Ser99-phospho-blocking mutant leads to a prometaphase/metaphase-like arrest due to the activation of the spindle assembly checkpoint. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1–Ser99 phosphorylation and delays metaphase to anaphase transition. Plk1–Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner. Therefore, mitotic Plk1 activity is regulated not only by Plk1–Thr210 phosphorylation, but also by Plk1 binding to 14-3-3γ following Plk1–Ser99 phosphorylation downstream of the PI3K–Akt signalling pathway. This novel Plk1 activation pathway controls proper progression from metaphase to anaphase. PMID:23695676

  19. TIGRFAMS: The TIGRFAMs database of protein families

    DOE Data Explorer

    TIGRFAMs are protein families based on Hidden Markov Models or HMMs. Use this page to see the curated seed alignmet for each TIGRFam, the full alignment of all family members and the cutoff scores for inclusion in each of the TIGRFAMs. Also use this page to search through the TIGRFAMs and HMMs for text in the TIGRFAMs Text Search or search for specific sequences in the TIGRFAMs Sequence Search.[Copied from the Overview at http://www.jcvi.org/cms/research/projects/tigrfams/overview/] See also TIGRFAMs ordered by the roles they play at http://cmr.jcvi.org/tigr-scripts/CMR/shared/EvidenceList.cgi?ev_type=TIGRFAM&order_type=role.

  20. On the Entropy of Protein Families

    NASA Astrophysics Data System (ADS)

    Barton, John P.; Chakraborty, Arup K.; Cocco, Simona; Jacquin, Hugo; Monasson, Rémi

    2016-03-01

    Proteins are essential components of living systems, capable of performing a huge variety of tasks at the molecular level, such as recognition, signalling, copy, transport, ... The protein sequences realizing a given function may largely vary across organisms, giving rise to a protein family. Here, we estimate the entropy of those families based on different approaches, including Hidden Markov Models used for protein databases and inferred statistical models reproducing the low-order (1- and 2-point) statistics of multi-sequence alignments. We also compute the entropic cost, that is, the loss in entropy resulting from a constraint acting on the protein, such as the mutation of one particular amino-acid on a specific site, and relate this notion to the escape probability of the HIV virus. The case of lattice proteins, for which the entropy can be computed exactly, allows us to provide another illustration of the concept of cost, due to the competition of different folds. The relevance of the entropy in relation to directed evolution experiments is stressed.

  1. Physiological functions of MTA family of proteins.

    PubMed

    Sen, Nirmalya; Gui, Bin; Kumar, Rakesh

    2014-12-01

    Although the functional significance of the metastasic tumor antigen (MTA) family of chromatin remodeling proteins in the pathobiology of cancer is fairly well recognized, the physiological role of MTA proteins continues to be an understudied research area and is just beginning to be recognized. Similar to cancer cells, MTA1 also modulates the expression of target genes in normal cells either by acting as a corepressor or coactivator. In addition, physiological functions of MTA proteins are likely to be influenced by its differential expression, subcellular localization, and regulation by upstream modulators and extracellular signals. This review summarizes our current understanding of the physiological functions of the MTA proteins in model systems. In particular, we highlight recent advances of the role MTA proteins play in the brain, eye, circadian rhythm, mammary gland biology, spermatogenesis, liver, immunomodulation and inflammation, cellular radio-sensitivity, and hematopoiesis and differentiation. Based on the growth of knowledge regarding the exciting new facets of the MTA family of proteins in biology and medicine, we speculate that the next burst of findings in this field may reveal further molecular regulatory insights of non-redundant functions of MTA coregulators in the normal physiology as well as in pathological conditions outside cancer.

  2. ErbB2, FoxM1 and 14-3-3ζ prime breast cancer cells for invasion in response to ionizing radiation.

    PubMed

    Kambach, D M; Sodi, V L; Lelkes, P I; Azizkhan-Clifford, J; Reginato, M J

    2014-01-30

    ErbB2 is frequently highly expressed in premalignant breast cancers, including ductal carcinoma in situ (DCIS); however, little is known about the signals or pathways it contributes to progression into the invasive/malignant state. Radiotherapy is often used to treat early premalignant lesions regardless of ErbB2 status. Here, we show that clinically relevant doses of ionizing radiation (IR)-induce cellular invasion of ErbB2-expressing breast cancer cells, as well as MCF10A cells overexpressing ErbB2. ErbB2-negative breast cancer cells, such as MCF7 and T47D, do not invade following treatment with IR nor do MCF10A cells overexpressing epidermal growth factor receptor. ErbB2 becomes phosphorylated at tyrosine 877 in a dose- and time- dependent manner following exposure to X-rays, and activates downstream signaling cascades including PI3K/Akt. Inhibition of these pathways, as well as inhibition of reactive oxygen species (ROS) with antioxidants, prevents IR-induced invasion. Activation of ErbB2-dependent signaling results in upregulation of the forkhead family transcription factor, FoxM1, and its transcriptional targets, including matrix metalloproteinase 2 (MMP2). Inhibition of FoxM1 by RNA interference prevented induction of invasion by IR, and overexpression of FoxM1 in MCF10A cells was sufficient to promote IR-induced invasion. Moreover, we found that 14-3-3ζ is also upregulated by IR in cancer cells in a ROS-dependent manner, is required for IR-induced invasion in ErbB2-positive breast cancer cells and together with FoxM1 is sufficient for invasion in ErbB2-negative breast cancer cells. Thus, our data show that IR-mediated activation of ErbB2 and induction of 14-3-3ζ collaborate to regulate FoxM1 and promote invasion of breast cancer cells and furthermore, may serve as therapeutic targets to enhance radiosensitivity of breast cancers.

  3. SUMOylation of Myc-Family Proteins

    PubMed Central

    Sabò, Arianna; Doni, Mirko; Amati, Bruno

    2014-01-01

    Myc-family proteins are key controllers of the metabolic and proliferative status of the cell, and are subjected to a complex network of regulatory events that guarantee their efficient and fast modulation by extracellular stimuli. Hence, unbalances in regulatory mechanisms leading to altered Myc levels or activities are often reported in cancer cells. Here we show that c- and N-Myc are conjugated to SUMO proteins at conserved lysines in their C-terminal domain. No obvious effects of SUMOylation were detected on bulk N-Myc stability or activities, including the regulation of transcription, proliferation or apoptosis. N-Myc SUMOylation could be induced by cellular stresses, such as heat shock and proteasome inhibition, and in all instances concerned a small fraction of the N-Myc protein. We surmise that, as shown for other substrates, SUMOylation may be part of a quality-control mechanism acting on misfolded Myc proteins. PMID:24608896

  4. Inhibition of Specific NF-κB Activity Contributes to the Tumor Suppressor Function of 14-3-3σ in Breast Cancer

    PubMed Central

    Inglés-Esteve, Julia; Morales, Mònica; Dalmases, Alba; Garcia-Carbonell, Ricard; Jené-Sanz, Alba; López-Bigas, Núria; Iglesias, Mar; Ruiz-Herguido, Cristina; Rovira, Ana; Rojo, Federico; Albanell, Joan; Gomis, Roger R.

    2012-01-01

    14-3-3σ is frequently lost in human breast cancers by genetic deletion or promoter methylation. We have now investigated the involvement of 14-3-3σ in the termination of NF-κB signal in mammary cells and its putative role in cancer relapse and metastasis. Our results show that 14-3-3σ regulates nuclear export of p65-NF-κB following chronic TNFα stimulation. Restoration of 14-3-3σ in breast cancer cells reduces migration capacity and metastatic abilities in vivo. By microarray analysis, we have identified a genetic signature that responds to TNFα in a 14-3-3σ-dependent manner and significantly associates with different breast and other types of cancer. By interrogating public databases, we have found that over-expression of this signature correlates with poor relapse-free survival in breast cancer patients. Finally, screening of 96 human breast tumors showed that NF-κB activation strictly correlates with the absence of 14-3-3σ and it is significantly associated with worse prognosis in the multivariate analysis. Our findings identify a genetic signature that is important for breast cancer prognosis and for future personalized treatments based on NF-κB targeting. PMID:22675457

  5. High frequency of hypermethylation at the 14-3-3 σ locus leads to gene silencing in breast cancer

    PubMed Central

    Ferguson, Anne T.; Evron, Ella; Umbricht, Christopher B.; Pandita, Tej K.; Chan, Timothy A.; Hermeking, Heiko; Marks, Jeffrey R.; Lambers, Anouk R.; Futreal, P. Andrew; Stampfer, Martha R.; Sukumar, Saraswati

    2000-01-01

    Expression of 14-3-3 σ (σ) is induced in response to DNA damage, and causes cells to arrest in G2. By SAGE (serial analysis of gene expression) analysis, we identified σ as a gene whose expression is 7-fold lower in breast carcinoma cells than in normal breast epithelium. We verified this finding by Northern blot analysis. Remarkably, σ mRNA was undetectable in 45 of 48 primary breast carcinomas. Genetic alterations at σ such as loss of heterozygosity were rare (1/20 informative cases), and no mutations were detected (0/34). On the other hand, hypermethylation of CpG islands in the σ gene was detected in 91% (75/82) of breast tumors and was associated with lack of gene expression. Hypermethylation of σ is functionally important, because treatment of σ-non-expressing breast cancer cell lines with the drug 5-aza-2′-deoxycytidine resulted in demethylation of the gene and synthesis of σ mRNA. Breast cancer cells lacking σ expression showed increased number of chromosomal breaks and gaps when exposed to γ-irradiation. Therefore, it is possible that loss of σ expression contributes to malignant transformation by impairing the G2 cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Hypermethylation and loss of σ expression are the most consistent molecular alterations in breast cancer identified so far. PMID:10811911

  6. The family of LSU-like proteins

    PubMed Central

    Sirko, Agnieszka; Wawrzyńska, Anna; Rodríguez, Milagros Collados; Sęktas, Pawel

    2015-01-01

    The plant response to sulfur deficiency includes extensive metabolic changes which can be monitored at various levels (transcriptome, proteome, metabolome) even before the first visible symptoms of sulfur starvation appear. Four members of the plant-specific LSU (response to Low SUlfur) gene family occur in Arabidopsis thaliana (LSU1-4). Variable numbers of LSU genes occur in other plant species but they were studied only in Arabidopsis and tobacco. Three out of four of the Arabidopsis LSU genes are induced by sulfur deficiency. The LSU-like genes in tobacco were characterized as UP9 (UPregulated by sulfur deficit 9). LSU-like proteins do not have characteristic domains that provide clues to their function. Despite having only moderate primary sequence conservation they share several common features including small size, a coiled–coil secondary structure and short conserved motifs in specific positions. Although the precise function of LSU-like proteins is still unknown there is some evidence that members of the LSU family are involved in plant responses to environmental challenges, such as sulfur deficiency, and possibly in plant immune responses. Various bioinformatic approaches have identified LSU-like proteins as important hubs for integration of signals from environmental stimuli. In this paper we review a variety of published data on LSU gene expression, the properties of lsu mutants and features of LSU-like proteins in the hope of shedding some light on their possible role in plant metabolism. PMID:25628631

  7. The family of LSU-like proteins.

    PubMed

    Sirko, Agnieszka; Wawrzyńska, Anna; Rodríguez, Milagros Collados; Sęktas, Pawel

    2014-01-01

    The plant response to sulfur deficiency includes extensive metabolic changes which can be monitored at various levels (transcriptome, proteome, metabolome) even before the first visible symptoms of sulfur starvation appear. Four members of the plant-specific LSU (response to Low SUlfur) gene family occur in Arabidopsis thaliana (LSU1-4). Variable numbers of LSU genes occur in other plant species but they were studied only in Arabidopsis and tobacco. Three out of four of the Arabidopsis LSU genes are induced by sulfur deficiency. The LSU-like genes in tobacco were characterized as UP9 (UPregulated by sulfur deficit 9). LSU-like proteins do not have characteristic domains that provide clues to their function. Despite having only moderate primary sequence conservation they share several common features including small size, a coiled-coil secondary structure and short conserved motifs in specific positions. Although the precise function of LSU-like proteins is still unknown there is some evidence that members of the LSU family are involved in plant responses to environmental challenges, such as sulfur deficiency, and possibly in plant immune responses. Various bioinformatic approaches have identified LSU-like proteins as important hubs for integration of signals from environmental stimuli. In this paper we review a variety of published data on LSU gene expression, the properties of lsu mutants and features of LSU-like proteins in the hope of shedding some light on their possible role in plant metabolism.

  8. Targeting functional motifs of a protein family

    NASA Astrophysics Data System (ADS)

    Bhadola, Pradeep; Deo, Nivedita

    2016-10-01

    The structural organization of a protein family is investigated by devising a method based on the random matrix theory (RMT), which uses the physiochemical properties of the amino acid with multiple sequence alignment. A graphical method to represent protein sequences using physiochemical properties is devised that gives a fast, easy, and informative way of comparing the evolutionary distances between protein sequences. A correlation matrix associated with each property is calculated, where the noise reduction and information filtering is done using RMT involving an ensemble of Wishart matrices. The analysis of the eigenvalue statistics of the correlation matrix for the β -lactamase family shows the universal features as observed in the Gaussian orthogonal ensemble (GOE). The property-based approach captures the short- as well as the long-range correlation (approximately following GOE) between the eigenvalues, whereas the previous approach (treating amino acids as characters) gives the usual short-range correlations, while the long-range correlations are the same as that of an uncorrelated series. The distribution of the eigenvector components for the eigenvalues outside the bulk (RMT bound) deviates significantly from RMT observations and contains important information about the system. The information content of each eigenvector of the correlation matrix is quantified by introducing an entropic estimate, which shows that for the β -lactamase family the smallest eigenvectors (low eigenmodes) are highly localized as well as informative. These small eigenvectors when processed gives clusters involving positions that have well-defined biological and structural importance matching with experiments. The approach is crucial for the recognition of structural motifs as shown in β -lactamase (and other families) and selectively identifies the important positions for targets to deactivate (activate) the enzymatic actions.

  9. Targeting functional motifs of a protein family.

    PubMed

    Bhadola, Pradeep; Deo, Nivedita

    2016-10-01

    The structural organization of a protein family is investigated by devising a method based on the random matrix theory (RMT), which uses the physiochemical properties of the amino acid with multiple sequence alignment. A graphical method to represent protein sequences using physiochemical properties is devised that gives a fast, easy, and informative way of comparing the evolutionary distances between protein sequences. A correlation matrix associated with each property is calculated, where the noise reduction and information filtering is done using RMT involving an ensemble of Wishart matrices. The analysis of the eigenvalue statistics of the correlation matrix for the β-lactamase family shows the universal features as observed in the Gaussian orthogonal ensemble (GOE). The property-based approach captures the short- as well as the long-range correlation (approximately following GOE) between the eigenvalues, whereas the previous approach (treating amino acids as characters) gives the usual short-range correlations, while the long-range correlations are the same as that of an uncorrelated series. The distribution of the eigenvector components for the eigenvalues outside the bulk (RMT bound) deviates significantly from RMT observations and contains important information about the system. The information content of each eigenvector of the correlation matrix is quantified by introducing an entropic estimate, which shows that for the β-lactamase family the smallest eigenvectors (low eigenmodes) are highly localized as well as informative. These small eigenvectors when processed gives clusters involving positions that have well-defined biological and structural importance matching with experiments. The approach is crucial for the recognition of structural motifs as shown in β-lactamase (and other families) and selectively identifies the important positions for targets to deactivate (activate) the enzymatic actions.

  10. Correlated rigid modes in protein families

    NASA Astrophysics Data System (ADS)

    Striegel, D. A.; Wojtowicz, D.; Przytycka, T. M.; Periwal, V.

    2016-04-01

    A great deal of evolutionarily conserved information is contained in genomes and proteins. Enormous effort has been put into understanding protein structure and developing computational tools for protein folding, and many sophisticated approaches take structure and sequence homology into account. Several groups have applied statistical physics approaches to extracting information about proteins from sequences alone. Here, we develop a new method for sequence analysis based on first principles, in information theory, in statistical physics and in Bayesian analysis. We provide a complete derivation of our approach and we apply it to a variety of systems, to demonstrate its utility and its limitations. We show in some examples that phylogenetic alignments of amino-acid sequences of families of proteins imply the existence of a small number of modes that appear to be associated with correlated global variation. These modes are uncovered efficiently in our approach by computing a non-perturbative effective potential directly from the alignment. We show that this effective potential approaches a limiting form inversely with the logarithm of the number of sequences. Mapping symbol entropy flows along modes to underlying physical structures shows that these modes arise due to correlated compensatory adjustments. In the protein examples, these occur around functional binding pockets.

  11. Thiol Dioxygenases: Unique Families of Cupin Proteins

    PubMed Central

    Simmons, C. R.; Karplus, P. A.; Dominy, J. E.

    2011-01-01

    Proteins in the cupin superfamily have a wide range of biological functions in archaea, bacteria and eukaryotes. Although proteins in the cupin superfamily show very low overall sequence similarity, they all contain two short but partially conserved cupin sequence motifs separated by a less conserved intermotif region that varies both in length and amino acid sequence. Furthermore, these proteins all share a common architecture described as a 6-stranded β-barrel core, and this canonical cupin or “jelly roll” β-barrel is formed with cupin motif 1, the intermotif region, and cupin motif 2 each forming two of the core six β-strands in the folded protein structure. The recently obtained crystal structures of cysteine dioxygenase (CDO), with contains conserved cupin motifs, show that it has the predicted canonical cupin β-barrel fold. Although there had been no reports of CDO activity in prokaryotes, we identified a number of bacterial cupin proteins of unknown function that share low similarity with mammalian CDO and that conserve many residues in the active site pocket of CDO. Putative bacterial CDOs predicted to have CDO activity were shown to have similar substrate specificity and kinetic parameters as eukaryotic CDOs. Information gleaned from crystal structures of mammalian CDO along with sequence information for homologs shown to have CDO activity facilitated the identification of a CDO family fingerprint motif. One key feature of the CDO fingerprint motif is that the canonical metal-binding glutamate residue in cupin motif 1 is replaced by a cysteine (in mammalian CDOs) or by a glycine (bacterial CDOs). The recent report that some putative bacterial CDO homologs are actually 3-mercaptopropionate dioxygenases suggests that the CDO family may include proteins with specificities for other thiol substrates. A paralog of CDO in mammals was also identified and shown to be the other mammalian thiol dioxygenase, cysteamine dioxygenase (ADO). A tentative

  12. Sequence analysis of the AAA protein family.

    PubMed Central

    Beyer, A.

    1997-01-01

    The AAA protein family, a recently recognized group of Walker-type ATPases, has been subjected to an extensive sequence analysis. Multiple sequence alignments revealed the existence of a region of sequence similarity, the so-called AAA cassette. The borders of this cassette were localized and within it, three boxes of a high degree of conservation were identified. Two of these boxes could be assigned to substantial parts of the ATP binding site (namely, to Walker motifs A and B); the third may be a portion of the catalytic center. Phylogenetic trees were calculated to obtain insights into the evolutionary history of the family. Subfamilies with varying degrees of intra-relatedness could be discriminated; these relationships are also supported by analysis of sequences outside the canonical AAA boxes: within the cassette are regions that are strongly conserved within each subfamily, whereas little or even no similarity between different subfamilies can be observed. These regions are well suited to define fingerprints for subfamilies. A secondary structure prediction utilizing all available sequence information was performed and the result was fitted to the general 3D structure of a Walker A/GTPase. The agreement was unexpectedly high and strongly supports the conclusion that the AAA family belongs to the Walker superfamily of A/GTPases. PMID:9336829

  13. Evidence against a Role for the JIL-1 Kinase in H3S28 Phosphorylation and 14-3-3 Recruitment to Active Genes in Drosophila

    PubMed Central

    Li, Yeran; Cai, Weili; Bao, Xiaomin; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M.

    2013-01-01

    JIL-1 is the major kinase controlling phosphorylation of histone H3S10 and has been demonstrated to function to counteract heterochromatization and gene silencing. However, an alternative model has been proposed in which JIL-1 is required for transcription to occur, additionally phosphorylates H3S28, and recruits 14-3-3 to active genes. Since these findings are incompatible with our previous demonstration that there are robust levels of transcription in the complete absence of JIL-1 and that JIL-1 is not present at developmental or heat shock-induced polytene chromosome puffs, we have reexamined JIL-1’s possible role in H3S28 phosphorylation and 14-3-3 recruitment. Using two different H3S28ph antibodies we show by immunocytochemistry and immunoblotting that in Drosophila the H3S28ph mark is not present at detectable levels above background on polytene chromosomes at interphase but only on chromosomes at pro-, meta-, and anaphase during cell division in S2 cells and third instar larval neuroblasts. Moreover, this mitotic H3S28ph signal is also present in a JIL-1 null mutant background at undiminished levels suggesting that JIL-1 is not the mitotic H3S28ph kinase. We also demonstrate that H3S28ph is not enriched at heat shock puffs. Using two different pan-specific 14-3-3 antibodies as well as an enhancer trap 14-3-3ε-GFP line we show that 14-3-3, while present in salivary gland nuclei, does not localize to chromosomes but only to the nuclear matrix surrounding the chromosomes. In our hands 14-3-3 is not recruited to developmental or heat shock puffs. Furthermore, using a lacO repeat tethering system to target LacI-JIL-1 to ectopic sites on polytene chromosomes we show that only H3S10ph is present and upregulated at such sites, not H3S28ph or 14-3-3. Thus, our results argue strongly against a model where JIL-1 is required for H3S28 phosphorylation and 14-3-3 recruitment at active genes. PMID:23638096

  14. A 14-3-3γ dimer-based scaffold bridges CtBP1-S/BARS to PI(4)KIIIβ to regulate post-Golgi carrier formation.

    PubMed

    Valente, Carmen; Turacchio, Gabriele; Mariggiò, Stefania; Pagliuso, Alessandro; Gaibisso, Renato; Di Tullio, Giuseppe; Santoro, Michele; Formiggini, Fabio; Spanò, Stefania; Piccini, Daniele; Polishchuk, Roman S; Colanzi, Antonino; Luini, Alberto; Corda, Daniela

    2012-02-26

    Large pleiomorphic carriers leave the Golgi complex for the plasma membrane by en bloc extrusion of specialized tubular domains, which then undergo fission. Several components of the underlying molecular machinery have been identified, including those involved in the budding/initiation of tubular carrier precursors (for example, the phosphoinositide kinase PI(4)KIIIβ, the GTPase ARF, and FAPP2), and in the fission of these precursors (for example, PKD, CtBP1-S/BARS). However, how these proteins interact to bring about carrier formation is poorly understood. Here, we describe a protein complex that mediates carrier formation and contains budding and fission molecules, as well as other molecules, such as the adaptor protein 14-3-3γ. Specifically, we show that 14-3-3γ dimers bridge CtBP1-S/BARS with PI(4)KIIIβ, and that the resulting complex is stabilized by phosphorylation by PKD and PAK. Disrupting the association of these proteins inhibits the fission of elongating carrier precursors, indicating that this complex couples the carrier budding and fission processes.

  15. Learning generative models for protein fold families.

    PubMed

    Balakrishnan, Sivaraman; Kamisetty, Hetunandan; Carbonell, Jaime G; Lee, Su-In; Langmead, Christopher James

    2011-04-01

    We introduce a new approach to learning statistical models from multiple sequence alignments (MSA) of proteins. Our method, called GREMLIN (Generative REgularized ModeLs of proteINs), learns an undirected probabilistic graphical model of the amino acid composition within the MSA. The resulting model encodes both the position-specific conservation statistics and the correlated mutation statistics between sequential and long-range pairs of residues. Existing techniques for learning graphical models from MSA either make strong, and often inappropriate assumptions about the conditional independencies within the MSA (e.g., Hidden Markov Models), or else use suboptimal algorithms to learn the parameters of the model. In contrast, GREMLIN makes no a priori assumptions about the conditional independencies within the MSA. We formulate and solve a convex optimization problem, thus guaranteeing that we find a globally optimal model at convergence. The resulting model is also generative, allowing for the design of new protein sequences that have the same statistical properties as those in the MSA. We perform a detailed analysis of covariation statistics on the extensively studied WW and PDZ domains and show that our method out-performs an existing algorithm for learning undirected probabilistic graphical models from MSA. We then apply our approach to 71 additional families from the PFAM database and demonstrate that the resulting models significantly out-perform Hidden Markov Models in terms of predictive accuracy.

  16. Gene expressions levels of 14-3-3a, NKCCla, APO-14, and Na(+)-K(+)-ATPaseβ in gill tissue of Mugil cephalus acclimated to low salinity.

    PubMed

    Li, L; Jiang, M; Shen, X Q

    2017-02-16

    Fishes adapt to salinity changes primarily through osmotic pressure regulation, a process often associated with several genes, including 14-3-3a, NKCCla, APO-14, and Na(+)-K(+)-ATPaseβ. The present study investigated the differential expression of genes 14-3-3a, NKCCla, APO-14, and Na(+)-K(+)-ATPaseβ in the gill tissue of Mugil cephalus acclimated to low salinity. Susceptibility relationships between the four gene expressions levels and salinity were detected and analyzed using polymerase chain reaction-restriction fragment length polymorphism. Homology analysis results indicated significant differences in the correlation between gene expression and salinity. Under low-salt conditions, expression levels for genes Na(+)-K(+)-ATPaseβ and NKCC1a were significantly elevated (P < 0.05), whereas those of genes 14-3-3a and APO-14 were significantly reduced (P < 0.05). Thus, when compared to 14-3-3a and APO-14, Na(+)-K(+)-ATPaseβ, and NKCC1a may be better suited to promoting the development of osmotic-regulation mechanisms and increased resistance to environmental stress under low-salt conditions. Furthermore, Na(+)-K(+)-ATPaseβ and NKCC1a were identified as suitable potential molecular biomarkers for regulating and controlling genes in low-salinity aquatic environments.

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

    PubMed

    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.

  18. Annotation extension through protein family annotation coherence metrics

    PubMed Central

    Bastos, Hugo P.; Clarke, Luka A.; Couto, Francisco M.

    2013-01-01

    Protein functional annotation consists in associating proteins with textual descriptors elucidating their biological roles. The bulk of annotation is done via automated procedures that ultimately rely on annotation transfer. Despite a large number of existing protein annotation procedures the ever growing protein space is never completely annotated. One of the facets of annotation incompleteness derives from annotation uncertainty. Often when protein function cannot be predicted with enough specificity it is instead conservatively annotated with more generic terms. In a scenario of protein families or functionally related (or even dissimilar) sets this leads to a more difficult task of using annotations to compare the extent of functional relatedness among all family or set members. However, we postulate that identifying sub-sets of functionally coherent proteins annotated at a very specific level, can help the annotation extension of other incompletely annotated proteins within the same family or functionally related set. As an example we analyse the status of annotation of a set of CAZy families belonging to the Polysaccharide Lyase class. We show that through the use of visualization methods and semantic similarity based metrics it is possible to identify families and respective annotation terms within them that are suitable for possible annotation extension. Based on our analysis we then propose a semi-automatic methodology leading to the extension of single annotation terms within these partially annotated protein sets or families. PMID:24130572

  19. Genomic analysis of membrane protein families: abundance and conserved motifs

    PubMed Central

    Liu, Yang; Engelman, Donald M; Gerstein, Mark

    2002-01-01

    Background Polytopic membrane proteins can be related to each other on the basis of the number of transmembrane helices and sequence similarities. Building on the Pfam classification of protein domain families, and using transmembrane-helix prediction and sequence-similarity searching, we identified a total of 526 well-characterized membrane protein families in 26 recently sequenced genomes. To this we added a clustering of a number of predicted but unclassified membrane proteins, resulting in a total of 637 membrane protein families. Results Analysis of the occurrence and composition of these families revealed several interesting trends. The number of assigned membrane protein domains has an approximately linear relationship to the total number of open reading frames (ORFs) in 26 genomes studied. Caenorhabditis elegans is an apparent outlier, because of its high representation of seven-span transmembrane (7-TM) chemoreceptor families. In all genomes, including that of C. elegans, the number of distinct membrane protein families has a logarithmic relation to the number of ORFs. Glycine, proline, and tyrosine locations tend to be conserved in transmembrane regions within families, whereas isoleucine, valine, and methionine locations are relatively mutable. Analysis of motifs in putative transmembrane helices reveals that GxxxG and GxxxxxxG (which can be written GG4 and GG7, respectively; see Materials and methods) are among the most prevalent. This was noted in earlier studies; we now find these motifs are particularly well conserved in families, however, especially those corresponding to transporters, symporters, and channels. Conclusions We carried out a genome-wide analysis on patterns of the classified polytopic membrane protein families and analyzed the distribution of conserved amino acids and motifs in the transmembrane helix regions in these families. PMID:12372142

  20. Involvement of PCH family proteins in cytokinesis and actin distribution.

    PubMed

    Lippincott, J; Li, R

    2000-04-15

    Pombe Cdc15 homology (PCH) proteins constitute an extensive protein family whose members have been found in diverse eukaryotic organisms. These proteins are characterized by the presence of several conserved sequence and structural motifs. Recent studies in yeast and mammalian cultured cells have implicated these proteins in actin-based processes, in particular, cytokinesis. Here we review the recent findings on the in vivo localization, function, and binding partners of PCH family members. We also provide new microscopy data regarding the in vivo dynamics of a budding yeast PCH protein involved in cytokinesis.

  1. Novel protein-protein interaction family proteins involved in chloroplast movement response.

    PubMed

    Kodama, Yutaka; Suetsugu, Noriyuki; Wada, Masamitsu

    2011-04-01

    To optimize photosynthetic activity, chloroplasts change their intracellular location in response to ambient light conditions; chloroplasts move toward low intensity light to maximize light capture, and away from high intensity light to avoid photodamage. Although several proteins have been reported to be involved in the chloroplast photorelocation movement response, any physical interaction among them was not found so far. We recently found a physical interaction between two plant-specific coiled-coil proteins, WEB1 (Weak Chloroplast Movement under Blue Light 1) and PMI2 (Plastid Movement Impaired 2), that were identified to regulate chloroplast movement velocity. Since the both coiled-coil regions of WEB1 and PMI2 were classified into an uncharacterized protein family having DUF827 (DUF: Domain of Unknown Function) domain, it was the first report that DUF827 proteins could mediate protein-protein interaction. In this mini-review article, we discuss regarding molecular function of WEB1 and PMI2, and also define a novel protein family composed of WEB1, PMI2 and WEB1/PMI2-like proteins for protein-protein interaction in land plants.

  2. Mu-8: visualizing differences between proteins and their families

    PubMed Central

    2014-01-01

    Background A complete understanding of the relationship between the amino acid sequence and resulting protein function remains an open problem in the biophysical sciences. Current approaches often rely on diagnosing functionally relevant mutations by determining whether an amino acid frequently occurs at a specific position within the protein family. However, these methods do not account for the biophysical properties and the 3D structure of the protein. We have developed an interactive visualization technique, Mu-8, that provides researchers with a holistic view of the differences of a selected protein with respect to a family of homologous proteins. Mu-8 helps to identify areas of the protein that exhibit: (1) significantly different bio-chemical characteristics, (2) relative conservation in the family, and (3) proximity to other regions that have suspect behavior in the folded protein. Methods Our approach quantifies and communicates the difference between a reference protein and its family based on amino acid indices or principal components of amino acid index classes, while accounting for conservation, proximity amongst residues, and overall 3D structure. Results We demonstrate Mu-8 in a case study with data provided by the 2013 BioVis contest. When comparing the sequence of a dysfunctional protein to its functional family, Mu-8 reveals several candidate regions that may cause function to break down. PMID:25237392

  3. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    EPA Science Inventory

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  4. The KP4 killer protein gene family

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Killer protein 4 (KP4) is a well studied toxin secreted by the maize smut fungus Ustilago maydis that kills sensitive Ustilago strains as well as inhibits Fusarium and plant root growth. This small, cysteine rich protein is encoded by a virus that depends on host survival for replication. KP4 functi...

  5. Metagenome and Metatranscriptome Analyses Using Protein Family Profiles

    PubMed Central

    Zhong, Cuncong; Yooseph, Shibu

    2016-01-01

    Analyses of metagenome data (MG) and metatranscriptome data (MT) are often challenged by a paucity of complete reference genome sequences and the uneven/low sequencing depth of the constituent organisms in the microbial community, which respectively limit the power of reference-based alignment and de novo sequence assembly. These limitations make accurate protein family classification and abundance estimation challenging, which in turn hamper downstream analyses such as abundance profiling of metabolic pathways, identification of differentially encoded/expressed genes, and de novo reconstruction of complete gene and protein sequences from the protein family of interest. The profile hidden Markov model (HMM) framework enables the construction of very useful probabilistic models for protein families that allow for accurate modeling of position specific matches, insertions, and deletions. We present a novel homology detection algorithm that integrates banded Viterbi algorithm for profile HMM parsing with an iterative simultaneous alignment and assembly computational framework. The algorithm searches a given profile HMM of a protein family against a database of fragmentary MG/MT sequencing data and simultaneously assembles complete or near-complete gene and protein sequences of the protein family. The resulting program, HMM-GRASPx, demonstrates superior performance in aligning and assembling homologs when benchmarked on both simulated marine MG and real human saliva MG datasets. On real supragingival plaque and stool MG datasets that were generated from healthy individuals, HMM-GRASPx accurately estimates the abundances of the antimicrobial resistance (AMR) gene families and enables accurate characterization of the resistome profiles of these microbial communities. For real human oral microbiome MT datasets, using the HMM-GRASPx estimated transcript abundances significantly improves detection of differentially expressed (DE) genes. Finally, HMM-GRASPx was used to

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

    PubMed Central

    Wang, Shou-Chieh; Lee, Shiow-Fen; Wang, Chau-Jong; Lee, Chao-Hsin; Lee, Wen-Chin; Lee, Huei-Jane

    2011-01-01

    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. PMID:19965962

  7. TRIM Family Proteins: Roles in Autophagy, Immunity, and Carcinogenesis.

    PubMed

    Hatakeyama, Shigetsugu

    2017-01-21

    Tripartite motif (TRIM) family proteins, most of which have E3 ubiquitin ligase activities, have various functions in cellular processes including intracellular signaling, development, apoptosis, protein quality control, innate immunity, autophagy, and carcinogenesis. The ubiquitin system is one of the systems for post-translational modifications, which play crucial roles not only as markers for degradation of target proteins by the proteasome but also as regulators of protein-protein interactions and of the activation of enzymes. Accumulating evidence has shown that TRIM family proteins have unique, important roles and that their dysregulation causes several diseases classified as cancer, immunological disease, or developmental disorders. In this review we focus on recent emerging topics on TRIM proteins in the regulation of autophagy, innate immunity, and carcinogenesis.

  8. Proteomic pathway analysis of the hippocampus in schizophrenia and bipolar affective disorder implicates 14-3-3 signaling, aryl hydrocarbon receptor signaling, and glucose metabolism: potential roles in GABAergic interneuron pathology.

    PubMed

    Schubert, Klaus Oliver; Föcking, Melanie; Cotter, David R

    2015-09-01

    Neuropathological changes of the hippocampus have been associated with psychotic disorders such as schizophrenia and bipolar disorder. Recent work has particularly implicated hippocampal GABAergic interneurons in the pathophysiology of these diseases. However, the molecular mechanisms underlying structural and cellular hippocampal pathology remain poorly understood. We used data from comprehensive difference-in-gel electrophoresis (2-D DIGE) investigations of postmortem human hippocampus of people with schizophrenia and bipolar disorder, covering the acidic (isoelectric point (pI) between pH4 and 7) and, separately, the basic (pI between pH6 and 11) sub-proteome, for Ingenuity Pathway Analysis (IPA) of implicated protein networks and pathways. Comparing disease and control cases, we identified 58 unique differentially expressed proteins in schizophrenia, and 70 differentially expressed proteins in bipolar disorder, using mass spectrometry. IPA implicated, most prominently, 14-3-3 and aryl hydrocarbon receptor signaling in schizophrenia, and gluconeogenesis/glycolysis in bipolar disorder. Both disorders were characterized by alterations of proteins involved in the oxidative stress response, mitochondrial function, and protein-endocytosis, -trafficking, -degradation, and -ubiquitination. These findings are interpreted with a focus on GABAergic interneuron pathology in the hippocampus.

  9. The FAD-dependent glycerol-3-phosphate dehydrogenase of Giardia duodenalis: an unconventional enzyme that interacts with the g14-3-3 and it is a target of the antitumoral compound NBDHEX

    PubMed Central

    Lalle, Marco; Camerini, Serena; Cecchetti, Serena; Finelli, Renata; Sferra, Gabriella; Müller, Joachim; Ricci, Giorgio; Pozio, Edoardo

    2015-01-01

    The flagellated protozoan Giardia duodenalis is a worldwide parasite causing giardiasis, an acute and chronic diarrheal disease. Metabolism in G. duodenalis has a limited complexity thus making metabolic enzymes ideal targets for drug development. However, only few metabolic pathways (i.e., carbohydrates) have been described so far. Recently, the parasite homolog of the mitochondrial-like glycerol-3-phosphate dehydrogenase (gG3PD) has been identified among the interactors of the g14-3-3 protein. G3PD is involved in glycolysis, electron transport, glycerophospholipids metabolism, and hyperosmotic stress response, and is emerging as promising target in tumor treatment. In this work, we demonstrate that gG3PD is a functional flavoenzyme able to convert glycerol-3-phosphate into dihydroxyacetone phosphate and that its activity and the intracellular glycerol level increase during encystation. Taking advantage of co-immunoprecipitation assays and deletion mutants, we provide evidence that gG3PD and g14-3-3 interact at the trophozoite stage, the intracellular localization of gG3PD is stage dependent and it partially co-localizes with mitosomes during cyst development. Finally, we demonstrate that the gG3PD activity is affected by the antitumoral compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, that results more effective in vitro at killing G. duodenalis trophozoites than the reference drug metronidazole. Overall, our results highlight the involvement of gG3PD in processes crucial for the parasite survival thus proposing this enzyme as target for novel antigiardial interventions. PMID:26082764

  10. The FAD-dependent glycerol-3-phosphate dehydrogenase of Giardia duodenalis: an unconventional enzyme that interacts with the g14-3-3 and it is a target of the antitumoral compound NBDHEX.

    PubMed

    Lalle, Marco; Camerini, Serena; Cecchetti, Serena; Finelli, Renata; Sferra, Gabriella; Müller, Joachim; Ricci, Giorgio; Pozio, Edoardo

    2015-01-01

    The flagellated protozoan Giardia duodenalis is a worldwide parasite causing giardiasis, an acute and chronic diarrheal disease. Metabolism in G. duodenalis has a limited complexity thus making metabolic enzymes ideal targets for drug development. However, only few metabolic pathways (i.e., carbohydrates) have been described so far. Recently, the parasite homolog of the mitochondrial-like glycerol-3-phosphate dehydrogenase (gG3PD) has been identified among the interactors of the g14-3-3 protein. G3PD is involved in glycolysis, electron transport, glycerophospholipids metabolism, and hyperosmotic stress response, and is emerging as promising target in tumor treatment. In this work, we demonstrate that gG3PD is a functional flavoenzyme able to convert glycerol-3-phosphate into dihydroxyacetone phosphate and that its activity and the intracellular glycerol level increase during encystation. Taking advantage of co-immunoprecipitation assays and deletion mutants, we provide evidence that gG3PD and g14-3-3 interact at the trophozoite stage, the intracellular localization of gG3PD is stage dependent and it partially co-localizes with mitosomes during cyst development. Finally, we demonstrate that the gG3PD activity is affected by the antitumoral compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, that results more effective in vitro at killing G. duodenalis trophozoites than the reference drug metronidazole. Overall, our results highlight the involvement of gG3PD in processes crucial for the parasite survival thus proposing this enzyme as target for novel antigiardial interventions.

  11. Expansion of the Protein Repertoire in Newly Explored Environments: Human Gut Microbiome Specific Protein Families

    PubMed Central

    Ellrott, Kyle; Jaroszewski, Lukasz; Li, Weizhong; Wooley, John C.; Godzik, Adam

    2010-01-01

    The microbes that inhabit particular environments must be able to perform molecular functions that provide them with a competitive advantage to thrive in those environments. As most molecular functions are performed by proteins and are conserved between related proteins, we can expect that organisms successful in a given environmental niche would contain protein families that are specific for functions that are important in that environment. For instance, the human gut is rich in polysaccharides from the diet or secreted by the host, and is dominated by Bacteroides, whose genomes contain highly expanded repertoire of protein families involved in carbohydrate metabolism. To identify other protein families that are specific to this environment, we investigated the distribution of protein families in the currently available human gut genomic and metagenomic data. Using an automated procedure, we identified a group of protein families strongly overrepresented in the human gut. These not only include many families described previously but also, interestingly, a large group of previously unrecognized protein families, which suggests that we still have much to discover about this environment. The identification and analysis of these families could provide us with new information about an environment critical to our health and well being. PMID:20532204

  12. BCL-2 family proteins as regulators of mitochondria metabolism.

    PubMed

    Gross, Atan

    2016-08-01

    The BCL-2 family proteins are major regulators of apoptosis, and one of their major sites of action are the mitochondria. Mitochondria are the cellular hubs for metabolism and indeed selected BCL-2 family proteins also possess roles related to mitochondria metabolism and dynamics. Here we discuss the link between mitochondrial metabolism/dynamics and the fate of stem cells, with an emphasis on the role of the BID-MTCH2 pair in regulating this link. We also discuss the possibility that BCL-2 family proteins act as metabolic sensors/messengers coming on and off of mitochondria to "sample" the cytosol and provide the mitochondria with up-to-date metabolic information. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

  13. Systems Proteomics View of the Endogenous Human Claudin Protein Family

    PubMed Central

    Liu, Fei; Koval, Michael; Ranganathan, Shoba; Fanayan, Susan; Hancock, William S.; Lundberg, Emma K.; Beavis, Ronald C.; Lane, Lydie; Duek, Paula; McQuade, Leon; Kelleher, Neil L.; Baker, Mark S.

    2016-01-01

    Claudins are the major transmembrane protein components of tight junctions in human endothelia and epithelia. Tissue-specific expression of claudin members suggests that this protein family is not only essential for sustaining the role of tight junctions in cell permeability control but also vital in organizing cell contact signaling by protein–protein interactions. How this protein family is collectively processed and regulated is key to understanding the role of junctional proteins in preserving cell identity and tissue integrity. The focus of this review is to first provide a brief overview of the functional context, on the basis of the extensive body of claudin biology research that has been thoroughly reviewed, for endogenous human claudin members and then ascertain existing and future proteomics techniques that may be applicable to systematically characterizing the chemical forms and interacting protein partners of this protein family in human. The ability to elucidate claudin-based signaling networks may provide new insight into cell development and differentiation programs that are crucial to tissue stability and manipulation. PMID:26680015

  14. DAZ Family Proteins, Key Players for Germ Cell Development.

    PubMed

    Fu, Xia-Fei; Cheng, Shun-Feng; Wang, Lin-Qing; Yin, Shen; De Felici, Massimo; Shen, Wei

    2015-01-01

    DAZ family proteins are found almost exclusively in germ cells in distant animal species. Deletion or mutations of their encoding genes usually severely impair either oogenesis or spermatogenesis or both. The family includes Boule (or Boll), Dazl (or Dazla) and DAZ genes. Boule and Dazl are situated on autosomes while DAZ, exclusive of higher primates, is located on the Y chromosome. Deletion of DAZ gene is the most common causes of infertility in humans. These genes, encoding for RNA binding proteins, contain a highly conserved RNA recognition motif and at least one DAZ repeat encoding for a 24 amino acids sequence able to bind other mRNA binding proteins. Basically, Daz family proteins function as adaptors for target mRNA transport and activators of their translation. In some invertebrate species, BOULE protein play a pivotal role in germline specification and a conserved regulatory role in meiosis. Depending on the species, DAZL is expressed in primordial germ cells (PGCs) and/or pre-meiotic and meiotic germ cells of both sexes. Daz is found in fetal gonocytes, spermatogonia and spermatocytes of adult testes. Here we discuss DAZ family genes in a phylogenic perspective, focusing on the common and distinct features of these genes, and their pivotal roles during gametogenesis evolved during evolution.

  15. A Comparison of Rosetta Stones in Adapter Protein Families

    PubMed Central

    Kumar, Hulikal Shivashankara Santosh; Kumar, Vadlapudi

    2016-01-01

    The inventory of proteins used in different kingdoms appears surprisingly similar in all sequenced eukaryotic genome. Protein domains represent the basic evolutionary units that form proteins. Domain duplication and shuffling by recombination are probably the most important forces driving protein evolution and hence the complexity of the proteome. While the duplication of whole genes as well as domain encoding exons increases the abundance of domains in the proteome, domain shuffling increases versatility, i.e. the number of distinct contexts in which a domain can occur. In this study we considered five important adapter domain families namely WD40, KELCH, Ankyrin, PDZ and Pleckstrin Homology (PH domain) family for the comparison of Domain versatility, Abundance and domain sharing between them. We used ecological statistics methods such as Jaccard’s Similarity Index (JSI), Detrended Correspondence Analysis, k-Means clustering for the domain distribution data. We found high propensity of domain sharing between PH and PDZ. We found higher abundance of only few selected domains in PH, PDZ, ANK and KELCH families. We also found WD40 family with high versatility and less redundant domain occurrence, with less domain sharing. Hence, the assignments of functions to more orphan WD40 proteins that will help in the identification of suitable drug targets. PMID:28246462

  16. Current Overview of Allergens of Plant Pathogenesis Related Protein Families

    PubMed Central

    Sinha, Mau; Singh, Rashmi Prabha; Kushwaha, Gajraj Singh; Iqbal, Naseer; Singh, Avinash; Kaushik, Sanket; Sharma, Sujata; Singh, Tej P.

    2014-01-01

    Pathogenesis related (PR) proteins are one of the major sources of plant derived allergens. These proteins are induced by the plants as a defense response system in stress conditions like microbial and insect infections, wounding, exposure to harsh chemicals, and atmospheric conditions. However, some plant tissues that are more exposed to environmental conditions like UV irradiation and insect or fungal attacks express these proteins constitutively. These proteins are mostly resistant to proteases and most of them show considerable stability at low pH. Many of these plant pathogenesis related proteins are found to act as food allergens, latex allergens, and pollen allergens. Proteins having similar amino acid sequences among the members of PR proteins may be responsible for cross-reactivity among allergens from diverse plants. This review analyzes the different pathogenesis related protein families that have been reported as allergens. Proteins of these families have been characterized in regard to their biological functions, amino acid sequence, and cross-reactivity. The three-dimensional structures of some of these allergens have also been evaluated to elucidate the antigenic determinants of these molecules and to explain the cross-reactivity among the various allergens. PMID:24696647

  17. An Ancient Family of RNA-Binding Proteins: Still Important!

    PubMed

    Wells, Melissa L; Perera, Lalith; Blackshear, Perry J

    2017-04-01

    RNA-binding proteins are important modulators of mRNA stability, a crucial process that determines the ultimate cellular levels of mRNAs and their encoded proteins. The tristetraprolin (TTP) family of RNA-binding proteins appeared early in the evolution of eukaryotes, and has persisted in modern eukaryotes. The domain structures and biochemical functions of family members from widely divergent lineages are remarkably similar, but their mRNA 'targets' can be very different, even in closely related species. Recent gene knockout studies in species as distantly related as plants, flies, yeasts, and mice have demonstrated crucial roles for these proteins in a wide variety of physiological processes. Inflammatory and hematopoietic phenotypes in mice have suggested potential therapeutic approaches for analogous human disorders.

  18. Altered Prion Protein Expression Pattern in CSF as a Biomarker for Creutzfeldt-Jakob Disease

    PubMed Central

    Torres, Mauricio; Cartier, Luis; Matamala, José Manuel; Hernández, Nancy; Woehlbier, Ute; Hetz, Claudio

    2012-01-01

    Creutzfeldt-Jakob disease (CJD) is the most frequent human Prion-related disorder (PrD). The detection of 14-3-3 protein in the cerebrospinal fluid (CSF) is used as a molecular diagnostic criterion for patients clinically compatible with CJD. However, there is a pressing need for the identification of new reliable disease biomarkers. The pathological mechanisms leading to accumulation of 14-3-3 protein in CSF are not fully understood, however neuronal loss followed by cell lysis is assumed to cause the increase in 14-3-3 levels, which also occurs in conditions such as brain ischemia. Here we investigated the relation between the levels of 14-3-3 protein, Lactate dehydrogenase (LDH) activity and expression of the prion protein (PrP) in CSF of sporadic and familial CJD cases. Unexpectedly, we found normal levels of LDH activity in CJD cases with moderate levels of 14-3-3 protein. Increased LDH activity was only observed in a percentage of the CSF samples that also exhibited high 14-3-3 levels. Analysis of the PrP expression pattern in CSF revealed a reduction in PrP levels in all CJD cases, as well as marked changes in its glycosylation pattern. PrP present in CSF of CJD cases was sensitive to proteases. The alterations in PrP expression observed in CJD cases were not detected in other pathologies affecting the nervous system, including cases of dementia and tropical spastic paraparesis/HTLV-1 associated myelopathy (HAM/TSP). Time course analysis in several CJD patients revealed that 14-3-3 levels in CSF are dynamic and show a high degree of variability during the end stage of the disease. Post-mortem analysis of brain tissue also indicated that 14-3-3 protein is upregulated in neuronal cells, suggesting that its expression is modulated during the course of the disease. These results suggest that a combined analysis of 14-3-3 and PrP expression pattern in CSF is a reliable biomarker to confirm the clinical diagnosis of CJD patients and follow disease progression

  19. Analysis of a cAMP regulated coactivator family reveals an alternative phosphorylation motif for AMPK family members

    PubMed Central

    Moresco, James J.; Vaughan, Joan M.; Matsumura, Shigenobu; Yates, John R.; Montminy, Marc

    2017-01-01

    The second messenger cAMP stimulates cellular gene expression via the PKA-mediated phosphorylation of the transcription factor CREB and through dephosphorylation of the cAMP-responsive transcriptional coactivators (CRTCs). Under basal conditions, CRTCs are phosphorylated by members of the AMPK family of Ser/Thr kinases and sequestered in the cytoplasm via a phosphorylation-dependent association with 14-3-3 proteins. Increases in cAMP promote the dephosphorylation and nuclear translocation of CRTCs, where they bind to CREB and stimulate relevant target genes. Although they share considerable sequence homology, members of the CRTC family exert non-overlapping effects on cellular gene expression through as yet unidentified mechanisms. Here we show that the three CRTCs exhibit distinct patterns of 14-3-3 binding at three conserved sites corresponding to S70, S171, and S275 (in CRTC2). S171 functions as the gatekeeper site for 14-3-3 binding; it acts cooperatively with S275 in stabilizing this interaction following its phosphorylation by the cAMP-responsive SIK and the cAMP-nonresponsive MARK kinases. Although S171 contains a consensus recognition site for phosphorylation by AMPK family members, S70 and S275 carry variant motifs (MNTGGS275LPDL), lacking basic residues that are otherwise critical for SIK/MARK recognition as well as 14-3-3 binding. Correspondingly, the activity of these motifs differs between CRTC family members. As the variant (SLPDL) motif is present and apparently phosphorylated in other mammalian proteins, our studies suggest that the regulation of cellular targets by AMPK family members is more extensive than previously appreciated. PMID:28235073

  20. Human erythrocyte membrane proteins of zone 4.5 exist as families of related proteins.

    PubMed

    Whitfield, C F; Coleman, D B; Kay, M M; Shiffer, K A; Miller, J; Goodman, S R

    1985-01-01

    An analysis of the polypeptide composition of zone 4.5 of human erythrocyte membranes has been done by immunoautoradiographic and two-dimensional peptide mapping techniques. Results of these studies demonstrated that the Coomassie blue profile was constant, with 14 well-resolved bands present. Zone 4.5 polypeptides existed as at least four families of two or more components with closely related polypeptide backbones. The families could be distinguished on the basis of their extraction characteristics, immunological cross-reactivity, and two-dimensional peptide maps. One family was related to protein 4.1, one family was related to band 3, and two families were independent and not similar to other larger membrane proteins. The data show that all of the visualized bands in zone 4.5 do not have the same protein composition and that several closely related forms of some polypeptides are present.

  1. The human cruciform-binding protein, CBP, is involved in DNA replication and associates in vivo with mammalian replication origins.

    PubMed

    Novac, Olivia; Alvarez, David; Pearson, Christopher E; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2002-03-29

    We previously identified and purified from human (HeLa) cells a 66-kDa cruciform-binding protein, CBP, with binding specificity for cruciform DNA regardless of its sequence. DNA cruciforms have been implicated in the regulation of initiation of DNA replication. CBP is a member of the 14-3-3 family of proteins, which are conserved regulatory molecules expressed in all eukaryotes. Here, the in vivo association of CBP/14-3-3 with mammalian origins of DNA replication was analyzed by studying its association with the monkey replication origins ors8 and ors12, as assayed by a chromatin immunoprecipitation assay and quantitative PCR analysis. The association of the 14-3-3beta, -epsilon, -gamma, and -zeta isoforms with these origins was found to be approximately 9-fold higher, compared with other portions of the genome, in logarithmically growing cells. In addition, the association of these isoforms with ors8 and ors12 was also analyzed as a function of the cell cycle. Higher binding of 14-3-3beta, -epsilon, -gamma, and -zeta isoforms with ors8 and ors12 was found at the G(1)/S border, by comparison with other stages of the cell cycle. The CBP/14-3-3 cruciform binding activity was also found to be maximal at the G(1)/S boundary. The involvement of 14-3-3 in mammalian DNA replication was analyzed by studying the effect of anti-14-3-3beta, -epsilon, -gamma, and -zeta antibodies in the in vitro replication of p186, a plasmid containing the minimal replication origin of ors8. Anti-14-3-3epsilon, -gamma, and -zeta antibodies alone or in combination inhibited p186 replication by approximately 50-80%, while anti-14-3-3beta antibodies had a lesser effect ( approximately 25-50%). All of the antibodies tested were also able to interfere with CBP binding to cruciform DNA. The results indicate that CBP/14-3-3 is an origin-binding protein, acting at the initiation step of DNA replication by binding to cruciform-containing molecules, and dissociates after origin firing.

  2. Ferritin family proteins and their use in bionanotechnology

    PubMed Central

    He, Didi; Marles-Wright, Jon

    2015-01-01

    Ferritin family proteins are found in all kingdoms of life and act to store iron within a protein cage and to protect the cell from oxidative damage caused by the Fenton reaction. The structural and biochemical features of the ferritins have been widely exploited in bionanotechnology applications: from the production of metal nanoparticles; as templates for semi-conductor production; and as scaffolds for vaccine design and drug delivery. In this review we first discuss the structural properties of the main ferritin family proteins, and describe how their organisation specifies their functions. Second, we describe materials science applications of ferritins that rely on their ability to sequester metal within their cavities. Finally, we explore the use of ferritin as a container for drug delivery and as a scaffold for the production of vaccines. PMID:25573765

  3. Molecular modeling of pathogenesis-related proteins of family 5.

    PubMed

    Thompson, Claudia E; Fernandes, Cláudia L; de Souza, Osmar N; Salzano, Francisco M; Bonatto, Sandro L; Freitas, Loreta B

    2006-01-01

    The family of pathogenesis-related (PR) 5 proteins have diverse functions, and some of them are classified as thaumatins, osmotins, and inhibitors of alpha-amylase or trypsin. Although the specific function of many PR5 in plants is unknown, they are involved in the acquired systemic resistance and response to biotic stress, causing the inhibition of hyphal growth and reduction of spore germination, probably by a membrane permeabilization mechanism or by interaction with pathogen receptors. We have constructed three-dimensional models of four proteins belonging to the Rosaceae and Fagaceae botanical families by using the technique of comparative molecular modelling by homology. There are four main structural differences between all the PR5, corresponding to regions with replacements of amino acids. Folding and the secondary structures are very similar for all of them. However, the isoelectric point and charge distributions differ for each protein.

  4. A family of human cdc2-related protein kinases.

    PubMed Central

    Meyerson, M; Enders, G H; Wu, C L; Su, L K; Gorka, C; Nelson, C; Harlow, E; Tsai, L H

    1992-01-01

    The p34cdc2 protein kinase is known to regulate important transitions in the eukaryotic cell cycle. We have identified 10 human protein kinases based on their structural relation to p34cdc2. Seven of these kinases are novel and the products of five share greater than 50% amino acid sequence identity with p34cdc2. The seven novel genes are broadly expressed in human cell lines and tissues with each displaying some cell type or tissue specificity. The cdk3 gene, like cdc2 and cdk2, can complement cdc28 mutants of Saccharomyces cerevisiae, suggesting that all three of these protein kinases can play roles in the regulation of the mammalian cell cycle. The identification of a large family of cdc2-related kinases opens the possibility of combinatorial regulation of the cell cycle together with the emerging large family of cyclins. Images PMID:1639063

  5. A database of protein structure families with common folding motifs.

    PubMed

    Holm, L; Ouzounis, C; Sander, C; Tuparev, G; Vriend, G

    1992-12-01

    The availability of fast and robust algorithms for protein structure comparison provides an opportunity to produce a database of three-dimensional comparisons, called families of structurally similar proteins (FSSP). The database currently contains an extended structural family for each of 154 representative (below 30% sequence identity) protein chains. Each data set contains: the search structure; all its relatives with 70-30% sequence identity, aligned structurally; and all other proteins from the representative set that contain substructures significantly similar to the search structure. Very close relatives (above 70% sequence identity) rarely have significant structural differences and are excluded. The alignments of remote relatives are the result of pairwise all-against-all structural comparisons in the set of 154 representative protein chains. The comparisons were carried out with each of three novel automatic algorithms that cover different aspects of protein structure similarity. The user of the database has the choice between strict rigid-body comparisons and comparisons that take into account interdomain motion or geometrical distortions; and, between comparisons that require strictly sequential ordering of segments and comparisons, which allow altered topology of loop connections or chain reversals. The data sets report the structurally equivalent residues in the form of a multiple alignment and as a list of matching fragments to facilitate inspection by three-dimensional graphics. If substructures are ignored, the result is a database of structure alignments of full-length proteins, including those in the twilight zone of sequence similarity.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Coincident inactivation of 14-3-3sigma and p16INK4a is an early event in vulval squamous neoplasia.

    PubMed

    Gasco, Milena; Sullivan, Alex; Repellin, Claire; Brooks, Louise; Farrell, Paul J; Tidy, John A; Dunne, Barbara; Gusterson, Barry; Evans, David J; Crook, Tim

    2002-03-14

    The structure and expression of 14-3-3 sigma(sigma) was analysed in squamous carcinomas (SCC) of the vulva and in the vulval pre-malignant lesion vulval intraepithelial neoplasia (VIN). Sequence analysis of the sigma coding region did not detect mutations in any case of SCC or VIN III and loss of heterozygosity (LOH) occurred in only 2 out of 27 informative cases. In contrast to the absence of genetic change, methylation-specific PCR (MSP) analysis revealed dense CpG methylation within the sigma gene in approximately 60% of cases of vulval SCC, but methylation was not detected in matched, normal epithelial tissue. Methylation was associated in all cases with reduced or absent expression of sigma mRNA. There was no correlation between sigma methylation and HPV or p53 status. Analysis of pre-malignant vulval intraepithelial neoplasia (VIN) revealed that sigma methylation was detectable early in neoplastic development. Co-incident methylation, accompanied by loss of expression, of sigma and p16INK4a was commonly detected in both SCC and VIN III, suggesting that epigenetic silencing of these two genes is an early and important event in vulval neoplasia.

  7. The APOBEC Protein Family: United by Structure, Divergent in Function.

    PubMed

    Salter, Jason D; Bennett, Ryan P; Smith, Harold C

    2016-07-01

    The APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) family of proteins have diverse and important functions in human health and disease. These proteins have an intrinsic ability to bind to both RNA and single-stranded (ss) DNA. Both function and tissue-specific expression varies widely for each APOBEC protein. We are beginning to understand that the activity of APOBEC proteins is regulated through genetic alterations, changes in their transcription and mRNA processing, and through their interactions with other macromolecules in the cell. Loss of cellular control of APOBEC activities leads to DNA hypermutation and promiscuous RNA editing associated with the development of cancer or viral drug resistance, underscoring the importance of understanding how APOBEC proteins are regulated.

  8. A novel family of small proteins that affect plant development

    SciTech Connect

    John Charles Walker

    2011-04-29

    The DVL genes represent a new group of plant proteins that influence plant growth and development. Overexpression of DVL1, and other members of the DVL family, causes striking phenotypic changes. The DVL proteins share sequence homology in their C-terminal half. Point mutations in the C-terminal domain show it is necessary and deletion studies demonstrate the C-terminal domain is sufficient to confer the overexpression phenotypes. The phenotypes observed, and the conservation of the protein sequence in the plant kingdom, does suggest the DVL proteins have a role in modulating plant growth and development. Our working hypothesis is the DVL proteins function as regulators of cellular signaling pathways that control growth and development.

  9. DNA-binding properties of ARID family proteins

    PubMed Central

    Patsialou, Antonia; Wilsker, Deborah; Moran, Elizabeth

    2005-01-01

    The ARID (A–T Rich Interaction Domain) is a helix–turn–helix motif-based DNA-binding domain, conserved in all eukaryotes and diagnostic of a family that includes 15 distinct human proteins with important roles in development, tissue-specific gene expression and proliferation control. The 15 human ARID family proteins can be divided into seven subfamilies based on the degree of sequence identity between individual members. Most ARID family members have not been characterized with respect to their DNA-binding behavior, but it is already apparent that not all ARIDs conform to the pattern of binding AT-rich sequences. To understand better the divergent characteristics of the ARID proteins, we undertook a survey of DNA-binding properties across the entire ARID family. The results indicate that the majority of ARID subfamilies (i.e. five out of seven) bind DNA without obvious sequence preference. DNA-binding affinity also varies somewhat between subfamilies. Site-specific mutagenesis does not support suggestions made from structure analysis that specific amino acids in Loop 2 or Helix 5 are the main determinants of sequence specificity. Most probably, this is determined by multiple interacting differences across the entire ARID structure. PMID:15640446

  10. Characterization of aryl hydrocarbon receptor interacting protein (AIP) mutations in familial isolated pituitary adenoma families.

    PubMed

    Igreja, Susana; Chahal, Harvinder S; King, Peter; Bolger, Graeme B; Srirangalingam, Umasuthan; Guasti, Leonardo; Chapple, J Paul; Trivellin, Giampaolo; Gueorguiev, Maria; Guegan, Katie; Stals, Karen; Khoo, Bernard; Kumar, Ajith V; Ellard, Sian; Grossman, Ashley B; Korbonits, Márta

    2010-08-01

    Familial isolated pituitary adenoma (FIPA) is an autosomal dominant condition with variable genetic background and incomplete penetrance. Germline mutations of the aryl hydrocarbon receptor interacting protein (AIP) gene have been reported in 15-40% of FIPA patients. Limited data are available on the functional consequences of the mutations or regarding the regulation of the AIP gene. We describe a large cohort of FIPA families and characterize missense and silent mutations using minigene constructs, luciferase and beta-galactosidase assays, as well as in silico predictions. Patients with AIP mutations had a lower mean age at diagnosis (23.6+/-11.2 years) than AIP mutation-negative patients (40.4+/-14.5 years). A promoter mutation showed reduced in vitro activity corresponding to lower mRNA expression in patient samples. Stimulation of the protein kinase A-pathway positively regulates the AIP promoter. Silent mutations led to abnormal splicing resulting in truncated protein or reduced AIP expression. A two-hybrid assay of protein-protein interaction of all missense variants showed variable disruption of AIP-phosphodiesterase-4A5 binding. In summary, exonic, promoter, splice-site, and large deletion mutations in AIP are implicated in 31% of families in our FIPA cohort. Functional characterization of AIP changes is important to identify the functional impact of gene sequence variants.

  11. Nme protein family evolutionary history, a vertebrate perspective

    PubMed Central

    Desvignes, Thomas; Pontarotti, Pierre; Fauvel, Christian; Bobe, Julien

    2009-01-01

    Background The Nme family, previously known as Nm23 or NDPK, is involved in various molecular processes including tumor metastasis and some members of the family, but not all, exhibit a Nucleoside Diphosphate Kinase (NDPK) activity. Ten genes are known in humans, in which some members have been extensively studied. In non-mammalian species, the Nme protein family has received, in contrast, far less attention. The picture of the vertebrate Nme family remains thus incomplete and orthology relationships with mammalian counterparts were only partially characterized. The present study therefore aimed at characterizing the Nme gene repertoire in vertebrates with special interest for teleosts, and providing a comprehensive overview of the Nme gene family evolutionary history in vertebrates. Results In the present study, we present the evolutionary history of the Nme family in vertebrates and characterize the gene family repertoire for the first time in several non-mammalian species. Our observations show that vertebrate Nme genes can be separated in two evolutionary distinct groups. Nme1, Nme2, Nme3, and Nme4 belong to Group I while vertebrate Nme5, Nme6, Nme7, Nme8, and Nme9 belong to Group II. The position of Nme10 is in contrast more debatable due to its very specific evolutionary history. The present study clearly indicates that Nme5, Nme6, Nme7, and Nme8 originate from duplication events that occurred before the chordate radiation. In contrast, Nme genes of the Group I have a very different evolutionary history as our results suggest that they all arise from a common gene present in the chordate ancestor. In addition, expression patterns of all zebrafish nme transcripts were studied in a broad range of tissues by quantitative PCR and discussed in the light of the function of their mammalian counterparts. Conclusion This work offers an evolutionary framework that will pave the way for future studies on vertebrate Nme proteins and provides a unified vertebrate Nme

  12. A novel family of plant nuclear envelope-associated proteins.

    PubMed

    Pawar, Vidya; Poulet, Axel; Détourné, Gwénaëlle; Tatout, Christophe; Vanrobays, Emmanuel; Evans, David E; Graumann, Katja

    2016-10-01

    This paper describes the characterisation of a new family of higher plant nuclear envelope-associated proteins (NEAPs) that interact with other proteins of the nuclear envelope. In the model plant Arabidopsis thaliana, the family consists of three genes expressed ubiquitously (AtNEAP1-3) and a pseudogene (AtNEAP4). NEAPs consist of extensive coiled-coil domains, followed by a nuclear localisation signal and a C-terminal predicted transmembrane domain. Domain deletion mutants confirm the presence of a functional nuclear localisation signal and transmembrane domain. AtNEAP proteins localise to the nuclear periphery as part of stable protein complexes, are able to form homo- and heteromers, and interact with the SUN domain proteins AtSUN1 and AtSUN2, involved in the linker of nucleoskeleton and cytoskeleton (LINC) complex. An A. thaliana cDNA library screen identified a putative transcription factor called AtbZIP18 as a novel interactor of AtNEAP1, which suggest a connection between NEAP and chromatin. An Atneap1 Atneap3 double-knockout mutant showed reduced root growth, and altered nuclear morphology and chromatin structure. Thus AtNEAPs are suggested as inner nuclear membrane-anchored coiled-coil proteins with roles in maintaining nuclear morphology and chromatin structure.

  13. The HMG-1 box protein family: classification and functional relationships.

    PubMed Central

    Baxevanis, A D; Landsman, D

    1995-01-01

    The abundant and highly-conserved nucleoproteins comprising the high mobility group-1/2 (HMG-1/2) family contains two homologous basic domains of about 75 amino acids. These basic domains, termed HMG-1 boxes, are highly structured and facilitate HMG-DNA interactions. Many proteins that regulate various cellular functions involving DNA binding and whose target DNA sequences share common structural characteristics have been identified as having an HMG-1 box; these proteins include the RNA polymerase I transcription factor UBF, the mammalian testis-determining factor SRY and the mitochondrial transcription factors ABF2 and mtTF1, among others. The sequences of 121 HMG-1 boxes have been compiled and aligned in accordance with thermodynamic results from homology model building (threading) experiments, basing the alignment on structure rather than by using traditional sequence homology methods. The classification of a representative subset of these proteins was then determined using standard least-squares distance methods. The proteins segregate into two groups, the first consisting of HMG-1/2 proteins and the second consisting of proteins containing the HMG-1 box but which are not canonical HMG proteins. The proteins in the second group further segregate based on their function, their ability to bind specific sequences of DNA, or their ability to recognize discrete non-B-DNA structures. The HMG-1 box provides an excellent example of how a specific protein motif, with slight alteration, can be used to recognize DNA in a variety of functional contexts. Images PMID:7784217

  14. The APSES family proteins in fungi: Characterizations, evolution and functions.

    PubMed

    Zhao, Yong; Su, Hao; Zhou, Jing; Feng, Huihua; Zhang, Ke-Qin; Yang, Jinkui

    2015-08-01

    The APSES protein family belongs to transcriptional factors of the basic helix-loop-helix (bHLH) class, the originally described members (APSES: Asm1p, Phd1p, Sok2p, Efg1p and StuAp) are used to designate this group of proteins, and they have been identified as key regulators of fungal development and other biological processes. APSES proteins share a highly conserved DNA-binding domain (APSES domain) of about 100 amino acids, whose central domain is predicted to form a typical bHLH structure. Besides APSES domain, several APSES proteins also contain additional domains, such as KilA-N and ankyrin repeats. In recent years, an increasing number of APSES proteins have been identified from diverse fungi, and they involve in numerous biological processes, such as sporulation, cellular differentiation, mycelial growth, secondary metabolism and virulence. Most fungi, including Aspergillus fumigatus, Aspergillus nidulans, Candida albicans, Fusarium graminearum, and Neurospora crassa, contain five APSES proteins. However, Cryptococcus neoformans only contains two APSES proteins, and Saccharomyces cerevisiae contains six APSES proteins. The phylogenetic analysis showed the APSES domains from different fungi were grouped into four clades (A, B, C and D), which is consistent with the result of homologous alignment of APSES domains using DNAman. The roles of APSES proteins in clade C have been studied in detail, while little is known about the roles of other APSES proteins in clades A, B and D. In this review, the biochemical properties and functional domains of APSES proteins are predicted and compared, and the phylogenetic relationship among APSES proteins from various fungi are analyzed based on the APSES domains. Moreover, the functions of APSES proteins in different fungi are summarized and discussed.

  15. Cbln and C1q family proteins: new transneuronal cytokines.

    PubMed

    Yuzaki, M

    2008-06-01

    The C1q family is characterized by a C-terminal conserved global C1q domain, which is structurally very similar to the tumor necrosis factor homology domain. Although some C1q family members are expressed in the central nervous system, their functions have not been well characterized. Cbln1, a member of the Cbln subfamily of the C1q family, is predominantly expressed in cerebellar granule cells. Interestingly, Cbln1 was recently shown to play two unique roles at excitatory synapses formed between cerebellar granule cells and Purkinje cells: the formation and stabilization of synaptic contact, and the control of functional synaptic plasticity by regulating the postsynaptic endocytosis pathway. Since other Cbln subfamily members, Cbln2-Cbln4, are expressed in various regions of developing and mature brains, Cbln subfamily proteins may generally serve as a new class of transneuronal regulators of synapse development and synaptic plasticity in various brain regions.

  16. [Immunodiffusion analysis of plasma proteins in the canine family].

    PubMed

    Baranov, O K; Iurishina, N A; Savina, M A

    1976-01-01

    Immunodiffusion studies have been made on the plasma of 9 species (Vulpes vulpes, V. corsak, Alopex lagopus, Canis aureus, C. lupus, C. familiaris, C. dingo, Nyctereutes procynoides, Fennecus zerde) from the family of Canidae using milk antisera. Unlike rabbit antisera used earlier, milk antisera make it possible to detect more significant antigenic divergency with respect to 5 alpha- and beta-globulins. These globulins seem to have a higher evolution rate of antigenic mosaics as compared to other plasma proteins in the family investigated. The family Canidae serologically may be divided into two main groups: 1) the genus Canis which includes the wolf, domestic dog, dingo, jackal and 2) species which significantly differ from the former (the fox, polar fox, dog fox, fennec). In relation to these two groups, the raccoon dog occupies special position.

  17. A widespread family of bacterial cell wall assembly proteins

    PubMed Central

    Kawai, Yoshikazu; Marles-Wright, Jon; Cleverley, Robert M; Emmins, Robyn; Ishikawa, Shu; Kuwano, Masayoshi; Heinz, Nadja; Bui, Nhat Khai; Hoyland, Christopher N; Ogasawara, Naotake; Lewis, Richard J; Vollmer, Waldemar; Daniel, Richard A; Errington, Jeff

    2011-01-01

    Teichoic acids and acidic capsular polysaccharides are major anionic cell wall polymers (APs) in many bacteria, with various critical cell functions, including maintenance of cell shape and structural integrity, charge and cation homeostasis, and multiple aspects of pathogenesis. We have identified the widespread LytR–Cps2A–Psr (LCP) protein family, of previously unknown function, as novel enzymes required for AP synthesis. Structural and biochemical analysis of several LCP proteins suggest that they carry out the final step of transferring APs from their lipid-linked precursor to cell wall peptidoglycan (PG). In Bacillus subtilis, LCP proteins are found in association with the MreB cytoskeleton, suggesting that MreB proteins coordinate the insertion of the major polymers, PG and AP, into the cell wall. PMID:21964069

  18. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    DOE PAGES

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; ...

    2016-05-16

    The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis;more » however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.« less

  19. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    PubMed Central

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; Lopez-Garrido, Javier; Muszewska, Anna; Dudkiewicz, Małgorzata; Grynberg, Marcin; Yee, Samantha; Pogliano, Kit; Tomchick, Diana R.; Pawłowski, Krzysztof; Dixon, Jack E.; Tagliabracci, Vincent S.

    2016-01-01

    The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology. PMID:27185916

  20. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    SciTech Connect

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; Lopez-Garrido, Javier; Muszewska, Anna; Dudkiewicz, Małgorzata; Grynberg, Marcin; Yee, Samantha; Pogliano, Kit; Tomchick, Diana R.; Pawłowski, Krzysztof; Dixon, Jack E.; Tagliabracci, Vincent S.

    2016-05-16

    The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.

  1. Investigating Structure and Dynamics of Atg8 Family Proteins.

    PubMed

    Weiergräber, O H; Schwarten, M; Strodel, B; Willbold, D

    2017-01-01

    Atg8 family members were the first autophagy-related proteins to be investigated in structural detail and continue to be among the best-understood molecules of the pathway. In this review, we will first provide a concise outline of the major methods that are being applied for structural characterization of these proteins and the complexes they are involved in. This includes a discussion of the strengths and limitations associated with each method, along with guidelines for successful adoption to a specific problem. Subsequently, we will present examples illustrating the application of these techniques, with a particular focus on the complementarity of information they provide.

  2. Characterization of Aryl Hydrocarbon Receptor Interacting Protein (AIP) Mutations in Familial Isolated Pituitary Adenoma Families

    PubMed Central

    Igreja, Susana; Chahal, Harvinder S; King, Peter; Bolger, Graeme B; Srirangalingam, Umasuthan; Guasti, Leonardo; Chapple, J Paul; Trivellin, Giampaolo; Gueorguiev, Maria; Guegan, Katie; Stals, Karen; Khoo, Bernard; Kumar, Ajith V; Ellard, Sian; Grossman, Ashley B; Korbonits, Márta

    2010-01-01

    Familial isolated pituitary adenoma (FIPA) is an autosomal dominant condition with variable genetic background and incomplete penetrance. Germline mutations of the aryl hydrocarbon receptor interacting protein (AIP) gene have been reported in 15–40% of FIPA patients. Limited data are available on the functional consequences of the mutations or regarding the regulation of the AIP gene. We describe a large cohort of FIPA families and characterize missense and silent mutations using minigene constructs, luciferase and β-galactosidase assays, as well as in silico predictions. Patients with AIP mutations had a lower mean age at diagnosis (23.6±11.2 years) than AIP mutation-negative patients (40.4±14.5 years). A promoter mutation showed reduced in vitro activity corresponding to lower mRNA expression in patient samples. Stimulation of the protein kinase A-pathway positively regulates the AIP promoter. Silent mutations led to abnormal splicing resulting in truncated protein or reduced AIP expression. A two-hybrid assay of protein–protein interaction of all missense variants showed variable disruption of AIP-phosphodiesterase-4A5 binding. In summary, exonic, promoter, splice-site, and large deletion mutations in AIP are implicated in 31% of families in our FIPA cohort. Functional characterization of AIP changes is important to identify the functional impact of gene sequence variants. Hum Mutat 31:1–11, 2010. © 2010 Wiley-Liss, Inc. PMID:20506337

  3. The ADF/cofilin family: actin-remodeling proteins.

    PubMed

    Maciver, Sutherland K; Hussey, Patrick J

    2002-01-01

    The ADF/cofilins are a family of actin-binding proteins expressed in all eukaryotic cells so far examined. Members of this family remodel the actin cytoskeleton, for example during cytokinesis, when the actin-rich contractile ring shrinks as it contracts through the interaction of ADF/cofilins with both monomeric and filamentous actin. The depolymerizing activity is twofold: ADF/cofilins sever actin filaments and also increase the rate at which monomers leave the filament's pointed end. The three-dimensional structure of ADF/cofilins is similar to a fold in members of the gelsolin family of actin-binding proteins in which this fold is typically repeated three or six times; although both families bind polyphosphoinositide lipids and actin in a pH-dependent manner, they share no obvious sequence similarity. Plants and animals have multiple ADF/cofilin genes, belonging in vertebrates to two types, ADF and cofilins. Other eukaryotes (such as yeast, Acanthamoeba and slime moulds) have a single ADF/cofilin gene. Phylogenetic analysis of the ADF/cofilins reveals that, with few exceptions, their relationships reflect conventional views of the relationships between the major groups of organisms.

  4. The latent transforming growth factor beta binding protein (LTBP) family.

    PubMed Central

    Oklü, R; Hesketh, R

    2000-01-01

    The transforming growth factor beta (TGFbeta) cytokines are a multi-functional family that exert a wide variety of effects on both normal and transformed mammalian cells. The secretion and activation of TGFbetas is regulated by their association with latency-associated proteins and latent TGFbeta binding proteins (LTBPs). Over the past few years, three members of the LTBP family have been identified, in addition to the protoype LTBP1 first sequenced in 1990. Three of the LTBP family are expressed in a variety of isoforms as a consequence of alternative splicing. This review summarizes the differences between the isoforms in terms of the effects on domain structure and hence possible function. The close identity between LTBPs and members of the fibrillin family, mutations in which have been linked directly to Marfan's syndrome, suggests that anomalous expression of LTBPs may be associated with disease. Recent data indicating that differential expression of LTBP1 isoforms occurs during the development of coronary heart disease is considered, together with evidence that modulation of LTBP function, and hence of TGFbeta activity, is associated with a variety of cancers. PMID:11104663

  5. In silico modeling of the yeast protein and protein family interaction network

    NASA Astrophysics Data System (ADS)

    Goh, K.-I.; Kahng, B.; Kim, D.

    2004-03-01

    Understanding of how protein interaction networks of living organisms have evolved or are organized can be the first stepping stone in unveiling how life works on a fundamental ground. Here we introduce an in silico ``coevolutionary'' model for the protein interaction network and the protein family network. The essential ingredient of the model includes the protein family identity and its robustness under evolution, as well as the three previously proposed: gene duplication, divergence, and mutation. This model produces a prototypical feature of complex networks in a wide range of parameter space, following the generalized Pareto distribution in connectivity. Moreover, we investigate other structural properties of our model in detail with some specific values of parameters relevant to the yeast Saccharomyces cerevisiae, showing excellent agreement with the empirical data. Our model indicates that the physical constraints encoded via the domain structure of proteins play a crucial role in protein interactions.

  6. Molecular evolution of the ependymin protein family: a necessary update

    PubMed Central

    Suárez-Castillo, Edna C; García-Arrarás, José E

    2007-01-01

    Background Ependymin (Epd), the predominant protein in the cerebrospinal fluid of teleost fishes, was originally associated with neuroplasticity and regeneration. Ependymin-related proteins (Epdrs) have been identified in other vertebrates, including amphibians and mammals. Recently, we reported the identification and characterization of an Epdr in echinoderms, showing that there are ependymin family members in non-vertebrate deuterostomes. We have now explored multiple databases to find Epdrs in different metazoan species. Using these sequences we have performed genome mapping, molecular phylogenetic analyses using Maximum Likelihood and Bayesian methods, and statistical tests of tree topologies, to ascertain the phylogenetic relationship among ependymin proteins. Results Our results demonstrate that ependymin genes are also present in protostomes. In addition, as a result of the putative fish-specific genome duplication event and posterior divergence, the ependymin family can be divided into four groups according to their amino acid composition and branching pattern in the gene tree: 1) a brain-specific group of ependymin sequences that is unique to teleost fishes and encompasses the originally described ependymin; 2) a group expressed in non-brain tissue in fishes; 3) a group expressed in several tissues that appears to be deuterostome-specific, and 4) a group found in invertebrate deuterostomes and protostomes, with a broad pattern of expression and that probably represents the evolutionary origin of the ependymins. Using codon-substitution models to statistically assess the selective pressures acting over the ependymin protein family, we found evidence of episodic positive Darwinian selection and relaxed selective constraints in each one of the postduplication branches of the gene tree. However, purifying selection (with among-site variability) appears to be the main influence on the evolution of each subgroup within the family. Functional divergence among the

  7. The PIN-FORMED (PIN) protein family of auxin transporters

    PubMed Central

    2009-01-01

    Summary The PIN-FORMED (PIN) proteins are secondary transporters acting in the efflux of the plant signal molecule auxin from cells. They are asymmetrically localized within cells and their polarity determines the directionality of intercellular auxin flow. PIN genes are found exclusively in the genomes of multicellular plants and play an important role in regulating asymmetric auxin distribution in multiple developmental processes, including embryogenesis, organogenesis, tissue differentiation and tropic responses. All PIN proteins have a similar structure with amino- and carboxy-terminal hydrophobic, membrane-spanning domains separated by a central hydrophilic domain. The structure of the hydrophobic domains is well conserved. The hydrophilic domain is more divergent and it determines eight groups within the protein family. The activity of PIN proteins is regulated at multiple levels, including transcription, protein stability, subcellular localization and transport activity. Different endogenous and environmental signals can modulate PIN activity and thus modulate auxin-distribution-dependent development. A large group of PIN proteins, including the most ancient members known from mosses, localize to the endoplasmic reticulum and they regulate the subcellular compartmentalization of auxin and thus auxin metabolism. Further work is needed to establish the physiological importance of this unexpected mode of auxin homeostasis regulation. Furthermore, the evolution of PIN-based transport, PIN protein structure and more detailed biochemical characterization of the transport function are important topics for further studies. PMID:20053306

  8. IQGAP1 Interaction with RHO Family Proteins Revisited

    PubMed Central

    Nouri, Kazem; Fansa, Eyad K.; Amin, Ehsan; Dvorsky, Radovan; Gremer, Lothar; Willbold, Dieter; Schmitt, Lutz; Timson, David J.; Ahmadian, Mohammad R.

    2016-01-01

    IQ motif-containing GTPase activating protein 1 (IQGAP1) plays a central role in the physical assembly of relevant signaling networks that are responsible for various cellular processes, including cell adhesion, polarity, and transmigration. The RHO family proteins CDC42 and RAC1 have been shown to mainly interact with the GAP-related domain (GRD) of IQGAP1. However, the role of its RASGAP C-terminal (RGCT) and C-terminal domains in the interactions with RHO proteins has remained obscure. Here, we demonstrate that IQGAP1 interactions with RHO proteins underlie a multiple-step binding mechanism: (i) a high affinity, GTP-dependent binding of RGCT to the switch regions of CDC42 or RAC1 and (ii) a very low affinity binding of GRD and a C terminus adjacent to the switch regions. These data were confirmed by phosphomimetic mutation of serine 1443 to glutamate within RGCT, which led to a significant reduction of IQGAP1 affinity for CDC42 and RAC1, clearly disclosing the critical role of RGCT for these interactions. Unlike CDC42, an extremely low affinity was determined for the RAC1-GRD interaction, suggesting that the molecular nature of IQGAP1 interaction with CDC42 partially differs from that of RAC1. Our study provides new insights into the interaction characteristics of IQGAP1 with RHO family proteins and highlights the complementary importance of kinetic and equilibrium analyses. We propose that the ability of IQGAP1 to interact with RHO proteins is based on a multiple-step binding process, which is a prerequisite for the dynamic functions of IQGAP1 as a scaffolding protein and a critical mechanism in temporal regulation and integration of IQGAP1-mediated cellular responses. PMID:27815503

  9. UBXD Proteins: A Family of Proteins with Diverse Functions in Cancer

    PubMed Central

    Rezvani, Khosrow

    2016-01-01

    The UBXD family is a diverse group of UBX (ubiquitin-regulatory X) domain-containing proteins in mammalian cells. Members of this family contain a UBX domain typically located at the carboxyl-terminal of the protein. In contrast to the UBX domain shared by all members of UBXD family, the amino-terminal domains are diverse and appear to carry out different roles in a subcellular localization-dependent manner. UBXD proteins are principally associated with the endoplasmic reticulum (ER), where they positively or negatively regulate the ER-associated degradation machinery (ERAD). The distinct protein interaction networks of UBXD proteins allow them to have specific functions independent of the ERAD pathway in a cell type- and tissue context-dependent manner. Recent reports have illustrated that a number of mammalian members of the UBXD family play critical roles in several proliferation and apoptosis pathways dysregulated in selected types of cancer. This review covers recent advances that elucidate the therapeutic potential of selected members of the UBXD family that can contribute to tumor growth. PMID:27754413

  10. Evolutionary plasticity of plasma membrane interaction in DREPP family proteins.

    PubMed

    Vosolsobě, Stanislav; Petrášek, Jan; Schwarzerová, Kateřina

    2017-05-01

    The plant-specific DREPP protein family comprises proteins that were shown to regulate the actin and microtubular cytoskeleton in a calcium-dependent manner. Our phylogenetic analysis showed that DREPPs first appeared in ferns and that DREPPs have a rapid and plastic evolutionary history in plants. Arabidopsis DREPP paralogues called AtMDP25/PCaP1 and AtMAP18/PCaP2 are N-myristoylated, which has been reported as a key factor in plasma membrane localization. Here we show that N-myristoylation is neither conserved nor ancestral for the DREPP family. Instead, by using confocal microscopy and a new method for quantitative evaluation of protein membrane localization, we show that DREPPs rely on two mechanisms ensuring their plasma membrane localization. These include N-myristoylation and electrostatic interaction of a polybasic amino acid cluster. We propose that various plasma membrane association mechanisms resulting from the evolutionary plasticity of DREPPs are important for refining plasma membrane interaction of these signalling proteins under various conditions and in various cells.

  11. Graphical models of residue coupling in protein families.

    PubMed

    Thomas, John; Ramakrishnan, Naren; Bailey-Kellogg, Chris

    2008-01-01

    Many statistical measures and algorithmic techniques have been proposed for studying residue coupling in protein families. Generally speaking, two residue positions are considered coupled if, in the sequence record, some of their amino acid type combinations are significantly more common than others. While the proposed approaches have proven useful in finding and describing coupling, a significant missing component is a formal probabilistic model that explicates and compactly represents the coupling, integrates information about sequence,structure, and function, and supports inferential procedures for analysis, diagnosis, and prediction.We present an approach to learning and using probabilistic graphical models of residue coupling. These models capture significant conservation and coupling constraints observable ina multiply-aligned set of sequences. Our approach can place a structural prior on considered couplings, so that all identified relationships have direct mechanistic explanations. It can also incorporate information about functional classes, and thereby learn a differential graphical model that distinguishes constraints common to all classes from those unique to individual classes. Such differential models separately account for class-specific conservation and family-wide coupling, two different sources of sequence covariation. They are then able to perform interpretable functional classification of new sequences, explaining classification decisions in terms of the underlying conservation and coupling constraints. We apply our approach in studies of both G protein-coupled receptors and PDZ domains, identifying and analyzing family-wide and class-specific constraints, and performing functional classification. The results demonstrate that graphical models of residue coupling provide a powerful tool for uncovering, representing, and utilizing significant sequence structure-function relationships in protein families.

  12. [The importance of ADAM family proteins in malignant tumors].

    PubMed

    Walkiewicz, Katarzyna; Gętek, Monika; Muc-Wierzgoń, Małgorzata; Kokot, Teresa; Nowakowska-Zajdel, Ewa

    2016-02-11

    Increasing numbers of reports about the role of adamalysins (ADAM) in malignant tumors are being published. To date, more than 30 representatives of this group, out of which about 20 occur in humans, have been described. The ADAM family is a homogeneous group of proteins which regulate, from the stage of embryogenesis, a series of processes such as cell migration, adhesion, and cell fusion. Half of them have proteolytic activity and are involved in the degradation of the extracellular matrix and the disintegration of certain protein complexes, thereby regulating the bioavailability of various growth factors. Many of these functions have a direct role in the processes of carcinogenesis and promoting the growth of tumor, which affect some signaling pathways, including those related to insulin-like growth factors (IGF1, IGF2), vascular growth factor (VEGF), tumor necrosis factor α (TNFα) and the EGFR/HER pathway. Another branch of studies is the evaluation of the possibility of using members of ADAM family proteins in the diagnosis, especially in breast, colon and non- small cell lung cancer. The detection of concentrations of adamalysin in serum, urine and pleural aspirates might contribute to the development of methods of early diagnosis of cancer and monitoring the therapy. However, both the role of adamalysins in the development and progression of tumors and their importance as a diagnostic and predictive further research still need to be checked on large groups of patients.

  13. Muscarinic receptor family interacting proteins: role in receptor function.

    PubMed

    Borroto-Escuela, Dasiel O; Correia, Patrícia A; Romero-Fernandez, Wilber; Narvaez, Manuel; Fuxe, Kjell; Ciruela, Francisco; Garriga, Pere

    2011-02-15

    G protein-coupled receptors constitute one of the most important families of membrane receptors through which cells respond to extracellular stimuli. Receptors of this superfamily likely function as signal transduction complexes. The identification and analysis of their components provide new insights into a better understanding of these receptors' function and regulation. We used tandem-affinity purification and mass spectrometry as a systematic approach to characterize multiprotein complexes in the acetylcholine muscarinic receptor subfamily. To overcome the limitations associated with membrane protein receptor solubilization with detergents, we developed a strategy in which receptors are co-expressed with a cytoplasmic minigene construct, encoding the third intracellular loop and the C-terminal tail tagged to the tandem-affinity-cassette of each receptor subtype. Numerous protein complexes were identified, including many new interactions in various signalling pathways. Systematic identification data set together with protein interactions reported in the literature revealed a high degree of connectivity. These allow the proposal, for the first time, of an outline of the muscarinic interactome as a network of protein complexes and a context for a more reasoned and informed approach to drug discovery and muscarinic receptor subtype specificities.

  14. Classification epitopes in groups based on their protein family

    PubMed Central

    2015-01-01

    Background The humoral immune system response is based on the interaction between antibodies and antigens for the clearance of pathogens and foreign molecules. The interaction between these proteins occurs at specific positions known as antigenic determinants or B-cell epitopes. The experimental identification of epitopes is costly and time consuming. Therefore the use of in silico methods, to help discover new epitopes, is an appealing alternative due the importance of biomedical applications such as vaccine design, disease diagnostic, anti-venoms and immune-therapeutics. However, the performance of predictions is not optimal been around 70% of accuracy. Further research could increase our understanding of the biochemical and structural properties that characterize a B-cell epitope. Results We investigated the possibility of linear epitopes from the same protein family to share common properties. This hypothesis led us to analyze physico-chemical (PCP) and predicted secondary structure (PSS) features of a curated dataset of epitope sequences available in the literature belonging to two different groups of antigens (metalloproteinases and neurotoxins). We discovered statistically significant parameters with data mining techniques which allow us to distinguish neurotoxin from metalloproteinase and these two from random sequences. After a five cross fold validation we found that PCP based models obtained area under the curve values (AUC) and accuracy above 0.9 for regression, decision tree and support vector machine. Conclusions We demonstrated that antigen's family can be inferred from properties within a single group of linear epitopes (metalloproteinases or neurotoxins). Also we discovered the characteristics that represent these two epitope groups including their similarities and differences with random peptides and their respective amino acid sequence. These findings open new perspectives to improve epitope prediction by considering the specific antigen

  15. Genealogy of an ancient protein family: the Sirtuins, a family of disordered members

    PubMed Central

    2013-01-01

    Background Sirtuins genes are widely distributed by evolution and have been found in eubacteria, archaea and eukaryotes. While prokaryotic and archeal species usually have one or two sirtuin homologs, in humans as well as in eukaryotes we found multiple versions and in mammals this family is comprised of seven different homologous proteins being all NAD-dependent de-acylases. 3D structures of human SIRT2, SIRT3, and SIRT5 revealed the overall conformation of the conserved core domain but they were unable to give a structural information about the presence of very flexible and dynamically disordered regions, the role of which is still structurally and functionally unclear. Recently, we modeled the 3D-structure of human SIRT1, the most studied member of this family, that unexpectedly emerged as a member of the intrinsically disordered proteins with its long disordered terminal arms. Despite clear similarities in catalytic cores between the human sirtuins little is known of the general structural characteristics of these proteins. The presence of disorder in human SIRT1 and the propensity of these proteins in promoting molecular interactions make it important to understand the underlying mechanisms of molecular recognition that reasonably should involve terminal segments. The mechanism of recognition, in turn, is a prerequisite for the understanding of any functional activity. Aim of this work is to understand what structural properties are shared among members of this family in humans as well as in other organisms. Results We have studied the distribution of the structural features of N- and C-terminal segments of sirtuins in all known organisms to draw their evolutionary histories by taking into account average length of terminal segments, amino acid composition, intrinsic disorder, presence of charged stretches, presence of putative phosphorylation sites, flexibility, and GC content of genes. Finally, we have carried out a comprehensive analysis of the putative

  16. Quantification of protein copy number in single mitochondria: The Bcl-2 family proteins.

    PubMed

    Chen, Chaoxiang; Zhang, Xiang; Zhang, Shuyue; Zhu, Shaobin; Xu, Jingyi; Zheng, Yan; Han, Jinyan; Zeng, Jin-Zhang; Yan, Xiaomei

    2015-12-15

    Bcl-2 family proteins, represented by antiapoptotic protein Bcl-2 and proapoptotic protein Bax, are key regulators of mitochondria-mediated apoptosis pathway. To build a quantitative model of how Bcl-2 family protein interactions control mitochondrial outer membrane permeabilization and subsequent cytochrome c release, it is essential to know the number of proteins in individual mitochondria. Here, we report an effective method to quantify the copy number and distribution of proteins in single mitochondria via immunofluorescent labeling and sensitive detection by a laboratory-built high sensitivity flow cytometer (HSFCM). Mitochondria isolated from HeLa cells were stained with Alexa Fluor 488 (AF488)-labeled monoclonal antibodies specifically targeting Bcl-2 or Bax and with nucleic acid dye. A series of fluorescent nanospheres with fluorescence intensity calibrated in the unit of molecules of equivalent soluble fluorochrome (MESF)-AF488 were used to construct a calibration curve for converting the immunofluorescence of a single mitochondrion to the number of antibodies bound to it and then to the number of proteins per mitochondrion. Under the normal condition, the measured mean copy numbers were 1300 and 220 per mitochondrion for Bcl-2 and Bax, respectively. A significant variation in protein copy number was identified, which ranged from 130 to 6000 (2.5-97.5%) for Bcl-2 and from 65 to 700 (2.5-97.5%) for Bax, respectively. We observed an approximately 4.4 fold increase of Bax copy number per mitochondrion upon 9h of apoptosis stimulation while the abundance of Bcl-2 remained almost unchanged. To the best of our knowledge, this is the first report of Bcl-2 family protein copy number and variance in single mitochondria. Collectively, we demonstrate that the HSFCM-based immunoassay provides a rapid and sensitive method for determining protein copy number distribution in single mitochondria.

  17. Targeting human SET1/MLL family of proteins

    PubMed Central

    Blazer, Levi; Eram, Mohammad S.; Barsyte‐Lovejoy, Dalia; Arrowsmith, Cheryl H.; Hajian, Taraneh

    2017-01-01

    Abstract The SET1 family of proteins, and in particular MLL1, are essential regulators of transcription and key mediators of normal development and disease. Here, we summarize the detailed characterization of the methyltransferase activity of SET1 complexes and the role of the key subunits, WDR5, RbBP5, ASH2L, and DPY30. We present new data on full kinetic characterization of human MLL1, MLL3, SET1A, and SET1B trimeric, tetrameric, and pentameric complexes to elaborate on substrate specificities and compare our findings with what has been reported before. We also review exciting recent work identifying potent inhibitors of oncogenic MLL1 function through disruption of protein–protein interactions within the MLL1 complex. PMID:28160335

  18. Palmitoylation of POTE family proteins for plasma membrane targeting

    SciTech Connect

    Das, Sudipto; Ise, Tomoko; Nagata, Satoshi; Maeda, Hiroshi; Bera, Tapan K.; Pastan, Ira

    2007-11-23

    The POTE gene family is composed of 13 paralogs and likely evolved by duplications and remodeling of the human genome. One common property of POTE proteins is their localization on the inner aspect of the plasma membrane. To determine the structural elements required for membrane localization, we expressed mutants of different POTEs in 293T cells as EGFP fusion proteins. We also tested their palmitoylation by a biotin-switch assay. Our data indicate that the membrane localizations of different POTEs are mediated by similar 3-4 short cysteine rich repeats (CRRs) near the amino-terminuses and that palmitoylation on paired cysteine residues in each CRR motif is responsible for the localization. Multiple palmitoylation in the small CRRs can result in the strong association of whole POTEs with plasma membrane.

  19. The aquaporin family of water channel proteins in clinical medicine.

    PubMed

    Lee, M D; King, L S; Agre, P

    1997-05-01

    The aquaporins are a family of membrane channel proteins that serve as selective pores through which water crosses the plasma membranes of many human tissues and cell types. The sites where aquaporins are expressed implicate these proteins in renal water reabsorption, cerebrospinal fluid secretion and reabsorption, generation of pulmonary secretions, aqueous humor secretion and reabsorption, lacrimation, and multiple other physiologic processes. Determination of the aquaporin gene sequences and their chromosomal locations has provided insight into the structure and pathophysiologic roles of these proteins, and primary and secondary involvement of aquaporins is becoming apparent in diverse clinical disorders. Aquaporin-1 (AQP1) is expressed in multiple tissues including red blood cells, and the Colton blood group antigens represent a polymorphism on the AQP1 protein. AQP2 is restricted to renal collecting ducts and has been linked to congenital nephrogenic diabetes insipidus in humans and to lithium-induced nephrogenic diabetes insipidus and fluid retention from congestive heart failure in rat models. Congenital cataracts result from mutations in the mouse gene encoding the lens homolog Aqp0 (Mip). The present understanding of aquaporin physiology is still incomplete; identification of additional members of the aquaporin family will affect future studies of multiple disorders of water distribution throughout the body. In some tissues, the aquaporins may participate in the transepithelial movement of fluid without being rate limiting, so aquaporins may be involved in clinical disorders without being causative. As outlined in this review, our challenge is to identify disease states in which aquaporins are involved, to define the aquaporins' roles mechanistically, and to search for ways to exploit this information therapeutically.

  20. 14-3-3ζ turns TGF-β’s function from tumor suppressor to metastasis promoter in breast cancer by contextual changes of Smad partners from p53 to Gli2

    PubMed Central

    Xu, Jia; Acharya, Sunil; Sahin, Ozgur; Zhang, Qingling; Saito, Yohei; Yao, Jun; Wang, Hai; Li, Ping; Zhang, Lin; Lowery, Frank J; Kuo, Wen-Ling; Xiao, Yi; Ensor, Joe; Sahin, Aysegul A; Zhang, Xiang H.-F.; Hung, Mien-Chie; Zhang, Jitao David; Yu, Dihua

    2015-01-01

    Summary Transforming growth factor-β (TGF-β) functions as a tumor suppressor in pre-malignant cells but as a metastasis promoter in cancer cells. The dichotomous functions of TGF-β are proposed to be dictated by different partners of its downstream effectors Smads. However, the mechanism for the contextual changes of Smad partners remained undefined. Here, we demonstrate that 14-3-3ζ destabilizes p53, a Smad partner in pre-malignant mammary epithelial cells, by downregulating 14-3-3σ, thus turning off TGF-β’s tumor suppression function. Conversely, 14-3-3ζ stabilizes Gli2 in breast cancer cells, and Gli2 partners with Smads to activate PTHrP and promote TGF-β-induced bone metastasis. The 14-3-3ζ-driven contextual changes of Smad partners from p53 to Gli2 may serve as biomarkers and therapeutic targets of TGF-β-mediated cancer progression. PMID:25670079

  1. IκB kinase-induced interaction of TPL-2 kinase with 14-3-3 is essential for Toll-like receptor activation of ERK-1 and -2 MAP kinases

    PubMed Central

    Ben-Addi, Abduelhakem; Mambole-Dema, Agnes; Brender, Christine; Martin, Stephen R.; Janzen, Julia; Kjaer, Sven; Smerdon, Stephen J.; Ley, Steven C.

    2014-01-01

    The MEK-1/2 kinase TPL-2 is critical for Toll-like receptor activation of the ERK-1/2 MAP kinase pathway during inflammatory responses, but it can transform cells following C-terminal truncation. IκB kinase (IKK) complex phosphorylation of the TPL-2 C terminus regulates full-length TPL-2 activation of ERK-1/2 by a mechanism that has remained obscure. Here, we show that TPL-2 Ser-400 phosphorylation by IKK and TPL-2 Ser-443 autophosphorylation cooperated to trigger TPL-2 association with 14-3-3. Recruitment of 14-3-3 to the phosphorylated C terminus stimulated TPL-2 MEK-1 kinase activity, which was essential for TPL-2 activation of ERK-1/2. The binding of 14-3-3 to TPL-2 was also indispensible for lipopolysaccharide-induced production of tumor necrosis factor by macrophages, which is regulated by TPL-2 independently of ERK-1/2 activation. Our data identify a key step in the activation of TPL-2 signaling and provide a mechanistic insight into how C-terminal deletion triggers the oncogenic potential of TPL-2 by rendering its kinase activity independent of 14-3-3 binding. PMID:24912162

  2. Docking validation resources: protein family and ligand flexibility experiments.

    PubMed

    Mukherjee, Sudipto; Balius, Trent E; Rizzo, Robert C

    2010-11-22

    A database consisting of 780 ligand-receptor complexes, termed SB2010, has been derived from the Protein Databank to evaluate the accuracy of docking protocols for regenerating bound ligand conformations. The goal is to provide easily accessible community resources for development of improved procedures to aid virtual screening for ligands with a wide range of flexibilities. Three core experiments using the program DOCK, which employ rigid (RGD), fixed anchor (FAD), and flexible (FLX) protocols, were used to gauge performance by several different metrics: (1) global results, (2) ligand flexibility, (3) protein family, and (4) cross-docking. Global spectrum plots of successes and failures vs rmsd reveal well-defined inflection regions, which suggest the commonly used 2 Å criteria is a reasonable choice for defining success. Across all 780 systems, success tracks with the relative difficulty of the calculations: RGD (82.3%) > FAD (78.1%) > FLX (63.8%). In general, failures due to scoring strongly outweigh those due to sampling. Subsets of SB2010 grouped by ligand flexibility (7-or-less, 8-to-15, and 15-plus rotatable bonds) reveal that success degrades linearly for FAD and FLX protocols, in contrast to RGD, which remains constant. Despite the challenges associated with FLX anchor orientation and on-the-fly flexible growth, success rates for the 7-or-less (74.5%) and, in particular, the 8-to-15 (55.2%) subset are encouraging. Poorer results for the very flexible 15-plus set (39.3%) indicate substantial room for improvement. Family-based success appears largely independent of ligand flexibility, suggesting a strong dependence on the binding site environment. For example, zinc-containing proteins are generally problematic, despite moderately flexible ligands. Finally, representative cross-docking examples, for carbonic anhydrase, thermolysin, and neuraminidase families, show the utility of family-based analysis for rapid identification of particularly good or bad

  3. The Golgin Family of Coiled-Coil Tethering Proteins

    PubMed Central

    Witkos, Tomasz M.; Lowe, Martin

    2016-01-01

    The golgins are a family of predominantly coiled-coil proteins that are localized to the Golgi apparatus. Golgins are present in all eukaryotes, suggesting an evolutionary conserved function. Golgins are anchored to the Golgi membrane by their carboxy terminus and are predicted to adopt an extended conformation that projects into the surrounding cytoplasm. This arrangement is ideal for the capture or tethering of nearby membranes or cytoskeletal elements. Golgin-mediated tethering is thought to be important for vesicular traffic at the Golgi apparatus, the maintenance of Golgi architecture, as well as the positioning of the Golgi apparatus within cells. In addition to acting as tethers, some golgins can also sequester various factors at the Golgi membrane, allowing for the spatiotemporal regulation of downstream cellular functions. Although it is now established that golgins are membrane and cytoskeleton tethers, the mechanisms underlying tethering remain poorly defined. Moreover, the importance of golgin-mediated tethering in a physiological context remains to be fully explored. This review will describe our current understanding of golgin function, highlighting recent progress that has been made, and goes on to discuss outstanding questions and potential avenues for future research with regard to this family of conserved Golgi-associated proteins. PMID:26793708

  4. Chemosensitization of Prostate Cancer by Modulating Bcl-2 Family Proteins

    PubMed Central

    Karnak, David; Xu, Liang

    2010-01-01

    A major challenge in oncology is the development of chemoresistance. This often occurs as cancer progresses and malignant cells acquire mechanisms to resist insults that would normally induce apoptosis. The onset of androgen independence in advanced prostate cancer is a prime example of this phenomenon. Overexpression of the pro-survival/anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 are hallmarks of this transition. Here we outline the evolution of therapeutics designed to either limit the source or disrupt the interactions of these pro-survival proteins. By either lessening the stoichiometric abundance of Bcl-2/xL/Mcl-1 in reference to their pro-apoptotic foils or freeing these pro-apoptotic proteins from their grip, these treatments aim to sensitize cells to chemotherapy by priming cells for death. DNA anti-sense and RNA interference have been effectively employed to decrease Bcl-2 family mRNA and protein levels in cell culture models of advanced prostate cancer. However, clinical studies are lagging due to in vivo delivery challenges. The burgeoning field of nanoparticle delivery holds great promise in helping to overcome the challenge of administering highly labile nucleic acid based therapeutics. On another front, small molecule inhibitors that block the hetero-dimerization of pro-survival with pro-apoptotic proteins have significant clinical advantages and have advanced farther in clinical trials with promising early results. Most recently, a peptide has been discovered that can convert Bcl-2 from a pro-survival to a pro-apoptotic protein. The future may lie in targeting multiple steps of the apoptotic pathway, including Bcl-2/xL/Mcl-1, to debilitate the survival capacity of cancer cells and make chemotherapy induced death their only option. PMID:20298153

  5. The Cbln family of proteins interact with multiple signaling pathways.

    PubMed

    Wei, Peng; Pattarini, Roberto; Rong, Yongqi; Guo, Hong; Bansal, Parmil K; Kusnoor, Sheila V; Deutch, Ariel Y; Parris, Jennifer; Morgan, James I

    2012-06-01

    Cerebellin precursor protein (Cbln1) is essential for synapse integrity in cerebellum through assembly into complexes that bridge pre-synaptic β-neurexins (Nrxn) to post-synaptic GluRδ2. However, GluRδ2 is largely cerebellum-specific, yet Cbln1 and its little studied family members, Cbln2 and Cbln4, are expressed throughout brain. Therefore, we investigated whether additional proteins mediate Cbln family actions. Whereas Cbln1 and Cbln2 bound to GluRδ2 and Nrxns1-3, Cbln4 bound weakly or not at all, suggesting it has distinct binding partners. In a candidate receptor-screening assay, Cbln4 (but not Cbln1 or Cbln2) bound selectively to the netrin receptor, (deleted in colorectal cancer (DCC) in a netrin-displaceable fashion. To determine whether Cbln4 had a netrin-like function, Cbln4-null mice were generated. Cbln4-null mice did not phenocopy netrin-null mice. Cbln1 and Cbln4 were likely co-localized in neurons thought to be responsible for synaptic changes in striatum of Cbln1-null mice. Furthermore, complexes containing Cbln1 and Cbln4 had greatly reduced affinity to DCC but increased affinity to Nrxns, suggesting a functional interaction. However, Cbln4-null mice lacked the striatal synaptic changes seen in Cbln null mice. Thus, Cbln family members interact with multiple receptors/signaling pathways in a subunit composition-dependent manner and have independent functions with Cbln4 potentially involved in the less well-characterized role of netrin/DCC in adult brain.

  6. Proteins on the catwalk: modelling the structural domains of the CCN family of proteins.

    PubMed

    Holbourn, Kenneth P; Perbal, Bernard; Ravi Acharya, K

    2009-03-01

    The CCN family of proteins (CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6) are multifunctional mosaic proteins that play keys roles in crucial areas of physiology such as angiogenesis, skeletal development tumourigenesis, cell proliferation, adhesion and survival. This expansive repertoire of functions comes through a modular structure of 4 discrete domains that act both independently and in concert. How these interactions with ligands and with neighbouring domains lead to the biological effects is still to be explored but the molecular structure of the domains is likely to play an important role in this. In this review we have highlighted some of the key features of the individual domains of CCN family of proteins based on their biological effects using a homology modelling approach.

  7. Analysis of membrane proteins in metagenomics: networks of correlated environmental features and protein families.

    PubMed

    Patel, Prianka V; Gianoulis, Tara A; Bjornson, Robert D; Yip, Kevin Y; Engelman, Donald M; Gerstein, Mark B

    2010-07-01

    Recent metagenomics studies have begun to sample the genomic diversity among disparate habitats and relate this variation to features of the environment. Membrane proteins are an intuitive, but thus far overlooked, choice in this type of analysis as they directly interact with the environment, receiving signals from the outside and transporting nutrients. Using global ocean sampling (GOS) data, we found nearly approximately 900,000 membrane proteins in large-scale metagenomic sequence, approximately a fifth of which are completely novel, suggesting a large space of hitherto unexplored protein diversity. Using GPS coordinates for the GOS sites, we extracted additional environmental features via interpolation from the World Ocean Database, the National Center for Ecological Analysis and Synthesis, and empirical models of dust occurrence. This allowed us to study membrane protein variation in terms of natural features, such as phosphate and nitrate concentrations, and also in terms of human impacts, such as pollution and climate change. We show that there is widespread variation in membrane protein content across marine sites, which is correlated with changes in both oceanographic variables and human factors. Furthermore, using these data, we developed an approach, protein families and environment features network (PEN), to quantify and visualize the correlations. PEN identifies small groups of covarying environmental features and membrane protein families, which we call "bimodules." Using this approach, we find that the affinity of phosphate transporters is related to the concentration of phosphate and that the occurrence of iron transporters is connected to the amount of shipping, pollution, and iron-containing dust.

  8. Non-apoptotic functions of BCL-2 family proteins.

    PubMed

    Gross, Atan; Katz, Samuel G

    2017-02-24

    The BCL-2 family proteins are major regulators of the apoptosis process, but the mechanisms by which they regulate this process are only partially understood. It is now well documented that these proteins play additional non-apoptotic roles that are likely to be related to their apoptotic roles and to provide important clues to cracking their mechanisms of action. It seems that these non-apoptotic roles are largely related to the activation of cellular survival pathways designated to maintain or regain cellular survival, but, if unsuccessful, will switch over into a pro-apoptotic mode. These non-apoptotic roles span a wide range of processes that include the regulation of mitochondrial physiology (metabolism, electron transport chain, morphology, permeability transition), endoplasmic reticulum physiology (calcium homeostasis, unfolded protein response (UPR)), nuclear processes (cell cycle, DNA damage response (DDR)), whole-cell metabolism (glucose and lipid), and autophagy. Here we review all these different non-apoptotic roles, make an attempt to link them to the apoptotic roles, and present many open questions for future research directions in this fascinating field.Cell Death and Differentiation advance online publication, 24 February 2017; doi:10.1038/cdd.2017.22.

  9. Yeast as a tool for studying proteins of the Bcl-2 family.

    PubMed

    Polčic, Peter; Jaká, Petra; Mentel, Marek

    2015-03-02

    Permeabilization of the outer mitochondrial membrane that leads to the release of cytochrome c and several other apoptogenic proteins from mitochondria into cytosol represents a commitment point of apoptotic pathway in mammalian cells. This crucial event is governed by proteins of the Bcl-2 family. Molecular mechanisms, by which Bcl-2 family proteins permeabilize mitochondrial membrane, remain under dispute. Although yeast does not have apparent homologues of these proteins, when mammalian members of Bcl-2 family are expressed in yeast, they retain their activity, making yeast an attractive model system, in which to study their action. This review focuses on using yeast expressing mammalian proteins of the Bcl-2 family as a tool to investigate mechanisms, by which these proteins permeabilize mitochondrial membranes, mechanisms, by which pro- and antiapoptotic members of this family interact, and involvement of other cellular components in the regulation of programmed cell death by Bcl-2 family proteins.

  10. Yeast as a tool for studying proteins of the Bcl-2 family

    PubMed Central

    Polčic, Peter; Jaká, Petra; Mentel, Marek

    2015-01-01

    Permeabilization of the outer mitochondrial membrane that leads to the release of cytochrome c and several other apoptogenic proteins from mitochondria into cytosol represents a commitment point of apoptotic pathway in mammalian cells. This crucial event is governed by proteins of the Bcl-2 family. Molecular mechanisms, by which Bcl-2 family proteins permeabilize mitochondrial membrane, remain under dispute. Although yeast does not have apparent homologues of these proteins, when mammalian members of Bcl-2 family are expressed in yeast, they retain their activity, making yeast an attractive model system, in which to study their action. This review focuses on using yeast expressing mammalian proteins of the Bcl-2 family as a tool to investigate mechanisms, by which these proteins permeabilize mitochondrial membranes, mechanisms, by which pro- and antiapoptotic members of this family interact, and involvement of other cellular components in the regulation of programmed cell death by Bcl-2 family proteins. PMID:28357280

  11. Structural and Functional Characterization of the VQ Protein Family and VQ Protein Variants from Soybean

    PubMed Central

    Zhou, Yuan; Yang, Yan; Zhou, Xinjian; Chi, Yingjun; Fan, Baofang; Chen, Zhixiang

    2016-01-01

    Proteins containing the FxxxVQxhTG or VQ motif interact with WRKY transcription factors. Although VQ proteins have been reported in several plants, knowledge about their structures, functions and evolution is still very limited. Here, we report structural and functional analysis of the VQ protein family from soybean. Like Arabidopsis homologues, soybean VQ proteins bind only Group I and IIc WRKY proteins and a substantial number of their genes are responsive to stress-associated phytohormones. Overexpression of some soybean VQ genes in Arabidopsis had strong effects on plant growth, development, disease resistance and heat tolerance. Phylogenetic analysis, sequence alignment and site-directed mutagenesis revealed that the region immediately upstream of the FxxxVQxhTG motif also affects binding to WRKY proteins. Consistent with a larger WRKY-binding VQ domain, soybean VQ22 protein from cultivated soybean contains a 4-amino acid deletion in the region preceding its VQ motif that completely abolishes its binding to WRKY proteins. By contrast, the 4-amino acid deletion is absent in the VQ22 protein from wild soybean species (Glycine soja). Overexpression of wild soybean VQ22 in cultivated soybean inhibited growth, particularly after cold treatment. Thus, the mutation of soybean VQ22 is associated with advantageous phenotypes and may have been positively selected during evolution. PMID:27708406

  12. The Overlap of Small Molecule and Protein Binding Sites within Families of Protein Structures

    PubMed Central

    Davis, Fred P.; Sali, Andrej

    2010-01-01

    Protein–protein interactions are challenging targets for modulation by small molecules. Here, we propose an approach that harnesses the increasing structural coverage of protein complexes to identify small molecules that may target protein interactions. Specifically, we identify ligand and protein binding sites that overlap upon alignment of homologous proteins. Of the 2,619 protein structure families observed to bind proteins, 1,028 also bind small molecules (250–1000 Da), and 197 exhibit a statistically significant (p<0.01) overlap between ligand and protein binding positions. These “bi-functional positions”, which bind both ligands and proteins, are particularly enriched in tyrosine and tryptophan residues, similar to “energetic hotspots” described previously, and are significantly less conserved than mono-functional and solvent exposed positions. Homology transfer identifies ligands whose binding sites overlap at least 20% of the protein interface for 35% of domain–domain and 45% of domain–peptide mediated interactions. The analysis recovered known small-molecule modulators of protein interactions as well as predicted new interaction targets based on the sequence similarity of ligand binding sites. We illustrate the predictive utility of the method by suggesting structural mechanisms for the effects of sanglifehrin A on HIV virion production, bepridil on the cellular entry of anthrax edema factor, and fusicoccin on vertebrate developmental pathways. The results, available at http://pibase.janelia.org, represent a comprehensive collection of structurally characterized modulators of protein interactions, and suggest that homologous structures are a useful resource for the rational design of interaction modulators. PMID:20140189

  13. New Functions for the Ancient DedA Membrane Protein Family

    PubMed Central

    Sikdar, Rakesh; Kumar, Sujeet; Boughner, Lisa A.

    2013-01-01

    The DedA protein family is a highly conserved and ancient family of membrane proteins with representatives in most sequenced genomes, including those of bacteria, archaea, and eukarya. The functions of the DedA family proteins remain obscure. However, recent genetic approaches have revealed important roles for certain bacterial DedA family members in membrane homeostasis. Bacterial DedA family mutants display such intriguing phenotypes as cell division defects, temperature sensitivity, altered membrane lipid composition, elevated envelope-related stress responses, and loss of proton motive force. The DedA family is also essential in at least two species of bacteria: Borrelia burgdorferi and Escherichia coli. Here, we describe the phylogenetic distribution of the family and summarize recent progress toward understanding the functions of the DedA membrane protein family. PMID:23086209

  14. MicroRNAs affect BCL-2 family proteins in the setting of cerebral ischemia.

    PubMed

    Ouyang, Yi-Bing; Giffard, Rona G

    2014-11-01

    The BCL-2 family is centrally involved in the mechanism of cell death after cerebral ischemia. It is well known that the proteins of the BCL-2 family are key regulators of apoptosis through controlling mitochondrial outer membrane permeabilization. Recent findings suggest that many BCL-2 family members are also directly involved in controlling transmission of Ca(2+) from the endoplasmic reticulum (ER) to mitochondria through a specialization called the mitochondria-associated ER membrane (MAM). Increasing evidence supports the involvement of microRNAs (miRNAs), some of them targeting BCL-2 family proteins, in the regulation of cerebral ischemia. In this mini-review, after highlighting current knowledge about the multiple functions of BCL-2 family proteins and summarizing their relationship to outcome from cerebral ischemia, we focus on the regulation of BCL-2 family proteins by miRNAs, especially miR-29 which targets multiple BCL-2 family proteins.

  15. PATtyFams: Protein Families for the Microbial Genomes in the PATRIC Database

    PubMed Central

    Davis, James J.; Gerdes, Svetlana; Olsen, Gary J.; Olson, Robert; Pusch, Gordon D.; Shukla, Maulik; Vonstein, Veronika; Wattam, Alice R.; Yoo, Hyunseung

    2016-01-01

    The ability to build accurate protein families is a fundamental operation in bioinformatics that influences comparative analyses, genome annotation, and metabolic modeling. For several years we have been maintaining protein families for all microbial genomes in the PATRIC database (Pathosystems Resource Integration Center, patricbrc.org) in order to drive many of the comparative analysis tools that are available through the PATRIC website. However, due to the burgeoning number of genomes, traditional approaches for generating protein families are becoming prohibitive. In this report, we describe a new approach for generating protein families, which we call PATtyFams. This method uses the k-mer-based function assignments available through RAST (Rapid Annotation using Subsystem Technology) to rapidly guide family formation, and then differentiates the function-based groups into families using a Markov Cluster algorithm (MCL). This new approach for generating protein families is rapid, scalable and has properties that are consistent with alignment-based methods. PMID:26903996

  16. PATtyFams: Protein families for the microbial genomes in the PATRIC database

    SciTech Connect

    Davis, James J.; Gerdes, Svetlana; Olsen, Gary J.; Olson, Robert; Pusch, Gordon D.; Shukla, Maulik; Vonstein, Veronika; Wattam, Alice R.; Yoo, Hyunseung

    2016-02-08

    The ability to build accurate protein families is a fundamental operation in bioinformatics that influences comparative analyses, genome annotation, and metabolic modeling. For several years we have been maintaining protein families for all microbial genomes in the PATRIC database (Pathosystems Resource Integration Center, patricbrc.org) in order to drive many of the comparative analysis tools that are available through the PATRIC website. However, due to the burgeoning number of genomes, traditional approaches for generating protein families are becoming prohibitive. In this report, we describe a new approach for generating protein families, which we call PATtyFams. This method uses the k-mer-based function assignments available through RAST (Rapid Annotation using Subsystem Technology) to rapidly guide family formation, and then differentiates the function-based groups into families using a Markov Cluster algorithm (MCL). In conclusion, this new approach for generating protein families is rapid, scalable and has properties that are consistent with alignment-based methods.

  17. The secretory carrier membrane protein family: structure and membrane topology.

    PubMed

    Hubbard, C; Singleton, D; Rauch, M; Jayasinghe, S; Cafiso, D; Castle, D

    2000-09-01

    Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain-binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is approximately 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an approximately 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane

  18. Deciphering the molecular and functional basis of Dbl family proteins: a novel systematic approach toward classification of selective activation of the Rho family proteins.

    PubMed

    Jaiswal, Mamta; Dvorsky, Radovan; Ahmadian, Mohammad Reza

    2013-02-08

    The diffuse B-cell lymphoma (Dbl) family of the guanine nucleotide exchange factors is a direct activator of the Rho family proteins. The Rho family proteins are involved in almost every cellular process that ranges from fundamental (e.g. the establishment of cell polarity) to highly specialized processes (e.g. the contraction of vascular smooth muscle cells). Abnormal activation of the Rho proteins is known to play a crucial role in cancer, infectious and cognitive disorders, and cardiovascular diseases. However, the existence of 74 Dbl proteins and 25 Rho-related proteins in humans, which are largely uncharacterized, has led to increasing complexity in identifying specific upstream pathways. Thus, we comprehensively investigated sequence-structure-function-property relationships of 21 representatives of the Dbl protein family regarding their specificities and activities toward 12 Rho family proteins. The meta-analysis approach provides an unprecedented opportunity to broadly profile functional properties of Dbl family proteins, including catalytic efficiency, substrate selectivity, and signaling specificity. Our analysis has provided novel insights into the following: (i) understanding of the relative differences of various Rho protein members in nucleotide exchange; (ii) comparing and defining individual and overall guanine nucleotide exchange factor activities of a large representative set of the Dbl proteins toward 12 Rho proteins; (iii) grouping the Dbl family into functionally distinct categories based on both their catalytic efficiencies and their sequence-structural relationships; (iv) identifying conserved amino acids as fingerprints of the Dbl and Rho protein interaction; and (v) defining amino acid sequences conserved within, but not between, Dbl subfamilies. Therefore, the characteristics of such specificity-determining residues identified the regions or clusters conserved within the Dbl subfamilies.

  19. Phylogenetic analysis of the Argonaute protein family in platyhelminths.

    PubMed

    Zheng, Yadong

    2013-03-01

    Argonaute proteins (AGOs) are mediators of gene silencing via recruitment of small regulatory RNAs to induce translational regression or degradation of targeted molecules. Platyhelminths have been reported to express microRNAs but the diversity of AGOs in the phylum has not been explored. Phylogenetic relationships of members of this protein family were studied using data from six platyhelminth genomes. Phylogenetic analysis showed that all cestode and trematode AGOs, along with some triclad planarian AGOs, were grouped into the Ago subfamily and its novel sister clade, here referred to as Cluster 1. These were very distant from Piwi and Class 3 subfamilies. By contrast, a number of planarian Piwi-like AGOs formed a novel sister clade to the Piwi subfamily. Extensive sequence searching revealed the presence of an additional locus for AGO2 in the cestode Echinococcus granulosus and exon expansion in this species and E. multilocularis. The current study suggests the absence of the Piwi subfamily and Class 3 AGOs in cestodes and trematodes and the Piwi-like AGO expansion in a free-living triclad planarian and the occurrence of exon expansion prior to or during the evolution of the most-recent common ancestor of the Echinococcus species studied.

  20. A large family of anti-activators accompanying XylS/AraC family regulatory proteins.

    PubMed

    Santiago, Araceli E; Yan, Michael B; Tran, Minh; Wright, Nathan; Luzader, Deborah H; Kendall, Melissa M; Ruiz-Perez, Fernando; Nataro, James P

    2016-07-01

    AraC Negative Regulators (ANR) suppress virulence genes by directly down-regulating AraC/XylS members in Gram-negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR-activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC-like member AggR. ANR-AggR binding disrupted AggR dimerization and prevented AggR-DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α-helices. Site-directed mutagenesis studies suggest that at least predicted α-helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners.

  1. A large family of anti‐activators accompanying XylS/AraC family regulatory proteins

    PubMed Central

    Yan, Michael B.; Tran, Minh; Wright, Nathan; Luzader, Deborah H.; Kendall, Melissa M.; Ruiz‐Perez, Fernando; Nataro, James P.

    2016-01-01

    Summary AraC Negative Regulators (ANR) suppress virulence genes by directly down‐regulating AraC/XylS members in Gram‐negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR‐activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC‐like member AggR. ANR‐AggR binding disrupted AggR dimerization and prevented AggR‐DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α‐helices. Site‐directed mutagenesis studies suggest that at least predicted α‐helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners. PMID:27038276

  2. YAP and 14-3-3γ are involved in HS-OA-induced growth inhibition of hepatocellular carcinoma cells: A novel mechanism for hydrogen sulfide releasing oleanolic acid

    PubMed Central

    Xu, Guanglin; Wang, Jing; Wu, Fangfang; Wang, Na; Zhou, Wenli; Wang, Qian; Pan, Wang; Ao, Guizhen; Yang, Jiquan

    2016-01-01

    Hydrogen sulfide-releasing oleanolic acid (HS-OA) is an emerging novel class of compounds and consists of an oleanolic acid (OA) and a H2S-releasing moiety. Although it exhibits improved anti-inflammatory activity, its potency in human cancers has not been understood yet. In this study, we examined the effects of HS-OA on the growth of liver cancer cell lines and the underlying mechanisms. HS-OA inhibited the growth of all four cancer cell lines studied, with potencies of 10- to 30-fold greater than that of its counterpart (OA). HS-OA induced significant apoptosis and decreased viability, clonogenic activity and migration of Hep G2 cells. Further studies showed that HS-OA resulted in the reduction of YAP expression and its downstream targets, CTGF and CYR 61, thus promoting cell apoptosis. In addition, HS-OA caused a decrease of 14-3-3γ expression, which led to Bad translocation to the mitochondria, ΔΨm loss, cytochrome c release, caspase activation and a recovery of 14-3-3γ reversed these effects induced by HS-OA. These findings indicate that YAP and 14-3-3γ are involved in HS-OA's effects on liver cancer cells and identifying HS-OA as a potential new drug candidate for cancer therapy. PMID:27437776

  3. Homeodomain-interacting protein kinase (Hipk) phosphorylates the small SPOC family protein Spenito.

    PubMed

    Dewald, D N; Steinmetz, E L; Walldorf, U

    2014-12-01

    The Drosophila homeodomain-interacting protein kinase (Hipk) is a versatile regulator involved in a variety of pathways, such as Notch and Wingless signalling, thereby acting in processes including the promotion of eye development or control of cell numbers in the nervous system. In vertebrates, extensive studies have related its homologue HIPK2 to important roles in the control of p53-mediated apoptosis and tumour suppression. Spenito (Nito) belongs to the group of small SPOC family proteins and has a role, amongst others, as a regulator of Wingless signalling downstream of Armadillo. In the present study, we show that both proteins have an enzyme-substrate relationship, adding a new interesting component to the broad range of Hipk interactions, and we map several phosphorylation sites of Nito. Furthermore, we were able to define a preliminary consensus motif for Hipk target sites, which will simplify the identification of new substrates of this kinase.

  4. Small G-protein Signaling in Neuronal Plasticity and Memory Formation: the Specific Role of Ras Family Proteins

    PubMed Central

    Ye, Xiaojing; Carew, Thomas J.

    2010-01-01

    Small G-proteins are an extensive family of proteins that bind and hydrolyze GTP. They are ubiquitous inside cells, regulating a wide range of cellular processes. Recently, many studies have examined the role of small G-proteins, particularly the Ras family of G-proteins, in memory formation. Once thought to be primarily involved in the transduction of a variety of extracellular signals during development, it is now clear that Ras family proteins also play critical roles in molecular processing underlying neuronal and behavioral plasticity. We here review a number of recent studies that explore how the signaling of Ras family proteins contributes to memory formation. Understanding these signaling processes is of fundamental importance both from a basic scientific perspective, with the goal of providing mechanistic insights into a critical aspect of cognitive behavior, and from a clinical perspective, with the goal of providing effective therapies for a range of disorders involving cognitive impairments. PMID:21040840

  5. Spermadhesins: a new protein family. Facts, hypotheses and perspectives.

    PubMed

    Töpfer-Petersen, E; Romero, A; Varela, P F; Ekhlasi-Hundrieser, M; Dostàlovà, Z; Sanz, L; Calvete, J J

    1998-01-01

    Spermadhesins are a novel family of secretory proteins expressed in the male genital tract of pig, horse and bull. They are major products of the seminal plasma and have been found to be peripherally associated to the sperm surface. The structure and function of spermadhesins have been thoroughly investigated in the pig, which exhibits the greatest diversity of members: AWN, AQN-1, AQN-2, PSP-I and PSP-II and its glycosylated isoforms. They are multifunctional proteins showing a range of ligand-binding abilities, e.g. carbohydrates, sulfated glycosaminoglycans, phospholipids and protease inhibitors, suggesting that they may be involved in different steps of fertilization. Isolated porcine spermadhesins bind the zona pellucida glycoproteins in a cation-dependent manner with a Kd in a low micromolar range, and AWN, AQN-1 and AQN-3 display similar binding affinity for glycoproteins containing Gal beta(1-3)-GalNAc and Gal beta(1-4)-GlcNAc sequences in O-linked and N-linked oligosaccharides, respectively. During sperm passage through the epididymis AQN-3 and AWN have been shown to bind tightly to the sperm surface by interaction with the phospholipids of the membrane bilayer. At ejaculation the spermadhesins form a protective coat around the sensitive acrosomal region of the sperm head, thus possibly preventing premature acrosome reaction. During in vitro capacitation most of these aggregated sperm adhesins are lost, with the exception of phospholipid-bound spermadhesins. AWN and AQN-3 may now serve as a primary receptor for the oocyte zona pellucida, thus contributing to initial binding and recognition between sperm and egg. The amino acid sequence of spermadhesins does not show any discernible similarity with known carbohydrate recognition domains (CRD). However, they belong to the superfamily of proteins with a CUB domain with a predicted all-beta structure. The crystal structure of the heterodimeric complex of the spermadhesins PSP-I/PSP-II has been solved, showing

  6. Proteins of the PIAS family enhance the sumoylation of the papillomavirus E1 protein*

    PubMed Central

    Rosas-Acosta, Germán; Wilson, Van G.

    2012-01-01

    SUMMARY The E1 protein, a papillomavirus (PV)-encoded origin-binding helicase essential for PV DNA replication, is post-translationally modified by sumoylation. As this modification is essential for the nuclear accumulation of the bovine PV E1 (BPV E1), factors modulating the sumoylation of E1 could ultimately alter the outcome of a papillomavirus infection. Therefore, we systematically tested the known sumoylation enhancing factors (E3 SUMO ligases), namely RanBP2 and PIAS family proteins, to determine their ability to bind to E1 and stimulate its sumoylation, using in vitro assays. We found that RanBP2 bound to BPV E1 but failed to bind to the E1 from a human PV (HPV11 E1), and lacked any sumoylation enhancing activity for both BPV E1 and HPV11 E1. In contrast, proteins of the PIAS family (except for PIASy) bound to both BPV E1 and HPV11 E1 and stimulated their sumoylation, with PIASxβ (Miz1) exerting the largest stimulatory effect. The structural integrity of the RING finger domain of the PIAS proteins was required for their E3 SUMO ligase activity on PV E1 sumoylation, but was dispensable for their PV E1 binding activity. Furthermore, the sumoylation enhancing activity exerted by the PIAS proteins on BPV E1 was more pronounced than on HPV11 E1, and appeared to favor SUMO1 versus SUMO2 as the SUMO modifier. Altogether, this study identifies PIAS proteins as possible modulators of PV E1 sumoylation during papillomavirus infections. PMID:15582666

  7. The protein-protein interaction network of the human Sirtuin family.

    PubMed

    Sharma, Ankush; Costantini, Susan; Colonna, Giovanni

    2013-10-01

    Protein-protein interaction networks are useful for studying human diseases and to look for possible health care through a holistic approach. Networks are playing an increasing and important role in the understanding of physiological processes such as homeostasis, signaling, spatial and temporal organizations, and pathological conditions. In this article we show the complex system of interactions determined by human Sirtuins (Sirt) largely involved in many metabolic processes as well as in different diseases. The Sirtuin family consists of seven homologous Sirt-s having structurally similar cores but different terminal segments, being rather variable in length and/or intrinsically disordered. Many studies have determined their cellular location as well as biological functions although molecular mechanisms through which they act are actually little known therefore, the aim of this work was to define, explore and understand the Sirtuin-related human interactome. As a first step, we have integrated the experimentally determined protein-protein interactions of the Sirtuin-family as well as their first and second neighbors to a Sirtuin-related sub-interactome. Our data showed that the second-neighbor network of Sirtuins encompasses 25% of the entire human interactome, and exhibits a scale-free degree distribution and interconnectedness among top degree nodes. Moreover, the Sirtuin sub interactome showed a modular structure around the core comprising mixed functions. Finally, we extracted from the Sirtuin sub-interactome subnets related to cancer, aging and post-translational modifications for information on key nodes and topological space of the subnets in the Sirt family network.

  8. Methionine-rich repeat proteins: a family of membrane-associated proteins which contain unusual repeat regions.

    PubMed

    Weiss, Jamie L; Evans, Nicholas A; Ahmed, Tanweer; Wrigley, Jonathan D J; Khan, Shukria; Wright, Charles; Keen, Jeffrey N; Holzenburg, Andreas; Findlay, John B C

    2005-03-01

    We report the protein isolation, cloning and characterization of members of an unusual protein family, which comprise the most abundant proteins present in the squid eye. The proteins in this family have a range of molecular weights from 32 to 36 kDa. Electron microscopy and detergent solubilization demonstrate that these proteins are tightly associated with membrane structures where they may form tetramers. Despite this, these proteins have no stretches of hydrophobic residues that could form typical transmembrane domains. They share an unusual protein sequence rich in methionine, and contain multiple repeating motifs. We have therefore named these proteins Methionine-Rich Repeat Proteins (MRRPs). The use of structure prediction algorithms suggest very little recognized secondary structure elements. At the time of cloning no sequence or structural homologues have been found in any database. We have isolated three closely related cDNA clones from the MRRP family. Coupled in vitro transcription/translation of the MRRP clones shows that they encode proteins with molecular masses similar to components of native MRRPs. Immunoblot analysis of these proteins reveals that they are also present in squid brain, optic lobe, and heart, and also indicate that MRRP-like protein motifs may also exist in mammalian tissues. We propose that MRRPs define a family of important proteins that have an unusual mode of attachment or insertion into cell membranes and are found in evolutionarily diverse organisms.

  9. Adhesion family of G protein-coupled receptors and cancer.

    PubMed

    Lin, Hsi-Hsien

    2012-01-01

    The adhesion-class G protein-coupled receptors (adhesion-GPCRs) constitute the second largest GPCR sub-family in humans. Adhesion-GPCRs are defined by the chimeric structure of an unusually large extracellular cell-adhesion domain and a GPCR-like seven-pass transmembrane domain. Adhesion-GPCRs are hence expected to display both cellular adhesion and signaling functions in many biological systems. Adhesion-GPCRs are normally expressed in the central nervous, immune, and reproductive systems in a cell type- or tissue-restricted fashion. However, aberrant expression of distinct adhesion-GPCR molecules has been identified in various human cancers with some of the receptors closely associated with cancer development. Tumor-associated adhesion-GPCRs are thought to involve in tumorigenesis by affecting the growth of tumor cells, angiogenesis, tumor cell migration, invasion and metastasis either positively or negatively. Furthermore, some adhesion-GPCRs are considered potential biomarkers for specific types of cancers. In this review article, the expressional characteristics and functional role of cancer-associated adhesion-GPCRs are discussed in depth.

  10. Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

    PubMed Central

    Frost, J A; Xu, S; Hutchison, M R; Marcus, S; Cobb, M H

    1996-01-01

    The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway. PMID:8668187

  11. Internal organization of large protein families: relationship between the sequence, structure, and function-based clustering.

    PubMed

    Cai, Xiao-Hui; Jaroszewski, Lukasz; Wooley, John; Godzik, Adam

    2011-08-01

    The protein universe can be organized in families that group proteins sharing common ancestry. Such families display variable levels of structural and functional divergence, from homogenous families, where all members have the same function and very similar structure, to very divergent families, where large variations in function and structure are observed. For practical purposes of structure and function prediction, it would be beneficial to identify sub-groups of proteins with highly similar structures (iso-structural) and/or functions (iso-functional) within divergent protein families. We compared three algorithms in their ability to cluster large protein families and discuss whether any of these methods could reliably identify such iso-structural or iso-functional groups. We show that clustering using profile-sequence and profile-profile comparison methods closely reproduces clusters based on similarities between 3D structures or clusters of proteins with similar biological functions. In contrast, the still commonly used sequence-based methods with fixed thresholds result in vast overestimates of structural and functional diversity in protein families. As a result, these methods also overestimate the number of protein structures that have to be determined to fully characterize structural space of such families. The fact that one can build reliable models based on apparently distantly related templates is crucial for extracting maximal amount of information from new sequencing projects.

  12. Monoubiquitination of Tob/BTG family proteins competes with degradation-targeting polyubiquitination

    SciTech Connect

    Suzuki, Toru; Kim, Minsoo; Kozuka-Hata, Hiroko; Watanabe, Masato; Oyama, Masaaki; Tsumoto, Kouhei; Yamamoto, Tadashi

    2011-05-27

    Highlights: {yields} Tob/BTG family proteins are monoubiquitinated in the absence of E3s in vitro. {yields} Monoubiquitination sites of Tob are identified by mass spectrometry. {yields} The monoubiquitination event correlates with lower levels of polyubiquitination. -- Abstract: Tob belongs to the anti-proliferative Tob/BTG protein family. The expression level of Tob family proteins is strictly regulated both transcriptionally and through post-translational modification. Ubiquitin (Ub)/proteosome-dependent degradation of Tob family proteins is critical in controlling cell cycle progression and DNA damage responses. Various Ub ligases (E3s) are responsible for degradation of Tob protein. Here, we show that Tob family proteins undergo monoubiquitination even in the absence of E3s in vitro. Determination of the ubiquitination site(s) in Tob by mass spectrometric analysis revealed that two lysine residues (Lys48 and Lys63) located in Tob/BTG homology domain are ubiquitinated. A mutant Tob, in which both Lys48 and Lys63 are substituted with alanine, is more strongly polyubiquitinated than wild-type Tob in vivo. These data suggest that monoubiquitination of Tob family proteins confers resistance against polyubiquitination, which targets proteins for degradation. The strategy for regulating the stability of Tob family proteins suggests a novel role for monoubiquitination.

  13. Exploring metazoan evolution through dynamic and holistic changes in protein families and domains

    PubMed Central

    2012-01-01

    Background Proteins convey the majority of biochemical and cellular activities in organisms. Over the course of evolution, proteins undergo normal sequence mutations as well as large scale mutations involving domain duplication and/or domain shuffling. These events result in the generation of new proteins and protein families. Processes that affect proteome evolution drive species diversity and adaptation. Herein, change over the course of metazoan evolution, as defined by birth/death and duplication/deletion events within protein families and domains, was examined using the proteomes of 9 metazoan and two outgroup species. Results In studying members of the three major metazoan groups, the vertebrates, arthropods, and nematodes, we found that the number of protein families increased at the majority of lineages over the course of metazoan evolution where the magnitude of these increases was greatest at the lineages leading to mammals. In contrast, the number of protein domains decreased at most lineages and at all terminal lineages. This resulted in a weak correlation between protein family birth and domain birth; however, the correlation between domain birth and domain member duplication was quite strong. These data suggest that domain birth and protein family birth occur via different mechanisms, and that domain shuffling plays a role in the formation of protein families. The ratio of protein family birth to protein domain birth (domain shuffling index) suggests that shuffling had a more demonstrable effect on protein families in nematodes and arthropods than in vertebrates. Through the contrast of high and low domain shuffling indices at the lineages of Trichinella spiralis and Gallus gallus, we propose a link between protein redundancy and evolutionary changes controlled by domain shuffling; however, the speed of adaptation among the different lineages was relatively invariant. Evaluating the functions of protein families that appeared or disappeared at the

  14. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    PubMed Central

    Nissen, Klaus B.; Haugaard-Kedström, Linda M.; Wilbek, Theis S.; Nielsen, Line S.; Åberg, Emma; Kristensen, Anders S.; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins. PMID:25658767

  15. Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

    PubMed

    Nissen, Klaus B; Haugaard-Kedström, Linda M; Wilbek, Theis S; Nielsen, Line S; Åberg, Emma; Kristensen, Anders S; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.

  16. Bcl-2-related protein family gene expression during oligodendroglial differentiation.

    PubMed

    Itoh, Takayuki; Itoh, Aki; Pleasure, David

    2003-06-01

    Oligodendroglial lineage cells (OLC) vary in susceptibility to both necrosis and apoptosis depending on their developmental stages, which might be regulated by differential expression of Bcl-2-related genes. As an initial step to test this hypothesis, we examined the expression of 19 Bcl-2-related genes in purified cultures of rat oligodendroglial progenitors, immature and mature oligodendrocytes. All 'multidomain' anti-apoptotic members (Bcl-x, Bcl-2, Mcl-1, Bcl-w and Bcl2l10/Diva/Boo) except Bcl2a1/A1 are expressed in OLC. Semiquantitative and real-time RT-PCR revealed that Bcl-xL and Mcl-1 mRNAs are the dominant anti-apoptotic members and increase four- and twofold, respectively, with maturation. Bcl-2 mRNA is less abundant than Bcl-xL mRNA in progenitors and falls an additional 10-fold during differentiation. Bcl-w mRNA also increases, with significant changes in its splicing pattern, as OLC mature. Transfection studies demonstrated that Bcl-xL overexpression protects against kainate-induced excitotoxicity, whereas Bcl-2 overexpression does not. As for 'multidomain' pro-apoptotic members (Bax, Bad and Bok/Mtd), Bax and Bak are highly expressed throughout differentiation. Among 'BH3 domain-only' members examined (Bim, Biklk, DP5/Hrk, Bad, Bid, Noxa, Puma/Bbc3, Bmf, BNip3 and BNip3L), BNip3 and Bmf mRNAs increase markedly during differentiation. These results provide basic information to guide further studies on the roles for Bcl-2-related family proteins in OLC death.

  17. An unbiased expression screen for synaptogenic proteins identifies the LRRTM protein family as synaptic organizers.

    PubMed

    Linhoff, Michael W; Laurén, Juha; Cassidy, Robert M; Dobie, Frederick A; Takahashi, Hideto; Nygaard, Haakon B; Airaksinen, Matti S; Strittmatter, Stephen M; Craig, Ann Marie

    2009-03-12

    Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1(-/-) mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia.

  18. The CCN Family Proteins: Modulators of Bone Development and Novel Targets in Bone-Associated Tumors

    PubMed Central

    Chen, Po-Chun; Cheng, Hsu-Chen; Yang, Shun-Fa; Tang, Chih-Hsin

    2014-01-01

    The CCN family of proteins is composed of six extracellular matrix-associated proteins that play crucial roles in skeletal development, wound healing, fibrosis, and cancer. Members of the CCN family share four conserved cysteine-rich modular domains that trigger signal transduction in cell adhesion, migration, proliferation, differentiation, and survival through direct binding to specific integrin receptors and heparan sulfate proteoglycans. In the present review, we discuss the roles of the CCN family proteins in regulating resident cells of the bone microenvironment. In vertebrate development, the CCN family plays a critical role in osteo/chondrogenesis and vasculo/angiogenesis. These effects are regulated through signaling via integrins, bone morphogenetic protein, vascular endothelial growth factor, Wnt, and Notch via direct binding to CCN family proteins. Due to the important roles of CCN family proteins in skeletal development, abnormal expression of CCN proteins is related to the tumorigenesis of primary bone tumors such as osteosarcoma, Ewing sarcoma, and chondrosarcoma. Additionally, emerging studies have suggested that CCN proteins may affect progression of secondary metastatic bone tumors by moderating the bone microenvironment. CCN proteins could therefore serve as potential therapeutic targets for drug development against primary and metastatic bone tumors. PMID:24551846

  19. The CCN family proteins: modulators of bone development and novel targets in bone-associated tumors.

    PubMed

    Chen, Po-Chun; Cheng, Hsu-Chen; Yang, Shun-Fa; Lin, Chiao-Wen; Tang, Chih-Hsin

    2014-01-01

    The CCN family of proteins is composed of six extracellular matrix-associated proteins that play crucial roles in skeletal development, wound healing, fibrosis, and cancer. Members of the CCN family share four conserved cysteine-rich modular domains that trigger signal transduction in cell adhesion, migration, proliferation, differentiation, and survival through direct binding to specific integrin receptors and heparan sulfate proteoglycans. In the present review, we discuss the roles of the CCN family proteins in regulating resident cells of the bone microenvironment. In vertebrate development, the CCN family plays a critical role in osteo/chondrogenesis and vasculo/angiogenesis. These effects are regulated through signaling via integrins, bone morphogenetic protein, vascular endothelial growth factor, Wnt, and Notch via direct binding to CCN family proteins. Due to the important roles of CCN family proteins in skeletal development, abnormal expression of CCN proteins is related to the tumorigenesis of primary bone tumors such as osteosarcoma, Ewing sarcoma, and chondrosarcoma. Additionally, emerging studies have suggested that CCN proteins may affect progression of secondary metastatic bone tumors by moderating the bone microenvironment. CCN proteins could therefore serve as potential therapeutic targets for drug development against primary and metastatic bone tumors.

  20. The Sorcerer II Global Ocean Sampling Expedition: Expanding theUniverse of Protein Families

    SciTech Connect

    Yooseph, Shibu; Sutton, Granger; Rusch, Douglas B.; Halpern,Aaron L.; Williamson, Shannon J.; Remington, Karin; Eisen, Jonathan A.; Heidelberg, Karla B.; Manning, Gerard; Li, Weizhong; Jaroszewski, Lukasz; Cieplak, Piotr; Miller, Christopher S.; Li, Huiying; Mashiyama, Susan T.; Joachimiak, Marcin P.; van Belle, Christopher; Chandonia, John-Marc; Soergel, David A.; Zhai, Yufeng; Natarajan, Kannan; Lee, Shaun; Raphael,Benjamin J.; Bafna, Vineet; Friedman, Robert; Brenner, Steven E.; Godzik,Adam; Eisenberg, David; Dixon, Jack E.; Taylor, Susan S.; Strausberg,Robert L.; Frazier, Marvin; Venter, J.Craig

    2006-03-23

    Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

  1. The Sorcerer II Global Ocean Sampling expedition: expanding the universe of protein families.

    PubMed

    Yooseph, Shibu; Sutton, Granger; Rusch, Douglas B; Halpern, Aaron L; Williamson, Shannon J; Remington, Karin; Eisen, Jonathan A; Heidelberg, Karla B; Manning, Gerard; Li, Weizhong; Jaroszewski, Lukasz; Cieplak, Piotr; Miller, Christopher S; Li, Huiying; Mashiyama, Susan T; Joachimiak, Marcin P; van Belle, Christopher; Chandonia, John-Marc; Soergel, David A; Zhai, Yufeng; Natarajan, Kannan; Lee, Shaun; Raphael, Benjamin J; Bafna, Vineet; Friedman, Robert; Brenner, Steven E; Godzik, Adam; Eisenberg, David; Dixon, Jack E; Taylor, Susan S; Strausberg, Robert L; Frazier, Marvin; Venter, J Craig

    2007-03-01

    Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

  2. The Sorcerer II Global Ocean Sampling Expedition: Expanding the Universe of Protein Families

    PubMed Central

    Yooseph, Shibu; Sutton, Granger; Rusch, Douglas B; Halpern, Aaron L; Williamson, Shannon J; Remington, Karin; Eisen, Jonathan A; Heidelberg, Karla B; Manning, Gerard; Li, Weizhong; Jaroszewski, Lukasz; Cieplak, Piotr; Miller, Christopher S; Li, Huiying; Mashiyama, Susan T; Joachimiak, Marcin P; van Belle, Christopher; Chandonia, John-Marc; Soergel, David A; Zhai, Yufeng; Natarajan, Kannan; Lee, Shaun; Raphael, Benjamin J; Bafna, Vineet; Friedman, Robert; Brenner, Steven E; Godzik, Adam; Eisenberg, David; Dixon, Jack E; Taylor, Susan S; Strausberg, Robert L; Frazier, Marvin; Venter, J. Craig

    2007-01-01

    Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature. PMID:17355171

  3. Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade.

    PubMed

    Yamaguchi, Ayami; Tanaka, Sae; Yamaguchi, Shiho; Kuwahara, Hirokazu; Takamura, Chizuko; Imajoh-Ohmi, Shinobu; Horikawa, Daiki D; Toyoda, Atsushi; Katayama, Toshiaki; Arakawa, Kazuharu; Fujiyama, Asao; Kubo, Takeo; Kunieda, Takekazu

    2012-01-01

    Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.

  4. Nucleo-cytoplasmic functions of the PDZ-LIM protein family: new insights in organ development

    PubMed Central

    Krcmery, Jennifer; Camarata, Troy; Kulisz, Andre; Simon, Hans-Georg

    2010-01-01

    Summary Recent work on the PDZ-LIM protein family has revealed important activities at the cellular level, mediating signals between the nucleus and the cytoskeleton, with significant impact on organ development. We review and integrate current knowledge about the PDZ-LIM protein family and propose a new functional role, sequestering nuclear factors in the cytoplasm. Characterized by their PDZ and LIM domains, the PDZ-LIM family is comprised of evolutionarily conserved proteins found throughout the animal kingdom, from worms to humans. Combining two functional domains in one protein, PDZ-LIM proteins have wide-ranging and multi-compartmental cell functions during development and homeostasis while, in contrast, misregulation can lead to cancer formation and progression. New emerging roles include interactions with integrins, T-box transcription factors, and receptor tyrosine kinases. Facilitating the assembly of protein complexes, PDZ-LIM proteins can act as signal modulators, influence actin dynamics, regulate cell architecture and control gene transcription. PMID:20091751

  5. A family of plasmodesmal proteins with receptor-like properties for plant viral movement proteins.

    PubMed

    Amari, Khalid; Boutant, Emmanuel; Hofmann, Christina; Schmitt-Keichinger, Corinne; Fernandez-Calvino, Lourdes; Didier, Pascal; Lerich, Alexander; Mutterer, Jérome; Thomas, Carole L; Heinlein, Manfred; Mély, Yves; Maule, Andrew J; Ritzenthaler, Christophe

    2010-09-23

    Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.

  6. Wnt family proteins are secreted and associated with the cell surface.

    PubMed Central

    Smolich, B D; McMahon, J A; McMahon, A P; Papkoff, J

    1993-01-01

    Members of the Wnt gene family are proposed to function in both normal development and differentiation as well as in mammary tumorigenesis. To understand the function of Wnt proteins in these two processes, we present here a biochemical characterization of seven Wnt family members. For these studies, AtT-20 cells, a neuroendocrine cell line previously shown to efficiently process and secrete Wnt-1, was transfected with expression vectors encoding Wnt family members. All of the newly characterized Wnt proteins are glycosylated, secreted proteins that are tightly associated with the cell surface or extracellular matrix. We have also identified native Wnt proteins in retinoic acid-treated P19 embryonal carcinoma cells, and they exhibit the same biochemical characteristics as the recombinant proteins. These data suggest that Wnt family members function in cell to cell signaling in a fashion similar to Wnt-1. Images PMID:8167409

  7. ELMO Domains, Evolutionary and Functional Characterization of a Novel GTPase-activating Protein (GAP) Domain for Arf Protein Family GTPases*

    PubMed Central

    East, Michael P.; Bowzard, J. Bradford; Dacks, Joel B.; Kahn, Richard A.

    2012-01-01

    The human family of ELMO domain-containing proteins (ELMODs) consists of six members and is defined by the presence of the ELMO domain. Within this family are two subclassifications of proteins, based on primary sequence conservation, protein size, and domain architecture, deemed ELMOD and ELMO. In this study, we used homology searching and phylogenetics to identify ELMOD family homologs in genomes from across eukaryotic diversity. This demonstrated not only that the protein family is ancient but also that ELMOs are potentially restricted to the supergroup Opisthokonta (Metazoa and Fungi), whereas proteins with the ELMOD organization are found in diverse eukaryotes and thus were likely the form present in the last eukaryotic common ancestor. The segregation of the ELMO clade from the larger ELMOD group is consistent with their contrasting functions as unconventional Rac1 guanine nucleotide exchange factors and the Arf family GTPase-activating proteins, respectively. We used unbiased, phylogenetic sorting and sequence alignments to identify the most highly conserved residues within the ELMO domain to identify a putative GAP domain within the ELMODs. Three independent but complementary assays were used to provide an initial characterization of this domain. We identified a highly conserved arginine residue critical for both the biochemical and cellular GAP activity of ELMODs. We also provide initial evidence of the function of human ELMOD1 as an Arf family GAP at the Golgi. These findings provide the basis for the future study of the ELMOD family of proteins and a new avenue for the study of Arf family GTPases. PMID:23014990

  8. Monoubiquitination of Tob/BTG family proteins competes with degradation-targeting polyubiquitination.

    PubMed

    Suzuki, Toru; Kim, Minsoo; Kozuka-Hata, Hiroko; Watanabe, Masato; Oyama, Masaaki; Tsumoto, Kouhei; Yamamoto, Tadashi

    2011-05-27

    Tob belongs to the anti-proliferative Tob/BTG protein family. The expression level of Tob family proteins is strictly regulated both transcriptionally and through post-translational modification. Ubiquitin (Ub)/proteosome-dependent degradation of Tob family proteins is critical in controlling cell cycle progression and DNA damage responses. Various Ub ligases (E3s) are responsible for degradation of Tob protein. Here, we show that Tob family proteins undergo monoubiquitination even in the absence of E3s in vitro. Determination of the ubiquitination site(s) in Tob by mass spectrometric analysis revealed that two lysine residues (Lys48 and Lys63) located in Tob/BTG homology domain are ubiquitinated. A mutant Tob, in which both Lys48 and Lys63 are substituted with alanine, is more strongly polyubiquitinated than wild-type Tob in vivo. These data suggest that monoubiquitination of Tob family proteins confers resistance against polyubiquitination, which targets proteins for degradation. The strategy for regulating the stability of Tob family proteins suggests a novel role for monoubiquitination.

  9. Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer

    PubMed Central

    Berger, Stephanie; Procko, Erik; Margineantu, Daciana; Lee, Erinna F; Shen, Betty W; Zelter, Alex; Silva, Daniel-Adriano; Chawla, Kusum; Herold, Marco J; Garnier, Jean-Marc; Johnson, Richard; MacCoss, Michael J; Lessene, Guillaume; Davis, Trisha N; Stayton, Patrick S; Stoddard, Barry L; Fairlie, W Douglas; Hockenbery, David M; Baker, David

    2016-01-01

    Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes. DOI: http://dx.doi.org/10.7554/eLife.20352.001 PMID:27805565

  10. Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer.

    PubMed

    Berger, Stephanie; Procko, Erik; Margineantu, Daciana; Lee, Erinna F; Shen, Betty W; Zelter, Alex; Silva, Daniel-Adriano; Chawla, Kusum; Herold, Marco J; Garnier, Jean-Marc; Johnson, Richard; MacCoss, Michael J; Lessene, Guillaume; Davis, Trisha N; Stayton, Patrick S; Stoddard, Barry L; Fairlie, W Douglas; Hockenbery, David M; Baker, David

    2016-11-02

    Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes.

  11. Structure and evolutionary history of a large family of NLR proteins in the zebrafish

    PubMed Central

    Zielinski, Julia; Kondrashov, Fyodor

    2016-01-01

    Multicellular eukaryotes have evolved a range of mechanisms for immune recognition. A widespread family involved in innate immunity are the NACHT-domain and leucine-rich-repeat-containing (NLR) proteins. Mammals have small numbers of NLR proteins, whereas in some species, mostly those without adaptive immune systems, NLRs have expanded into very large families. We describe a family of nearly 400 NLR proteins encoded in the zebrafish genome. The proteins share a defining overall structure, which arose in fishes after a fusion of the core NLR domains with a B30.2 domain, but can be subdivided into four groups based on their NACHT domains. Gene conversion acting differentially on the NACHT and B30.2 domains has shaped the family and created the groups. Evidence of positive selection in the B30.2 domain indicates that this domain rather than the leucine-rich repeats acts as the pathogen recognition module. In an unusual chromosomal organization, the majority of the genes are located on one chromosome arm, interspersed with other large multigene families, including a new family encoding zinc-finger proteins. The NLR-B30.2 proteins represent a new family with diversity in the specific recognition module that is present in fishes in spite of the parallel existence of an adaptive immune system. PMID:27248802

  12. Eos and pegasus, two members of the Ikaros family of proteins with distinct DNA binding activities.

    PubMed

    Perdomo, J; Holmes, M; Chong, B; Crossley, M

    2000-12-08

    Members of the Ikaros family of transcription factors, Ikaros, Aiolos, and Helios, are expressed in lymphocytes and have been implicated in controlling lymphoid development. These proteins contain two characteristic clusters of zinc fingers, an N-terminal domain important for DNA recognition, and a C-terminal domain that mediates homo- and heterotypic associations between family members. The conservation of these domains is such that all three proteins recognize related DNA sequences, and all are capable of dimerizing with other family members. Here we describe two additional Ikaros family proteins, Eos and Pegasus. Eos is most highly related to Helios and shares its DNA binding and protein association properties. Pegasus is related to other Ikaros proteins in its C-terminal dimerization domain but contains a divergent N-terminal zinc finger domain. Pegasus self-associates and binds to other family members but recognizes distinct DNA-binding sites. Eos and Pegasus repress the expression of reporter genes containing their recognition elements. Our results suggest that these proteins may associate with previously described Ikaros family proteins in lymphoid cells and play additional roles in other tissues.

  13. Genome-scale phylogenetic function annotation of large and diverse protein families.

    PubMed

    Engelhardt, Barbara E; Jordan, Michael I; Srouji, John R; Brenner, Steven E

    2011-11-01

    The Statistical Inference of Function Through Evolutionary Relationships (SIFTER) framework uses a statistical graphical model that applies phylogenetic principles to automate precise protein function prediction. Here we present a revised approach (SIFTER version 2.0) that enables annotations on a genomic scale. SIFTER 2.0 produces equivalently precise predictions compared to the earlier version on a carefully studied family and on a collection of 100 protein families. We have added an approximation method to SIFTER 2.0 and show a 500-fold improvement in speed with minimal impact on prediction results in the functionally diverse sulfotransferase protein family. On the Nudix protein family, previously inaccessible to the SIFTER framework because of the 66 possible molecular functions, SIFTER achieved 47.4% accuracy on experimental data (where BLAST achieved 34.0%). Finally, we used SIFTER to annotate all of the Schizosaccharomyces pombe proteins with experimental functional characterizations, based on annotations from proteins in 46 fungal genomes. SIFTER precisely predicted molecular function for 45.5% of the characterized proteins in this genome, as compared with four current function prediction methods that precisely predicted function for 62.6%, 30.6%, 6.0%, and 5.7% of these proteins. We use both precision-recall curves and ROC analyses to compare these genome-scale predictions across the different methods and to assess performance on different types of applications. SIFTER 2.0 is capable of predicting protein molecular function for large and functionally diverse protein families using an approximate statistical model, enabling phylogenetics-based protein function prediction for genome-wide analyses. The code for SIFTER and protein family data are available at http://sifter.berkeley.edu.

  14. Vaccinia Virus Protein A49 Is an Unexpected Member of the B-cell Lymphoma (Bcl)-2 Protein Family*

    PubMed Central

    Neidel, Sarah; Maluquer de Motes, Carlos; Mansur, Daniel S.; Strnadova, Pavla; Smith, Geoffrey L.; Graham, Stephen C.

    2015-01-01

    Vaccinia virus (VACV) encodes several proteins that inhibit activation of the proinflammatory transcription factor nuclear factor κB (NF-κB). VACV protein A49 prevents translocation of NF-κB to the nucleus by sequestering cellular β-TrCP, a protein required for the degradation of the inhibitor of κB. A49 does not share overall sequence similarity with any protein of known structure or function. We solved the crystal structure of A49 from VACV Western Reserve to 1.8 Å resolution and showed, surprisingly, that A49 has the same three-dimensional fold as Bcl-2 family proteins despite lacking identifiable sequence similarity. Whereas Bcl-2 family members characteristically modulate cellular apoptosis, A49 lacks a surface groove suitable for binding BH3 peptides and does not bind proapoptotic Bcl-2 family proteins Bax or Bak. The N-terminal 17 residues of A49 do not adopt a single well ordered conformation, consistent with their proposed role in binding β-TrCP. Whereas pairs of A49 molecules interact symmetrically via a large hydrophobic surface in crystallo, A49 does not dimerize in solution or in cells, and we propose that this hydrophobic interaction surface may mediate binding to a yet undefined cellular partner. A49 represents the eleventh VACV Bcl-2 family protein and, despite these proteins sharing very low sequence identity, structure-based phylogenetic analysis shows that all poxvirus Bcl-2 proteins are structurally more similar to each other than they are to any cellular or herpesvirus Bcl-2 proteins. This is consistent with duplication and diversification of a single BCL2 family gene acquired by an ancestral poxvirus. PMID:25605733

  15. Regulation and roles for claudin-family tight junction proteins

    PubMed Central

    Findley, Mary K.; Koval, Michael

    2009-01-01

    Transmembrane proteins known as claudins play a critical role in tight junctions by regulating paracellular barrier permeability. The control of claudin assembly into tight junctions requires a complex interplay between several classes of claudins, other transmembrane proteins and scaffold proteins. Claudins are also subject to regulation by post-translational modifications including phosphorylation and palmitoylation. Several human diseases have been linked to claudin mutations, underscoring the physiologic function of these proteins. Roles for claudins in regulating cell phenotype and growth control also are beginning to emerge, suggesting a multifaceted role for claudins in regulation of cells beyond serving as a simple structural element of tight junctions. PMID:19319969

  16. TRIM family proteins and their emerging roles in innate immunity.

    PubMed

    Ozato, Keiko; Shin, Dong-Mi; Chang, Tsung-Hsien; Morse, Herbert C

    2008-11-01

    The superfamily of tripartite motif-containing (TRIM) proteins is conserved throughout the metazoan kingdom and has expanded rapidly during vertebrate evolution; there are now more than 60 TRIM proteins known in humans and mice. Many TRIM proteins are induced by type I and type II interferons, which are crucial for many aspects of resistance to pathogens, and several are known to be required for the restriction of infection by lentiviruses. In this Review, we describe recent data that reveal broader antiviral and antimicrobial activities of TRIM proteins and discuss their involvement in the regulation of pathogen-recognition and transcriptional pathways in host defence.

  17. Phylogenetic analyses provide the first insights into the evolution of OVATE family proteins in land plants

    PubMed Central

    Liu, Di; Sun, Wei; Yuan, Yaowu; Zhang, Ning; Hayward, Alice; Liu, Yongliang; Wang, Ying

    2014-01-01

    Background and Aims The OVATE gene encodes a nuclear-localized regulatory protein belonging to a distinct family of plant-specific proteins known as the OVATE family proteins (OFPs). OVATE was first identified as a key regulator of fruit shape in tomato, with nonsense mutants displaying pear-shaped fruits. However, the role of OFPs in plant development has been poorly characterized. Methods Public databases were searched and a total of 265 putative OVATE protein sequences were identified from 13 sequenced plant genomes that represent the major evolutionary lineages of land plants. A phylogenetic analysis was conducted based on the alignment of the conserved OVATE domain from these 13 selected plant genomes. The expression patterns of tomato SlOFP genes were analysed via quantitative real-time PCR. The pattern of OVATE gene duplication resulting in the expansion of the gene family was determined in arabidopsis, rice and tomato. Key Results Genes for OFPs were found to be present in all the sampled land plant genomes, including the early-diverged lineages, mosses and lycophytes. Phylogenetic analysis based on the amino acid sequences of the conserved OVATE domain defined 11 sub-groups of OFPs in angiosperms. Different evolutionary mechanisms are proposed for OVATE family evolution, namely conserved evolution and divergent expansion. Characterization of the AtOFP family in arabidopsis, the OsOFP family in rice and the SlOFP family in tomato provided further details regarding the evolutionary framework and revealed a major contribution of tandem and segmental duplications towards expansion of the OVATE gene family. Conclusions This first genome-wide survey on OFPs provides new insights into the evolution of the OVATE protein family and establishes a solid base for future functional genomics studies on this important but poorly characterized regulatory protein family in plants. PMID:24812252

  18. Immune regulatory functions of DOCK family proteins in health and disease.

    PubMed

    Nishikimi, Akihiko; Kukimoto-Niino, Mutsuko; Yokoyama, Shigeyuki; Fukui, Yoshinori

    2013-09-10

    DOCK proteins constitute a family of evolutionarily conserved guanine nucleotide exchange factors (GEFs) for Rho family of GTPases. Although DOCK family proteins do not contain the Dbl homology domain typically found in GEFs, they mediate the GTP-GDP exchange reaction through DHR-2 domain. Accumulating evidence indicates that the DOCK proteins act as major GEFs in varied biological settings. For example, DOCK2, which is predominantly expressed in hematopoietic cells, regulates migration and activation of leukocytes through Rac activation. On the other hand, it was recently reported that mutations of DOCK8, another member of the DOCK family proteins, cause a combined immunodeficiency syndrome in humans. This article reviews the structure, functions and signaling of DOCK2 and DOCK8, especially focusing on their roles in immune responses.

  19. Four Members of Heat Shock Protein 70 Family in Korean Rose Bitterling (Rhodeus uyekii)

    PubMed Central

    Kim, Jung Hyun; Dong, Chun Mae; Kim, Julan; An, Cheul Min; Baek, Hae Ja; Kong, Hee Jeong

    2015-01-01

    Heat shock protein (HSP) 70, the highly conserved stress protein families, plays important roles in protecting cells against heat and other stresses in most animal species. In the present study, we identified and characterized four Hsp70 (RuHSP4, RuHSC70, RuHSP12A, RuGRP78) family proteins based on the expressed sequence tag (EST) analysis of the Korean rose bitterling R. uyekii cDNA library. The deduced RuHSP70 family has high amino acid identities of 72-99% with those of other species. Phylogenetic analysis revealed that RuHsp70 family clustered with fish groups (HSP4, HSC70, HSP12A, GRP78) proteins. Quantitative RT-PCR analysis showed the specific expression patterns of RuHsp70 family members in the early developmental stages and several tissues in Korean rose bitterling. The expression of 4 groups of Hsp70 family was detected in all tested tissue. Particularly, Hsp70 family of Korean rose bitterling is highly expressed in hepatopancreas and sexual gonad (testis and ovary). The expression of Hsp70 family was differentially regulated in accordance with early development stage of Rhodeus uyekii. PMID:27004270

  20. PATtyFams: Protein families for the microbial genomes in the PATRIC database

    DOE PAGES

    Davis, James J.; Gerdes, Svetlana; Olsen, Gary J.; ...

    2016-02-08

    The ability to build accurate protein families is a fundamental operation in bioinformatics that influences comparative analyses, genome annotation, and metabolic modeling. For several years we have been maintaining protein families for all microbial genomes in the PATRIC database (Pathosystems Resource Integration Center, patricbrc.org) in order to drive many of the comparative analysis tools that are available through the PATRIC website. However, due to the burgeoning number of genomes, traditional approaches for generating protein families are becoming prohibitive. In this report, we describe a new approach for generating protein families, which we call PATtyFams. This method uses the k-mer-based functionmore » assignments available through RAST (Rapid Annotation using Subsystem Technology) to rapidly guide family formation, and then differentiates the function-based groups into families using a Markov Cluster algorithm (MCL). In conclusion, this new approach for generating protein families is rapid, scalable and has properties that are consistent with alignment-based methods.« less

  1. The Classical Arabinogalactan Protein Gene Family of Arabidopsis

    PubMed Central

    Schultz, Carolyn J.; Johnson, Kim L.; Currie, Graeme; Bacic, Antony

    2000-01-01

    Arabinogalactan proteins (AGPs) are extracellular proteoglycans implicated in plant growth and development. We searched for classical AGPs in Arabidopsis by identifying expressed sequence tags based on the conserved domain structure of the predicted protein backbone. To confirm that these genes encoded bona fide AGPs, we purified native AGPs and then deglycosylated and deblocked them for N-terminal protein sequencing. In total, we identified 15 genes encoding the protein backbones of classical AGPs, including genes for AG peptides—AGPs with very short backbones (10 to 13 amino acid residues). Seven of the AGPs were verified as AGPs by protein sequencing. A gene encoding a putative cell adhesion molecule with AGP-like domains was also identified. This work provides a firm foundation for beginning functional analysis by using a genetic approach. PMID:11006345

  2. Protein Methyltransferases: A Distinct, Diverse, and Dynamic Family of Enzymes.

    PubMed

    Boriack-Sjodin, P Ann; Swinger, Kerren K

    2016-03-22

    Methyltransferase proteins make up a superfamily of enzymes that add one or more methyl groups to substrates that include protein, DNA, RNA, and small molecules. The subset of proteins that act upon arginine and lysine side chains are characterized as epigenetic targets because of their activity on histone molecules and their ability to affect transcriptional regulation. However, it is now clear that these enzymes target other protein substrates, as well, greatly expanding their potential impact on normal and disease biology. Protein methyltransferases are well-characterized structurally. In addition to revealing the overall architecture of the subfamilies of enzymes, structures of complexes with substrates and ligands have permitted detailed analysis of biochemical mechanism, substrate recognition, and design of potent and selective inhibitors. This review focuses on how knowledge gained from structural studies has impacted the understanding of this large class of epigenetic enzymes.

  3. Molecular evolution and expression of the CRAL_TRIO protein family in insects.

    PubMed

    Smith, Gilbert; Briscoe, Adriana D

    2015-07-01

    CRAL_TRIO domain proteins are known to bind small lipophilic molecules such as retinal, inositol and Vitamin E and include such gene family members as PINTA, α-tocopherol transfer (ATT) proteins, retinoid binding proteins, and clavesins. In insects, very little is known about either the molecular evolution of this family of proteins or their ligand specificity. Here we characterize insect CRAL_TRIO domain proteins and present the first insect CRAL_TRIO protein phylogeny constructed by performing reciprocal BLAST searches of the reference genomes of Drosophila melanogaster, Anopheles gambiae, Apis mellifera, Tribolium castaneum, Bombyx mori, Manduca sexta and Danaus plexippus. We find several highly conserved amino acid residues in the CRAL_TRIO domain-containing genes across insects and a gene expansion resulting in more than twice as many gene family members in lepidopterans than in other surveyed insect species, but no lepidopteran homolog of the PINTA gene in Drosophila. In addition, we examined the expression pattern of CRAL_TRIO domain genes in Manduca sexta heads using RNA-Seq data. Of the 42 gene family members found in the M. sexta reference genome, we found 30 expressed in the head tissue with similar expression profiles between males and females. Our results suggest this gene family underwent a large expansion in lepidopteran, making the lepidopteran CRAL_TRIO domain family distinct from other holometabolous insect lineages.

  4. Three new members of the RNP protein family in Xenopus.

    PubMed Central

    Good, P J; Rebbert, M L; Dawid, I B

    1993-01-01

    Many RNP proteins contain one or more copies of the RNA recognition motif (RRM) and are thought to be involved in cellular RNA metabolism. We have previously characterized in Xenopus a nervous system specific gene, nrp1, that is more similar to the hnRNP A/B proteins than to other known proteins (K. Richter, P. J. Good, and I. B. Dawid (1990), New Biol. 2, 556-565). PCR amplification with degenerate primers was used to identify additional cDNAs encoding two RRMs in Xenopus. Three previously uncharacterized genes were identified. Two genes encode hnRNP A/B proteins with two RRMs and a glycine-rich domain. One of these is the Xenopus homolog of the human A2/B1 gene; the other, named hnRNP A3, is similar to both the A1 and A2 hnRNP genes. The Xenopus hnRNP A1, A2 and A3 genes are expressed throughout development and in all adult tissues. Multiple protein isoforms for the hnRNP A2 gene are predicted that differ by the insertion of short peptide sequences in the glycine-rich domain. The third newly isolated gene, named xrp1, encodes a protein that is related by sequence to the nrp1 protein but is expressed ubiquitously. Despite the similarity to nuclear RNP proteins, both the nrp1 and xrp1 proteins are localized to the cytoplasm in the Xenopus oocyte. The xrp1 gene may have a function in all cells that is similar to that executed by nrp1 specifically within the nervous system. Images PMID:8451200

  5. CPF and CPFL, two related gene families encoding cuticular proteins of Anopheles gambiae and other insects.

    PubMed

    Togawa, Toru; Augustine Dunn, W; Emmons, Aaron C; Willis, Judith H

    2007-07-01

    Cuticular proteins (CPs) are structural proteins of insects as well as other arthropods. Several CP families have been described, among them a small family defined by a 51 amino acid motif [Andersen, S.O., Rafn, K., Roepstorff, P., 1997. Sequence studies of proteins from larval and pupal cuticle of the yellow meal worm, Tenebrio molitor. Insect Biochem. Mol. Biol. 27, 121-131]. We identified four proteins of this family in Anopheles gambiae that we have named CPF. We have also identified CPFs from other insects by searching databases. Alignment of these CPF proteins showed that the conserved region is only 44 aa long and revealed another conserved motif at the C-terminus. A dendrogram divided the CPF proteins into four groups, one basal and three specialized. We also identified several proteins of another CP family, CPFL, which has similarities to CPFs. CPFs and CPFLs share some protein motifs. Expression studies with real-time qRT-PCR of the A. gambiae CPFs and CPFLs showed that the four CPFs and one CPFL gene are expressed just before pupal or adult ecdysis, suggesting that they are components of the outer layer of pupal and adult cuticles. The other CPFLs appear to contribute to larval cuticle. Recombinant CPF proteins did not bind to chitin in the assay we used.

  6. The mammalian START domain protein family in lipid transport in health and disease.

    PubMed

    Clark, Barbara J

    2012-03-01

    Lipid transfer proteins of the steroidogenic acute regulatory protein-related lipid transfer (START) domain family are defined by the presence of a conserved ∼210 amino acid sequence that folds into an α/β helix-grip structure forming a hydrophobic pocket for ligand binding. The mammalian START proteins bind diverse ligands, such as cholesterol, oxysterols, phospholipids, sphingolipids, and possibly fatty acids, and have putative roles in non-vesicular lipid transport, thioesterase enzymatic activity, and tumor suppression. However, the biological functions of many members of the START domain protein family are not well established. Recent research has focused on characterizing the cell-type distribution and regulation of the START proteins, examining the specificity and directionality of lipid transport, and identifying disease states associated with dysregulation of START protein expression. This review summarizes the current concepts of the proposed physiological and pathological roles for the mammalian START domain proteins in cholesterol and lipid trafficking.

  7. Clustering of protein families into functional subtypes using Relative Complexity Measure with reduced amino acid alphabets

    PubMed Central

    2010-01-01

    Background Phylogenetic analysis can be used to divide a protein family into subfamilies in the absence of experimental information. Most phylogenetic analysis methods utilize multiple alignment of sequences and are based on an evolutionary model. However, multiple alignment is not an automated procedure and requires human intervention to maintain alignment integrity and to produce phylogenies consistent with the functional splits in underlying sequences. To address this problem, we propose to use the alignment-free Relative Complexity Measure (RCM) combined with reduced amino acid alphabets to cluster protein families into functional subtypes purely on sequence criteria. Comparison with an alignment-based approach was also carried out to test the quality of the clustering. Results We demonstrate the robustness of RCM with reduced alphabets in clustering of protein sequences into families in a simulated dataset and seven well-characterized protein datasets. On protein datasets, crotonases, mandelate racemases, nucleotidyl cyclases and glycoside hydrolase family 2 were clustered into subfamilies with 100% accuracy whereas acyl transferase domains, haloacid dehalogenases, and vicinal oxygen chelates could be assigned to subfamilies with 97.2%, 96.9% and 92.2% accuracies, respectively. Conclusions The overall combination of methods in this paper is useful for clustering protein families into subtypes based on solely protein sequence information. The method is also flexible and computationally fast because it does not require multiple alignment of sequences. PMID:20718947

  8. Fundamental Characteristics of AAA+ Protein Family Structure and Function

    PubMed Central

    2016-01-01

    Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily (ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis. In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with an emphasis on pivotal aspects elucidated with archaeal proteins. PMID:27703410

  9. Matricellular proteins of the Cyr61/CTGF/NOV (CCN) family and the nervous system

    PubMed Central

    Malik, Anna R.; Liszewska, Ewa; Jaworski, Jacek

    2015-01-01

    Matricellular proteins are secreted proteins that exist at the border of cells and the extracellular matrix (ECM). However, instead of playing a role in structural integrity of the ECM, these proteins, that act as modulators of various surface receptors, have a regulatory function and instruct a multitude of cellular responses. Among matricellular proteins are members of the Cyr61/CTGF/NOV (CCN) protein family. These proteins exert their activity by binding directly to integrins and heparan sulfate proteoglycans and activating multiple intracellular signaling pathways. CCN proteins also influence the activity of growth factors and cytokines and integrate their activity with integrin signaling. At the cellular level, CCN proteins regulate gene expression and cell survival, proliferation, differentiation, senescence, adhesion, and migration. To date, CCN proteins have been extensively studied in the context of osteo- and chondrogenesis, angiogenesis, and carcinogenesis, but the expression of these proteins is also observed in a variety of tissues. The role of CCN proteins in the nervous system has not been systematically studied or described. Thus, the major aim of this review is to introduce the CCN protein family to the neuroscience community. We first discuss the structure, interactions, and cellular functions of CCN proteins and then provide a detailed review of the available data on the neuronal expression and contribution of CCN proteins to nervous system development, function, and pathology. PMID:26157362

  10. Characterization of a New Family of Metal Transport Proteins

    SciTech Connect

    Guerinot, Mary Lou; Eide, David

    1999-06-01

    Soils at many DOE sites are contaminated with metals and radionuclides. Such soils obviously pose a risk to human and animal health. Unlike organic wastes, which can be metabolized, metals are immutable and cannot be degraded into harmless constituents. Phytoremediation, the use of plants to remove toxic materials from soil and water, may prove to be an environmentally friendly and cost effective solution for cleaning up metal contaminated sites. The success of phytoremediation will rely on the availability of plants that absorb, translocate, and tolerate the contaminating metals. However, before we can engineer such plants, we need more basic information on how plants acquire metals. An important long term goal of our research program is to understand how metals such as zinc, cadmium and iron are transported across membranes. Our research is focused on a new family of metal transporters, which we have identified through combined studies in the yeast Saccharomyces cerevisiae and in the model plant Arabidopsis thaliana. We have identified a family of 24 presumptive metal transport genes in a variety of organisms including yeast, trypanosomes, plants, nematodes, and humans. This family, which we have designated the ''ZIP'' genes, provides a rich source of material with which to undertake studies on metal transport in eukar

  11. Review of the GAS3 Family of Proteins and their Relevance to Cancer

    PubMed Central

    Ashki, Negin; Gordon, Lynn; Wadehra, Madhuri

    2017-01-01

    The GAS3 family of tetraspan proteins has recently been implicated in the progression of cancer. Currently, six members of the GAS3 family have been identified in humans and mice, and while their expressions in disease vary, data suggest that they play a role in epithelial cell structure and function. In this review, we highlight the studies implicating four of the members in disease pathogenesis as well as probe the structural similarities between the family members. Finally, the impact of targeting select members of the family such as PMP22 and EMP2 is discussed. PMID:27279240

  12. Structure-based function inference using protein family-specific fingerprints

    PubMed Central

    Bandyopadhyay, Deepak; Huan, Jun; Liu, Jinze; Prins, Jan; Snoeyink, Jack; Wang, Wei; Tropsha, Alexander

    2006-01-01

    We describe a method to assign a protein structure to a functional family using family-specific fingerprints. Fingerprints represent amino acid packing patterns that occur in most members of a family but are rare in the background, a nonredundant subset of PDB; their information is additional to sequence alignments, sequence patterns, structural superposition, and active-site templates. Fingerprints were derived for 120 families in SCOP using Frequent Subgraph Mining. For a new structure, all occurrences of these family-specific fingerprints may be found by a fast algorithm for subgraph isomorphism; the structure can then be assigned to a family with a confidence value derived from the number of fingerprints found and their distribution in background proteins. In validation experiments, we infer the function of new members added to SCOP families and we discriminate between structurally similar, but functionally divergent TIM barrel families. We then apply our method to predict function for several structural genomics proteins, including orphan structures. Some predictions have been corroborated by other computational methods and some validated by subsequent functional characterization. PMID:16731985

  13. Under lock and key: Spatiotemporal regulation of WASP family proteins coordinates separate dynamic cellular processes

    PubMed Central

    Burianek, Lauren E.; Soderling, Scott H.

    2013-01-01

    WASP family proteins are nucleation promoting factors that bind to and activate the Arp2/3 complex in order to stimulate nucleation of branched actin filaments. The WASP family consists of WASP, N-WASP, WAVE1-3, WASH, and the novel family members WHAMM and JMY. Each of the family members contains a C-terminus responsible for their nucleation promoting activity and unique N-termini that allow for them to be regulated in a spatiotemporal manner. Upon activation they reorganize the cytoskeleton for different cellular functions depending on their subcellular localization and regulatory protein interactions. Emerging evidence indicates that WASH, WHAMM, and JMY have functions that require the coordination of both actin polymerization and microtubule dynamics. Here, we review the mechanisms of regulation for each family member and their associated in vivo functions including cell migration, vesicle trafficking, and neuronal development. PMID:23291261

  14. Physical and functional interactions of histone deacetylase 3 with TFII-I family proteins and PIASxbeta.

    PubMed

    Tussié-Luna, María Isabel; Bayarsaihan, Dashzeveg; Seto, Edward; Ruddle, Frank H; Roy, Ananda L

    2002-10-01

    TFII-I family proteins are characterized structurally by the presence of multiple reiterated I-repeats, each containing a putative helix-loop-helix domain. Functionally, they behave as multifunctional transcription factors that are activated by a variety of extracellular signals. In studying their subcellular localization, we noticed that these transcription factors frequently reside in subnuclear domains/dots. Because nuclear dots are believed often to harbor components of histone deacetylase enzymes (HDACs), we investigated whether TFII-I family proteins colocalize and interact with HDACs. Here, we show that TFII-I and its related member hMusTRD1/BEN physically and functionally interact with HDAC3. The TFII-I family proteins and HDAC3 also show nearly identical expression patterns in early mouse development. Consistent with our earlier observation that TFII-I family proteins also interact with PIASxbeta, a member of the E3 ligase family involved in the small ubiquitin-like modifier (SUMO) pathway, we show further that PIASxbeta physically and functionally interacts with HDAC3 and relieves the transcriptional repression exerted by HDAC3 upon TFII-I-mediated gene activation. These results suggest a complex interplay between two posttranslational pathways-histone modification and SUMOylation-brokered in part by TFII-I family proteins.

  15. Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides

    PubMed Central

    Safavi-Hemami, Helena; Li, Qing; Jackson, Ronneshia L.; Song, Albert S.; Boomsma, Wouter; Bandyopadhyay, Pradip K.; Gruber, Christian W.; Purcell, Anthony W.; Yandell, Mark; Olivera, Baldomero M.

    2016-01-01

    Formation of correct disulfide bonds in the endoplasmic reticulum is a crucial step for folding proteins destined for secretion. Protein disulfide isomerases (PDIs) play a central role in this process. We report a previously unidentified, hypervariable family of PDIs that represents the most diverse gene family of oxidoreductases described in a single genus to date. These enzymes are highly expressed specifically in the venom glands of predatory cone snails, animals that synthesize a remarkably diverse set of cysteine-rich peptide toxins (conotoxins). Enzymes in this PDI family, termed conotoxin-specific PDIs, significantly and differentially accelerate the kinetics of disulfide-bond formation of several conotoxins. Our results are consistent with a unique biological scenario associated with protein folding: The diversification of a family of foldases can be correlated with the rapid evolution of an unprecedented diversity of disulfide-rich structural domains expressed by venomous marine snails in the superfamily Conoidea. PMID:26957604

  16. Analysis of rice Snf2 family proteins and their potential roles in epigenetic regulation.

    PubMed

    Hu, Yongfeng; Zhu, Ning; Wang, Xuemin; Yi, Qingping; Zhu, Deyan; Lai, Yan; Zhao, Yu

    2013-09-01

    Snf2 family proteins are ATP-dependent chromatin remodeling factors that control many aspects of DNA events such as transcription, replication, homologous recombination and DNA repair. In animals several members in this family have been revealed to control gene expression in concert with other epigenetic mechanisms including histone modification, histone variants and DNA methylation. Their function in regulating genome expression in plant has hardly been disclosed before except in Arabidopsis. Here we identified 40 members of this family in the rice (Oryza Sativa) genome and constructed a phylogenetic tree together with Arabidopsis 41 Snf2 proteins. Sequence alignment of the Snf2 helicase regions revealed conserved motifs and blocks in most proteins. Expression profile analysis indicates that many rice Snf2 family genes show a tissue-specific expression pattern and some of them respond to abiotic stresses including drought, salt and cold. The results provide a basis for further analysis of their roles in epigenetic regulation to control rice development.

  17. Exploring metazoan evolution through dynamic and holistic changes in protein families and domains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding proteome evolution is important for deciphering processes that drive species diversity and adaptation. Herein, the dynamics of change in protein families and protein domains over the course of metazoan evolution was explored. Change, as defined by birth/death and duplication/deletion ...

  18. Members of the evolutionarily conserved PMT family of protein O-mannosyltransferases form distinct protein complexes among themselves.

    PubMed

    Girrbach, Verena; Strahl, Sabine

    2003-04-04

    Protein O-mannosyltransferases (PMTs) initiate the assembly of O-mannosyl glycans, an essential protein modification. Since PMTs are evolutionarily conserved in fungi but are absent in green plants, the PMT family is a putative target for new antifungal drugs, particularly in fighting the threat of phytopathogenic fungi. The PMT family is phylogenetically classified into PMT1, PMT2, and PMT4 subfamilies, which differ in protein substrate specificity. In the model organism Saccharomyces cerevisiae as well as in many other fungi the PMT family is highly redundant, and only the simultaneous deletion of PMT1/PMT2 and PMT4 subfamily members is lethal. In this study we analyzed the molecular organization of PMT family members in S. cerevisiae. We show that members of the PMT1 subfamily (Pmt1p and Pmt5p) interact in pairs with members of the PMT2 subfamily (Pmt2p and Pmt3p) and that Pmt1p-Pmt2p and Pmt5p-Pmt3p complexes represent the predominant forms. Under certain physiological conditions, however, Pmt1p interacts also with Pmt3p, and Pmt5p with Pmt2p, suggesting a compensatory cooperation that guarantees the maintenance of O-mannosylation. Unlike the PMT1/PMT2 subfamily members, the single member of the PMT4 subfamily (Pmt4p) acts as a homomeric complex. Using mutational analyses we demonstrate that the same conserved protein domains underlie both heteromeric and homomeric interactions, and we identify an invariant arginine residue of transmembrane domain two as essential for the formation and/or stability of PMT complexes in general. Our data suggest that protein-protein interactions between the PMT family members offer a point of attack to shut down overall protein O-mannosylation in fungi.

  19. Regulation of cytokine signaling by the SOCS and Spred family proteins.

    PubMed

    Yoshimura, Akihiko

    2009-06-01

    Various cytokines are involved in the regulation of the immune system and of hematopoiesis. Most cytokines utilize the so-called JAK-STAT pathway, but others activate the Ras-ERK pathway, which is more important than the STAT pathway for the proliferation of hematopoietic cells. Dysregulation of cytokine signaling can cause a variety of diseases, including allergy, inflammation, and cancer. We have identified two important regulator families involved in cytokine signaling: the SOCS proteins and the Spred proteins. Suppressors of cytokine signaling (SOCS) proteins bind to JAK and to certain receptors, thereby suppressing further signaling events. Spred family proteins interact with Ras and Raf, thereby suppressing ERK activation. Studies have shown that SOCS and Spred proteins are key physiological regulators of immunity, hematopoiesis, and angiogenesis. Evidence is also emerging for the involvement of these proteins in human diseases.

  20. From plant genomes to protein families: computational tools

    PubMed Central

    Martinez, Manuel

    2013-01-01

    The development of new high-throughput sequencing technologies has increased dramatically the number of successful genomic projects. Thus, draft genomic sequences of more than 60 plant species are currently available. Suitable bioinformatics tools are being developed to assemble, annotate and analyze the enormous number of sequences produced. In this context, specific plant comparative genomic databases are become powerful tools for gene family annotation in plant clades. In this mini-review, the current state-of-art of genomic projects is glossed. Besides, the computational tools developed to compare genomic data are compiled. PMID:24688740

  1. Structural Features and Chaperone Activity of the NudC Protein Family

    SciTech Connect

    Zheng, Meiying; Cierpicki, Tomasz; Burdette, Alexander J.; Utepbergenov, Darkhan; Janczyk, Pawe; #322; #321; .; Derewenda, Urszula; Stukenberg, P. Todd; Caldwell, Kim A.; Derewenda, Zygmunt S.

    2012-05-25

    The NudC family consists of four conserved proteins with representatives in all eukaryotes. The archetypal nudC gene from Aspergillus nidulans is a member of the nud gene family that is involved in the maintenance of nuclear migration. This family also includes nudF, whose human orthologue, Lis1, codes for a protein essential for brain cortex development. Three paralogues of NudC are known in vertebrates: NudC, NudC-like (NudCL), and NudC-like 2 (NudCL2). The fourth distantly related member of the family, CML66, contains a NudC-like domain. The three principal NudC proteins have no catalytic activity but appear to play as yet poorly defined roles in proliferating and dividing cells. We present crystallographic and NMR studies of the human NudC protein and discuss the results in the context of structures recently deposited by structural genomics centers (i.e., NudCL and mouse NudCL2). All proteins share the same core CS domain characteristic of proteins acting either as cochaperones of Hsp90 or as independent small heat shock proteins. However, while NudC and NudCL dimerize via an N-terminally located coiled coil, the smaller NudCL2 lacks this motif and instead dimerizes as a result of unique domain swapping. We show that NudC and NudCL, but not NudCL2, inhibit the aggregation of several target proteins, consistent with an Hsp90-independent heat shock protein function. Importantly, and in contrast to several previous reports, none of the three proteins is able to form binary complexes with Lis1. The availability of structural information will be of help in further studies on the cellular functions of the NudC family.

  2. Molecular Basis for Bcl-2 Homology 3 Domain Recognition in the Bcl-2 Protein Family

    PubMed Central

    Moroy, Gautier; Martin, Elyette; Dejaegere, Annick; Stote, Roland H.

    2009-01-01

    The proteins of the Bcl-2 family are important regulators of apoptosis, or programmed cell death. These proteins regulate this fundamental biological process via the formation of heterodimers involving both pro- and anti-apoptotic family members. Disruption of the balance between anti- and pro-apoptotic Bcl-2 proteins is the cause of numerous pathologies. Bcl-xl, an anti-apoptotic protein of this family, is known to form heterodimers with multiple pro-apoptotic proteins, such as Bad, Bim, Bak, and Bid. To elucidate the molecular basis of this recognition process, we used molecular dynamics simulations coupled with the Molecular Mechanics/Poisson-Boltzmann Surface Area approach to identify the amino acids that make significant energetic contributions to the binding free energy of four complexes formed between Bcl-xl and pro-apoptotic Bcl-2 homology 3 peptides. A fifth protein-peptide complex composed of another anti-apoptotic protein, Bcl-w, in complex with the peptide from Bim was also studied. The results identified amino acids of both the anti-apoptotic proteins as well as the Bcl-2 homology 3 (BH3) domains of the pro-apoptotic proteins that make strong, recurrent interactions in the protein complexes. The calculations show that the two anti-apoptotic proteins, Bcl-xl and Bcl-w, share a similar recognition mechanism. Our results provide insight into the molecular basis for the promiscuous nature of this molecular recognition process by members of the Bcl-2 protein family. These amino acids could be targeted in the design of new mimetics that serve as scaffolds for new antitumoral molecules. PMID:19293158

  3. The Pfam protein families database: towards a more sustainable future

    PubMed Central

    Finn, Robert D.; Coggill, Penelope; Eberhardt, Ruth Y.; Eddy, Sean R.; Mistry, Jaina; Mitchell, Alex L.; Potter, Simon C.; Punta, Marco; Qureshi, Matloob; Sangrador-Vegas, Amaia; Salazar, Gustavo A.; Tate, John; Bateman, Alex

    2016-01-01

    In the last two years the Pfam database (http://pfam.xfam.org) has undergone a substantial reorganisation to reduce the effort involved in making a release, thereby permitting more frequent releases. Arguably the most significant of these changes is that Pfam is now primarily based on the UniProtKB reference proteomes, with the counts of matched sequences and species reported on the website restricted to this smaller set. Building families on reference proteomes sequences brings greater stability, which decreases the amount of manual curation required to maintain them. It also reduces the number of sequences displayed on the website, whilst still providing access to many important model organisms. Matches to the full UniProtKB database are, however, still available and Pfam annotations for individual UniProtKB sequences can still be retrieved. Some Pfam entries (1.6%) which have no matches to reference proteomes remain; we are working with UniProt to see if sequences from them can be incorporated into reference proteomes. Pfam-B, the automatically-generated supplement to Pfam, has been removed. The current release (Pfam 29.0) includes 16 295 entries and 559 clans. The facility to view the relationship between families within a clan has been improved by the introduction of a new tool. PMID:26673716

  4. Essential role of CREB family proteins during Xenopus embryogenesis.

    PubMed

    Lutz, B; Schmid, W; Niehrs, C; Schütz, G

    1999-10-01

    The leucine zipper transcription factors cAMP response element binding protein (CREB), cAMP response element modulatory protein (CREM) and activating transcription factor 1 (ATF1) bind to the cAMP response element (CRE) with the palindromic consensus sequence TGACGTCA. Their transcriptional activities are dependent on serine phosphorylation induced by various extracellular signals such as hormones, growth factors and neurotransmitters. Here we show that CREB is the predominant CRE-binding protein in Xenopus embryos and that it plays an essential role during early development. The importance of CREB for morphogenetic processes was assessed by injection of RNA encoding a dominant-negative form of CREB that is fused to a truncated progesterone receptor ligand binding domain. In this fusion protein, a dominant-negative function can be induced by application of the synthetic steroid RU486 at given developmental stages. The inhibition of CREB at blastula and early gastrula stages leads to severe posterior defects of the embryos reflected by strong spina bifida, whereas the inhibition of CREB at the beginning of neurulation resulted in stunted embryos with microcephaly. In these embryos, initial induction of neural and mesodermal tissues is not dependent on CREB function, as genes such as Otx2, Krox20, Shh and MyoD are still expressed in injected embryos. But the expression domains of Otx2 and MyoD were found to be distorted reflecting the abnormal development in both neural and somitic derivatives. In summary, our data show that CREB is essential during several developmental stages of Xenopus embryogenesis.

  5. The Protein Kinase RSK Family - Roles in Prostate Cancer

    DTIC Science & Technology

    2005-02-01

    of the prodrug, kaempferol 3-O-(2",3",4"-tri-O-acetyl-a-L-rhamnopyranoside)(3Ac- SL0101), a novel inhibitor of the Ser/Thr protein kinase, RSK...Y, Holman NJ, Hecht SM, Lannigan DA. Synthesis of the prodrug, kaempferol 3-O-(2", 3", 4"-tri-O-acetyl-a-L-rhamnopyranoside) (3Ac-SLO1 01), a novel

  6. Vps10 family proteins and the retromer complex in aging-related neurodegeneration and diabetes.

    PubMed

    Lane, Rachel F; St George-Hyslop, Peter; Hempstead, Barbara L; Small, Scott A; Strittmatter, Stephen M; Gandy, Sam

    2012-10-10

    Members of the vacuolar protein sorting 10 (Vps10) family of receptors (including sortilin, SorL1, SorCS1, SorCS2, and SorCS3) play pleiotropic functions in protein trafficking and intracellular and intercellular signaling in neuronal and non-neuronal cells. Interactions have been documented between Vps10 family members and the retromer coat complex, a key component of the intracellular trafficking apparatus that sorts cargo from the early endosome to the trans-Golgi network. In recent years, genes encoding several members of the Vps10 family of proteins, as well as components of the retromer coat complex, have been implicated as genetic risk factors for sporadic and autosomal dominant forms of neurodegenerative diseases, including Alzheimer's disease, frontotemporal lobar degeneration, and Parkinson's disease, with risk for type 2 diabetes mellitus and atherosclerosis. In addition to their functions in protein trafficking, the Vps10 family proteins modulate neurotrophic signaling pathways. Sortilin can impact the intracellular response to brain-derived neurotrophic factor (BDNF) by regulating anterograde trafficking of Trk receptors to the synapse and direct control of BDNF levels, while both sortilin and SorCS2 function as cell surface receptors to mediate acute responses to proneurotrophins. This mini-review and symposium will highlight the emerging data from this rapidly growing area of research implicating the Vps10 family of receptors and the retromer in physiological intracellular trafficking signaling by neurotrophins and in the pathogenesis of neurodegeneration.

  7. p23 and HSP20/alpha-crystallin proteins define a conserved sequence domain present in other eukaryotic protein families.

    PubMed

    Garcia-Ranea, J A; Mirey, Gladys; Camonis, Jacques; Valencia, Alfonso

    2002-10-09

    We identified families of proteins characterized by the presence of a domain similar to human p23 protein, which include proteins such as Sgt1, involved in the yeast kinetochore assembly; melusin, involved in specific interactions with the cytoplasmic integrin beta1 domain; Rar1, related to pathogenic resistance in plants, and to development in animals; B5+B5R flavo-hemo cytochrome NAD(P)H oxidoreductase type B in humans and mice; and NudC, involved in nucleus migration during mitosis. We also found that p23 and the HSP20/alpha-crystallin family of heat shock proteins, which share the same three-dimensional folding, show a pattern of conserved residues that points to a common origin in the evolution of both protein domains. The p23 and HSP20/alpha-crystallin phylogenetic relationship and their similar role in chaperone activity suggest a common function, probably involving protein-protein interaction, for those proteins containing p23-like domains.

  8. The VQ Motif-Containing Protein Family of Plant-Specific Transcriptional Regulators1

    PubMed Central

    Jing, Yanjun; Lin, Rongcheng

    2015-01-01

    The VQ motif-containing proteins (designated as VQ proteins) are a class of plant-specific proteins with a conserved and single short FxxhVQxhTG amino acid sequence motif. VQ proteins regulate diverse developmental processes, including responses to biotic and abiotic stresses, seed development, and photomorphogenesis. In this Update, we summarize and discuss recent advances in our understanding of the regulation and function of VQ proteins and the role of the VQ motif in mediating transcriptional regulation and protein-protein interactions in signaling pathways. Based on the accumulated evidence, we propose a general mechanism of action for the VQ protein family, which likely defines a novel class of transcriptional regulators specific to plants. PMID:26220951

  9. Dead-box proteins: a family affair—active and passive players in RNP-remodeling

    PubMed Central

    Linder, Patrick

    2006-01-01

    DEAD-box proteins are characterized by nine conserved motifs. According to these criteria, several hundreds of these proteins can be identified in databases. Many different DEAD-box proteins can be found in eukaryotes, whereas prokaryotes have small numbers of different DEAD-box proteins. DEAD-box proteins play important roles in RNA metabolism, and they are very specific and cannot mutually be replaced. In vitro, many DEAD-box proteins have been shown to have RNA-dependent ATPase and ATP-dependent RNA helicase activities. From the genetic and biochemical data obtained mainly in yeast, it has become clear that these proteins play important roles in remodeling RNP complexes in a temporally controlled fashion. Here, I shall give a general overview of the DEAD-box protein family. PMID:16936318

  10. Arabidopsis Ovate Family Proteins, a Novel Transcriptional Repressor Family, Control Multiple Aspects of Plant Growth and Development

    SciTech Connect

    Wang, Shucai; Chang, Ying; Guo, Jianjun; Zeng, Qingning; Ellis, Brian; Chen, Jay

    2011-01-01

    BACKGROUND: The Arabidopsis genome contains 18 genes that are predicted to encode Ovate Family Proteins (AtOFPs), a protein family characterized by a conserved OVATE domain, an approximately 70-amino acid domain that was originally found in tomato OVATE protein. Among AtOFP family members, AtOFP1 has been shown to suppress cell elongation, in part, by suppressing the expression of AtGA20ox1, AtOFP4 has been shown to regulate secondary cell wall formation by interact with KNOTTED1-LIKE HOMEODOMAIN PROTEIN 7 (KNAT7), and AtOFP5 has been shown to regulate the activity of a BEL1-LIKEHOMEODOMAIN 1(BLH1)-KNAT3 complex during early embryo sac development, but little is known about the function of other AtOFPs. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated here that AtOFP proteins could function as effective transcriptional repressors in the Arabidopsis protoplast transient expression system. The analysis of loss-of-function alleles of AtOFPs suggested AtOFP genes may have overlapping function in regulating plant growth and development, because none of the single mutants identified, including T-DNA insertion mutants in AtOFP1, AtOFP4, AtOFP8, AtOFP10, AtOFP15 and AtOFP16, displayed any apparent morphological defects. Further, Atofp1 Atofp4 and Atofp15 Atofp16 double mutants still did not differ significantly from wild-type. On the other hand, plants overexpressing AtOFP genes displayed a number of abnormal phenotypes, which could be categorized into three distinct classes, suggesting that AtOFP genes may also have diverse functions in regulating plant growth and development. Further analysis suggested that AtOFP1 regulates cotyledon development in a postembryonic manner, and global transcript profiling revealed that it suppress the expression of many other genes. CONCLUSIONS/SIGNIFICANCE: Our results showed that AtOFPs function as transcriptional repressors and they regulate multiple aspects of plant growth and development. These results provided the first overview of a

  11. The retinoblastoma family of proteins directly represses transcription in Saccharomyces cerevisiae.

    PubMed

    Arnerić, Milica; Traven, Ana; Staresincić, Lidija; Sopta, Mary

    2002-03-15

    The retinoblastoma family of proteins are key cell cycle regulatory molecules important for the differentiation of various mammalian cell types. The retinoblastoma protein regulates transcription of a variety of genes either by blocking the activation domain of various activators or by active repression via recruitment to appropriate promoters. We show here that the retinoblastoma family of proteins functions as direct transcriptional repressors in a heterologous yeast system when fused to the DNA binding domain of Gal4. Mapping experiments indicate that either the A or the B domain of the pocket region is sufficient for repression in vivo. As is the case in mammalian cells, a phosphorylation site mutant of the retinoblastoma protein is a stronger transcriptional repressor than the wild type protein. We show that transcriptional repression by pRb is dependent on CLN3 in vivo. Furthermore, the yeast histone deacetylase components, RPD3 and SIN3, are required for transcriptional repression.

  12. The J-domain proteins of Arabidopsis thaliana: an unexpectedly large and diverse family of chaperones.

    PubMed

    Miernyk, J A

    2001-07-01

    A total of 89 J-domain proteins were identified in the genome of the model flowering plant Arabidopsis thaliana. The deduced amino acid sequences of the J-domain proteins were analyzed for an assortment of structural features and motifs. Based on the results of sequence comparisons and structure and function predictions, 51 distinct families were identified. The families ranged in size from 1 to 6 members. Subcellular localizations of the A thaliana J-domain proteins were predicted; species were found in both the soluble and membrane compartments of all cellular organelles. Based on digital Northern analysis, the J-domain proteins could be separated into groups of low, medium, and moderate expression levels. This genomics-based analysis of the A thaliana J-domain proteins establishes a framework for detailed studies of biological function and specificity. It additionally provides a comprehensive basis for evolutionary comparisons.

  13. Systematic genome-wide annotation of spliceosomal proteins reveals differential gene family expansion

    PubMed Central

    Barbosa-Morais, Nuno L.; Carmo-Fonseca, Maria; Aparício, Samuel

    2006-01-01

    Although more than 200 human spliceosomal and splicing-associated proteins are known, the evolution of the splicing machinery has not been studied extensively. The recent near-complete sequencing and annotation of distant vertebrate and chordate genomes provides the opportunity for an exhaustive comparative analysis of splicing factors across eukaryotes. We describe here our semiautomated computational pipeline to identify and annotate splicing factors in representative species of eukaryotes. We focused on protein families whose role in splicing is confirmed by experimental evidence. We visually inspected 1894 proteins and manually curated 224 of them. Our analysis shows a general conservation of the core spliceosomal proteins across the eukaryotic lineage, contrasting with selective expansions of protein families known to play a role in the regulation of splicing, most notably of SR proteins in metazoans and of heterogeneous nuclear ribonucleoproteins (hnRNP) in vertebrates. We also observed vertebrate-specific expansion of the CLK and SRPK kinases (which phosphorylate SR proteins), and the CUG-BP/CELF family of splicing regulators. Furthermore, we report several intronless genes amongst splicing proteins in mammals, suggesting that retrotransposition contributed to the complexity of the mammalian splicing apparatus. PMID:16344558

  14. The Dishevelled Protein Family: Still Rather a Mystery After Over 20 Years of Molecular Studies

    PubMed Central

    Mlodzik, Marek

    2016-01-01

    Dishevelled (Dsh) is a key component of Wnt-signaling pathways and possibly also has other functional requirements. Dsh appears to be a key factor to interpret Wnt signals coming via the Wnt-receptor family, the Frizzled proteins, from the plasma membrane and route them into the correct intracellular pathways. However, how Dsh is regulated to relay signal flow to specific and distinct cellular responses upon interaction with the same Wnt-receptor family remains very poorly understood. PMID:26969973

  15. Inference of Hopfield-Potts patterns from covariation in protein families: calculation and statistical error bars

    NASA Astrophysics Data System (ADS)

    Cocco, Simona; Monasson, Rémi; Weigt, Martin

    2013-12-01

    We consider the Hopfield-Potts model for the covariation between residues in protein families recently introduced in Cocco, Monasson, Weigt (2013). The patterns of the model are inferred from the data within a new gauge, more symmetric in the residues. We compute the statistical error bars on the pattern components. Results are illustrated on real data for a response regulator receiver domain (Pfam ID PF00072) family.

  16. The Rh protein family: gene evolution, membrane biology, and disease association.

    PubMed

    Huang, Cheng-Han; Ye, Mao

    2010-04-01

    The Rh (Rhesus) genes encode a family of conserved proteins that share a structural fold of 12 transmembrane helices with members of the major facilitator superfamily. Interest in this family has arisen from the discovery of Rh factor's involvement in hemolytic disease in the fetus and newborn, and of its homologs widely expressed in epithelial tissues. The Rh factor and Rh-associated glycoprotein (RhAG), with epithelial cousins RhBG and RhCG, form four subgroups conferring upon vertebrates a genealogical commonality. The past decade has heralded significant advances in understanding the phylogenetics, allelic diversity, crystal structure, and biological function of Rh proteins. This review describes recent progress on this family and the molecular insights gleaned from its gene evolution, membrane biology, and disease association. The focus is on its long evolutionary history and surprising structural conservation from prokaryotes to humans, pointing to the importance of its functional role, related to but distinct from ammonium transport proteins.

  17. ADAM and ADAMTS family proteins and their role in the colorectal cancer etiopathogenesis

    PubMed Central

    Przemyslaw, Leszczynski; Boguslaw, Hendrich Andrzej; Elzbieta, Szmida; Malgorzata, Sasiadek Maria

    2013-01-01

    The ADAM and ADAMTS families, also called adamalysins belong to an important group of extracellular matrix proteins. The ADAMs family belong to both the transmembrane and secreted proteins, while ADAMTS family only contains secreted forms. Adamalysins play an important role in the cell phenotype regulation via their activities in signaling pathways, cell adhesion and migration. The human proteome contains 21 ADAM, and 19 ADAMTS proteins, which are involved in extracellular matrix remodeling, shedding of various substrates such as: adhesion ligands, growth factors, their receptors and diverse cytokines. Recent studies provide evidence that adamalysins play a crucial role in colorectal cancer (CRC) etiopathogenesis. It seems possible that adamalysins might be used as CRC prediction markers or potential pharmaceutical targets. [BMB Reports 2013; 46(3): 139-150] PMID:23527857

  18. The HIR protein family: isolation and characterization of a complete murine cDNA.

    PubMed

    Scamps, C; Lorain, S; Lamour, V; Lipinski, M

    1996-04-10

    A full-length cDNA has been isolated for the murine homolog of the human HIRA protein, a member of the HIR family of nuclear proteins that is encoded from the chromosome 22 region critical for the DiGeorge syndrome. This family also contains Hir1p and Hir2p, two proteins identified as regulators of histone gene transcription in yeast. The murine and human amino acid sequences are 95.3% identical, with a striking 99.2% identity in the N-terminal WD repeat domain that is characteristic of the family. The two cDNAs are highly conserved within the coding regions, but also in the entire 5' untranslated region and in a strikingly long stretch of nucleotides in the 3' untranslated region.

  19. SEDS proteins are a widespread family of bacterial cell wall polymerases.

    PubMed

    Meeske, Alexander J; Riley, Eammon P; Robins, William P; Uehara, Tsuyoshi; Mekalanos, John J; Kahne, Daniel; Walker, Suzanne; Kruse, Andrew C; Bernhardt, Thomas G; Rudner, David Z

    2016-09-29

    Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan-synthetizing machinery called the Rod complex. Here we report that, in Bacillus subtilis, this complex is functional in the absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases the expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS (shape, elongation, division and sporulation) family of proteins, which have essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a family of peptidoglycan polymerases. Thus, B. subtilis and probably most bacteria use two distinct classes of polymerase to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development.

  20. SEDS proteins are a widespread family of bacterial cell wall polymerases

    PubMed Central

    Meeske, Alexander J.; Riley, Eammon P.; Robins, William P.; Uehara, Tsuyoshi; Mekelanos, John J.; Kahne, Daniel; Walker, Suzanne; Kruse, Andrew C.; Bernhardt, Thomas G.; Rudner, David Z.

    2016-01-01

    Summary Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan synthetic machinery called the Rod complex. We report that in Bacillus subtilis this complex is functional in the absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS family of proteins that play essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a new family of peptidoglycan polymerases. Thus, B. subtilis and likely most bacteria use two distinct classes of polymerases to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development. PMID:27525505

  1. Genome-wide Analysis of WD40 Protein Family in Human

    PubMed Central

    Zou, Xu-Dong; Hu, Xue-Jia; Ma, Jing; Li, Tuan; Ye, Zhi-Qiang; Wu, Yun-Dong

    2016-01-01

    The WD40 proteins, often acting as scaffolds to form functional complexes in fundamental cellular processes, are one of the largest families encoded by the eukaryotic genomes. Systematic studies of this family on genome scale are highly required for understanding their detailed functions, but are currently lacking in the animal lineage. Here we present a comprehensive in silico study of the human WD40 family. We have identified 262 non-redundant WD40 proteins, and grouped them into 21 classes according to their domain architectures. Among them, 11 animal-specific domain architectures have been recognized. Sequence alignment indicates the complicated duplication and recombination events in the evolution of this family. Through further phylogenetic analysis, we have revealed that the WD40 family underwent more expansion than the overall average in the evolutionary early stage, and the early emerged WD40 proteins are prone to domain architectures with fundamental cellular roles and more interactions. While most widely and highly expressed human WD40 genes originated early, the tissue-specific ones often have late origin. These results provide a landscape of the human WD40 family concerning their classification, evolution, and expression, serving as a valuable complement to the previous studies in the plant lineage. PMID:27991561

  2. In Arabidopsis thaliana, 1% of the genome codes for a novel protein family unique to plants.

    PubMed

    Aubourg, S; Boudet, N; Kreis, M; Lecharny, A

    2000-03-01

    In the sequences released by the Arabidopsis Genome Initiative (AGI), we discovered a new and unexpectedly large family of orphan genes (127 genes by 01.08.99), named AtPCMP. The distribution of the AtPCMP genes on the five chromosomes suggests that the genome of Arabidopsis thaliana contains more than 200 genes of this family (1% of the whole genome). The deduced AtPCMP proteins are characterized by a surprising combinatorial organization of sequence motifs. The amino-terminal domain is made of a succession of three conserved motifs which generate an important diversity. These proteins are classified into three subfamilies based on the length and nature of their carboxy-terminal domain constituted by 1-6 motifs. All the motifs characterized have an important level of conservation in both sequence and spacing. A specific signature of this large family is defined. The presence of ESTs in databases and the detection of clones in A. thaliana cDNA libraries indicate that most of the genes of this family are expressed. The absence of similar sequences outside the plant kingdom strongly suggests that this unusually large orphan family is unique to plants. Features, the genesis, the potential function and the evolution of this plant combinatorial and modular protein family are discussed.

  3. Using the SUBcellular database for Arabidopsis proteins to localize the Deg protease family

    PubMed Central

    Tanz, Sandra K.; Castleden, Ian; Hooper, Cornelia M.; Small, Ian; Millar, A. Harvey

    2014-01-01

    Sub-functionalization during the expansion of gene families in eukaryotes has occurred in part through specific subcellular localization of different family members. To better understand this process in plants, compiled records of large-scale proteomic and fluorescent protein localization datasets can be explored and bioinformatic predictions for protein localization can be used to predict the gaps in experimental data. This process can be followed by targeted experiments to test predictions. The SUBA3 database is a free web-service at http://suba.plantenergy.uwa.edu.au that helps users to explore reported experimental data and predictions concerning proteins encoded by gene families and to define the experiments required to locate these homologous sets of proteins. Here we show how SUBA3 can be used to explore the subcellular location of the Deg protease family of ATP-independent serine endopeptidases (Deg1–Deg16). Combined data integration and new experiments refined location information for Deg1 and Deg9, confirmed Deg2, Deg5, and Deg8 in plastids and Deg 15 in peroxisomes and provide substantial experimental evidence for mitochondrial localized Deg proteases. Two of these, Deg3 and Deg10, additionally localized to the plastid, revealing novel dual-targeted Deg proteases in the plastid and the mitochondrion. SUBA3 is continually updated to ensure that researchers can use the latest published data when planning the experimental steps remaining to localize gene family functions. PMID:25161662

  4. Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins.

    PubMed

    Saritas-Yildirim, Banu; Pliner, Hannah A; Ochoa, Angelica; Silva, Elena M

    2015-01-01

    Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.

  5. S18 family of mitochondrial ribosomal proteins: evolutionary history and Gly132 polymorphism in colon carcinoma.

    PubMed

    Mushtaq, Muhammad; Ali, Raja Hashim; Kashuba, Vladimir; Klein, George; Kashuba, Elena

    2016-08-23

    S18 family of mitochondrial ribosomal proteins (MRPS18, S18) consists of three members, S18-1 to -3. Earlier, we found that overexpression of S18-2 protein resulted in immortalization and eventual transformation of primary rat fibroblasts. The S18-1 and -3 have not exhibited such abilities. To understand the differences in protein properties, the evolutionary history of S18 family was analyzed. The S18-3, followed by S18-1 and S18-2 emerged as a result of ancient gene duplication in the root of eukaryotic species tree, followed by two metazoan-specific gene duplications. However, the most conserved metazoan S18 homolog is the S18-1; it shares the most sequence similarity with S18 proteins of bacteria and of other eukaryotic clades. Evolutionarily conserved residues of S18 proteins were analyzed in various cancers. S18-2 is mutated at a higher rate, compared with S18-1 and -3 proteins. Moreover, the evolutionarily conserved residue, Gly132 of S18-2, shows genetic polymorphism in colon adenocarcinomas that was confirmed by direct DNA sequencing.Concluding, S18 family represents the yet unexplored important mitochondrial ribosomal proteins.

  6. S18 family of mitochondrial ribosomal proteins: evolutionary history and Gly132 polymorphism in colon carcinoma

    PubMed Central

    Mushtaq, Muhammad; Ali, Raja Hashim; Kashuba, Vladimir; Klein, George; Kashuba, Elena

    2016-01-01

    S18 family of mitochondrial ribosomal proteins (MRPS18, S18) consists of three members, S18-1 to −3. Earlier, we found that overexpression of S18-2 protein resulted in immortalization and eventual transformation of primary rat fibroblasts. The S18-1 and −3 have not exhibited such abilities. To understand the differences in protein properties, the evolutionary history of S18 family was analyzed. The S18-3, followed by S18-1 and S18-2 emerged as a result of ancient gene duplication in the root of eukaryotic species tree, followed by two metazoan-specific gene duplications. However, the most conserved metazoan S18 homolog is the S18-1; it shares the most sequence similarity with S18 proteins of bacteria and of other eukaryotic clades. Evolutionarily conserved residues of S18 proteins were analyzed in various cancers. S18-2 is mutated at a higher rate, compared with S18-1 and −3 proteins. Moreover, the evolutionarily conserved residue, Gly132 of S18-2, shows genetic polymorphism in colon adenocarcinomas that was confirmed by direct DNA sequencing. Concluding, S18 family represents the yet unexplored important mitochondrial ribosomal proteins. PMID:27489352

  7. Identification of a novel SNF2/SWI2 protein family member, SRCAP, which interacts with CREB-binding protein.

    PubMed

    Johnston, H; Kneer, J; Chackalaparampil, I; Yaciuk, P; Chrivia, J

    1999-06-04

    The ability of cAMP response-element binding protein (CREB)-binding protein (CBP) to function as a co-activator for a number of transcription factors appears to be mediated by its ability to act as a histone acetyltransferase and through its interaction with a number of other proteins (general transcription factors, histone acetyltransferases, and other co-activators). Here we report that CBP also interacts with a novel ATPase termed Snf2-Related CBP Activator Protein (SRCAP). Consistent with this activity, SRCAP contains the conserved ATPase domain found within members of the Snf2 family. Transfection experiments demonstrate that SRCAP is able to activate transcription when expressed as a Gal-SRCAP chimera and that SRCAP also enhances the ability of CBP to activate transcription. The adenoviral protein E1A was found to disrupt interaction between SRCAP and CBP possibly representing a mechanism for E1A-mediated transcriptional repression.

  8. Two distinct SSB protein families in nucleo-cytoplasmic large DNA viruses

    PubMed Central

    Venclovas, Česlovas

    2012-01-01

    Motivation: Eukaryote-infecting nucleo-cytoplasmic large DNA viruses (NCLDVs) feature some of the largest genomes in the viral world. These viruses typically do not strongly depend on the host DNA replication systems. In line with this observation, a number of essential DNA replication proteins, such as DNA polymerases, primases, helicases and ligases, have been identified in the NCLDVs. One other ubiquitous component of DNA replisomes is the single-stranded DNA-binding (SSB) protein. Intriguingly, no NCLDV homologs of canonical OB-fold-containing SSB proteins had previously been detected. Only in poxviruses, one of seven NCLDV families, I3 was identified as the SSB protein. However, whether I3 is related to any known protein structure has not yet been established. Results: Here, we addressed the case of ‘missing’ canonical SSB proteins in the NCLDVs and also probed evolutionary origins of the I3 family. Using advanced computational methods, in four NCLDV families, we detected homologs of the bacteriophage T7 SSB protein (gp2.5). We found the properties of these homologs to be consistent with the SSB function. Moreover, we implicated specific residues in single-stranded DNA binding. At the same time, we found no evolutionary link between the T7 gp2.5-like NCLDV SSB homologs and the poxviral SSB protein (I3). Instead, we identified a distant relationship between I3 and small protein B (SmpB), a bacterial RNA-binding protein. Thus, apparently, the NCLDVs have the two major distinct sets of SSB proteins having bacteriophage and bacterial origins, respectively. Contact: venclovas@ibt.lt Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23097418

  9. Structural biology of the Bcl-2 family and its mimicry by viral proteins

    PubMed Central

    Kvansakul, M; Hinds, M G

    2013-01-01

    Intrinsic apoptosis in mammals is regulated by protein–protein interactions among the B-cell lymphoma-2 (Bcl-2) family. The sequences, structures and binding specificity between pro-survival Bcl-2 proteins and their pro-apoptotic Bcl-2 homology 3 motif only (BH3-only) protein antagonists are now well understood. In contrast, our understanding of the mode of action of Bax and Bak, the two necessary proteins for apoptosis is incomplete. Bax and Bak are isostructural with pro-survival Bcl-2 proteins and also interact with BH3-only proteins, albeit weakly. Two sites have been identified; the in-groove interaction analogous to the pro-survival BH3-only interaction and a site on the opposite molecular face. Interaction of Bax or Bak with activator BH3-only proteins and mitochondrial membranes triggers a series of ill-defined conformational changes initiating their oligomerization and mitochondrial outer membrane permeabilization. Many actions of the mammalian pro-survival Bcl-2 family are mimicked by viruses. By expressing proteins mimicking mammalian pro-survival Bcl-2 family proteins, viruses neutralize death-inducing members of the Bcl-2 family and evade host cell apoptosis during replication. Remarkably, structural elements are preserved in viral Bcl-2 proteins even though there is in many cases little discernible sequence conservation with their mammalian counterparts. Some viral Bcl-2 proteins are dimeric, but they have distinct structures to those observed for mammalian Bcl-2 proteins. Furthermore, viral Bcl-2 proteins modulate innate immune responses regulated by NF-κB through an interface separate from the canonical BH3-binding groove. Our increasing structural understanding of the viral Bcl-2 proteins is leading to new insights in the cellular Bcl-2 network by exploring potential alternate functional modes in the cellular context. We compare the cellular and viral Bcl-2 proteins and discuss how alterations in their structure, sequence and binding specificity

  10. The OmpA family of proteins: roles in bacterial pathogenesis and immunity.

    PubMed

    Confer, Anthony W; Ayalew, Sahlu

    2013-05-03

    The OmpA family of outer membrane proteins is a group of genetically related, heat-modifiable, surface-exposed, porin proteins that are in high-copy number in the outer membrane of mainly Gram-negative bacteria. OmpA proteins are characterized by an N-terminal domain that forms an eight-stranded, anti-parallel β barrel, which is embedded in the outer membrane. The C-terminal domain is globular and located in the periplasmic space. Escherichia coli OmpA is the best characterized of the proteins. Other well-characterized OmpA-equivalent proteins from pathogenic bacteria include Pseudomonas aeruginosa OprF, Haemophilus influenzae P5, Klebsiella pneumoniae OmpA, and Chlamydia trachomatis major outer membrane protein (MOMP). OmpA from the veterinary pathogens Mannheimia haemolytica, Haemophilus parasuis, Leptospira interrogans, and Pasteurella multocida have been studied to a lesser extent. Among many of the pathogenic bacteria, OmpA proteins have important pathogenic roles including bacterial adhesion, invasion, or intracellular survival as well as evasion of host defenses or stimulators of pro-inflammatory cytokine production. These pathogenic roles are most commonly associated with central nervous system, respiratory and urogenital diseases. Alternatively, OmpA family proteins can serve as targets of the immune system with immunogenicity related to surface-exposed loops of the molecule. In several cases, OmpA proteins are under evaluation as potential vaccine candidates.

  11. FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells

    PubMed Central

    Nalbant, Demet; Youn, Hyewon; Nalbant, S Isil; Sharma, Savitha; Cobos, Everardo; Beale, Elmus G; Du, Yang; Williams, Simon C

    2005-01-01

    Background Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line. Results One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20) with three members (FAM20A, FAM20B and FAM20C) in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c) were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members. Conclusions The FAM20 family represents a new