Transcriptome Dynamics during Maize Endosperm Development

PubMed Central

Feng, Jiaojiao; Xu, Shutu; Wang, Lei; Li, Feifei; Li, Yibo; Zhang, Renhe; Zhang, Xinghua; Xue, Jiquan; Guo, Dongwei

2016-01-01

The endosperm is a major organ of the seed that plays vital roles in determining seed weight and quality. However, genome-wide transcriptome patterns throughout maize endosperm development have not been comprehensively investigated to date. Accordingly, we performed a high-throughput RNA sequencing (RNA-seq) analysis of the maize endosperm transcriptome at 5, 10, 15 and 20 days after pollination (DAP). We found that more than 11,000 protein-coding genes underwent alternative splicing (AS) events during the four developmental stages studied. These genes were mainly involved in intracellular protein transport, signal transmission, cellular carbohydrate metabolism, cellular lipid metabolism, lipid biosynthesis, protein modification, histone modification, cellular amino acid metabolism, and DNA repair. Additionally, 7,633 genes, including 473 transcription factors (TFs), were differentially expressed among the four developmental stages. The differentially expressed TFs were from 50 families, including the bZIP, WRKY, GeBP and ARF families. Further analysis of the stage-specific TFs showed that binding, nucleus and ligand-dependent nuclear receptor activities might be important at 5 DAP, that immune responses, signalling, binding and lumen development are involved at 10 DAP, that protein metabolic processes and the cytoplasm might be important at 15 DAP, and that the responses to various stimuli are different at 20 DAP compared with the other developmental stages. This RNA-seq analysis provides novel, comprehensive insights into the transcriptome dynamics during early endosperm development in maize. PMID:27695101

Activation by Insulin and Amino Acids of Signaling Components Leading to Translation Initiation in Skeletal Muscle of Neonatal Pigs Is Developmentally Regulated

PubMed Central

Suryawan, Agus; Orellana, Renan A.; Nguyen, Hanh V.; Jeyapalan, Asumthia S.; Fleming, Jillian R.; Davis, Teresa A.

2009-01-01

Insulin (INS) and amino acids (AA) act independently to stimulate protein synthesis in skeletal muscle of neonatal pigs and the responses decrease with development. The purpose of this study was to compare the separate effects of fed levels of INS and AA on the activation of signaling components leading to translation initiation and how these responses change with development. Overnight fasted 6-day-old (n=4/group) and 26-day-old (n=6/group) pigs were studied during: 1) euinsulinemic-euglycemic-euaminoacidemic conditions (controls), 2) euinsulinemic-euglycemic-hyperaminoacidemic clamps (AA), and 3) hyperinsulinemic-euglycemic-euaminoacidemic clamps (INS). INS, but not AA, increased the phosphorylation of protein kinase B (PKB) and tuberous sclerosis 2 (TSC2). Both INS and AA increased protein synthesis and the phosphorylation of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase-1, and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) and these responses were higher in 6-day-old compared to 26-day-old pigs. Both INS and AA decreased the binding of 4E-BP1 to eIF4E and increased eIF4E binding to eIF4G; these effects were greater in 6-day-old than in 26-day-old pigs. Neither INS nor AA altered the composition of mTORC1 (raptor, mTOR, and GβL) or mTORC2 (rictor, mTOR, and GβL) complexes. Furthermore, neither INS, AA, nor age had any effect on the abundance of Rheb and the phosphorylation of AMP-activated kinase (AMPK) and eukaryotic elongation factor 2 (eEF2). Our results suggest that the activation by insulin and amino acids of signaling components leading to translation initiation is developmentally regulated and parallels the developmental decline in protein synthesis in skeletal muscle of neonatal pigs. PMID:17878222

Structural and Functional Features of a Developmentally Regulated Lipopolysaccharide-Binding Protein

PubMed Central

Krasity, Benjamin C.; Troll, Joshua V.; Lehnert, Erik M.; Hackett, Kathleen T.; Dillard, Joseph P.; Apicella, Michael A.; Goldman, William E.

2015-01-01

ABSTRACT Mammalian lipopolysaccharide (LPS) binding proteins (LBPs) occur mainly in extracellular fluids and promote LPS delivery to specific host cell receptors. The function of LBPs has been studied principally in the context of host defense; the possible role of LBPs in nonpathogenic host-microbe interactions has not been well characterized. Using the Euprymna scolopes-Vibrio fischeri model, we analyzed the structure and function of an LBP family protein, E. scolopes LBP1 (EsLBP1), and provide evidence for its role in triggering a symbiont-induced host developmental program. Previous studies showed that, during initial host colonization, the LPS of V. fischeri synergizes with peptidoglycan (PGN) monomer to induce morphogenesis of epithelial tissues of the host animal. Computationally modeled EsLBP1 shares some but not all structural features of mammalian LBPs that are thought important for LPS binding. Similar to human LBP, recombinant EsLBP1 expressed in insect cells bound V. fischeri LPS and Neisseria meningitidis lipooligosaccharide (LOS) with nanomolar or greater affinity but bound Francisella tularensis LPS only weakly and did not bind PGN monomer. Unlike human LBP, EsLBP1 did not bind N. meningitidis LOS:CD14 complexes. The eslbp1 transcript was upregulated ~22-fold by V. fischeri at 24 h postinoculation. Surprisingly, this upregulation was not induced by exposure to LPS but, rather, to the PGN monomer alone. Hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) and immunocytochemistry (ICC) localized eslbp1 transcript and protein in crypt epithelia, where V. fischeri induces morphogenesis. The data presented here provide a window into the evolution of LBPs and the scope of their roles in animal symbioses. PMID:26463160

Bovine oocytes and early embryos express Staufen and ELAVL RNA-binding proteins.

PubMed

Calder, M D; Madan, P; Watson, A J

2008-05-01

RNA-binding proteins (RBP) influence RNA editing, localization, stability and translation and may contribute to oocyte developmental competence by regulating the stability and turnover of oogenetic mRNAs. The expression of Staufen 1 and 2 and ELAVL1, ELAVL2 RNA-binding proteins during cow early development was characterized. Cumulus-oocyte complexes were collected from slaughterhouse ovaries, matured, inseminated and subjected to embryo culture in vitro. Oocyte or preimplantation embryo pools were processed for RT-PCR and whole-mount immunofluorescence analysis of mRNA expression and protein distribution. STAU1 and STAU2 and ELAVL1 mRNAs and proteins were detected throughout cow preimplantation development from the germinal vesicle (GV) oocyte to the blastocyst stage. ELAVL2 mRNAs were detectable from the GV to the morula stage, whereas ELAVL2 protein was in all stages examined and localized to both cytoplasm and nuclei. The findings provide a foundation for investigating the role of RBPs during mammalian oocyte maturation and early embryogenesis.

Redox modulation of plant developmental regulators from the class I TCP transcription factor family.

PubMed

Viola, Ivana L; Güttlein, Leandro N; Gonzalez, Daniel H

2013-07-01

TEOSINTE BRANCHED1-CYCLOIDEA-PROLIFERATING CELL FACTOR1 (TCP) transcription factors participate in plant developmental processes associated with cell proliferation and growth. Most members of class I, one of the two classes that compose the family, have a conserved cysteine at position 20 (Cys-20) of the TCP DNA-binding and dimerization domain. We show that Arabidopsis (Arabidopsis thaliana) class I proteins with Cys-20 are sensitive to redox conditions, since their DNA-binding activity is inhibited after incubation with the oxidants diamide, oxidized glutathione, or hydrogen peroxide or with nitric oxide-producing agents. Inhibition can be reversed by treatment with the reductants dithiothreitol or reduced glutathione or by incubation with the thioredoxin/thioredoxin reductase system. Mutation of Cys-20 in the class I protein TCP15 abolished its redox sensitivity. Under oxidizing conditions, covalently linked dimers were formed, suggesting that inactivation is associated with the formation of intermolecular disulfide bonds. Inhibition of class I TCP protein activity was also observed in vivo, in yeast (Saccharomyces cerevisiae) cells expressing TCP proteins and in plants after treatment with redox agents. This inhibition was correlated with modifications in the expression of the downstream CUC1 gene in plants. Modeling studies indicated that Cys-20 is located at the dimer interface near the DNA-binding surface. This places this residue in the correct orientation for intermolecular disulfide bond formation and explains the sensitivity of DNA binding to the oxidation of Cys-20. The redox properties of Cys-20 and the observed effects of cellular redox agents both in vitro and in vivo suggest that class I TCP protein action is under redox control in plants.

Redox Modulation of Plant Developmental Regulators from the Class I TCP Transcription Factor Family1[W][OA

PubMed Central

Viola, Ivana L.; Güttlein, Leandro N.; Gonzalez, Daniel H.

2013-01-01

TEOSINTE BRANCHED1-CYCLOIDEA-PROLIFERATING CELL FACTOR1 (TCP) transcription factors participate in plant developmental processes associated with cell proliferation and growth. Most members of class I, one of the two classes that compose the family, have a conserved cysteine at position 20 (Cys-20) of the TCP DNA-binding and dimerization domain. We show that Arabidopsis (Arabidopsis thaliana) class I proteins with Cys-20 are sensitive to redox conditions, since their DNA-binding activity is inhibited after incubation with the oxidants diamide, oxidized glutathione, or hydrogen peroxide or with nitric oxide-producing agents. Inhibition can be reversed by treatment with the reductants dithiothreitol or reduced glutathione or by incubation with the thioredoxin/thioredoxin reductase system. Mutation of Cys-20 in the class I protein TCP15 abolished its redox sensitivity. Under oxidizing conditions, covalently linked dimers were formed, suggesting that inactivation is associated with the formation of intermolecular disulfide bonds. Inhibition of class I TCP protein activity was also observed in vivo, in yeast (Saccharomyces cerevisiae) cells expressing TCP proteins and in plants after treatment with redox agents. This inhibition was correlated with modifications in the expression of the downstream CUC1 gene in plants. Modeling studies indicated that Cys-20 is located at the dimer interface near the DNA-binding surface. This places this residue in the correct orientation for intermolecular disulfide bond formation and explains the sensitivity of DNA binding to the oxidation of Cys-20. The redox properties of Cys-20 and the observed effects of cellular redox agents both in vitro and in vivo suggest that class I TCP protein action is under redox control in plants. PMID:23686421

Effects of altered gravity on the expression of Calcium -binding and matrix proteins in the inner ear of developing fish following ∆g-expositions.

NASA Astrophysics Data System (ADS)

Hilbig, Reinhard; Hendrik Anken, Ralf; Weigele, Jochen

The results of the Foton-M3 mission (OmegaHab) give evidence that the otoliths of the fish form OmegaHab were larger as compared to the ground control. Additionally the shape (raphe) and morphology especially the mode of crystallization of the otoliths were affected during growth in weightlessness. The reason for these changes is assumed to originate from changes in the composition of the otolith matrix and Ca-binding proteins (OMP). The OMPs play an important role in controlling the crystallization process and additionally the morphology of crystals, determining the crystallpolymorph and the strength of the crystals. The matrix of otoliths is a complex functional structure containing several calcium-binding proteins, structural proteins and protease inhibitors. Furthermore it is composed of otolith matrix protein-1, Otolin, Otoconin, SPARC and Neuroserpin, which is a specific expression in the otolth matrix for chichlid fish. During embryonic development of the fish inner ear, these proteins show a spacial and temporal expression pattern. The formation of the inner ear -including otoliths and sensory cells -starting from the otocyst-anlage -can be subdivided in several major developmental stages e.g. the forming of the otic cavity (stage 7/8), the tetha cell or seeding stage (stage 8, 9), the development of the semicircular channels (stage 12), the transition to further daily growth (post stage15) and the development of the third otolith, asteriscus (stage 23). These developmental phases contain different constitutions or involvements of matrix proteins. We investigated the matrixprotein composition of the chichlid fish Oreochromis mossambicus and found that the otolith matrix differentiate between other fishes. In this case some matrix proteins seem to be uniform in fishes, other known matrix proteins are lacking and we have also references to new candidates for matrix proteins chichlids. In this case we investigated the expression of the matrix proteins otolith matrix protein 1, Otolin, Otoconin, SPARC and Neuroserpin during the genesis of Oeochromis mossambicus with its particular regard to the certain developmental stages of the inner ear. The profiles of these compounds were followed in experiments using hypergravity and simulated gravity and thus should be the basis for new microgravity experiments

Profiles of embryonic nuclear protein binding to the proximal promoter region of the soybean β-conglycinin α subunit gene.

PubMed

Yoshino, M; Tsutsumi, K; Kanazawa, A

2015-01-01

β-Conglycinin, a major component of seed storage protein in soybean, comprises three subunits: α, α' and β. The expression of genes for these subunits is strictly controlled during embryogenesis. The proximal promoter region up to 245 bp upstream of the transcription start site of the α subunit gene sufficiently confers spatial and temporal control of transcription in embryos. Here, the binding profile of nuclear proteins in the proximal promoter region of the α subunit gene was analysed. DNase I footprinting analysis indicated binding of proteins to the RY element and DNA regions including box I, a region conserved in cognate gene promoters. An electrophoretic mobility shift assay (EMSA) using different portions of box I as a probe revealed that multiple portions of box I bind to nuclear proteins. In addition, an EMSA using nuclear proteins extracted from embryos at different developmental stages indicated that the levels of major DNA-protein complexes on box I increased during embryo maturation. These results are consistent with the notion that box I is important for the transcriptional control of seed storage protein genes. Furthermore, the present data suggest that nuclear proteins bind to novel motifs in box I including 5'-TCAATT-3' rather than to predicted cis-regulatory elements. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Polyamine binding to proteins in oat and Petunia protoplasts

NASA Technical Reports Server (NTRS)

Mizrahi, Y.; Applewhite, P. B.; Galston, A. W.

1989-01-01

Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozen-thawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.

Polyamine binding to proteins in oat and Petunia protoplasts.

PubMed

Mizrahi, Y; Applewhite, P B; Galston, A W

1989-01-01

Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozen-thawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.

Trimeric association of Hox and TALE homeodomain proteins mediates Hoxb2 hindbrain enhancer activity.

PubMed

Jacobs, Y; Schnabel, C A; Cleary, M L

1999-07-01

Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (three-amino-acid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. In this study, we demonstrated that Pbx and Meis bind DNA as heterotrimeric complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which we showed to be required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to interact through their amino termini and bind DNA without stringent half-site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. Our data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element.

Uncoupling of Obesity from Insulin Resistance Through a Targeted Mutation in aP2, the Adipocyte Fatty Acid Binding Protein

NASA Astrophysics Data System (ADS)

Hotamisligil, Gokhan S.; Johnson, Randall S.; Distel, Robert J.; Ellis, Ramsey; Papaioannou, Virginia E.; Spiegelman, Bruce M.

1996-11-01

Fatty acid binding proteins (FABPs) are small cytoplasmic proteins that are expressed in a highly tissue-specific manner and bind to fatty acids such as oleic and retinoic acid. Mice with a null mutation in aP2, the gene encoding the adipocyte FABP, were developmentally and metabolically normal. The aP2-deficient mice developed dietary obesity but, unlike control mice, they did not develop insulin resistance or diabetes. Also unlike their obese wild-type counterparts, obese aP2-/- animals failed to express in adipose tissue tumor necrosis factor-α (TNF-α), a molecule implicated in obesity-related insulin resistance. These results indicate that aP2 is central to the pathway that links obesity to insulin resistance, possibly by linking fatty acid metabolism to expression of TNF-α.

Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

PubMed Central

Meyer, Katja; Koester, Tino; Staiger, Dorothee

2015-01-01

Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance. PMID:26213982

Developmental roles of 21 Drosophila transcription factors are determined by quantitative differences in binding to an overlapping set of thousands of genomic regions

DOE Office of Scientific and Technical Information (OSTI.GOV)

MacArthur, Stewart; Li, Xiao-Yong; Li, Jingyi

2009-05-15

BACKGROUND: We previously established that six sequence-specific transcription factors that initiate anterior/posterior patterning in Drosophila bind to overlapping sets of thousands of genomic regions in blastoderm embryos. While regions bound at high levels include known and probable functional targets, more poorly bound regions are preferentially associated with housekeeping genes and/or genes not transcribed in the blastoderm, and are frequently found in protein coding sequences or in less conserved non-coding DNA, suggesting that many are likely non-functional. RESULTS: Here we show that an additional 15 transcription factors that regulate other aspects of embryo patterning show a similar quantitative continuum of functionmore » and binding to thousands of genomic regions in vivo. Collectively, the 21 regulators show a surprisingly high overlap in the regions they bind given that they belong to 11 DNA binding domain families, specify distinct developmental fates, and can act via different cis-regulatory modules. We demonstrate, however, that quantitative differences in relative levels of binding to shared targets correlate with the known biological and transcriptional regulatory specificities of these factors. CONCLUSIONS: It is likely that the overlap in binding of biochemically and functionally unrelated transcription factors arises from the high concentrations of these proteins in nuclei, which, coupled with their broad DNA binding specificities, directs them to regions of open chromatin. We suggest that most animal transcription factors will be found to show a similar broad overlapping pattern of binding in vivo, with specificity achieved by modulating the amount, rather than the identity, of bound factor.« less

Structural and functional features of a developmentally regulated lipopolysaccharide-binding protein.

PubMed

Krasity, Benjamin C; Troll, Joshua V; Lehnert, Erik M; Hackett, Kathleen T; Dillard, Joseph P; Apicella, Michael A; Goldman, William E; Weiss, Jerrold P; McFall-Ngai, Margaret J

2015-10-13

Mammalian lipopolysaccharide (LPS) binding proteins (LBPs) occur mainly in extracellular fluids and promote LPS delivery to specific host cell receptors. The function of LBPs has been studied principally in the context of host defense; the possible role of LBPs in nonpathogenic host-microbe interactions has not been well characterized. Using the Euprymna scolopes-Vibrio fischeri model, we analyzed the structure and function of an LBP family protein, E. scolopes LBP1 (EsLBP1), and provide evidence for its role in triggering a symbiont-induced host developmental program. Previous studies showed that, during initial host colonization, the LPS of V. fischeri synergizes with peptidoglycan (PGN) monomer to induce morphogenesis of epithelial tissues of the host animal. Computationally modeled EsLBP1 shares some but not all structural features of mammalian LBPs that are thought important for LPS binding. Similar to human LBP, recombinant EsLBP1 expressed in insect cells bound V. fischeri LPS and Neisseria meningitidis lipooligosaccharide (LOS) with nanomolar or greater affinity but bound Francisella tularensis LPS only weakly and did not bind PGN monomer. Unlike human LBP, EsLBP1 did not bind N. meningitidis LOS:CD14 complexes. The eslbp1 transcript was upregulated ~22-fold by V. fischeri at 24 h postinoculation. Surprisingly, this upregulation was not induced by exposure to LPS but, rather, to the PGN monomer alone. Hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) and immunocytochemistry (ICC) localized eslbp1 transcript and protein in crypt epithelia, where V. fischeri induces morphogenesis. The data presented here provide a window into the evolution of LBPs and the scope of their roles in animal symbioses. Mammalian lipopolysaccharide (LPS)-binding protein (LBP) is implicated in conveying LPS to host cells and potentiating its signaling activity. In certain disease states, such as obesity, the overproduction of this protein has been a reliable biomarker of chronic inflammation. Here, we describe a symbiosis-induced invertebrate LBP whose tertiary structure and LPS-binding characteristics are similar to those of mammalian LBPs; however, the primary structure of this distantly related squid protein (EsLBP1) differs in key residues previously believed to be essential for LPS binding, suggesting that an alternative strategy exists. Surprisingly, symbiotic expression of eslbp1 is induced by peptidoglycan derivatives, not LPS, a pattern converse to that of RegIIIγ, an important mammalian immunity protein that binds peptidoglycan but whose gene expression is induced by LPS. Finally, EsLBP1 occurs along the apical surfaces of all the host's epithelia, suggesting that it was recruited from a general defensive role to one that mediates specific interactions with its symbiont. Copyright © 2015 Krasity et al.

The Arabidopsis thaliana TCP transcription factors: A broadening horizon beyond development

PubMed Central

Li, Shutian

2015-01-01

The TCP family of transcription factors is named after the first 4 characterized members, namely TEOSINTE BRANCHED1 (TB1) from maize (Zea mays), CYCLOIDEA (CYC) from snapdragon (Antirrhinum majus), as well as PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR1 (PCF1) and PCF2 from rice (Oryza sativa). Phylogenic analysis of this plant-specific protein family unveils a conserved bHLH-containing DNA-binding motif known as the TCP domain. In accordance with the structure of this shared domain, TCP proteins are grouped into class I (TCP-P) and class II (TCP-C), which are suggested to antagonistically modulate plant growth and development via competitively binding similar cis-regulatory modules called site II elements. Over the last decades, TCPs across the plant kingdom have been demonstrated to control a plethora of plant processes. Notably, TCPs also regulate plant development and defense responses via stimulating the biosynthetic pathways of bioactive metabolites, such as brassinosteroid (BR), jasmonic acid (JA) and flavonoids. Besides, mutagenesis analysis coupled with biochemical experiments identifies several crucial amino acids located within the TCP domain, which confer the redox sensitivity of class I TCPs and determine the distinct DNA-binding properties of TCPs. In this review, developmental functions of TCPs in various biological pathways are briefly described with an emphasis on their involvement in the synthesis of bioactive substances. Furthermore, novel biochemical aspects of TCPs with respect to redox regulation and DNA-binding preferences are elaborated. In addition, the unexpected participation of TCPs in effector-triggered immunity (ETI) and defense against insects indicates that the widely recognized developmental regulators are capable of fine-tuning defense signaling and thereby enable plants to evade deleterious developmental phenotypes. Altogether, these recent impressive breakthroughs remarkably advance our understanding as to how TCPs integrate internal developmental cues with external environmental stimuli to orchestrate plant development. PMID:26039357

The Arabidopsis thaliana TCP transcription factors: A broadening horizon beyond development.

PubMed

Li, Shutian

2015-01-01

The TCP family of transcription factors is named after the first 4 characterized members, namely TEOSINTE BRANCHED1 (TB1) from maize (Zea mays), CYCLOIDEA (CYC) from snapdragon (Antirrhinum majus), as well as PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR1 (PCF1) and PCF2 from rice (Oryza sativa). Phylogenic analysis of this plant-specific protein family unveils a conserved bHLH-containing DNA-binding motif known as the TCP domain. In accordance with the structure of this shared domain, TCP proteins are grouped into class I (TCP-P) and class II (TCP-C), which are suggested to antagonistically modulate plant growth and development via competitively binding similar cis-regulatory modules called site II elements. Over the last decades, TCPs across the plant kingdom have been demonstrated to control a plethora of plant processes. Notably, TCPs also regulate plant development and defense responses via stimulating the biosynthetic pathways of bioactive metabolites, such as brassinosteroid (BR), jasmonic acid (JA) and flavonoids. Besides, mutagenesis analysis coupled with biochemical experiments identifies several crucial amino acids located within the TCP domain, which confer the redox sensitivity of class I TCPs and determine the distinct DNA-binding properties of TCPs. In this review, developmental functions of TCPs in various biological pathways are briefly described with an emphasis on their involvement in the synthesis of bioactive substances. Furthermore, novel biochemical aspects of TCPs with respect to redox regulation and DNA-binding preferences are elaborated. In addition, the unexpected participation of TCPs in effector-triggered immunity (ETI) and defense against insects indicates that the widely recognized developmental regulators are capable of fine-tuning defense signaling and thereby enable plants to evade deleterious developmental phenotypes. Altogether, these recent impressive breakthroughs remarkably advance our understanding as to how TCPs integrate internal developmental cues with external environmental stimuli to orchestrate plant development.

Developmental changes in the role of a pertussis toxin sensitive guanine nucleotide binding protein in the rat cardiac alpha sub 1 -adrenergic system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, H.M.

1989-01-01

During development, the cardiac alpha{sub 1}-adrenergic chronotropic response changes from positive in the neonate to negative in the adult. This thesis examined the possibility of a developmental change in coupling of a PT-sensitive G-protein to the alpha{sub 1}-adrenergic receptor. Radioligand binding experiments performed with the iodinated alpha{sub 1}-selective radioligand ({sup 125}I)-I-2-({beta}-(4-hydroxphenyl)ethylaminomethyl)tetralone (({sup 125}I)-IBE 2254) demonstrated that the alpha{sub 1}-adrenergic receptor is coupled to a G-protein in both neonatal and adult rat hearts. However, in the neonate the alpha{sub 1}-adrenergic receptor is coupled to a PT-insensitive G-protein, whereas in the adult the alpha{sub 1}-adrenergic receptor is coupled to both a PT-insensitivemore » and a PT-sensitive G-protein. Consistent with the results from binding experiments, PT did not have any effect on the alpha{sub 1}-mediated positive chronotropic response in the neonate, whereas in the adult the alpha{sub 1}-mediated negative chronotropic response was completely converted to a positive one after PT-treatment. This thesis also examined the possibility of an alteration in coupling of the alpha{sub 1}-adrenergic receptor to its effector under certain circumstances such as high potassium (K{sup +}) depolarization in nerve-muscle (NM) co-cultures, a system which has been previously shown to be a convenient in vitro model to study the mature inhibitory alpha{sub 1}-response.« less

Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

PubMed

Dong, Yongbin; Wang, Qilei; Zhang, Long; Du, Chunguang; Xiong, Wenwei; Chen, Xinjian; Deng, Fei; Ma, Zhiyan; Qiao, Dahe; Hu, Chunhui; Ren, Yangliu; Li, Yuling

2015-01-01

The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize.

Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn

PubMed Central

Du, Chunguang; Xiong, Wenwei; Chen, Xinjian; Deng, Fei; Ma, Zhiyan; Qiao, Dahe; Hu, Chunhui; Ren, Yangliu; Li, Yuling

2015-01-01

The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize. PMID:26587848

Comparison of developmental gradients for growth, ATPase, and fusicoccin-binding activity in mung bean hypocotyls

NASA Technical Reports Server (NTRS)

Basel, L. E.; Cleland, R. E.

1992-01-01

A comparison has been made of the developmental gradients along a mung bean (Vigna radiata L.) hypocotyl of the growth rate, plasma membrane ATPase, and fusicoccin-binding protein (FCBP) activity to determine whether they are interrelated. The hook and four sequential 7.5 millimeter segments of the hypocotyl below the hook were cut. A plasma membrane-enriched fraction was isolated from each section by aqueous two-phase partitioning and assayed for vanadate-sensitive ATPase and FCBP activity. Each gradient had a distinctive and different pattern. Endogenous growth rate was maximal in the second section and much lower in the others. Vanadate-sensitive ATPase activity was maximal in the third section, but remained high in the older sections. Amounts of ATPase protein, shown by specific antibody binding, did not correlate with the amount of vanadate-sensitive ATPase activity in the three youngest sections. FCBP activity was almost absent in the first section, then increased to a maximum in the oldest sections. These data show that the growth rate is not determined by the ATPase activity, and that there are no fixed ratios between the ATPase and FCBP.

Structural and evolutionary analysis of Leishmania Alba proteins.

PubMed

da Costa, Kauê Santana; Galúcio, João Marcos Pereira; Leonardo, Elvis Santos; Cardoso, Guelber; Leal, Élcio; Conde, Guilherme; Lameira, Jerônimo

2017-10-01

The Alba superfamily proteins share a common RNA-binding domain. These proteins participate in a variety of regulatory pathways by controlling developmental gene expression. They also interact with ribosomal subunits, translation factors, and other RNA-binding proteins. The Leishmania infantum genome encodes two Alba-domain proteins, LiAlba1 and LiAlba3. In this work, we used homology modeling, protein-protein docking, and molecular dynamics (MD) simulations to explore the details of the Alba1-Alba3-RNA complex from Leishmania infantum at the molecular level. In addition, we compared the structure of LiAlba3 with the human ribonuclease P component, Rpp20. We also mapped the ligand-binding residues on the Alba3 surface to analyze its druggability and performed mutational analyses in Alba3 using alanine scanning to identify residues involved in its function and structural stability. These results suggest that the RGG-box motif of LiAlba1 is important for protein function and stability. Finally, we discuss the function of Alba proteins in the context of pathogen adaptation to host cells. The data provided herein will facilitate further translational research regarding Alba structure and function. Copyright © 2017 Elsevier B.V. All rights reserved.

The poly(rC)-binding protein αCP2 is a noncanonical factor in X. laevis cytoplasmic polyadenylation

PubMed Central

Vishnu, Melanie R.; Sumaroka, Marina; Klein, Peter S.; Liebhaber, Stephen A.

2011-01-01

Post-transcriptional control of mRNA stability and translation is central to multiple developmental pathways. This control can be linked to cytoplasmic polyadenylation in certain settings. In maturing Xenopus oocytes, specific mRNAs are targeted for polyadenylation via recruitment of the Cytoplasmic Polyadenylation Element (CPE) binding protein (CPEB) to CPE(s) within the 3′ UTR. Cytoplasmic polyadenylation is also critical to early embryonic events, although corresponding determinants are less defined. Here, we demonstrate that the Xenopus ortholog of the poly(rC) binding protein αCP2 can recruit cytoplasmic poly(A) polymerase activity to mRNAs in Xenopus post-fertilization embryos, and that this recruitment relies on cis sequences recognized by αCP2. We find that the hα-globin 3′ UTR, a validated mammalian αCP2 target, constitutes an effective target for cytoplasmic polyadenylation in Xenopus embryos, but not during Xenopus oocyte maturation. We further demonstrate that the cytoplasmic polyadenylation activity is dependent on the action of the C-rich αCP-binding site in conjunction with the adjacent AAUAAA. Consistent with its ability to target mRNA for poly(A) addition, we find that XαCP2 associates with core components of the Xenopus cytoplasmic polyadenylation complex, including the cytoplasmic poly(A) polymerase XGLD2. Furthermore, we observe that the C-rich αCP-binding site can robustly enhance the activity of a weak canonical oocyte maturation CPE in early embryos, possibly via a direct interaction between XαCP2 and CPEB1. These studies establish XαCP2 as a novel cytoplasmic polyadenylation trans factor, indicate that C-rich sequences can function as noncanonical cytoplasmic polyadenylation elements, and expand our understanding of the complexities underlying cytoplasmic polyadenylation in specific developmental settings. PMID:21444632

Developmental Effects and Estrogenicity of Bisphenol A Alternatives in a Zebrafish Embryo Model.

PubMed

Mu, Xiyan; Huang, Ying; Li, Xuxing; Lei, Yunlei; Teng, Miaomiao; Li, Xuefeng; Wang, Chengju; Li, Yingren

2018-03-06

In order to understand the negative effects of bisphenol A (BPA) alternatives comprehensively, zebrafish embryos were used to assess the lethality, developmental effects, and estrogenic activity of bisphenol analogues. The in silico estrogenic activities of bisphenol analogues were assayed by binding simulation. According to our results, the lethality of bisphenol analogues decreased in order of bisphenol AF (BPAF) > BPA > bisphenol F (BPF) > bisphenol S (BPS). BPAF and BPF induced significant effects on zebrafish embryos, including decreased heart rate, hatching inhibition, and teratogenic effects. The binding potentials of bisphenol analogues toward zebrafish ERs (zfERS) decreased in the following order: BPAF > BPA > BPF > BPS. Among the three subtypes of zfERs, zfERβ2 showed the highest binding activity toward the bisphenols, followed by zfERα and zfERβ1. In vivo estrogenic activity tests showed that BPAF, BPA, and BPF significantly enhanced the protein levels of ERα along with the mRNA levels of esr1, esr2a, esr2b, and vtg1 in zebrafish embryos. Esr2b showed the strongest response to BPAF and BPA exposure among the three esrs. In contrast, BPS did not significantly regulate ER protein level or ER transcription. In conclusion, BPAF showed the highest lethality, developmental effects, and estrogenic activity (both in silico and in vivo) followed by BPA and BPF. BPS showed the weakest toxicity and estrogenic activity. zfERβ2 might act as the main target among the three ER subtypes of zebrafish after exposure to BPAF and BPA.

Hey bHLH transcription factors.

PubMed

Weber, David; Wiese, Cornelia; Gessler, Manfred

2014-01-01

Hey bHLH transcription factors are direct targets of canonical Notch signaling. The three mammalian Hey proteins are closely related to Hes proteins and they primarily repress target genes by either directly binding to core promoters or by inhibiting other transcriptional activators. Individual candidate gene approaches and systematic screens identified a number of Hey target genes, which often encode other transcription factors involved in various developmental processes. Here, we review data on interaction partners and target genes and conclude with a model for Hey target gene regulation. Furthermore, we discuss how expression of Hey proteins affects processes like cell fate decisions and differentiation, e.g., in cardiovascular, skeletal, and neural development or oncogenesis and how this relates to the observed developmental defects and phenotypes observed in various knockout mice. © 2014 Elsevier Inc. All rights reserved.

Lune/eye gone, a Pax-like protein, uses a partial paired domain and a homeodomain for DNA recognition.

PubMed

Jun, S; Wallen, R V; Goriely, A; Kalionis, B; Desplan, C

1998-11-10

Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix-turn-helix domains joined by a linker region. Each of the subdomains, the PAI and RED domains, has been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. We describe a Pax protein, originally called Lune, that is the product of the Drosophila eye gone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. eyg seems to play a role both in the organogenesis of the salivary gland during embryogenesis and in the development of the eye. A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes.

Lune/eye gone, a Pax-like protein, uses a partial paired domain and a homeodomain for DNA recognition

PubMed Central

Jun, Susie; Wallen, Robert V.; Goriely, Anne; Kalionis, Bill; Desplan, Claude

1998-01-01

Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix–turn–helix domains joined by a linker region. Each of the subdomains, the PAI and RED domains, has been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. We describe a Pax protein, originally called Lune, that is the product of the Drosophila eye gone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. eyg seems to play a role both in the organogenesis of the salivary gland during embryogenesis and in the development of the eye. A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes. PMID:9811867

DEVELOPMENTAL HYPOTHYROIDISM REDUCES PARVALBUMIN EXPRESSION IN GABAERGIC NEURONS OF CORTEX AND HIPPOCAMPUS: IMMUNOHISTOCHEMICAL FINDINGS AND FUNCTIONAL CORRELATES.

EPA Science Inventory

GABAergic interneurons comprise the bulk of local inhibitory neuronal circuitry in cortex and hippocampus and a subpopulation of these interneurons contain the calcium binding protein, parvalbumin (PV). A previous report indicated that severe hypothyroidism reduced PV immunoreact...

Identification of a primary target of thalidomide teratogenicity.

PubMed

Ito, Takumi; Ando, Hideki; Suzuki, Takayuki; Ogura, Toshihiko; Hotta, Kentaro; Imamura, Yoshimasa; Yamaguchi, Yuki; Handa, Hiroshi

2010-03-12

Half a century ago, thalidomide was widely prescribed to pregnant women as a sedative but was found to be teratogenic, causing multiple birth defects. Today, thalidomide is still used in the treatment of leprosy and multiple myeloma, although how it causes limb malformation and other developmental defects is unknown. Here, we identified cereblon (CRBN) as a thalidomide-binding protein. CRBN forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1) and Cul4A that is important for limb outgrowth and expression of the fibroblast growth factor Fgf8 in zebrafish and chicks. Thalidomide initiates its teratogenic effects by binding to CRBN and inhibiting the associated ubiquitin ligase activity. This study reveals a basis for thalidomide teratogenicity and may contribute to the development of new thalidomide derivatives without teratogenic activity.

Developmentally regulated GTP-binding protein 2 depletion leads to mitochondrial dysfunction through downregulation of dynamin-related protein 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vo, Mai-Tram; Ko, Myoung Seok; Lee, Unn Hwa

Mitochondrial dynamics, including constant fusion and fission, play critical roles in maintaining mitochondrial morphology and function. Here, we report that developmentally regulated GTP-binding protein 2 (DRG2) regulates mitochondrial morphology by modulating the expression of the mitochondrial fission gene dynamin-related protein 1 (Drp1). shRNA-mediated silencing of DRG2 induced mitochondrial swelling, whereas expression of an shRNA-resistant version of DRG2 decreased mitochondrial swelling in DRG2-depleted cells. Analysis of the expression levels of genes involved in mitochondrial fusion and fission revealed that DRG2 depletion significantly decreased the level of Drp1. Overexpression of Drp1 rescued the defect in mitochondrial morphology induced by DRG2 depletion. DRG2more » depletion reduced the mitochondrial membrane potential, oxygen consumption rate (OCR), and amount of mitochondrial DNA (mtDNA), whereas it increased reactive oxygen species (ROS) production and apoptosis. Taken together, our data demonstrate that DRG2 acts as a regulator of mitochondrial fission by controlling the expression of Drp1. - Highlights: • DRG2 depletion increased mitochondrial swelling. • DRG2 depletion inhibited the expression of Drp1. • Overexpression of DRG2 or Drp1 rescued mitochondrial shape in DRG2 depleted cells. • DRG2 depletion induced mitochondrial dysfunction.« less

Extensive Use of RNA-Binding Proteins in Drosophila Sensory Neuron Dendrite Morphogenesis

PubMed Central

Olesnicky, Eugenia C.; Killian, Darrell J.; Garcia, Evelyn; Morton, Mary C.; Rathjen, Alan R.; Sola, Ismail E.; Gavis, Elizabeth R.

2013-01-01

The large number of RNA-binding proteins and translation factors encoded in the Drosophila and other metazoan genomes predicts widespread use of post-transcriptional regulation in cellular and developmental processes. Previous studies identified roles for several RNA-binding proteins in dendrite branching morphogenesis of Drosophila larval sensory neurons. To determine the larger contribution of post-transcriptional gene regulation to neuronal morphogenesis, we conducted an RNA interference screen to identify additional Drosophila proteins annotated as either RNA-binding proteins or translation factors that function in producing the complex dendritic trees of larval class IV dendritic arborization neurons. We identified 88 genes encoding such proteins whose knockdown resulted in aberrant dendritic morphology, including alterations in dendritic branch number, branch length, field size, and patterning of the dendritic tree. In particular, splicing and translation initiation factors were associated with distinct and characteristic phenotypes, suggesting that different morphogenetic events are best controlled at specific steps in post-transcriptional messenger RNA metabolism. Many of the factors identified in the screen have been implicated in controlling the subcellular distributions and translation of maternal messenger RNAs; thus, common post-transcriptional regulatory strategies may be used in neurogenesis and in the generation of asymmetry in the female germline and embryo. PMID:24347626

HnRNP-like proteins as post-transcriptional regulators.

PubMed

Yeap, Wan-Chin; Namasivayam, Parameswari; Ho, Chai-Ling

2014-10-01

Plant cells contain a diverse repertoire of RNA-binding proteins (RBPs) that coordinate a network of post-transcriptional regulation. RBPs govern diverse developmental processes by modulating the gene expression of specific transcripts. Recent gene annotation and RNA sequencing clearly showed that heterogeneous nuclear ribonucleoprotein (hnRNP)-like proteins which form a family of RBPs, are also expressed in higher plants and serve specific plant functions. In addition to their involvement in post-transcriptional regulation from mRNA capping to translation, they are also involved in telomere regulation, gene silencing and regulation in chloroplast. Here, we review the involvement of plant hnRNP-like proteins in post-transcription regulation of RNA processes and their functional roles in control of plant developmental processes especially plant-specific functions including flowering, chloroplastic-specific mRNA regulation, long-distance phloem transportation and plant responses to environmental stresses. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

A Novel Kinesin-Like Protein with a Calmodulin-Binding Domain

NASA Technical Reports Server (NTRS)

Wang, W.; Takezawa, D.; Narasimhulu, S. B.; Reddy, A. S. N.; Poovaiah, B. W.

1996-01-01

Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with S-35-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca(2+)-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCKI is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca(2+)/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.

Trimeric Association of Hox and TALE Homeodomain Proteins Mediates Hoxb2 Hindbrain Enhancer Activity

PubMed Central

Jacobs, Yakop; Schnabel, Catherine A.; Cleary, Michael L.

1999-01-01

Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (three-amino-acid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. In this study, we demonstrated that Pbx and Meis bind DNA as heterotrimeric complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which we showed to be required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to interact through their amino termini and bind DNA without stringent half-site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. Our data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element. PMID:10373562

Systematic discovery of Xist RNA binding proteins

PubMed Central

Chu, Ci; Zhang, Qiangfeng Cliff; da Rocha, Simão Teixeira; Flynn, Ryan A.; Bharadwaj, Maheetha; Calabrese, J. Mauro; Magnuson, Terry; Heard, Edith; Chang, Howard Y.

2015-01-01

Summary Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA- protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3′ RNA processing machinery. Xist, an essential lncRNA for X-chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK that participates in Xist-mediated gene silencing and histone modifications, but not Xist localization and Drosophila Split ends homolog Spen that interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing. PMID:25843628

Critical period plasticity is disrupted in the barrel cortex of Fmr1 knockout mice

PubMed Central

Harlow, Emily G.; Till, Sally M.; Russell, Theron A.; Wijetunge, Lasani S.; Kind, Peter; Contractor, Anis

2010-01-01

Summary Alterations in sensory processing constitute prominent symptoms of Fragile X syndrome; however, little is known about how disrupted synaptic and circuit development in sensory cortex contributes to these deficits. To investigate how the loss of fragile X mental retardation protein (FMRP) impacts the development of cortical synapses, we examined excitatory thalamocortical synapses in somatosensory cortex during the perinatal critical period in Fmr1 knockout mice. FMRP ablation resulted in dysregulation of glutamatergic signaling maturation. The fraction of silent synapses persisting to later developmental times was increased, there was a temporal delay in the window for synaptic plasticity, while other forms of developmental plasticity were not altered in Fmr1 knockout mice. Our results indicate that FMRP is required for the normal developmental progression of synaptic maturation, and loss of this important RNA binding protein impacts the timing of the critical period for layer IV synaptic plasticity. PMID:20159451

Developmental Regulation of Genes Encoding Universal Stress Proteins in Schistosoma mansoni

PubMed Central

Isokpehi, Raphael D.; Mahmud, Ousman; Mbah, Andreas N.; Simmons, Shaneka S.; Avelar, Lívia; Rajnarayanan, Rajendram V.; Udensi, Udensi K.; Ayensu, Wellington K.; Cohly, Hari H.; Brown, Shyretha D.; Dates, Centdrika R.; Hentz, Sonya D.; Hughes, Shawntae J.; Smith-McInnis, Dominique R.; Patterson, Carvey O.; Sims, Jennifer N.; Turner, Kelisha T.; Williams, Baraka S.; Johnson, Matilda O.; Adubi, Taiwo; Mbuh, Judith V.; Anumudu, Chiaka I.; Adeoye, Grace O.; Thomas, Bolaji N.; Nashiru, Oyekanmi; Oliveira, Guilherme

2011-01-01

The draft nuclear genome sequence of the snail-transmitted, dimorphic, parasitic, platyhelminth Schistosoma mansoni revealed eight genes encoding proteins that contain the Universal Stress Protein (USP) domain. Schistosoma mansoni is a causative agent of human schistosomiasis, a severe and debilitating Neglected Tropical Disease (NTD) of poverty, which is endemic in at least 76 countries. The availability of the genome sequences of Schistosoma species presents opportunities for bioinformatics and genomics analyses of associated gene families that could be targets for understanding schistosomiasis ecology, intervention, prevention and control. Proteins with the USP domain are known to provide bacteria, archaea, fungi, protists and plants with the ability to respond to diverse environmental stresses. In this research investigation, the functional annotations of the USP genes and predicted nucleotide and protein sequences were initially verified. Subsequently, sequence clusters and distinctive features of the sequences were determined. A total of twelve ligand binding sites were predicted based on alignment to the ATP-binding universal stress protein from Methanocaldococcus jannaschii. In addition, six USP sequences showed the presence of ATP-binding motif residues indicating that they may be regulated by ATP. Public domain gene expression data and RT-PCR assays confirmed that all the S. mansoni USP genes were transcribed in at least one of the developmental life cycle stages of the helminth. Six of these genes were up-regulated in the miracidium, a free-swimming stage that is critical for transmission to the snail intermediate host. It is possible that during the intra-snail stages, S. mansoni gene transcripts for universal stress proteins are low abundant and are induced to perform specialized functions triggered by environmental stressors such as oxidative stress due to hydrogen peroxide that is present in the snail hemocytes. This report serves to catalyze the formation of a network of researchers to understand the function and regulation of the universal stress proteins encoded in genomes of schistosomes and their snail intermediate hosts. PMID:22084571

In vivo interference with AtTCP20 function induces severe plant growth alterations and deregulates the expression of many genes important for development.

PubMed

Hervé, Christine; Dabos, Patrick; Bardet, Claude; Jauneau, Alain; Auriac, Marie Christine; Ramboer, Agnès; Lacout, Fabrice; Tremousaygue, Dominique

2009-03-01

AtTCP20 is a transcription factor belonging to the Arabidopsis (Arabidopsis thaliana) TCP-P subfamily, characterized by its capacity to bind to site II motifs (TGGGCY). Our aim was to understand the role of AtTCP20 in plant development. The expression pattern of a translational fusion of Prom(TCP20):CDS20GUSGFP suggested a function for AtTCP20 in several plant organs and stages of development. The role of AtTCP20 was challenged in planta by inducing expression of AtTCP20 proteins fused with either a transcriptional activator domain (VP16) or a repressor domain (EAR). Expression of both modified proteins led to severe developmental phenotypes. In-depth analysis suggested that AtTCP20 may participate in the regulation of cell expansion, cell division, and cell differentiation. Gene expression profiling in roots and hypocotyls revealed that 252 genes were down-regulated in both organs after induction of the AtTCP20EAR repressor gene. Site II motifs (TGGGCY) were underrepresented in their promoters. Conversely, GG(A/T)CCC sequences related to binding sites identified for TCP proteins in rice (Oryza sativa) were overrepresented, and a TCP20 fusion protein was shown to bind to these sequences in vitro. Gene ontology indicated that many targeted genes were involved in cell wall biogenesis and modification during expansion and also encoded numerous transcription factors controlling plant development. Our results are consistent with the previous proposal that AtTCP20 is involved in cell division and growth coordination. Moreover, they further suggest that AtTCP20 also contributes to cell expansion control and indicate a different involvement of this protein in plant morphogenesis depending on the organ and the developmental stage.

Biochemistry of the tale transcription factors PREP, MEIS, and PBX in vertebrates.

PubMed

Longobardi, E; Penkov, D; Mateos, D; De Florian, G; Torres, M; Blasi, Francesco

2014-01-01

TALE (three amino acids loop extension) homeodomain transcription factors are required in various steps of embryo development, in many adult physiological functions, and are involved in important pathologies. This review focuses on the PREP, MEIS, and PBX sub-families of TALE factors and aims at giving information on their biochemical properties, i.e., structure, interactors, and interaction surfaces. Members of the three sets of protein form dimers in which the common partner is PBX but they can also directly interact with other proteins forming higher-order complexes, in particular HOX. Finally, recent advances in determining the genome-wide DNA-binding sites of PREP1, MEIS1, and PBX1, and their partial correspondence with the binding sites of some HOX proteins, are reviewed. These studies have generated a few general rules that can be applied to all members of the three gene families. PREP and MEIS recognize slightly different consensus sequences: PREP prefers to bind to promoters and to have PBX as a DNA-binding partner; MEIS prefers HOX as partner, and both PREP and MEIS drive PBX to their own binding sites. This outlines the clear individuality of the PREP and MEIS proteins, the former mostly devoted to basic cellular functions, the latter more to developmental functions. Copyright © 2013 Wiley Periodicals, Inc.

Glimpse into Hox and tale regulation of cell differentiation and reprogramming.

PubMed

Cerdá-Esteban, Nuria; Spagnoli, Francesca M

2014-01-01

During embryonic development, cells become gradually restricted in their developmental potential and start elaborating lineage-specific transcriptional networks to ultimately acquire a unique differentiated state. Hox genes play a central role in specifying regional identities, thereby providing the cell with critical information on positional value along its differentiation path. The exquisite DNA-binding specificity of the Hox proteins is frequently dependent upon their interaction with members of the TALE family of homeodomain proteins. In addition to their function as Hox-cofactors, TALE homeoproteins control multiple crucial developmental processes through Hox-independent mechanisms. Here, we will review recent findings on the function of both Hox and TALE proteins in cell differentiation, referring mostly to vertebrate species. In addition, we will discuss the direct implications of this knowledge on cell plasticity and cell reprogramming. Copyright © 2013 Wiley Periodicals, Inc.

A Recurrent De Novo PACS2 Heterozygous Missense Variant Causes Neonatal-Onset Developmental Epileptic Encephalopathy, Facial Dysmorphism, and Cerebellar Dysgenesis.

PubMed

Olson, Heather E; Jean-Marçais, Nolwenn; Yang, Edward; Heron, Delphine; Tatton-Brown, Katrina; van der Zwaag, Paul A; Bijlsma, Emilia K; Krock, Bryan L; Backer, E; Kamsteeg, Erik-Jan; Sinnema, Margje; Reijnders, Margot R F; Bearden, David; Begtrup, Amber; Telegrafi, Aida; Lunsing, Roelineke J; Burglen, Lydie; Lesca, Gaetan; Cho, Megan T; Smith, Lacey A; Sheidley, Beth R; Moufawad El Achkar, Christelle; Pearl, Phillip L; Poduri, Annapurna; Skraban, Cara M; Tarpinian, Jennifer; Nesbitt, Addie I; Fransen van de Putte, Dietje E; Ruivenkamp, Claudia A L; Rump, Patrick; Chatron, Nicolas; Sabatier, Isabelle; De Bellescize, Julitta; Guibaud, Laurent; Sweetser, David A; Waxler, Jessica L; Wierenga, Klaas J; Donadieu, Jean; Narayanan, Vinodh; Ramsey, Keri M; Nava, Caroline; Rivière, Jean-Baptiste; Vitobello, Antonio; Tran Mau-Them, Frédéric; Philippe, Christophe; Bruel, Ange-Line; Duffourd, Yannis; Thomas, Laurel; Lelieveld, Stefan H; Schuurs-Hoeijmakers, Janneke; Brunner, Han G; Keren, Boris; Thevenon, Julien; Faivre, Laurence; Thomas, Gary; Thauvin-Robinet, Christel

2018-05-03

Developmental and epileptic encephalopathies (DEEs) represent a large clinical and genetic heterogeneous group of neurodevelopmental diseases. The identification of pathogenic genetic variants in DEEs remains crucial for deciphering this complex group and for accurately caring for affected individuals (clinical diagnosis, genetic counseling, impacting medical, precision therapy, clinical trials, etc.). Whole-exome sequencing and intensive data sharing identified a recurrent de novo PACS2 heterozygous missense variant in 14 unrelated individuals. Their phenotype was characterized by epilepsy, global developmental delay with or without autism, common cerebellar dysgenesis, and facial dysmorphism. Mixed focal and generalized epilepsy occurred in the neonatal period, controlled with difficulty in the first year, but many improved in early childhood. PACS2 is an important PACS1 paralog and encodes a multifunctional sorting protein involved in nuclear gene expression and pathway traffic regulation. Both proteins harbor cargo(furin)-binding regions (FBRs) that bind cargo proteins, sorting adaptors, and cellular kinase. Compared to the defined PACS1 recurrent variant series, individuals with PACS2 variant have more consistently neonatal/early-infantile-onset epilepsy that can be challenging to control. Cerebellar abnormalities may be similar but PACS2 individuals exhibit a pattern of clear dysgenesis ranging from mild to severe. Functional studies demonstrated that the PACS2 recurrent variant reduces the ability of the predicted autoregulatory domain to modulate the interaction between the PACS2 FBR and client proteins, which may disturb cellular function. These findings support the causality of this recurrent de novo PACS2 heterozygous missense in DEEs with facial dysmorphim and cerebellar dysgenesis. Copyright © 2018 American Society of Human Genetics. All rights reserved.

The Fibroblast Growth Factor signaling pathway.

PubMed

Ornitz, David M; Itoh, Nobuyuki

2015-01-01

The signaling component of the mammalian Fibroblast Growth Factor (FGF) family is comprised of eighteen secreted proteins that interact with four signaling tyrosine kinase FGF receptors (FGFRs). Interaction of FGF ligands with their signaling receptors is regulated by protein or proteoglycan cofactors and by extracellular binding proteins. Activated FGFRs phosphorylate specific tyrosine residues that mediate interaction with cytosolic adaptor proteins and the RAS-MAPK, PI3K-AKT, PLCγ, and STAT intracellular signaling pathways. Four structurally related intracellular non-signaling FGFs interact with and regulate the family of voltage gated sodium channels. Members of the FGF family function in the earliest stages of embryonic development and during organogenesis to maintain progenitor cells and mediate their growth, differentiation, survival, and patterning. FGFs also have roles in adult tissues where they mediate metabolic functions, tissue repair, and regeneration, often by reactivating developmental signaling pathways. Consistent with the presence of FGFs in almost all tissues and organs, aberrant activity of the pathway is associated with developmental defects that disrupt organogenesis, impair the response to injury, and result in metabolic disorders, and cancer. For further resources related to this article, please visit the WIREs website. © 2015 The Authors. WIREs Developmental Biology published by Wiley Periodicals, Inc.

A Genomic Response to Trace Fear Conditioning in the Amygdala of Female Rats After Developmental Exposure to Manganese

EPA Science Inventory

Increases in brain-derived neurotrophic factor (Bdnf), Ca2+/calmodulin-dependent protein kinase II alpha (Camk2a), and cyclic adenosine monophosphate (cAMP) response element binding (Creb1) gene expression have been associated with learning in a variety of different rodent studie...

Identification of secretaglobin Scgb2a1 as a target for developmental reprogramming by BPA in the rat prostate.

PubMed

Wong, Rebecca Lee Yean; Wang, Quan; Treviño, Lindsey S; Bosland, Maarten C; Chen, Jing; Medvedovic, Mario; Prins, Gail S; Kannan, Kurunthachalam; Ho, Shuk-Mei; Walker, Cheryl Lyn

2015-01-01

Secretoglobins are a superfamily of secreted proteins thought to participate in inflammation, tissue repair, and tumorigenesis. Secretoglobin family 2A member 1 (Scgb2a1) is a component of prostatein, a major androgen-binding protein secreted by the rat prostate. Using a rat model for developmental reprogramming of susceptibility to prostate carcinogenesis, we identified, by RNA-seq, that Scgb2a1 is significantly upregulated (>100-fold) in the prostate of adult rats neonatally exposed to bisphenol A (BPA), with increased gene expression confirmed by quantitative RT-PCR and chromatin immunoprecipitation for histone H3 lysine 9 acetylation. Bisulfite analysis of both CpG islands located within 10 kb of the Scgb2a1 promoter identified significant hypomethylation of the CpG island upstream of the transcription start site of this gene in the reprogrammed prostate. These data suggest that expression of Scgb2a1 in the adult prostate could be epigenetically reprogrammed by BPA exposure during prostate development, with potential implications for cancer risk and response to chemotherapeutics associated with prostatein binding.

Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli.

PubMed

Levin, M S; Locke, B; Yang, N C; Li, E; Gordon, J I

1988-11-25

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) are 132-residue cytosolic proteins which have 56% amino acid sequence identity and bind all-trans-retinol as their endogenous ligand. They belong to a family of cytoplasmic proteins which have evolved to bind distinct hydrophobic ligands. Their patterns of tissue-specific and developmental regulation are distinct. We have compared the ligand binding properties of rat apo-CRBP and apo-CRBP II that have been expressed in Escherichia coli. Several observations indicate that the E. coli-derived apoproteins are structurally similar to the native rat proteins: they co-migrate on isoelectric focusing gels; and when complexed with all-trans-retinol, their absorption and excitation/emission spectra are nearly identical to those of the authentic rat holoproteins. Comparative lifetime and acrylamide quenching studies suggest that there are differences in the conformations of apo-CRBP and apo-CRBP II. The interaction of E. coli-derived apo-CRBP and apo-CRBP II with a variety of retinoids was analyzed using spectroscopic techniques. Both apoproteins formed high affinity complexes with all-trans-retinol (K'd approximately 10 nM). In direct binding assays, all-trans-retinal bound to both apoproteins (K'd approximately 50 nM for CRBP; K'd approximately 90 nM for CRBP II). However, all-trans-retinal could displace all-trans-retinol bound to CRBP II but not to CRBP. These observations suggests that there is a specific yet distinct interaction between these two proteins and all-trans-retinal. Apo-CRBP and apo-CRBP II did not demonstrate significant binding to either retinoic acid or methyl retinoate, an uncharged derivative of all-trans-retinoic acid. This indicates that the carboxymethyl group of methyl retinoate cannot be sterically accommodated in their binding pockets and that failure to bind retinoic acid probably is not simply due to the negative charge of its C-15 carboxylate group. Finally, neither all-trans-retinol nor retinoic acid bound to E. coli-derived rat intestinal fatty acid-binding protein, a homologous protein whose tertiary structure is known. Together, the data suggest that these three family members have acquired unique functional capabilities.

Genome-wide analysis of Polycomb targets in Drosophila

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schwartz, Yuri B.; Kahn, Tatyana G.; Nix, David A.

2006-04-01

Polycomb Group (PcG) complexes are multiprotein assemblages that bind to chromatin and establish chromatin states leading to epigenetic silencing. PcG proteins regulate homeotic genes in flies and vertebrates but little is known about other PcG targets and the role of the PcG in development, differentiation and disease. We have determined the distribution of the PcG proteins PC, E(Z) and PSC and of histone H3K27 trimethylation in the Drosophila genome. At more than 200 PcG target genes, binding sites for the three PcG proteins colocalize to presumptive Polycomb Response Elements (PREs). In contrast, H3 me3K27 forms broad domains including the entiremore » transcription unit and regulatory regions. PcG targets are highly enriched in genes encoding transcription factors but receptors, signaling proteins, morphogens and regulators representing all major developmental pathways are also included.« less

Human HOX Proteins Use Diverse and Context-Dependent Motifs to Interact with TALE Class Cofactors.

PubMed

Dard, Amélie; Reboulet, Jonathan; Jia, Yunlong; Bleicher, Françoise; Duffraisse, Marilyne; Vanaker, Jean-Marc; Forcet, Christelle; Merabet, Samir

2018-03-13

HOX proteins achieve numerous functions by interacting with the TALE class PBX and MEIS cofactors. In contrast to this established partnership in development and disease, how HOX proteins could interact with PBX and MEIS remains unclear. Here, we present a systematic analysis of HOX/PBX/MEIS interaction properties, scanning all paralog groups with human and mouse HOX proteins in vitro and in live cells. We demonstrate that a previously characterized HOX protein motif known to be critical for HOX-PBX interactions becomes dispensable in the presence of MEIS in all except the two most anterior paralog groups. We further identify paralog-specific TALE-binding sites that are used in a highly context-dependent manner. One of these binding sites is involved in the proliferative activity of HOXA7 in breast cancer cells. Together these findings reveal an extraordinary level of interaction flexibility between HOX proteins and their major class of developmental cofactors. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Receptor tyrosine kinase mutations in developmental syndromes and cancer: two sides of the same coin

PubMed Central

McDonell, Laura M.; Kernohan, Kristin D.; Boycott, Kym M.; Sawyer, Sarah L.

2015-01-01

Receptor tyrosine kinases (RTKs) are a family of ligand-binding cell surface receptors that regulate a wide range of essential cellular activities, including proliferation, differentiation, cell-cycle progression, survival and apoptosis. As such, these proteins play an important role during development and throughout life; germline mutations in genes encoding RTKs cause several developmental syndromes, while somatic alterations contribute to the pathogenesis of many aggressive cancers. This creates an interesting paradigm in which mutation timing, type and location in a gene leads to different cell signaling and biological responses, and ultimately phenotypic outcomes. In this review, we highlight the roles of RTKs in developmental disorders and cancer. The multifaceted roles of these receptors, their genetic signatures and their signaling during developmental morphogenesis and oncogenesis are discussed. Additionally, we propose that comparative analysis of RTK mutations responsible for developmental syndromes may shed light on those driving tumorigenesis. PMID:26152202

Wash functions downstream of Rho1 GTPase in a subset of Drosophila immune cell developmental migrations

PubMed Central

Verboon, Jeffrey M.; Rahe, Travis K.; Rodriguez-Mesa, Evelyn; Parkhurst, Susan M.

2015-01-01

Drosophila immune cells, the hemocytes, undergo four stereotypical developmental migrations to populate the embryo, where they provide immune reconnoitering, as well as a number of non–immune-related functions necessary for proper embryogenesis. Here, we describe a role for Rho1 in one of these developmental migrations in which posteriorly located hemocytes migrate toward the head. This migration requires the interaction of Rho1 with its downstream effector Wash, a Wiskott–Aldrich syndrome family protein. Both Wash knockdown and a Rho1 transgene harboring a mutation that prevents Wash binding exhibit the same developmental migratory defect as Rho1 knockdown. Wash activates the Arp2/3 complex, whose activity is needed for this migration, whereas members of the WASH regulatory complex (SWIP, Strumpellin, and CCDC53) are not. Our results suggest a WASH complex–independent signaling pathway to regulate the cytoskeleton during a subset of hemocyte developmental migrations. PMID:25739458

Biotype Characterization, Developmental Profiling, Insecticide Response and Binding Property of Bemisia tabaci Chemosensory Proteins: Role of CSP in Insect Defense

PubMed Central

Liu, Guoxia; Ma, Hongmei; Xie, Hongyan; Xuan, Ning; Guo, Xia; Fan, Zhongxue; Rajashekar, Balaji; Arnaud, Philippe; Offmann, Bernard; Picimbon, Jean-François

2016-01-01

Chemosensory proteins (CSPs) are believed to play a key role in the chemosensory process in insects. Sequencing genomic DNA and RNA encoding CSP1, CSP2 and CSP3 in the sweet potato whitefly Bemisia tabaci showed strong variation between B and Q biotypes. Analyzing CSP-RNA levels showed not only biotype, but also age and developmental stage-specific expression. Interestingly, applying neonicotinoid thiamethoxam insecticide using twenty-five different dose/time treatments in B and Q young adults showed that Bemisia CSP1, CSP2 and CSP3 were also differentially regulated over insecticide exposure. In our study one of the adult-specific gene (CSP1) was shown to be significantly up-regulated by the insecticide in Q, the most highly resistant form of B. tabaci. Correlatively, competitive binding assays using tryptophan fluorescence spectroscopy and molecular docking demonstrated that CSP1 protein preferentially bound to linoleic acid, while CSP2 and CSP3 proteins rather associated to another completely different type of chemical, i.e. α-pentyl-cinnamaldehyde (jasminaldehyde). This might indicate that some CSPs in whiteflies are crucial to facilitate the transport of fatty acids thus regulating some metabolic pathways of the insect immune response, while some others are tuned to much more volatile chemicals known not only for their pleasant odor scent, but also for their potent toxic insecticide activity. PMID:27167733

De novo truncating variants in the AHDC1 gene encoding the AT-hook DNA-binding motif-containing protein 1 are associated with intellectual disability and developmental delay.

PubMed

Yang, Hui; Douglas, Ganka; Monaghan, Kristin G; Retterer, Kyle; Cho, Megan T; Escobar, Luis F; Tucker, Megan E; Stoler, Joan; Rodan, Lance H; Stein, Diane; Marks, Warren; Enns, Gregory M; Platt, Julia; Cox, Rachel; Wheeler, Patricia G; Crain, Carrie; Calhoun, Amy; Tryon, Rebecca; Richard, Gabriele; Vitazka, Patrik; Chung, Wendy K

2015-10-01

Whole-exome sequencing (WES) represents a significant breakthrough in clinical genetics, and identifies a genetic etiology in up to 30% of cases of intellectual disability (ID). Using WES, we identified seven unrelated patients with a similar clinical phenotype of severe intellectual disability or neurodevelopmental delay who were all heterozygous for de novo truncating variants in the AT-hook DNA-binding motif-containing protein 1 (AHDC1). The patients were all minimally verbal or nonverbal and had variable neurological problems including spastic quadriplegia, ataxia, nystagmus, seizures, autism, and self-injurious behaviors. Additional common clinical features include dysmorphic facial features and feeding difficulties associated with failure to thrive and short stature. The AHDC1 gene has only one coding exon, and the protein contains conserved regions including AT-hook motifs and a PDZ binding domain. We postulate that all seven variants detected in these patients result in a truncated protein missing critical functional domains, disrupting interactions with other proteins important for brain development. Our study demonstrates that truncating variants in AHDC1 are associated with ID and are primarily associated with a neurodevelopmental phenotype.

Glycoconjugate distribution in early human notochord and axial mesenchyme.

PubMed

Götz, W; Quondamatteo, F

2001-02-01

Glycosylation patterns of cells and tissues give insights into spatially and temporally regulated developmental processes and can be detected histochemically using plant lectins with specific affinities for sugar moieties. The early development of the vertebral column in man is a process which has never been investigated by lectin histochemistry. Therefore, we studied binding of several lectins (AIA, Con A, GSA II, LFA, LTA, PNA, RCA I, SBA, SNA, WGA) in formaldehyde-fixed sections of the axial mesenchyme of 5 human embryos in Carnegie stages 12-15. During these developmental stages, an unsegmented mesenchyme covers the notochord. Staining patterns did not show striking temporal variations except for SBA which stained the cranial axial mesenchyme only in the early stage 12 embryo and for PNA, of which the staining intensity in the mesenchyme decreased with age. The notochord appeared as a highly glycosylated tissue. Carbohydrates detected may correspond to adhesion molecules or to secreted substances like proteoglycans or proteins which could play an inductive role, for example, for the neural tube. The axial perinotochordal unsegmented mesenchyme showed strong PNA binding. Therefore, its function as a PNA-positive "barrier" tissue is discussed. The endoderm of the primitive gut showed a lectin-binding pattern that was similar to that of the notochord, which may correlate with interactions between these tissues during earlier developmental stages.

Sequence Discrimination by Alternatively Spliced Isoforms of a DNA Binding Zinc Finger Domain

NASA Astrophysics Data System (ADS)

Gogos, Joseph A.; Hsu, Tien; Bolton, Jesse; Kafatos, Fotis C.

1992-09-01

Two major developmentally regulated isoforms of the Drosophila chorion transcription factor CF2 differ by an extra zinc finger within the DNA binding domain. The preferred DNA binding sites were determined and are distinguished by an internal duplication of TAT in the site recognized by the isoform with the extra finger. The results are consistent with modular interactions between zinc fingers and trinucleotides and also suggest rules for recognition of AT-rich DNA sites by zinc finger proteins. The results show how modular finger interactions with trinucleotides can be used, in conjunction with alternative splicing, to alter the binding specificity and increase the spectrum of sites recognized by a DNA binding domain. Thus, CF2 may potentially regulate distinct sets of target genes during development.

Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

DOE PAGES

Barbaglia, Allison M.; Tamot, Banita; Greve, Veronica; ...

2016-04-28

Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and thatmore » they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all three proteins are indeed lipid-binding and act in the vasculature possibly in a function related to long-distance signaling, the three proteins do not act in the same but rather in distinct pathways. Furthermore, it points toward PLAFP as a prime candidate to investigate long-distance lipid signaling in the plant drought response.« less

Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barbaglia, Allison M.; Tamot, Banita; Greve, Veronica

Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and thatmore » they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all three proteins are indeed lipid-binding and act in the vasculature possibly in a function related to long-distance signaling, the three proteins do not act in the same but rather in distinct pathways. Furthermore, it points toward PLAFP as a prime candidate to investigate long-distance lipid signaling in the plant drought response.« less

Phylogenetic and functional analyses of a plant protein related to human B-cell receptor-associated proteins.

PubMed

Atabekova, Anastasia K; Pankratenko, Anna V; Makarova, Svetlana S; Lazareva, Ekaterina A; Owens, Robert A; Solovyev, Andrey G; Morozov, Sergey Y

2017-01-01

Human B-cell receptor-associated protein BAP31 (HsBAP31) is the endoplasmic reticulum-resident protein involved in protein sorting and transport as well as pro-apoptotic signaling. Plant orthologs of HsBAP31 termed 'plant BAP-like proteins' (PBL proteins) have thus far remained unstudied. Recently, the PBL protein from Nicotiana tabacum (NtPBL) was identified as an interactor of Nt-4/1, a plant protein known to interact with plant virus movement proteins and affect the long-distance transport of potato spindle tuber viroid (PSTVd) via the phloem. Here, we have compared the sequences of PBL proteins and studied the biochemical properties of NtPBL. Analysis of a number of fully sequenced plant genomes revealed that PBL-encoding genes represent a small multigene family with up to six members per genome. Two conserved motifs were identified in the C-terminal region of PBL proteins. The NtPBL C-terminal hydrophilic region (NtPBL-C) was expressed in bacterial cells, purified, and used for analysis of its RNA binding properties in vitro. In gel shift experiments, NtPBL-C was found to bind several tested RNAs, showing the most efficient binding to microRNA precursors (pre-miRNA) and less efficient interaction with PSTVd. Mutational analysis suggested that NtPBL-C has a composite RNA-binding site, with two conserved lysine residues in the most C-terminal protein region being involved in binding of pre-miRNA but not PSTVd RNA. Virus-mediated transient expression of NtPBL-C in plants resulted in stunting and leaf malformation, developmental abnormalities similar to those described previously for blockage of miRNA biogenesis/function. We hypothesize that the NtPBL protein represents a previously undiscovered component of the miRNA pathway. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

DNA binding mechanism revealed by high resolution crystal structure of Arabidopsis thaliana WRKY1 protein

PubMed Central

Duan, Ming-Rui; Nan, Jie; Liang, Yu-He; Mao, Peng; Lu, Lu; Li, Lanfen; Wei, Chunhong; Lai, Luhua; Li, Yi; Su, Xiao-Dong

2007-01-01

WRKY proteins, defined by the conserved WRKYGQK sequence, are comprised of a large superfamily of transcription factors identified specifically from the plant kingdom. This superfamily plays important roles in plant disease resistance, abiotic stress, senescence as well as in some developmental processes. In this study, the Arabidopsis WRKY1 was shown to be involved in the salicylic acid signaling pathway and partially dependent on NPR1; a C-terminal domain of WRKY1, AtWRKY1-C, was constructed for structural studies. Previous investigations showed that DNA binding of the WRKY proteins was localized at the WRKY domains and these domains may define novel zinc-binding motifs. The crystal structure of the AtWRKY1-C determined at 1.6 Å resolution has revealed that this domain is composed of a globular structure with five β strands, forming an antiparallel β-sheet. A novel zinc-binding site is situated at one end of the β-sheet, between strands β4 and β5. Based on this high-resolution crystal structure and site-directed mutagenesis, we have defined and confirmed that the DNA-binding residues of AtWRKY1-C are located at β2 and β3 strands. These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins. PMID:17264121

UV-damaged DNA binding protein-1 and de-etiolated-1 regulate golden 2-like transcription factor by assembling a cullin 4-based ubiquitin ligase in tomato

USDA-ARS?s Scientific Manuscript database

Fleshy fruit undergo a novel developmental program that ends in the irreversible process of ripening and eventual tissue senescence. During these maturation processes, fruit undergo numerous physiological, biochemical and structural alterations, making them more attractive to seed dispersal organism...

Unusual Characteristics of the DNA Binding Domain of Epigenetic Regulatory Protein MeCP2 Determine Its Binding Specificity

PubMed Central

2015-01-01

The protein MeCP2 mediates epigenetic regulation by binding methyl-CpG (mCpG) sites on chromatin. MeCP2 consists of six domains of which one, the methyl binding domain (MBD), binds mCpG sites in duplex DNA. We show that solution conditions with physiological or greater salt concentrations or the presence of nonspecific competitor DNA is necessary for the MBD to discriminate mCpG from CpG with high specificity. The specificity for mCpG over CpG is >100-fold under these solution conditions. In contrast, the MBD does not discriminate hydroxymethyl-CpG from CpG. The MBD is unusual among site-specific DNA binding proteins in that (i) specificity is not conferred by the enhanced affinity for the specific site but rather by suppression of its affinity for generic DNA, (ii) its specific binding to mCpG is highly electrostatic, and (iii) it takes up as well as displaces monovalent cations upon DNA binding. The MBD displays an unusually high affinity for single-stranded DNA independent of modification or sequence. In addition, the MBD forms a discrete dimer on DNA via a noncooperative binding pathway. Because the affinity of the second monomer is 1 order of magnitude greater than that of nonspecific binding, the MBD dimer is a unique molecular complex. The significance of these results in the context of neuronal function and development and MeCP2-related developmental disorders such as Rett syndrome is discussed. PMID:24828757

The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs

PubMed Central

Hemmes, Hans; Lakatos, Lóránt; Goldbach, Rob; Burgyán, József; Prins, Marcel

2007-01-01

RNA silencing plays a key role in antiviral defense as well as in developmental processes in plants and insects. Negative strand RNA viruses such as the plant virus Rice hoja blanca tenuivirus (RHBV) replicate in plants and in their insect transmission vector. Like most plant-infecting viruses, RHBV encodes an RNA silencing suppressor, the NS3 protein, and here it is demonstrated that this protein is capable of suppressing RNA silencing in both plants and insect cells. Biochemical analyses showed that NS3 efficiently binds siRNA as well as miRNA molecules. Binding of NS3 is greatly influenced by the size of small RNA molecules, as 21 nucleotide (nt) siRNA molecules are bound > 100 times more efficiently than 26 nt species. Competition assays suggest that the activity of NS3 is based on binding to siRNAs prior to strand separation during the assembly of the RNA-induced silencing complex. In addition, NS3 has a high affinity for miRNA/miRNA* duplexes, indicating that its activity might also interfere with miRNA-regulated gene expression in both insects and plants. PMID:17513697

Transcriptome analysis of the epidermis of the purple quail-like (q-lp) mutant of silkworm, Bombyx mori.

PubMed

Wang, Pingyang; Qiu, Zhiyong; Xia, Dingguo; Tang, Shunming; Shen, Xingjia; Zhao, Qiaoling

2017-01-01

A new purple quail-like (q-lp) mutant found from the plain silkworm strain 932VR has pigment dots on the epidermis similar to the pigment mutant quail (q). In addition, q-lp mutant larvae are inactive, consume little and grow slowly, with a high death rate and other developmental abnormalities. Pigmentation of the silkworm epidermis consists of melanin, ommochrome and pteridine. Silkworm development is regulated by ecdysone and juvenile hormone. In this study, we performed RNA-Seq on the epidermis of the q-lp mutant in the 4th instar during molting, with 932VR serving as the control. The results showed 515 differentially expressed genes, of which 234 were upregulated and 281 downregulated in q-lp. BLASTGO analysis indicated that the downregulated genes mainly encode protein-binding proteins, membrane components, oxidation/reduction enzymes, and proteolytic enzymes, whereas the upregulated genes largely encode cuticle structural constituents, membrane components, transport related proteins, and protein-binding proteins. Quantitative reverse transcription PCR was used to verify the accuracy of the RNA-Seq data, focusing on key genes for biosynthesis of the three pigments and chitin as well as genes encoding cuticular proteins and several related nuclear receptors, which are thought to play key roles in the q-lp mutant. We drew three conclusions based on the results: 1) melanin, ommochrome and pteridine pigments are all increased in the q-lp mutant; 2) more cuticle proteins are expressed in q-lp than in 932VR, and the number of upregulated cuticular genes is significantly greater than downregulated genes; 3) the downstream pathway regulated by ecdysone is blocked in the q-lp mutant. Our research findings lay the foundation for further research on the developmental changes responsible for the q-lp mutant.

iTRAQ-Based Proteomics Analysis and Network Integration for Kernel Tissue Development in Maize

PubMed Central

Dong, Yongbin; Wang, Qilei; Du, Chunguang; Xiong, Wenwei; Li, Xinyu; Zhu, Sailan; Li, Yuling

2017-01-01

Grain weight is one of the most important yield components and a developmentally complex structure comprised of two major compartments (endosperm and pericarp) in maize (Zea mays L.), however, very little is known concerning the coordinated accumulation of the numerous proteins involved. Herein, we used isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative proteomic method to analyze the characteristics of dynamic proteomics for endosperm and pericarp during grain development. Totally, 9539 proteins were identified for both components at four development stages, among which 1401 proteins were non-redundant, 232 proteins were specific in pericarp and 153 proteins were specific in endosperm. A functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the tissue development. Three and 76 proteins involved in 49 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were integrated for the specific endosperm and pericarp proteins, respectively, reflecting their complex metabolic interactions. In addition, four proteins with important functions and different expression levels were chosen for gene cloning and expression analysis. Different concordance between mRNA level and the protein abundance was observed across different proteins, stages, and tissues as in previous research. These results could provide useful message for understanding the developmental mechanisms in grain development in maize. PMID:28837076

The multifunctional Staufen proteins: conserved roles from neurogenesis to synaptic plasticity

PubMed Central

Heraud-Farlow, Jacki E.; Kiebler, Michael A.

2014-01-01

Staufen (Stau) proteins belong to a family of RNA-binding proteins (RBPs) that are important for RNA localisation in many organisms. In this review we discuss recent findings on the conserved role played by Stau during both the early differentiation of neurons and in the synaptic plasticity of mature neurons. Recent molecular data suggest mechanisms for how Stau2 regulates mRNA localisation, mRNA stability, translation, and ribonucleoprotein (RNP) assembly. We offer a perspective on how this multifunctional RBP has been adopted to regulate mRNA localisation under several different cellular and developmental conditions. PMID:25012293

ChIP-Chip Identifies SEC23A, CFDP1, and NSD1 as TFII-I Target Genes in Human Neural Crest Progenitor Cells.

PubMed

Makeyev, Aleksandr V; Bayarsaihan, Dashzeveg

2013-05-01

Objectives :  GTF2I and GTF2IRD1 genes located in Williams-Beuren syndrome (WBS) critical region encode TFII-I family transcription factors. The aim of this study was to map genomic sites bound by these proteins across promoter regions of developmental regulators associated with craniofacial development. Design :  Chromatin was isolated from human neural crest progenitor cells and the DNA-binding profile was generated using the human RefSeq tiling promoter ChIP-chip arrays. Results :  TFII-I transcription factors are recruited to the promoters of SEC23A, CFDP1, and NSD1 previously defined as TFII-I target genes. Moreover, our analysis revealed additional binding elements that contain E-boxes and initiator-like motifs. Conclusions :  Genome-wide promoter binding studies revealed SEC23A, CFDP1, and NSD1 linked to craniofacial or dental development as direct TFII-I targets. Developmental regulation of these genes by TFII-I factors could contribute to the WBS-specific facial dysmorphism.

Drosophila Suppressor of Sable Protein [Su(s)] Promotes Degradation of Aberrant and Transposon-Derived RNAs▿

PubMed Central

Kuan, Yung-Shu; Brewer-Jensen, Paul; Bai, Wen-Li; Hunter, Cedric; Wilson, Carrie B.; Bass, Sarah; Abernethy, John; Wing, James S.; Searles, Lillie L.

2009-01-01

RNA-binding proteins act at various stages of gene expression to regulate and fine-tune patterns of mRNA accumulation. One protein in this class is Drosophila Su(s), a nuclear protein that has been previously shown to inhibit the accumulation of mutant transcripts by an unknown mechanism. Here, we have identified several additional RNAs that are downregulated by Su(s). These Su(s) targets include cryptic wild-type transcripts from the developmentally regulated Sgs4 and ng1 genes, noncoding RNAs derived from tandemly repeated αβ/αγ elements within an Hsp70 locus, and aberrant transcripts induced by Hsp70 promoter transgenes inserted at ectopic sites. We used the αβ RNAs to investigate the mechanism of Su(s) function and obtained evidence that these transcripts are degraded by the nuclear exosome and that Su(s) promotes this process. Furthermore, we showed that the RNA binding domains of Su(s) are important for this effect and mapped the sequences involved to a 267-nucleotide region of an αβ element. Taken together, these results suggest that Su(s) binds to certain nascent transcripts and stimulates their degradation by the nuclear exosome. PMID:19687295

Conservation of Matrix Attachment Region-Binding Filament-Like Protein 1 among Higher Plants1

PubMed Central

Harder, Patricia A.; Silverstein, Rebecca A.; Meier, Iris

2000-01-01

The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) on the DNA is considered to be of fundamental importance for higher-order chromatin organization and the regulation of gene expression. We have previously isolated a novel nuclear matrix-localized protein (MFP1) from tomato (Lycopersicon esculentum) that preferentially binds to MAR DNA. Tomato MFP1 has a predicted filament-protein-like structure and is associated with the nuclear envelope via an N-terminal targeting domain. Based on the antigenic relationship, we report here that MFP1 is conserved in a large number of dicot and monocot species. Several cDNAs were cloned from tobacco (Nicotiana tabacum) and shown to correspond to two tobacco MFP1 genes. Comparison of the primary and predicted secondary structures of MFP1 from tomato, tobacco, and Arabidopsis indicates a high degree of conservation of the N-terminal targeting domain, the overall putative coiled-coil structure of the protein, and the C-terminal DNA-binding domain. In addition, we show that tobacco MFP1 is regulated in an organ-specific and developmental fashion, and that this regulation occurs at the level of transcription or RNA stability. PMID:10631266

Molecular analysis of nicotinic receptor expression in autism.

PubMed

Martin-Ruiz, C M; Lee, M; Perry, R H; Baumann, M; Court, J A; Perry, E K

2004-04-07

Autism is a developmental disorder of unknown aetiopathology and lacking any specific pharmacological therapeutic intervention. Neurotransmitters such as serotonin, gamma-aminobutyric acid (GABA) and acetylcholine have been implicated. Abnormalities in nicotinic acetylcholine receptors have been identified including cortical loss of binding to the alpha4/beta2 subtype and increase in cerebellar alpha7 binding. Receptor expression (mRNA) has not so far been systematically examined. This study aims to further explore the role of nicotinic receptors in autism by analysing nicotinic receptor subunit mRNA in conjunction with protein levels and receptor binding in different brain areas. Quantitative RT-PCR for alpha4, alpha7 and beta2 subunit mRNA expression levels; alpha3, alpha4, alpha7 and beta2 subunit protein expression immunochemistry and specific radioligand receptor binding were performed in adult autism and control brain samples from cerebral cortex and cerebellum. Alpha4 and beta2 protein expression and receptor binding density as well as alpha4 mRNA levels were lower in parietal cortex in autism, while alpha7 did not change for any of these parameters. In cerebellum, alpha4 mRNA expression was increased, whereas subunit protein and receptor levels were decreased. Alpha7 receptor binding in cerebellum was increased alongside non-significant elevations in mRNA and protein expression levels. No significant changes were found for beta2 in cerebellum. The data obtained, using complementary measures of receptor expression, indicate that reduced gene expression of the alpha4beta2 nicotinic receptor in the cerebral cortex is a major feature of the neurochemical pathology of autism, whilst post-transcriptional abnormalities of both this and the alpha7 subtype are apparent in the cerebellum. The findings point to dendritic and/or synaptic nicotinic receptor abnormalities that may relate to disruptions in cerebral circuitry development.

FBXL19 recruits CDK-Mediator to CpG islands of developmental genes priming them for activation during lineage commitment

PubMed Central

Dimitrova, Emilia; Nakayama, Manabu; Koseki, Yoko; Konietzny, Rebecca; Kessler, Benedikt M; Koseki, Haruhiko

2018-01-01

CpG islands are gene regulatory elements associated with the majority of mammalian promoters, yet how they regulate gene expression remains poorly understood. Here, we identify FBXL19 as a CpG island-binding protein in mouse embryonic stem (ES) cells and show that it associates with the CDK-Mediator complex. We discover that FBXL19 recruits CDK-Mediator to CpG island-associated promoters of non-transcribed developmental genes to prime these genes for activation during cell lineage commitment. We further show that recognition of CpG islands by FBXL19 is essential for mouse development. Together this reveals a new CpG island-centric mechanism for CDK-Mediator recruitment to developmental gene promoters in ES cells and a requirement for CDK-Mediator in priming these developmental genes for activation during cell lineage commitment. PMID:29809150

Neuron class-specific requirements for Fragile X Mental Retardation Protein in critical period development of calcium signaling in learning and memory circuitry.

PubMed

Doll, Caleb A; Broadie, Kendal

2016-05-01

Neural circuit optimization occurs through sensory activity-dependent mechanisms that refine synaptic connectivity and information processing during early-use developmental critical periods. Fragile X Mental Retardation Protein (FMRP), the gene product lost in Fragile X syndrome (FXS), acts as an activity sensor during critical period development, both as an RNA-binding translation regulator and channel-binding excitability regulator. Here, we employ a Drosophila FXS disease model to assay calcium signaling dynamics with a targeted transgenic GCaMP reporter during critical period development of the mushroom body (MB) learning/memory circuit. We find FMRP regulates depolarization-induced calcium signaling in a neuron-specific manner within this circuit, suppressing activity-dependent calcium transients in excitatory cholinergic MB input projection neurons and enhancing calcium signals in inhibitory GABAergic MB output neurons. Both changes are restricted to the developmental critical period and rectified at maturity. Importantly, conditional genetic (dfmr1) rescue of null mutants during the critical period corrects calcium signaling defects in both neuron classes, indicating a temporally restricted FMRP requirement. Likewise, conditional dfmr1 knockdown (RNAi) during the critical period replicates constitutive null mutant defects in both neuron classes, confirming cell-autonomous requirements for FMRP in developmental regulation of calcium signaling dynamics. Optogenetic stimulation during the critical period enhances depolarization-induced calcium signaling in both neuron classes, but this developmental change is eliminated in dfmr1 null mutants, indicating the activity-dependent regulation requires FMRP. These results show FMRP shapes neuron class-specific calcium signaling in excitatory vs. inhibitory neurons in developing learning/memory circuitry, and that FMRP mediates activity-dependent regulation of calcium signaling specifically during the early-use critical period. Copyright © 2016 Elsevier Inc. All rights reserved.

Cooperative interactions between paired domain and homeodomain.

PubMed

Jun, S; Desplan, C

1996-09-01

The Pax proteins are a family of transcriptional regulators involved in many developmental processes in all higher eukaryotes. They are characterized by the presence of a paired domain (PD), a bipartite DNA binding domain composed of two helix-turn-helix (HTH) motifs,the PAI and RED domains. The PD is also often associated with a homeodomain (HD) which is itself able to form homo- and hetero-dimers on DNA. Many of these proteins therefore contain three HTH motifs each able to recognize DNA. However, all PDs recognize highly related DNA sequences, and most HDs also recognize almost identical sites. We show here that different Pax proteins use multiple combinations of their HTHs to recognize several types of target sites. For instance, the Drosophila Paired protein can bind, in vitro, exclusively through its PAI domain, or through a dimer of its HD, or through cooperative interaction between PAI domain and HD. However, prd function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein, Lune, may rely on the RED domain and HD. We propose a model by which Pax proteins recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire.

Characterization of a Smad motif similar to Drosophila mad in the mouse Msx 1 promoter.

PubMed

Alvarez Martinez, Cristina E; Binato, Renata; Gonzalez, Sayonara; Pereira, Monica; Robert, Benoit; Abdelhay, Eliana

2002-03-01

Mouse Msx 1 gene, orthologous of the Drosophila msh, is involved in several developmental processes. BMP family members are major proteins in the regulation of Msx 1 expression. BMP signaling activates Smad 1/5/8 proteins, which associate to Smad 4 before translocating to the nucleus. Analysis of Msx 1 promoter revealed the presence of three elements similar to the consensus established for Mad, the Smad 1 Drosophila counterpart. Notably, such an element was identified in an enhancer important for Msx 1 regulation. Gel shift analysis demonstrated that proteins from 13.5 dpc embryo associate to this enhancer. Remarkably, supershift assays showed that Smad proteins are present in the complex. Purified Smad 1 and 4 also bind to this fragment. We demonstrate that functional binding sites in this enhancer are confined to the Mad motif and flanking region. Our data suggest that this Mad motif may be functional in response to BMP signaling. ©2002 Elsevier Science (USA).

Identification of pathways directly regulated by SHORT VEGETATIVE PHASE during vegetative and reproductive development in Arabidopsis

PubMed Central

2013-01-01

Background MADS-domain transcription factors play important roles during plant development. The Arabidopsis MADS-box gene SHORT VEGETATIVE PHASE (SVP) is a key regulator of two developmental phases. It functions as a repressor of the floral transition during the vegetative phase and later it contributes to the specification of floral meristems. How these distinct activities are conferred by a single transcription factor is unclear, but interactions with other MADS domain proteins which specify binding to different genomic regions is likely one mechanism. Results To compare the genome-wide DNA binding profile of SVP during vegetative and reproductive development we performed ChIP-seq analyses. These ChIP-seq data were combined with tiling array expression analysis, induction experiments and qRT-PCR to identify biologically relevant binding sites. In addition, we compared genome-wide target genes of SVP with those published for the MADS domain transcription factors FLC and AP1, which interact with SVP during the vegetative and reproductive phases, respectively. Conclusions Our analyses resulted in the identification of pathways that are regulated by SVP including those controlling meristem development during vegetative growth and flower development whereas floral transition pathways and hormonal signaling were regulated predominantly during the vegetative phase. Thus, SVP regulates many developmental pathways, some of which are common to both of its developmental roles whereas others are specific to only one of them. PMID:23759218

The insulator protein Suppressor of Hairy-wing is an essential transcriptional repressor in the Drosophila ovary.

PubMed

Soshnev, Alexey A; Baxley, Ryan M; Manak, J Robert; Tan, Kai; Geyer, Pamela K

2013-09-01

Suppressor of Hairy-wing [Su(Hw)] is a DNA-binding factor required for gypsy insulator function and female germline development in Drosophila. The insulator function of the gypsy retrotransposon depends on Su(Hw) binding to clustered Su(Hw) binding sites (SBSs) and recruitment of the insulator proteins Centrosomal Protein 190 kD (CP190) and Modifier of mdg4 67.2 kD (Mod67.2). By contrast, the Su(Hw) germline function involves binding to non-clustered SBSs and does not require CP190 or Mod67.2. Here, we identify Su(Hw) target genes, using genome-wide analyses in the ovary to uncover genes with an ovary-bound SBS that are misregulated upon Su(Hw) loss. Most Su(Hw) target genes demonstrate enriched expression in the wild-type CNS. Loss of Su(Hw) leads to increased expression of these CNS-enriched target genes in the ovary and other tissues, suggesting that Su(Hw) is a repressor of neural genes in non-neural tissues. Among the Su(Hw) target genes is RNA-binding protein 9 (Rbp9), a member of the ELAV/Hu gene family. Su(Hw) regulation of Rbp9 appears to be insulator independent, as Rbp9 expression is unchanged in a genetic background that compromises the functions of the CP190 and Mod67.2 insulator proteins, even though both localize to Rbp9 SBSs. Rbp9 misregulation is central to su(Hw)(-/-) sterility, as Rbp9(+/-), su(Hw)(-/-) females are fertile. Eggs produced by Rbp9(+/-), su(Hw)(-/-) females show patterning defects, revealing a somatic requirement for Su(Hw) in the ovary. Our studies demonstrate that Su(Hw) is a versatile transcriptional regulatory protein with an essential developmental function involving transcriptional repression.

Target gene analysis by microarrays and chromatin immunoprecipitation identifies HEY proteins as highly redundant bHLH repressors.

PubMed

Heisig, Julia; Weber, David; Englberger, Eva; Winkler, Anja; Kneitz, Susanne; Sung, Wing-Kin; Wolf, Elmar; Eilers, Martin; Wei, Chia-Lin; Gessler, Manfred

2012-01-01

HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression.

Target Gene Analysis by Microarrays and Chromatin Immunoprecipitation Identifies HEY Proteins as Highly Redundant bHLH Repressors

PubMed Central

Englberger, Eva; Winkler, Anja; Kneitz, Susanne; Sung, Wing-Kin; Wolf, Elmar; Eilers, Martin; Wei, Chia-Lin; Gessler, Manfred

2012-01-01

HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression. PMID:22615585

Crystal structure of LGR4-Rspo1 complex: insights into the divergent mechanisms of ligand recognition by leucine-rich repeat G-protein-coupled receptors (LGRs).

PubMed

Xu, Jin-Gen; Huang, Chunfeng; Yang, Zhengfeng; Jin, Mengmeng; Fu, Panhan; Zhang, Ni; Luo, Jian; Li, Dali; Liu, Mingyao; Zhou, Yan; Zhu, Yongqun

2015-01-23

Leucine-rich repeat G-protein-coupled receptors (LGRs) are a unique class of G-protein-coupled receptors characterized by a large extracellular domain to recognize ligands and regulate many important developmental processes. Among the three groups of LGRs, group B members (LGR4-6) recognize R-spondin family proteins (Rspo1-4) to stimulate Wnt signaling. In this study, we successfully utilized the "hybrid leucine-rich repeat technique," which fused LGR4 with the hagfish VLR protein, to obtain two recombinant human LGR4 proteins, LGR415 and LGR49. We determined the crystal structures of ligand-free LGR415 and the LGR49-Rspo1 complex. LGR4 exhibits a twisted horseshoe-like structure. Rspo1 adopts a flat and β-fold architecture and is bound in the concave surface of LGR4 in the complex through electrostatic and hydrophobic interactions. All the Rspo1-binding residues are conserved in LGR4-6, suggesting that LGR4-6 bind R-spondins through an identical surface. Structural analysis of our LGR4-Rspo1 complex with the previously determined LGR4 and LGR5 structures revealed that the concave surface of LGR4 is the sole binding site for R-spondins, suggesting a one-site binding model of LGR4-6 in ligand recognition. The molecular mechanism of LGR4-6 is distinct from the two-step mechanism of group A receptors LGR1-3 and the multiple-interface binding model of group C receptors LGR7-8, suggesting LGRs utilize the divergent mechanisms for ligand recognition. Our structures, together with previous reports, provide a comprehensive understanding of the ligand recognition by LGRs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Four Amino Acids within a Tandem QxVx Repeat in a Predicted Extended α-Helix of the Smad-Binding Domain of Sip1 Are Necessary for Binding to Activated Smad Proteins

PubMed Central

Conidi, Andrea; van den Berghe, Veronique; Leslie, Kris; Stryjewska, Agata; Xue, Hua; Chen, Ye-Guang; Seuntjens, Eve; Huylebroeck, Danny

2013-01-01

The zinc finger transcription factor Smad-interacting protein-1 (Sip1; Zeb2, Zfhx1b) plays an important role during vertebrate embryogenesis in various tissues and differentiating cell types, and during tumorigenesis. Previous biochemical analysis suggests that interactions with several partner proteins, including TGFβ family receptor-activated Smads, regulate the activities of Sip1 in the nucleus both as a DNA-binding transcriptional repressor and activator. Using a peptide aptamer approach we mapped in Sip1 its Smad-binding domain (SBD), initially defined as a segment of 51 amino acids, to a shorter stretch of 14 amino acids within this SBD. Modelling suggests that this short SBD stretch is part of an extended α-helix that may fit the binding to a hydrophobic corridor within the MH2 domain of activated Smads. Four amino acids (two polar Q residues and two non-polar V residues) that form the tandem repeat (QxVx)2 in this 14-residue stretch were found to be crucial for binding to both TGFβ/Nodal/Activin-Smads and BMP-Smads. A full-length Sip1 with collective mutation of these Q and V residues (to A) no longer binds to Smads, while it retains its binding activity to its cognate bipartite target DNA sequence. This missense mutant Sip1(AxAx)2 provides a new molecular tool to identify SBD (in)dependent target genes in Sip1-controlled TGFβ and/or BMP (de)regulated cellular, developmental and pathological processes. PMID:24146916

Cell type-specific translational repression of Cyclin B during meiosis in males.

PubMed

Baker, Catherine Craig; Gim, Byung Soo; Fuller, Margaret T

2015-10-01

The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. Here, we show that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, we show that Fest is required for proper execution of meiosis I. © 2015. Published by The Company of Biologists Ltd.

A Recessive Mutation Resulting in a Disabling Amino Acid Substitution (T194R) in the LHX3 Homeodomain Causes Combined Pituitary Hormone Deficiency

PubMed Central

Bechtold-Dalla Pozza, Susanne; Hiedl, Stefan; Roeb, Julia; Lohse, Peter; Malik, Raleigh E.; Park, Soyoung; Durán-Prado, Mario; Rhodes, Simon J.

2012-01-01

Background/Aims Recessive mutations in the LHX3 ho-meodomain transcription factor gene are associated with developmental disorders affecting the pituitary and nervous system. We describe pediatric patients with combined pituitary hormone deficiency (CPHD) who harbor a novel mutation in LHX3. Methods Two female siblings from related parents were examined. Both patients had neonatal complications. The index patient had CPHD featuring deficiencies of GH, LH, FSH, PRL, and TSH, with later onset of ACTH deficiency. She also had a hypoplastic anterior pituitary, respiratory distress, hearing impairment, and limited neck rotation. The LHX3 gene was sequenced and the biochemical properties of the predicted altered proteins were characterized. Results A novel homozygous mutation predicted to change amino acid 194 from threonine to arginine (T194R) was detected in both patients. This amino acid is conserved in the DNA-binding homeodomain. Computer modeling predicted that the T194R change would alter the homeodomain structure. The T194R protein did not bind tested LHX3 DNA recognition sites and did not activate the α-glycoprotein and PRL target genes. Conclusion The T194R mutation affects a critical residue in the LHX3 protein. This study extends our understanding of the phenotypic features, molecular mechanism, and developmental course associated with mutations in the LHX3 gene. PMID:22286346

Identification, characterization, and expression analysis of calmodulin and calmodulin-like genes in grapevine (Vitis vinifera) reveal likely roles in stress responses.

PubMed

Vandelle, Elodie; Vannozzi, Alessandro; Wong, Darren; Danzi, Davide; Digby, Anne-Marie; Dal Santo, Silvia; Astegno, Alessandra

2018-06-04

Calcium (Ca 2+ ) is an ubiquitous key second messenger in plants, where it modulates many developmental and adaptive processes in response to various stimuli. Several proteins containing Ca 2+ binding domain have been identified in plants, including calmodulin (CaM) and calmodulin-like (CML) proteins, which play critical roles in translating Ca 2+ signals into proper cellular responses. In this work, a genome-wide analysis conducted in Vitis vinifera identified three CaM- and 62 CML-encoding genes. We assigned gene family nomenclature, analyzed gene structure, chromosomal location and gene duplication, as well as protein motif organization. The phylogenetic clustering revealed a total of eight subgroups, including one unique clade of VviCaMs distinct from VviCMLs. VviCaMs were found to contain four EF-hand motifs whereas VviCML proteins have one to five. Most of grapevine CML genes were intronless, while VviCaMs were intron rich. All the genes were well spread among the 19 grapevine chromosomes and displayed a high level of duplication. The expression profiling of VviCaM/VviCML genes revealed a broad expression pattern across all grape organs and tissues at various developmental stages, and a significant modulation in biotic stress-related responses. Our results highlight the complexity of CaM/CML protein family also in grapevine, supporting the versatile role of its different members in modulating cellular responses to various stimuli, in particular to biotic stresses. This work lays the foundation for further functional and structural studies on specific grapevine CaMs/CMLs in order to better understand the role of Ca 2+ -binding proteins in grapevine and to explore their potential for further biotechnological applications. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Differential Subcellular Localization of Leishmania Alba-Domain Proteins throughout the Parasite Development.

PubMed

Dupé, Aurélien; Dumas, Carole; Papadopoulou, Barbara

2015-01-01

Alba-domain proteins are RNA-binding proteins found in archaea and eukaryotes and recently studied in protozoan parasites where they play a role in the regulation of virulence factors and stage-specific proteins. This work describes in silico structural characterization, cellular localization and biochemical analyses of Alba-domain proteins in Leishmania infantum. We show that in contrast to other protozoa, Leishmania have two Alba-domain proteins, LiAlba1 and LiAlba3, representative of the Rpp20- and the Rpp25-like eukaryotic subfamilies, respectively, which share several sequence and structural similarities but also important differences with orthologs in other protozoa, especially in sequences targeted for post-translational modifications. LiAlba1 and LiAlba3 proteins form a complex interacting with other RNA-binding proteins, ribosomal subunits, and translation factors as supported by co-immunoprecipitation and sucrose gradient sedimentation analysis. A higher co-sedimentation of Alba proteins with ribosomal subunits was seen upon conditions of decreased translation, suggesting a role of these proteins in translational repression. The Leishmania Alba-domain proteins display differential cellular localization throughout the parasite development. In the insect promastigote stage, Alba proteins co-localize predominantly to the cytoplasm but they translocate to the nucleolus and the flagellum upon amastigote differentiation in the mammalian host and are found back to the cytoplasm once amastigote differentiation is completed. Heat-shock, a major signal of amastigote differentiation, triggers Alba translocation to the nucleolus and the flagellum. Purification of the Leishmania flagellum confirmed LiAlba3 enrichment in this organelle during amastigote differentiation. Moreover, partial characterization of the Leishmania flagellum proteome of promastigotes and differentiating amastigotes revealed the presence of other RNA-binding proteins, as well as differences in the flagellum composition between these two parasite lifestages. Shuttling of Alba-domain proteins between the cytoplasm and the nucleolus or the flagellum throughout the parasite life cycle suggests that these RNA-binding proteins participate in several distinct regulatory pathways controlling developmental gene expression in Leishmania.

Differential Subcellular Localization of Leishmania Alba-Domain Proteins throughout the Parasite Development

PubMed Central

Dupé, Aurélien; Dumas, Carole; Papadopoulou, Barbara

2015-01-01

Alba-domain proteins are RNA-binding proteins found in archaea and eukaryotes and recently studied in protozoan parasites where they play a role in the regulation of virulence factors and stage-specific proteins. This work describes in silico structural characterization, cellular localization and biochemical analyses of Alba-domain proteins in Leishmania infantum. We show that in contrast to other protozoa, Leishmania have two Alba-domain proteins, LiAlba1 and LiAlba3, representative of the Rpp20- and the Rpp25-like eukaryotic subfamilies, respectively, which share several sequence and structural similarities but also important differences with orthologs in other protozoa, especially in sequences targeted for post-translational modifications. LiAlba1 and LiAlba3 proteins form a complex interacting with other RNA-binding proteins, ribosomal subunits, and translation factors as supported by co-immunoprecipitation and sucrose gradient sedimentation analysis. A higher co-sedimentation of Alba proteins with ribosomal subunits was seen upon conditions of decreased translation, suggesting a role of these proteins in translational repression. The Leishmania Alba-domain proteins display differential cellular localization throughout the parasite development. In the insect promastigote stage, Alba proteins co-localize predominantly to the cytoplasm but they translocate to the nucleolus and the flagellum upon amastigote differentiation in the mammalian host and are found back to the cytoplasm once amastigote differentiation is completed. Heat-shock, a major signal of amastigote differentiation, triggers Alba translocation to the nucleolus and the flagellum. Purification of the Leishmania flagellum confirmed LiAlba3 enrichment in this organelle during amastigote differentiation. Moreover, partial characterization of the Leishmania flagellum proteome of promastigotes and differentiating amastigotes revealed the presence of other RNA-binding proteins, as well as differences in the flagellum composition between these two parasite lifestages. Shuttling of Alba-domain proteins between the cytoplasm and the nucleolus or the flagellum throughout the parasite life cycle suggests that these RNA-binding proteins participate in several distinct regulatory pathways controlling developmental gene expression in Leishmania. PMID:26334886

Mycobacterium tuberculosis WhiB1 is an essential DNA-binding protein with a nitric oxide sensitive iron-sulphur cluster

PubMed Central

Smith, Laura J.; Stapleton, Melanie R.; Fullstone, Gavin J. M.; Crack, Jason C.; Thomson, Andrew J.; Le Brun, Nick E.; Hunt, Debbie M.; Harvey, Evelyn; Adinolfi, Salvatore; Buxton, Roger S.; Green, Jeffrey

2010-01-01

Mycobacterium tuberculosis is a major pathogen that has the ability to establish, and emerge from, a persistent state. Wbl family proteins are associated with developmental processes in actinomycetes, and M. tuberculosis has seven such proteins. Here it is shown that the M. tuberculosis H37Rv whiB1 gene is essential. The WhiB1 protein possesses a [4Fe-4S]2+ cluster that is stable in air but reacts rapidly with eight equivalents of nitric oxide to yield two dinuclear dinitrosyl-iron thiol complexes. The [4Fe-4S] form of WhiB1 did not bind whiB1 promoter DNA, but the reduced and oxidized apo-WhiB1, and nitric oxide-treated holo-WhiB1 did bind to DNA. Mycobacterium smegmatis RNA polymerase induced transcription of whiB1 in vitro; however in the presence of apo-WhiB1 transcription was severely inhibited, irrespective of the presence or absence of the CRP protein Rv3676, which is known to activate whiB1 expression. Footprinting suggested that autorepression of whiB1 is achieved by apo-WhiB1 binding at a region that overlaps the core promoter elements. A model incorporating regulation of whiB1 expression in response to nitric oxide and cAMP is discussed with implications for sensing two important signals in establishing M. tuberculosis infections. PMID:20929442

Dense Bicoid hubs accentuate binding along the morphogen gradient

PubMed Central

Mir, Mustafa; Reimer, Armando; Haines, Jenna E.; Li, Xiao-Yong; Stadler, Michael; Garcia, Hernan

2017-01-01

Morphogen gradients direct the spatial patterning of developing embryos; however, the mechanisms by which these gradients are interpreted remain elusive. Here we used lattice light-sheet microscopy to perform in vivo single-molecule imaging in early Drosophila melanogaster embryos of the transcription factor Bicoid that forms a gradient and initiates patterning along the anteroposterior axis. In contrast to canonical models, we observed that Bicoid binds to DNA with a rapid off rate throughout the embryo such that its average occupancy at target loci is on-rate-dependent. We further observed Bicoid forming transient “hubs” of locally high density that facilitate binding as factor levels drop, including in the posterior, where we observed Bicoid binding despite vanishingly low protein levels. We propose that localized modulation of transcription factor on rates via clustering provides a general mechanism to facilitate binding to low-affinity targets and that this may be a prevalent feature of other developmental transcription factors. PMID:28982761

The Fibroblast Growth Factor signaling pathway

PubMed Central

Ornitz, David M; Itoh, Nobuyuki

2015-01-01

The signaling component of the mammalian Fibroblast Growth Factor (FGF) family is comprised of eighteen secreted proteins that interact with four signaling tyrosine kinase FGF receptors (FGFRs). Interaction of FGF ligands with their signaling receptors is regulated by protein or proteoglycan cofactors and by extracellular binding proteins. Activated FGFRs phosphorylate specific tyrosine residues that mediate interaction with cytosolic adaptor proteins and the RAS-MAPK, PI3K-AKT, PLCγ, and STAT intracellular signaling pathways. Four structurally related intracellular non-signaling FGFs interact with and regulate the family of voltage gated sodium channels. Members of the FGF family function in the earliest stages of embryonic development and during organogenesis to maintain progenitor cells and mediate their growth, differentiation, survival, and patterning. FGFs also have roles in adult tissues where they mediate metabolic functions, tissue repair, and regeneration, often by reactivating developmental signaling pathways. Consistent with the presence of FGFs in almost all tissues and organs, aberrant activity of the pathway is associated with developmental defects that disrupt organogenesis, impair the response to injury, and result in metabolic disorders, and cancer. © 2015 Wiley Periodicals, Inc. PMID:25772309

A novel bifunctional histone protein in Streptomyces: a candidate for structural coupling between DNA conformation and transcription during development and stress?

PubMed Central

Aldridge, Matthew; Facey, Paul; Francis, Lewis; Bayliss, Sion; Del Sol, Ricardo; Dyson, Paul

2013-01-01

Antibiotic-producing Streptomyces are complex bacteria that remodel global transcription patterns and their nucleoids during development. Here, we describe a novel developmentally regulated nucleoid-associated protein, DdbA, of the genus that consists of an N-terminal DNA-binding histone H1-like domain and a C-terminal DksA-like domain that can potentially modulate RNA polymerase activity in conjunction with ppGpp. Owing to its N-terminal domain, the protein can efficiently bind and condense DNA in vitro. Loss of function of this DNA-binding protein results in changes in both DNA condensation during development and the ability to adjust DNA supercoiling in response to osmotic stress. Initial analysis of the DksA-like activity of DdbA indicates that overexpression of the protein suppresses a conditional deficiency in antibiotic production of relA mutants that are unable to synthesise ppGpp, just as DksA overexpression in Escherichia coli can suppress ppGpp0 phenotypes. The null mutant is also sensitive to oxidative stress owing to impaired upregulation of transcription of sigR, encoding an alternative sigma factor. Consequently, we propose this bifunctional histone-like protein as a candidate that could structurally couple changes in DNA conformation and transcription during the streptomycete life-cycle and in response to stress. PMID:23525459

Thermodynamic characterization of two homologous protein complexes: Associations of the semaphorin receptor plexin-B1 RhoGTPase binding domain with Rnd1 and active Rac1

PubMed Central

Hota, Prasanta K; Buck, Matthias

2009-01-01

Plexin receptors function in response to semaphorin guidance cues in a variety of developmental processes involving cell motility. Interactions with Rho, as well as Ras family small GTPases are critical events in the cell signaling mechanism. We have recently determined the structure of a cytoplasmic domain (RBD) of plexin-B1 and mapped its binding interface with several Rho-GTPases, Rac1, Rnd1, and RhoD. All three GTPases associate with a similar region of this plexin domain, but show different functional behavior in cells. To understand whether thermodynamic properties of the GTPase–RBD interaction contribute to such different behavior, we have examined the interaction at different temperatures, buffer, and pH conditions. Although the binding affinity of both Rnd1 and Rac1 with the plexin-B1 RBD is similar, the detailed thermodynamic properties of the interactions are considerably different. These data suggest that on Rac1 binding to the plexin-B1 RBD, the proteins become more rigid in the complex. By contrast, Rnd1 binding is consistent with unchanged or slightly increased flexibility in one or both proteins. Both GTPases show an appreciable reduction in affinity for the dimeric plexin-B1 RBD indicating that GTPase binding is not cooperative with dimer formation, but that a partial steric hindrance destabilizes the dimer. However, a reduced affinity binding mode to a disulphide stabilized model for the dimeric RBD is also possible. Consistent with cellular studies, the interaction thermodynamics imply that further levels of regulation involving additional binding partners and/or regions outside of the RhoGTPase binding domain are required for receptor activation. PMID:19388051

Promoter activity of polypyrimidine tract-binding protein genes of potato responds to environmental cues.

PubMed

Butler, Nathaniel M; Hannapel, David J

2012-12-01

Polypyrimidine tract-binding (PTB) proteins are RNA-binding proteins that target specific RNAs for post-transcriptional processing by binding cytosine/uracil motifs. PTBs have established functions in a range of RNA processes including splicing, translation, stability and long-distance transport. Six PTB-like genes identified in potato have been grouped into two clades based on homology to other known plant PTBs. StPTB1 and StPTB6 are closely related to a PTB protein discovered in pumpkin, designated CmRBP50, and contain four canonical RNA-recognition motifs. CmRBP50 is expressed in phloem tissues and functions as the core protein of a phloem-mobile RNA/protein complex. Sequence from the potato genome database was used to clone the upstream sequence of these two PTB genes and analyzed to identify conserved cis-elements. The promoter of StPTB6 was enriched for regulatory elements for light and sucrose induction and defense. Upstream sequence of both PTB genes was fused to β-glucuronidase and monitored in transgenic potato lines. In whole plants, the StPTB1 promoter was most active in leaf veins and petioles, whereas StPTB6 was most active in leaf mesophyll. Both genes are active in new tubers and tuber sprouts. StPTB6 expression was induced in stems and stolon sections in response to sucrose and in leaves or petioles in response to light, heat, drought and mechanical wounding. These results show that CmRBP50-like genes of potato exhibit distinct expression patterns and respond to both developmental and environmental cues.

Global changes of the RNA-bound proteome during the maternal-to-zygotic transition in Drosophila

PubMed Central

Sysoev, Vasiliy O.; Fischer, Bernd; Frese, Christian K.; Gupta, Ishaan; Krijgsveld, Jeroen; Hentze, Matthias W.; Castello, Alfredo; Ephrussi, Anne

2016-01-01

The maternal-to-zygotic transition (MZT) is a process that occurs in animal embryos at the earliest developmental stages, during which maternally deposited mRNAs and other molecules are degraded and replaced by products of the zygotic genome. The zygotic genome is not activated immediately upon fertilization, and in the pre-MZT embryo post-transcriptional control by RNA-binding proteins (RBPs) orchestrates the first steps of development. To identify relevant Drosophila RBPs organism-wide, we refined the RNA interactome capture method for comparative analysis of the pre- and post-MZT embryos. We determine 523 proteins as high-confidence RBPs, half of which were not previously reported to bind RNA. Comparison of the RNA interactomes of pre- and post-MZT embryos reveals high dynamicity of the RNA-bound proteome during early development, and suggests active regulation of RNA binding of some RBPs. This resource provides unprecedented insight into the system of RBPs that govern the earliest steps of Drosophila development. PMID:27378189

Gibberellin Perception by the Gibberellin Receptor and its Effector Recognition

NASA Astrophysics Data System (ADS)

Hakoshima, Toshio; Murase, Kohji; Hirano, Yoshinori; Sun, Tai-Ping

Gibberellins control a diverse range of growth and developmental processes in higher plants and have been widely utilized in the agricultural industry. By binding to a nuclear receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1), gibberellins regulate gene expression by promoting degradation of the transcriptional regulator DELLA proteins. The precise manner in which GID1 discriminates and becomes activated by bioactive gibberellins for specific binding to DELLA proteins remains unclear. We present the crystal structure of a ternary complex of Arabidopsis thaliana GID1A, a bioactive gibberellin and the N-terminal DELLA domain of GAI. In this complex, GID1a occludes gibberellin in a deep binding pocket covered by its N-terminal helical switch region, which in turn interacts with the DELLA domain containing DELLA, VHYNP and LExLE motifs. Our results establish a structural model of a plant hormone receptor which is distinct from the hormone-perception mechanism and effector recognition of the known auxin receptors.

Coordination of flower development by homeotic master regulators.

PubMed

Ito, Toshiro

2011-02-01

Floral homeotic genes encode transcription factors and act as master regulators of flower development. The homeotic protein complex is expressed in a specific whorl of the floral primordium and determines floral organ identity by the combinatorial action. Homeotic proteins continue to be expressed until late in flower development to coordinate growth and organogenesis. Recent genomic studies have shown that homeotic proteins bind thousands of target sites in the genome and regulate the expression of transcription factors, chromatin components and various proteins involved in hormone biosynthesis and signaling and other physiological activities. Further, homeotic proteins program chromatin to direct the developmental coordination of stem cell maintenance and differentiation in shaping floral organs. Copyright © 2010 Elsevier Ltd. All rights reserved.

Widespread evidence of cooperative DNA binding by transcription factors in Drosophila development.

PubMed

Kazemian, Majid; Pham, Hannah; Wolfe, Scot A; Brodsky, Michael H; Sinha, Saurabh

2013-09-01

Regulation of eukaryotic gene transcription is often combinatorial in nature, with multiple transcription factors (TFs) regulating common target genes, often through direct or indirect mutual interactions. Many individual examples of cooperative binding by directly interacting TFs have been identified, but it remains unclear how pervasive this mechanism is during animal development. Cooperative TF binding should be manifest in genomic sequences as biased arrangements of TF-binding sites. Here, we explore the extent and diversity of such arrangements related to gene regulation during Drosophila embryogenesis. We used the DNA-binding specificities of 322 TFs along with chromatin accessibility information to identify enriched spacing and orientation patterns of TF-binding site pairs. We developed a new statistical approach for this task, specifically designed to accurately assess inter-site spacing biases while accounting for the phenomenon of homotypic site clustering commonly observed in developmental regulatory regions. We observed a large number of short-range distance preferences between TF-binding site pairs, including examples where the preference depends on the relative orientation of the binding sites. To test whether these binding site patterns reflect physical interactions between the corresponding TFs, we analyzed 27 TF pairs whose binding sites exhibited short distance preferences. In vitro protein-protein binding experiments revealed that >65% of these TF pairs can directly interact with each other. For five pairs, we further demonstrate that they bind cooperatively to DNA if both sites are present with the preferred spacing. This study demonstrates how DNA-binding motifs can be used to produce a comprehensive map of sequence signatures for different mechanisms of combinatorial TF action.

The multifunctional Staufen proteins: conserved roles from neurogenesis to synaptic plasticity.

PubMed

Heraud-Farlow, Jacki E; Kiebler, Michael A

2014-09-01

Staufen (Stau) proteins belong to a family of RNA-binding proteins (RBPs) that are important for RNA localisation in many organisms. In this review we discuss recent findings on the conserved role played by Stau during both the early differentiation of neurons and in the synaptic plasticity of mature neurons. Recent molecular data suggest mechanisms for how Stau2 regulates mRNA localisation, mRNA stability, translation, and ribonucleoprotein (RNP) assembly. We offer a perspective on how this multifunctional RBP has been adopted to regulate mRNA localisation under several different cellular and developmental conditions. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Caste- and development-associated gene expression in a lower termite

PubMed Central

Scharf, Michael E; Wu-Scharf, Dancia; Pittendrigh, Barry R; Bennett, Gary W

2003-01-01

Background Social insects such as termites express dramatic polyphenism (the occurrence of multiple forms in a species on the basis of differential gene expression) both in association with caste differentiation and between castes after differentiation. We have used cDNA macroarrays to compare gene expression between polyphenic castes and intermediary developmental stages of the termite Reticulitermes flavipes. Results We identified differentially expressed genes from nine ontogenic categories. Quantitative PCR was used to quantify precise differences in gene expression between castes and between intermediary developmental stages. We found worker and nymph-biased expression of transcripts encoding termite and endosymbiont cellulases; presoldier-biased expression of transcripts encoding the storage/hormone-binding protein vitellogenin; and soldier-biased expression of gene transcripts encoding two transcription/translation factors, two signal transduction factors and four cytoskeletal/muscle proteins. The two transcription/translation factors showed significant homology to the bicaudal and bric-a-brac developmental genes of Drosophila. Conclusions Our results show differential expression of regulatory, structural and enzyme-coding genes in association with termite castes and their developmental precursor stages. They also provide the first glimpse into how insect endosymbiont cellulase gene expression can vary in association with the caste of a host. These findings shed light on molecular processes associated with termite biology, polyphenism, caste differentiation and development and highlight potentially interesting variations in developmental themes between termites, other insects, and higher animals. PMID:14519197

Cross-Modal Binding in Developmental Dyslexia

ERIC Educational Resources Information Center

Jones, Manon W.; Branigan, Holly P.; Parra, Mario A.; Logie, Robert H.

2013-01-01

The ability to learn visual-phonological associations is a unique predictor of word reading, and individuals with developmental dyslexia show impaired ability in learning these associations. In this study, we compared developmentally dyslexic and nondyslexic adults on their ability to form cross-modal associations (or "bindings") based…

Cloning and characterization of a calcium binding EF-hand protein gene TaCab1 from wheat and its expression in response to Puccinia striiformis f. sp. tritici and abiotic stresses.

PubMed

Feng, Hao; Wang, Xiaomin; Sun, Yanfei; Wang, Xiaojie; Chen, Xianming; Guo, Jun; Duan, Yinghui; Huang, Lili; Kang, Zhensheng

2011-08-01

Calcium is a ubiquitous and essential secondary messenger in eukaryotic signal transduction pathways. Calcium binding protein, as a component of pathways, plays various roles in response to biotic and abiotic stresses, as well as in developmental processes in plants. In this study, a calcium binding protein gene, designated as TaCab1 (Triticum aestivum calcium binding EF-hand protein 1), was isolated and characterized from wheat leaves (cv. Suwon 11) infected by Puccinia striiformis f. sp. tritici by in silico cloning and reverse transcription PCR (RT-PCR). TaCab1 did not have an intron and was predicted to encode a 216 amino acid protein which possesses an N-terminal region with a signal peptide, a transmembrane domain, an EF-hand motif and a caleosin domain. The results of transient assays with constructs of TaCab1 with green fluorescent protein (GFP) gene indicated that TaCab1 encodes a transmembrane protein. Quantitative real-time PCR (qRT-PCR) analyses revealed that TaCab1 was highly expressed in leaves than roots and stems. Although up-regulated expression profiles of TaCab1 were quite similar in both incompatible and compatible interactions, its transcript accumulation in the compatible interaction was much higher than in the incompatible interaction. The transcription of TaCab1 was also up-regulated at different degrees after treated by phytohormones [abscisic acid, benzyl adenine, ethylene, methyl jasmonate and salicylic acid (SA)] and stress stimuli [wounding, low temperature, polyethylene glycol and high salinity]. These results suggest that TaCab1 is involved in the plant-pathogen recognition, symptom development, and the basal tolerance to biotic and abiotic stresses through the SA signaling pathway.

The pyruvate kinase of Stigmatella aurantiaca is an indole binding protein and essential for development.

PubMed

Stamm, Irmela; Lottspeich, Friedrich; Plaga, Wulf

2005-06-01

Myxospore formation of the myxobacterium Stigmatella aurantiaca can be uncoupled from the cooperative development i.e. fruiting body formation, by low concentrations of indole. Two putative indole receptor proteins were isolated by their capacity to bind indole and identified as pyruvate kinase (PK) and aldehyde dehydrogenase. The PK activity of Stigmatella crude extracts was stimulated by indole. Cloning of the PK gene (pykA) and the construction of a pykA disruption mutant strikingly revealed that PK is essential for multicellular development: Fruiting body formation was abolished in the mutant strain and indole-induced spore formation was delayed. The developmental defects could be complemented by insertion of the pykA gene at the mtaB locus of the Stigmatella genome excluding any polar effects of the pykA disruption.

[Epigenome: what we learned from Rett syndrome, a neurological disease caused by mutation of a methyl-CpG binding protein].

PubMed

Kubota, Takeo

2013-01-01

Epigenome is defined as DNA and histone modification-dependent gene regulation system. Abnormalities in this system are known to cause various neuro-developmental diseases. We recently reported that neurological symptoms of Rett syndrome, which is an autistic disorder caused by mutations in methyl-CpG binding protein 2 (MeCP2), was associated with failure of epigenomic gene regulation in neuronal cells, and that clinical differences in the identical twins with Rett syndrome in the differences in DNA methylation in neuronal genes, but not caused by DNA sequence differences. Since central nervus system requires precise gene regulation, neurological diseases including Alzheimer and Parkinson diseases may be caused by acquired DNA modification (epigenomic) changes that results in aberrant gene regulation as well as DNA sequence changes congenitally occurred (mutation).

Reciprocal expression of integration host factor and HU in the developmental cycle and infectivity of Legionella pneumophila.

PubMed

Morash, Michael G; Brassinga, Ann Karen C; Warthan, Michelle; Gourabathini, Poornima; Garduño, Rafael A; Goodman, Steven D; Hoffman, Paul S

2009-04-01

Legionella pneumophila is an intracellular parasite of protozoa that differentiates late in infection into metabolically dormant cysts that are highly infectious. Regulation of this process is poorly understood. Here we report that the small DNA binding regulatory proteins integration host factor (IHF) and HU are reciprocally expressed over the developmental cycle, with HU expressed during exponential phase and IHF expressed postexponentially. To assess the role of these regulatory proteins in development, chromosomal deletions were constructed. Single (ihfA or ihfB) and double deletion (Deltaihf) IHF mutants failed to grow in Acanthamoeba castellanii unless complemented in trans when expressed temporally from the ihfA promoter but not under P(tac) (isopropyl-beta-d-thiogalactopyranoside). In contrast, IHF mutants were infectious for HeLa cells, though electron microscopic examination revealed defects in late-stage cyst morphogenesis (thickened cell wall, intracytoplasmic membranes, and inclusions of poly-beta-hydroxybutyrate), and were depressed for the developmental marker MagA. Green fluorescent protein promoter fusion assays indicated that IHF and the stationary-phase sigma factor RpoS were required for full postexponential expression of magA. Finally, defects in cyst morphogenesis noted for Deltaihf mutants in HeLa cells correlated with a loss of both detergent resistance and hyperinfectivity compared with results for wild-type cysts. These studies establish IHF and HU as markers of developmental stages and show that IHF function is required for both differentiation and full virulence of L. pneumophila in natural amoebic hosts.

Ankyrin Repeat Domain Protein 2 and Inhibitor of DNA Binding 3 Cooperatively Inhibit Myoblast Differentiation by Physical Interaction*

PubMed Central

Mohamed, Junaith S.; Lopez, Michael A.; Cox, Gregory A.; Boriek, Aladin M.

2013-01-01

Ankyrin repeat domain protein 2 (ANKRD2) translocates from the nucleus to the cytoplasm upon myogenic induction. Overexpression of ANKRD2 inhibits C2C12 myoblast differentiation. However, the mechanism by which ANKRD2 inhibits myoblast differentiation is unknown. We demonstrate that the primary myoblasts of mdm (muscular dystrophy with myositis) mice (pMBmdm) overexpress ANKRD2 and ID3 (inhibitor of DNA binding 3) proteins and are unable to differentiate into myotubes upon myogenic induction. Although suppression of either ANKRD2 or ID3 induces myoblast differentiation in mdm mice, overexpression of ANKRD2 and inhibition of ID3 or vice versa is insufficient to inhibit myoblast differentiation in WT mice. We identified that ANKRD2 and ID3 cooperatively inhibit myoblast differentiation by physical interaction. Interestingly, although MyoD activates the Ankrd2 promoter in the skeletal muscles of wild-type mice, SREBP-1 (sterol regulatory element binding protein-1) activates the same promoter in the skeletal muscles of mdm mice, suggesting the differential regulation of Ankrd2. Overall, we uncovered a novel pathway in which SREBP-1/ANKRD2/ID3 activation inhibits myoblast differentiation, and we propose that this pathway acts as a critical determinant of the skeletal muscle developmental program. PMID:23824195

The myogenic repressor gene Holes in muscles is a direct transcriptional target of Twist and Tinman in the Drosophila embryonic mesoderm

PubMed Central

Elwell, Jennifer A.; Lovato, TyAnna L.; Adams, Melanie M.; Baca, Erica M.; Lee, Thai; Cripps, Richard M.

2015-01-01

Understanding the regulatory circuitry controlling myogenesis is critical to understanding developmental mechanisms and developmentally-derived diseases. We analyzed the transcriptional regulation of a Drosophila myogenic repressor gene, Holes in muscles (Him). Previously, Him was shown to inhibit Myocyte enhancer factor-2 (MEF2) activity, and is expressed in myoblasts but not differentiating myotubes. We demonstrate that different phases of Him embryonic expression arise through the actions of different enhancers, and we characterize the enhancer required for its early mesoderm expression. This Him early mesoderm enhancer contains two conserved binding sites for the basic helix-loop-helix regulator Twist, and one binding site for the NK homeodomain protein Tinman. The sites for both proteins are required for enhancer activity in early embryos. Twist and Tinman activate the enhancer in tissue culture assays, and ectopic expression of either factor is sufficient to direct ectopic expression of a Him-lacZ reporter, or of the endogenous Him gene. Moreover, sustained expression of twist expression in the mesoderm up-regulates mesodermal Him expression in late embryos. Our findings provide a model to define mechanistically how Twist can both promotes myogenesis through direct activation of Mef2, and can place a brake on myogenesis, through direct activation of Him. PMID:25704510

Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development.

PubMed

Suzuki, Atsushi; Niimi, Yuki; Shinmyozu, Kaori; Zhou, Zhi; Kiso, Makoto; Saga, Yumiko

2016-01-01

RNA-binding proteins (RBPs) play important roles for generating various cell types in many developmental processes, including eggs and sperms. Nanos is widely known as an evolutionarily conserved RNA-binding protein implicated in germ cell development. Mouse NANOS2 interacts directly with the CCR4-NOT (CNOT) deadenylase complex, resulting in the suppression of specific RNAs. However, the mechanisms involved in target specificity remain elusive. We show that another RBP, Dead end1 (DND1), directly interacts with NANOS2 to load unique RNAs into the CNOT complex. This interaction is mediated by the zinc finger domain of NANOS2, which is essential for its association with target RNAs. In addition, the conditional deletion of DND1 causes the disruption of male germ cell differentiation similar to that observed in Nanos2-KO mice. Thus, DND1 is an essential partner for NANOS2 that leads to the degradation of specific RNAs. We also present the first evidence that the zinc finger domain of Nanos acts as a protein-interacting domain for another RBP, providing a novel insight into Nanos-mediated germ cell development. © 2015 The Authors.

NtKRP, a kinesin-12 protein, regulates embryo/seed size and seed germination via involving in cell cycle progression at the G2/M transition.

PubMed

Tian, Shujuan; Wu, Jingjing; Li, Fen; Zou, Jianwei; Liu, Yuwen; Zhou, Bing; Bai, Yang; Sun, Meng-Xiang

2016-10-25

Kinesins comprise a superfamily of microtubule-based motor proteins involved in essential processes in plant development, but few kinesins have been functionally identified during seed development. Especially, few kinesins that regulate cell division during embryogenesis have been identified. Here we report the functional characterization of NtKRP, a motor protein of the kinesin-12 family. NtKRP is predominantly expressed in embryos and embryonic roots. NtKRP RNAi lines displayed reductions in cell numbers in the meristematic zone, in embryonic root length, and in mature embryo and seed sizes. Furthermore, we also show that CDKA;1 binds to NtKRP at the consensus phosphorylation sites and that the decreased cell numbers in NtKRP-silenced embryos are due to a delay in cell division cycle at the G2/M transition. In addition, binding between the cargo-binding tail domain of NtKRP and CDKA; 1 was also determined. Our results reveal a novel molecular pathway that regulates embryo/seed development and critical role of kinesin in temporal and spatial regulation of a specific issue of embryo developmental.

Genome-wide analysis of the DNA-binding with one zinc finger (Dof) transcription factor family in bananas.

PubMed

Dong, Chen; Hu, Huigang; Xie, Jianghui

2016-12-01

DNA-binding with one finger (Dof) domain proteins are a multigene family of plant-specific transcription factors involved in numerous aspects of plant growth and development. In this study, we report a genome-wide search for Musa acuminata Dof (MaDof) genes and their expression profiles at different developmental stages and in response to various abiotic stresses. In addition, a complete overview of the Dof gene family in bananas is presented, including the gene structures, chromosomal locations, cis-regulatory elements, conserved protein domains, and phylogenetic inferences. Based on the genome-wide analysis, we identified 74 full-length protein-coding MaDof genes unevenly distributed on 11 chromosomes. Phylogenetic analysis with Dof members from diverse plant species showed that MaDof genes can be classified into four subgroups (StDof I, II, III, and IV). The detailed genomic information of the MaDof gene homologs in the present study provides opportunities for functional analyses to unravel the exact role of the genes in plant growth and development.

Mapping of interaction domains between human repair proteins ERCC1 and XPF.

PubMed

de Laat, W L; Sijbers, A M; Odijk, H; Jaspers, N G; Hoeijmakers, J H

1998-09-15

ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-binding domain in XPF maps to C-terminal residues 814-905. ERCC1-XPF complex formation is established by a direct interaction between these two binding domains. A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1.

Mapping of interaction domains between human repair proteins ERCC1 and XPF.

PubMed Central

de Laat, W L; Sijbers, A M; Odijk, H; Jaspers, N G; Hoeijmakers, J H

1998-01-01

ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-binding domain in XPF maps to C-terminal residues 814-905. ERCC1-XPF complex formation is established by a direct interaction between these two binding domains. A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1. PMID:9722633

DNA-Binding Kinetics Determines the Mechanism of Noise-Induced Switching in Gene Networks

PubMed Central

Tse, Margaret J.; Chu, Brian K.; Roy, Mahua; Read, Elizabeth L.

2015-01-01

Gene regulatory networks are multistable dynamical systems in which attractor states represent cell phenotypes. Spontaneous, noise-induced transitions between these states are thought to underlie critical cellular processes, including cell developmental fate decisions, phenotypic plasticity in fluctuating environments, and carcinogenesis. As such, there is increasing interest in the development of theoretical and computational approaches that can shed light on the dynamics of these stochastic state transitions in multistable gene networks. We applied a numerical rare-event sampling algorithm to study transition paths of spontaneous noise-induced switching for a ubiquitous gene regulatory network motif, the bistable toggle switch, in which two mutually repressive genes compete for dominant expression. We find that the method can efficiently uncover detailed switching mechanisms that involve fluctuations both in occupancies of DNA regulatory sites and copy numbers of protein products. In addition, we show that the rate parameters governing binding and unbinding of regulatory proteins to DNA strongly influence the switching mechanism. In a regime of slow DNA-binding/unbinding kinetics, spontaneous switching occurs relatively frequently and is driven primarily by fluctuations in DNA-site occupancies. In contrast, in a regime of fast DNA-binding/unbinding kinetics, switching occurs rarely and is driven by fluctuations in levels of expressed protein. Our results demonstrate how spontaneous cell phenotype transitions involve collective behavior of both regulatory proteins and DNA. Computational approaches capable of simulating dynamics over many system variables are thus well suited to exploring dynamic mechanisms in gene networks. PMID:26488666

Poly(A) tail length regulates PABPC1 expression to tune translation in the heart.

PubMed

Chorghade, Sandip; Seimetz, Joseph; Emmons, Russell; Yang, Jing; Bresson, Stefan M; Lisio, Michael De; Parise, Gianni; Conrad, Nicholas K; Kalsotra, Auinash

2017-06-27

The rate of protein synthesis in the adult heart is one of the lowest in mammalian tissues, but it increases substantially in response to stress and hypertrophic stimuli through largely obscure mechanisms. Here, we demonstrate that regulated expression of cytosolic poly(A)-binding protein 1 (PABPC1) modulates protein synthetic capacity of the mammalian heart. We uncover a poly(A) tail-based regulatory mechanism that dynamically controls PABPC1 protein synthesis in cardiomyocytes and thereby titrates cellular translation in response to developmental and hypertrophic cues. Our findings identify PABPC1 as a direct regulator of cardiac hypertrophy and define a new paradigm of gene regulation in the heart, where controlled changes in poly(A) tail length influence mRNA translation.

Structural and functional characterizations of SsgB, a conserved activator of developmental cell division in morphologically complex actinomycetes.

PubMed

Xu, Qingping; Traag, Bjørn A; Willemse, Joost; McMullan, Daniel; Miller, Mitchell D; Elsliger, Marc-André; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Bakolitsa, Constantina; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Chruszcz, Maksymilian; Clayton, Thomas; Das, Debanu; Deller, Marc C; Duan, Lian; Ellrott, Kyle; Ernst, Dustin; Farr, Carol L; Feuerhelm, Julie; Grant, Joanna C; Grzechnik, Anna; Grzechnik, Slawomir K; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Krishna, S Sri; Kumar, Abhinav; Marciano, David; Minor, Wladek; Mommaas, A Mieke; Morse, Andrew T; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Puckett, Christina; Reyes, Ron; Rife, Christopher L; Sefcovic, Natasha; Tien, Henry J; Trame, Christine B; van den Bedem, Henry; Wang, Shuren; Weekes, Dana; Hodgson, Keith O; Wooley, John; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A; van Wezel, Gilles P

2009-09-11

SsgA-like proteins (SALPs) are a family of homologous cell division-related proteins that occur exclusively in morphologically complex actinomycetes. We show that SsgB, a subfamily of SALPs, is the archetypal SALP that is functionally conserved in all sporulating actinomycetes. Sporulation-specific cell division of Streptomyces coelicolor ssgB mutants is restored by introduction of distant ssgB orthologues from other actinomycetes. Interestingly, the number of septa (and spores) of the complemented null mutants is dictated by the specific ssgB orthologue that is expressed. The crystal structure of the SsgB from Thermobifida fusca was determined at 2.6 A resolution and represents the first structure for this family. The structure revealed similarities to a class of eukaryotic "whirly" single-stranded DNA/RNA-binding proteins. However, the electro-negative surface of the SALPs suggests that neither SsgB nor any of the other SALPs are likely to interact with nucleotide substrates. Instead, we show that a conserved hydrophobic surface is likely to be important for SALP function and suggest that proteins are the likely binding partners.

Molecular Cloning of Drebrin: Progress and Perspectives.

PubMed

Kojima, Nobuhiko

2017-01-01

Chicken drebrin isoforms were first identified in the optic tectum of developing brain. Although the time course of protein expression was different in each drebrin isoform, the similarity between their protein structures was suggested by biochemical analysis of purified protein. To determine their protein structures, the cloning of drebrin cDNAs was conducted. Comparison between the cDNA sequences shows that all drebrin cDNAs are identical except that the internal insertion sequences are present or absent in their sequences. Chicken drebrin are now classified into three isoforms, namely, drebrins E1, E2, and A. Genomic cloning demonstrated that the three isoforms are generated by an alternative splicing of individual exons encoding the insertion sequences from single drebrin gene. The mechanism should be precisely regulated in cell-type-specific and developmental stage-specific fashion. Drebrin protein, which is well conserved in various vertebrate species, although mammalian drebrin has only two isoforms, namely, drebrin E and drebrin A, is different from chicken drebrin that has three isoforms. Drebrin belongs to an actin-depolymerizing factor homology (ADF-H) domain protein family. Besides the ADF-H domain, drebrin has other domains, including the actin-binding domain and Homer-binding motifs. Diversity of protein isoform and multiple domains of drebrin could interact differentially with the actin cytoskeleton and other intracellular proteins and regulate diverse cellular processes.

FUNCTION OF PHLOEM-BORNE INFORMATION MACROMOLECULES IN INTEGRATING PLANT GROWTH & DEVELOPMENT

DOE Office of Scientific and Technical Information (OSTI.GOV)

William J. Lucas

2012-11-12

Studies on higher plants have revealed the operation of cell-to-cell and long-distance communication networks that mediate the transport of information macromolecules, such as proteins and RNA. Based on the findings from this DOE-funded project and results from other groups, it is now well established that the enucleate sieve tube system of the angiosperms contains a complex set of proteins including RNA binding proteins as well as a unique population of RNA molecules, comprised of both mRNA and small RNA species. Hetero-grafting experiments demonstrated that delivery of such RNA molecules, into the scion, is highly correlated with changes in developmental phenotypes.more » Furthermore, over the course of this project, our studies showed that plasmodesmata and the phloem are intimately involved in the local and systemic spread of sequence-specific signals that underlie gene silencing in plants. Major advances were also made in elucidating the underlying mechanisms that operate to mediate the selective entry and exit of proteins and RNA into and out of the phloem translocation stream. Our pioneering studies identified the first plant protein with the capacity to both bind specifically to small RNA molecules (si-RNA) and mediate in the cell-to-cell movement of such siRNA. Importantly, studies conducted with support from this DOE program also yielded a detailed characterization of the first phloem-mobile RNP complex isolated from pumpkin, namely the CmRBP50-RNP complex. This RNP complex was shown to bind, in a sequence-specific manner, to a set of transcripts encoding for transcription factors. The remarkable stability of this CmRBP50-RNP complex allows for long-distance delivery of bound transcripts from mature leaves into developing tissues and organs. Knowledge gained from this project can be used to exert control over the long-distance signaling networks used by plants to integrate their physiological and developmental programs at a whole plant level. Eventually, this information will aid in the engineering of elite plant lines with optimal traits for plant growth under non-ideal conditions, enhanced biomass and/or seed yield, and directed carbon allocation for efficient and sustainable biofuels production.« less

Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays

PubMed Central

Brand, Luise H.; Fischer, Nina M.; Harter, Klaus; Kohlbacher, Oliver; Wanke, Dierk

2013-01-01

WRKY transcription factors constitute a large protein family in plants that is involved in the regulation of developmental processes and responses to biotic or abiotic stimuli. The question arises how stimulus-specific responses are mediated given that the highly conserved WRKY DNA-binding domain (DBD) exclusively recognizes the ‘TTGACY’ W-box consensus. We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. The phylogenetic analysis of WRKY DBDs suggests that they evolved from an ancestral group IIc-like WRKY early in the eukaryote lineage. A direct descent of group IIc WRKYs supports a monophyletic origin of all other group II and III WRKYs from group I by loss of an N-terminal DBD. Group I WRKYs are of paraphyletic descent and evolved multiple times independently. By homology modeling, molecular dynamics simulations and in vitro DNA–protein interaction-enzyme-linked immunosorbent assay with AtWRKY50 (IIc), AtWRKY33 (I) and AtWRKY11 (IId) DBDs, we revealed differences in DNA-binding specificities. Our data imply that other components are essentially required besides the W-box-specific binding to DNA to facilitate a stimulus-specific WRKY function. PMID:23975197

PAPERCLIP identifies microRNA targets and a role of CstF64/64tau in promoting non-canonical poly(A) site usage

PubMed Central

Hwang, Hun-Way; Park, Christopher Y.; Goodarzi, Hani; Fak, John J.; Mele, Aldo; Moore, Michael J.; Saito, Yuhki; Darnell, Robert B.

2016-01-01

Accurate and precise annotation of the 3′ untranslated regions (3′ UTRs) is critical in understanding how mRNAs are regulated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). Here we describe a method, PAPERCLIP (Poly(A) binding Protein-mediated mRNA 3′ End Retrieval by CrossLinking ImmunoPrecipitation), which shows high specificity for the mRNA 3′ ends and compares favorably to existing 3′ end mapping methods. PAPERCLIP uncovers a previously unrecognized role of CstF64/64tau in promoting the usage of a selected group of non-canonical poly(A) sites, the majority of them containing a downstream GUKKU motif. Furthermore, in mouse brain, PAPERCLIP discovers extended 3′ UTR sequences harboring functional miRNA binding sites and reveals developmentally regulated APA shifts including one in Atp2b2 that is evolutionarily conserved in human and results in a gain of a functional binding site of miR-137. PAPERCLIP provides a powerful tool to decipher post-transcriptional regulation of mRNAs through APA in vivo. PMID:27050522

Ankyrin-binding activity of nervous system cell adhesion molecules expressed in adult brain.

PubMed

Davis, J Q; Bennett, V

1993-01-01

A family of ankyrin-binding glycoproteins have been identified in adult rat brain that include alternatively spliced products of the same pre-mRNA. A composite sequence of ankyrin-binding glycoprotein (ABGP) shares 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides and ankyrin associate as pure proteins in a 1:1 molar stoichiometry at a site located in the predicted cytoplasmic domain. ABGP polypeptides are expressed late in postnatal development to approximately the same levels as ankyrin, and comprise a significant fraction of brain membrane proteins. Immunofluorescence studies have shown that ABGP polypeptides are co-localized with ankyrinB. Major differences in developmental expression have been reported for neurofascin in embryos compared with the late postnatal expression of ABGP, suggesting that ABGP and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. Predicted cytoplasmic domains of rat ABGP and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, including L1, Nr-CAM and Ng-CAM of vertebrates, and neuroglian of Drosophila. A hypothesis to be evaluated is that ankyrin-binding activity is shared by all of these proteins.

Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention

PubMed Central

Yap, Karen; Lim, Zhao Qin; Khandelia, Piyush; Friedman, Brad; Makeyev, Eugene V.

2012-01-01

Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and nonneuronal cells. However, in nonneuronal cells, the splicing of 3′-terminal introns within these genes is repressed by the polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery, including the nuclear pore-associated protein Tpr and the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context. PMID:22661231

Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention.

PubMed

Yap, Karen; Lim, Zhao Qin; Khandelia, Piyush; Friedman, Brad; Makeyev, Eugene V

2012-06-01

Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and nonneuronal cells. However, in nonneuronal cells, the splicing of 3'-terminal introns within these genes is repressed by the polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery, including the nuclear pore-associated protein Tpr and the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context.

Sex determination in insects: a binary decision based on alternative splicing.

PubMed

Salz, Helen K

2011-08-01

The gene regulatory networks that control sex determination vary between species. Despite these differences, comparative studies in insects have found that alternative splicing is reiteratively used in evolution to control expression of the key sex-determining genes. Sex determination is best understood in Drosophila where activation of the RNA binding protein-encoding gene Sex-lethal is the central female-determining event. Sex-lethal serves as a genetic switch because once activated it controls its own expression by a positive feedback splicing mechanism. Sex fate choice in is also maintained by self-sustaining positive feedback splicing mechanisms in other dipteran and hymenopteran insects, although different RNA binding protein-encoding genes function as the binary switch. Studies exploring the mechanisms of sex-specific splicing have revealed the extent to which sex determination is integrated with other developmental regulatory networks. Copyright © 2011 Elsevier Ltd. All rights reserved.

Modelling a 3D structure for EgDf1 from shape Echinococcus granulosus: putative epitopes, phosphorylation motifs and ligand

NASA Astrophysics Data System (ADS)

Paulino, M.; Esteves, A.; Vega, M.; Tabares, G.; Ehrlich, R.; Tapia, O.

1998-07-01

EgDf1 is a developmentally regulated protein from the parasite Echinococcus granulosus related to a family of hydrophobic ligand binding proteins. This protein could play a crucial role during the parasite life cycle development since this organism is unable to synthetize most of their own lipids de novo. Furthermore, it has been shown that two related protein from other parasitic platyhelminths (Fh15 from Fasciola hepatica and Sm14 from Schistosoma mansoni) are able to confer protective inmunity against experimental infection in animal models. A three-dimensional structure would help establishing structure/function relationships on a knowledge based manner. 3D structures for EgDf1 protein were modelled by using myelin P2 (mP2) and intestine fatty acid binding protein (I-FABP) as templates. Molecular dynamics techniques were used to validate the models. Template mP2 yielded the best 3D structure for EgDf1. Palmitic and oleic acids were docked inside EgDf1. The present theoretical results suggest definite location in the secondary structure of the epitopic regions, consensus phosphorylation motifs and oleic acid as a good ligand candidate to EgDf1. This protein might well be involved in the process of supplying hydrophobic metabolites for membrane biosynthesis and for signaling pathways.

Regulation of nucleosome positioning by a CHD Type III chromatin remodeler and its relationship to developmental gene expression in Dictyostelium.

PubMed

Platt, James L; Kent, Nicholas A; Kimmel, Alan R; Harwood, Adrian J

2017-04-01

Nucleosome placement and repositioning can direct transcription of individual genes; however, the precise interactions of these events are complex and largely unresolved at the whole-genome level. The Chromodomain-Helicase-DNA binding (CHD) Type III proteins are a subfamily of SWI2/SNF2 proteins that control nucleosome positioning and are associated with several complex human disorders, including CHARGE syndrome and autism. Type III CHDs are required for multicellular development of animals and Dictyostelium but are absent in plants and yeast. These CHDs can mediate nucleosome translocation in vitro, but their in vivo mechanism is unknown. Here, we use genome-wide analysis of nucleosome positioning and transcription profiling to investigate the in vivo relationship between nucleosome positioning and gene expression during development of wild-type (WT) Dictyostelium and mutant cells lacking ChdC, a Type III CHD protein ortholog. We demonstrate major nucleosome positional changes associated with developmental gene regulation in WT. Loss of chdC caused an increase of intragenic nucleosome spacing and misregulation of gene expression, affecting ∼50% of the genes that are repositioned during WT development. These analyses demonstrate active nucleosome repositioning during Dictyostelium multicellular development, establish an in vivo function of CHD Type III chromatin remodeling proteins in this process, and reveal the detailed relationship between nucleosome positioning and gene regulation, as cells transition between developmental states. © 2017 Platt et al.; Published by Cold Spring Harbor Laboratory Press.

Noonan syndrome-associated SHP2/PTPN11 mutants cause EGF-dependent prolonged GAB1 binding and sustained ERK2/MAPK1 activation.

PubMed

Fragale, Alessandra; Tartaglia, Marco; Wu, Jie; Gelb, Bruce D

2004-03-01

Noonan syndrome is a developmental disorder with dysmorphic facies, short stature, cardiac defects, and skeletal anomalies, which can be caused by missense PTPN11 mutations. PTPN11 encodes Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2 or SHP-2), a protein tyrosine phosphatase that acts in signal transduction downstream to growth factor, hormone, and cytokine receptors. We compared the functional effects of three Noonan syndrome-causative PTPN11 mutations on SHP2's phosphatase activity, interaction with a binding partner, and signal transduction. All SHP2 mutants had significantly increased basal phosphatase activity compared to wild type, but that activity varied significantly between mutants and was further increased after epidermal growth factor stimulation. Cells expressing SHP2 mutants had prolonged extracellular signal-regulated kinase 2 activation, which was ligand-dependent. Binding of SHP2 mutants to Grb2-associated binder-1 was increased and sustained, and tyrosine phosphorylation of both proteins was prolonged. Coexpression of Grb2-associated binder-1-FF, which lacks SHP2 binding motifs, blocked the epidermal growth factor-mediated increase in SHP2's phosphatase activity and resulted in a dramatic reduction of extracellular signal-regulated kinase 2 activation. Taken together, these results document that Noonan syndrome-associated PTPN11 mutations increase SHP2's basal phosphatase activity, with greater activation when residues directly involved in binding at the interface between the N-terminal Src homology 2 and protein tyrosine phosphatase domains are altered. The SHP2 mutants prolonged signal flux through the RAS/mitogen-activated protein kinase (ERK2/MAPK1) pathway in a ligand-dependent manner that required docking through Grb2-associated binder-1 (GAB1), leading to increased cell proliferation. Copyright 2004 Wiley-Liss, Inc.

Different forms of soluble cytoplasmic mRNA binding proteins and particles in Xenopus laevis oocytes and embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murray, M.T.; Krohne, G.; Franke, W.W.

1991-01-01

To gain insight into the mechanisms involved in the formation of maternally stored mRNPs during Xenopus laevis development, we searched for soluble cytoplasmic proteins of the oocyte that are able to selectively bind mRNAs, using as substrate radiolabeled mRNA. In vitro mRNP assembly in solution was followed by UV-cross-linking and RNase digestion, resulting in covalent tagging of polypeptides by nucleotide transfer. Five polypeptides of approximately 54, 56 60, 70, and 100 kD (p54, p56, p60, p70, and p100) have been found to selectively bind mRNA and assemble into mRNPs. These polypeptides, which correspond to previously described native mRNP components, occurmore » in three different particle classes of approximately 4.5S, approximately 6S, and approximately 15S, as also determined by their reactions with antibodies against p54 and p56. Whereas the approximately 4.5S class contains p42, p60, and p70, probably each in the form of individual molecules or small complexes, the approximately 6S particles appears to consist only of p54 and p56, which occur in a near-stoichiometric ratio suggestive of a heterodimer complex. The approximately 15S particles contain, in addition to p54 and p56, p60 and p100 and this is the single occurring form of RNA-binding p100. We have also observed changes in the in vitro mRNA binding properties of these polypeptides during oogenesis and early embryonic development, in relation to their phosphorylation state and to the activity of an approximately 15S particle-associated protein kinase, suggesting that these proteins are involved in the developmental translational regulation of maternal mRNAs.« less

Intramolecular control of transcriptional activity by the NK2-specific domain in NK-2 homeodomain proteins

PubMed Central

Watada, Hirotaka; Mirmira, Raghavendra G.; Kalamaras, Julie; German, Michael S.

2000-01-01

The developmentally important homeodomain transcription factors of the NK-2 class contain a highly conserved region, the NK2-specific domain (NK2-SD). The function of this domain, however, remains unknown. The primary structure of the NK2-SD suggests that it might function as an accessory DNA-binding domain or as a protein–protein interaction interface. To assess the possibility that the NK2-SD may contribute to DNA-binding specificity, we used a PCR-based approach to identify a consensus DNA-binding sequences for Nkx2.2, an NK-2 family member involved in pancreas and central nervous system development. The consensus sequence (TCTAAGTGAGCTT) is similar to the known binding sequences for other NK-2 homeodomain proteins, but we show that the NK2-SD does not contribute significantly to specific DNA binding to this sequence. To determine whether the NK2-SD contributes to transactivation, we used GAL4-Nkx2.2 fusion constructs to map a powerful transcriptional activation domain in the C-terminal region beyond the conserved NK2-SD. Interestingly, this C-terminal region functions as a transcriptional activator only in the absence of an intact NK2-SD. The NK2-SD also can mask transactivation from the paired homeodomain transcription factor Pax6, but it has no effect on transcription by itself. These results demonstrate that the NK2-SD functions as an intramolecular regulator of the C-terminal activation domain in Nkx2.2 and support a model in which interactions through the NK2-SD regulate the ability of NK-2-class proteins to activate specific genes during development. PMID:10944215

Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition

PubMed Central

Madina, Bhaskara R.; Kumar, Vikas; Metz, Richard; Mooers, Blaine H.M.; Bundschuh, Ralf; Cruz-Reyes, Jorge

2014-01-01

Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3′-to-5′ in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3′ ends and strain-specific alternative 3′ editing within 3′ UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs. PMID:24865612

Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

PubMed Central

Galka, Marek M.; Rajagopalan, Nandhakishore; Buhrow, Leann M.; Nelson, Ken M.; Switala, Jacek; Cutler, Adrian J.; Palmer, David R. J.; Loewen, Peter C.; Abrams, Suzanne R.; Loewen, Michele C.

2015-01-01

Abscisic acid ((+)-ABA) is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC), x-ray crystallography and in silico modelling to identify putative (+)-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+)-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP) substrate. Functionally, (+)-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM), but more potent inhibition of Rubisco activation (Ki of ~ 130 μM). Comparative structural analysis of Rubisco in the presence of (+)-ABA with RuBP in the active site revealed only a putative low occupancy (+)-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+)-ABA binding site in the RuBP binding pocket. Overall we conclude that (+)-ABA interacts with Rubisco. While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation. PMID:26197050

Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

PubMed

Galka, Marek M; Rajagopalan, Nandhakishore; Buhrow, Leann M; Nelson, Ken M; Switala, Jacek; Cutler, Adrian J; Palmer, David R J; Loewen, Peter C; Abrams, Suzanne R; Loewen, Michele C

2015-01-01

Abscisic acid ((+)-ABA) is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC), x-ray crystallography and in silico modelling to identify putative (+)-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+)-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP) substrate. Functionally, (+)-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM), but more potent inhibition of Rubisco activation (Ki of ~ 130 μM). Comparative structural analysis of Rubisco in the presence of (+)-ABA with RuBP in the active site revealed only a putative low occupancy (+)-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+)-ABA binding site in the RuBP binding pocket. Overall we conclude that (+)-ABA interacts with Rubisco. While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.

Developmentally regulated HEART STOPPER, a mitochondrially targeted L18 ribosomal protein gene, is required for cell division, differentiation, and seed development in Arabidopsis

PubMed Central

Zhang, Hongyu; Luo, Ming; Day, Robert C.; Talbot, Mark J.; Ivanova, Aneta; Ashton, Anthony R.; Chaudhury, Abed M.; Macknight, Richard C.; Hrmova, Maria; Koltunow, Anna M.

2015-01-01

Evidence is presented for the role of a mitochondrial ribosomal (mitoribosomal) L18 protein in cell division, differentiation, and seed development after the characterization of a recessive mutant, heart stopper (hes). The hes mutant produced uncellularized endosperm and embryos arrested at the late globular stage. The mutant embryos differentiated partially on rescue medium with some forming callus. HES (At1g08845) encodes a mitochondrially targeted member of a highly diverged L18 ribosomal protein family. The substitution of a conserved amino residue in the hes mutant potentially perturbs mitoribosomal function via altered binding of 5S rRNA and/or influences the stability of the 50S ribosomal subunit, affecting mRNA binding and translation. Consistent with this, marker genes for mitochondrial dysfunction were up-regulated in the mutant. The slow growth of the endosperm and embryo indicates a defect in cell cycle progression, which is evidenced by the down-regulation of cell cycle genes. The down-regulation of other genes such as EMBRYO DEFECTIVE genes links the mitochondria to the regulation of many aspects of seed development. HES expression is developmentally regulated, being preferentially expressed in tissues with active cell division and differentiation, including developing embryos and the root tips. The divergence of the L18 family, the tissue type restricted expression of HES, and the failure of other L18 members to complement the hes phenotype suggest that the L18 proteins are involved in modulating development. This is likely via heterogeneous mitoribosomes containing different L18 members, which may result in differential mitochondrial functions in response to different physiological situations during development. PMID:26105995

Knockout of RNA Binding Protein MSI2 Impairs Follicle Development in the Mouse Ovary: Characterization of MSI1 and MSI2 during Folliculogenesis

PubMed Central

Sutherland, Jessie M.; Sobinoff, Alexander P.; Gunter, Kara M.; Fraser, Barbara A.; Pye, Victoria; Bernstein, Ilana R.; Boon, Evan; Siddall, Nicole A.; De Andres, Luisa I.; Hime, Gary R.; Holt, Janet E.; Graf, Thomas; McLaughlin, Eileen A.

2015-01-01

Characterizing the mechanisms underlying follicle development in the ovary is crucial to understanding female fertility and is an area of increasing research interest. The RNA binding protein Musashi is essential for post-transcriptional regulation of oocyte maturation in Xenopus and is expressed during ovarian development in Drosophila. In mammals Musashi is important for spermatogenesis and male fertility, but its role in the ovary has yet to be characterized. In this study we determined the expression of mammalian Musashi proteins MSI1 and MSI2 during mouse folliculogenesis, and through the use of a MSI2-specific knockout mouse model we identified that MSI2 is essential for normal follicle development. Time-course characterization of MSI1 and MSI2 revealed distinct differences in steady-state mRNA levels and protein expression/localization at important developmental time-points during folliculogenesis. Using a gene-trap mouse model that inactivates Msi2, we observed a significant decrease in ovarian mass, and change in follicle-stage composition due to developmental blocking of antral stage follicles and pre-antral follicle loss through atresia. We also confirmed that hormonally stimulated Msi2-deficient mice produce significantly fewer MII oocytes (60.9% less than controls, p < 0.05). Furthermore, the majority of these oocytes are of poor viability (62.2% non-viable/apoptotic, p < 0.05), which causes a reduction in female fertility evidenced by decreased litter size in Msi2-deficient animals (33.1% reduction to controls, p < 0.05). Our findings indicate that MSI1 and MSI2 display distinct expression profiles during mammalian folliculogenesis and that MSI2 is required for pre-antral follicle development. PMID:26131972

Polycomb group protein complexes exchange rapidly in living Drosophila.

PubMed

Ficz, Gabriella; Heintzmann, Rainer; Arndt-Jovin, Donna J

2005-09-01

Fluorescence recovery after photobleaching (FRAP) microscopy was used to determine the kinetic properties of Polycomb group (PcG) proteins in whole living Drosophila organisms (embryos) and tissues (wing imaginal discs and salivary glands). PcG genes are essential genes in higher eukaryotes responsible for the maintenance of the spatially distinct repression of developmentally important regulators such as the homeotic genes. Their absence, as well as overexpression, causes transformations in the axial organization of the body. Although protein complexes have been isolated in vitro, little is known about their stability or exact mechanism of repression in vivo. We determined the translational diffusion constants of PcG proteins, dissociation constants and residence times for complexes in vivo at different developmental stages. In polytene nuclei, the rate constants suggest heterogeneity of the complexes. Computer simulations with new models for spatially distributed protein complexes were performed in systems showing both diffusion and binding equilibria, and the results compared with our experimental data. We were able to determine forward and reverse rate constants for complex formation. Complexes exchanged within a period of 1-10 minutes, more than an order of magnitude faster than the cell cycle time, ruling out models of repression in which access of transcription activators to the chromatin is limited and demonstrating that long-term repression primarily reflects mass-action chemical equilibria.

Various Bee Pheromones Binding Affinity, Exclusive Chemosensillar Localization, and Key Amino Acid Sites Reveal the Distinctive Characteristics of Odorant-Binding Protein 11 in the Eastern Honey Bee, Apis cerana.

PubMed

Song, Xin-Mi; Zhang, Lin-Ya; Fu, Xiao-Bin; Wu, Fan; Tan, Jing; Li, Hong-Liang

2018-01-01

Odorant-binding proteins (OBPs) are the critical elements responsible for binding and transporting odors and pheromones in the sensitive olfactory system in insects. Honey bees are representative social insects that have complex odorants and pheromone communication systems relative to solitary insects. Here, we first cloned and characterized OBP11 ( AcerOBP11 ), from the worker bees antennae of Eastern honey bee, Apis cerana . Based on sequence and phylogenetic analysis, most sequences homologous to AcerOBP11 belong to the typical OBPs family. The transcriptional expression profiles showed that AcerOBP11 was expressed throughout the developmental stages and highly specifically expressed in adult antennae. Using immunofluorescence localization, AcerOBP11 in worker bee's antennae was only localized in the sensilla basiconica (SB) near the fringe of each segment. Fluorescence ligand-binding assay showed that AcerOBP11 protein had strong binding affinity with the tested various bee pheromones components, including the main queen mandibular pheromones (QMPs), methyl p-hydroxybenzoate (HOB), and ( E )-9-oxo-2-decanoic acid (9-ODA), alarm pheromone (n-hexanol), and worker pheromone components. AcerOBP11 also had strong binding affinity to plant volatiles, such as 4-Allylveratrole. Based on the docking and site-directed mutagenesis, two key amino acid residues (Ile97 and Ile140) were involved in the binding of AcerOBP11 to various bee pheromones. Taken together, we identified that AcerOBP11 was localized in a single type of antennal chemosensilla and had complex ligand-binding properties, which confer the dual-role with the primary characteristics of sensing various bee pheromones and secondary characteristics of sensing general odorants. This study not only prompts the theoretical basis of OBPs-mediated bee pheromones recognition of honey bee, but also extends the understanding of differences in pheromone communication between social and solitary insects.

An extensive requirement for transcription factor IID-specific TAF-1 in Caenorhabditis elegans embryonic transcription.

PubMed

Walker, Amy K; Shi, Yang; Blackwell, T Keith

2004-04-09

The general transcription factor TFIID sets the mRNA start site and consists of TATA-binding protein and associated factors (TAF(II)s), some of which are also present in SPT-ADA-GCN5 (SAGA)-related complexes. In yeast, results of multiple studies indicate that TFIID-specific TAF(II)s are not required for the transcription of most genes, implying that intact TFIID may have a surprisingly specialized role in transcription. Relatively little is known about how TAF(II)s contribute to metazoan transcription in vivo, especially at developmental and tissue-specific genes. Previously, we investigated functions of four shared TFIID/SAGA TAF(II)s in Caenorhabditis elegans. Whereas TAF-4 was required for essentially all embryonic transcription, TAF-5, TAF-9, and TAF-10 were dispensable at multiple developmental and other metazoan-specific promoters. Here we show evidence that in C. elegans embryos transcription of most genes requires TFIID-specific TAF-1. TAF-1 is not as universally required as TAF-4, but it is essential for a greater proportion of transcription than TAF-5, -9, or -10 and is important for transcription of many developmental and other metazoan-specific genes. TAF-2, which binds core promoters with TAF-1, appears to be required for a similarly substantial proportion of transcription. C. elegans TAF-1 overlaps functionally with the coactivator p300/CBP (CBP-1), and at some genes it is required along with the TBP-like protein TLF(TRF2). We conclude that during C. elegans embryogenesis TAF-1 and TFIID have broad roles in transcription and development and that TFIID and TLF may act together at certain promoters. Our findings imply that in metazoans TFIID may be of widespread importance for transcription and for expression of tissue-specific genes.

C. elegans microRNAs.

PubMed

Vella, Monica C; Slack, Frank J

2005-09-21

MicroRNAs (miRNAs) are small, non-coding regulatory RNAs found in many phyla that control such diverse events as development, metabolism, cell fate and cell death. They have also been implicated in human cancers. The C. elegans genome encodes hundreds of miRNAs, including the founding members of the miRNA family lin-4 and let-7. Despite the abundance of C. elegans miRNAs, few miRNA targets are known and little is known about the mechanism by which they function. However, C. elegans research continues to push the boundaries of discovery in this area. lin-4 and let-7 are the best understood miRNAs. They control the timing of adult cell fate determination in hypodermal cells by binding to partially complementary sites in the mRNA of key developmental regulators to repress protein expression. For example, lin-4 is predicted to bind to seven sites in the lin-14 3' untranslated region (UTR) to repress LIN-14, while let-7 is predicted to bind two let-7 complementary sites in the lin-41 3' UTR to down-regulate LIN-41. Two other miRNAs, lsy-6 and mir-273, control left-right asymmetry in neural development, and also target key developmental regulators for repression. Approximately one third of the C. elegans miRNAs are differentially expressed during development indicating a major role for miRNAs in C. elegans development. Given the remarkable conservation of developmental mechanism across phylogeny, many of the principles of miRNAs discovered in C. elegans are likely to be applicable to higher animals.

Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci.

PubMed

Amaral, Paulo P; Leonardi, Tommaso; Han, Namshik; Viré, Emmanuelle; Gascoigne, Dennis K; Arias-Carrasco, Raúl; Büscher, Magdalena; Pandolfini, Luca; Zhang, Anda; Pluchino, Stefano; Maracaja-Coutinho, Vinicius; Nakaya, Helder I; Hemberg, Martin; Shiekhattar, Ramin; Enright, Anton J; Kouzarides, Tony

2018-03-15

The mammalian genome is transcribed into large numbers of long noncoding RNAs (lncRNAs), but the definition of functional lncRNA groups has proven difficult, partly due to their low sequence conservation and lack of identified shared properties. Here we consider promoter conservation and positional conservation as indicators of functional commonality. We identify 665 conserved lncRNA promoters in mouse and human that are preserved in genomic position relative to orthologous coding genes. These positionally conserved lncRNA genes are primarily associated with developmental transcription factor loci with which they are coexpressed in a tissue-specific manner. Over half of positionally conserved RNAs in this set are linked to chromatin organization structures, overlapping binding sites for the CTCF chromatin organiser and located at chromatin loop anchor points and borders of topologically associating domains (TADs). We define these RNAs as topological anchor point RNAs (tapRNAs). Characterization of these noncoding RNAs and their associated coding genes shows that they are functionally connected: they regulate each other's expression and influence the metastatic phenotype of cancer cells in vitro in a similar fashion. Furthermore, we find that tapRNAs contain conserved sequence domains that are enriched in motifs for zinc finger domain-containing RNA-binding proteins and transcription factors, whose binding sites are found mutated in cancers. This work leverages positional conservation to identify lncRNAs with potential importance in genome organization, development and disease. The evidence that many developmental transcription factors are physically and functionally connected to lncRNAs represents an exciting stepping-stone to further our understanding of genome regulation.

Formation and biochemical characterization of tube/pelle death domain complexes: critical regulators of postreceptor signaling by the Drosophila toll receptor.

PubMed

Schiffmann, D A; White, J H; Cooper, A; Nutley, M A; Harding, S E; Jumel, K; Solari, R; Ray, K P; Gay, N J

1999-09-07

In Drosophila, the Toll receptor signaling pathway is required for embryonic dorso-ventral patterning and at later developmental stages for innate immune responses. It is thought that dimerization of the receptor by binding of the ligand spätzle causes the formation of a postreceptor activation complex at the cytoplasmic surface of the membrane. Two components of this complex are the adaptor tube and protein kinase pelle. These proteins both have "death domains", protein interaction motifs found in a number of signaling pathways, particularly those involved in apoptotic cell death. It is thought that pelle is bound by tube during formation of the activation complexes, and that this interaction is mediated by the death domains. In this paper, we show using the yeast two-hybrid system that the wild-type tube and pelle death domains bind together. Mutant tube proteins which do not support signaling in the embryo are also unable to bind pelle in the 2-hybrid assay. We have purified proteins corresponding to the death domains of tube and pelle and show that these form corresponding heterodimeric complexes in vitro. Partial proteolysis reveals a smaller core consisting of the minimal death domain sequences. We have studied the tube/pelle interaction with the techniques of surface plasmon resonance, analytical ultracentrifugation and isothermal titration calorimetry. These measurements produce a value of K(d) for the complex of about 0.5 microM.

Comparative proteomics analysis of silkworm hemolymph during the stages of metamorphosis via liquid chromatography and mass spectrometry.

PubMed

Hou, Yong; Zhang, Yan; Gong, Jing; Tian, Sha; Li, Jianwei; Dong, Zhaoming; Guo, Chao; Peng, Li; Zhao, Ping; Xia, Qingyou

2016-05-01

The silkworm is a lepidopteran insect that has an open circulatory system with hemolymph consisting of blood and lymph fluid. Hemolymph is not only considered as a depository of nutrients and energy, but it also plays a key role in substance transportation, immunity response, and proteolysis. In this study, we used LC-MS/MS to analyze the hemolymph proteins of four developmental stages during metamorphosis. A total of 728 proteins were identified from the hemolymph of the second day of wandering stage, first day of pupation, ninth day of pupation, and first day as an adult moth. GO annotations and categories showed that silkworm hemolymph proteins were enriched in carbohydrate metabolism, proteolysis, protein binding, and antibacterial humoral response. The levels of nutrient, immunity-related, and structural proteins changed significantly during development and metamorphosis. Some, such as cuticle, odorant-binding, and chemosensory proteins, showed stage-specific expression in the hemolymph. In addition, the expression of several antimicrobial peptides exhibited their highest level of abundance in the hemolymph of the early pupal stage. These findings provide a comprehensive proteomic insight of the silkworm hemolymph and suggest additional molecular targets for studying insect metamorphosis. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stochaj, W.R.; Grossman, A.R.

One- and two-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunological analyses were used to visualize differences in polypeptides synthesized by Symbiodinium sp. from the anemone Aiptasia pallida when grown in the cultured and endosymbiotic states (freshly isolated zooxanthellae). Surprisingly, a comparison of proteins in cultured and endosymbiotic Symbiodinium sp. revealed only four major polypeptides with similar isoelectric and molecular mass characteristics. Using monospecific antibodies, we demonstrated differences in specific proteins synthesized by the dinoflagellate in the two different growth states. The dimeric, 14 kDa form of the peripheral membrane peridinin-chlorophyll a binding protein predominates under endosymbiotic conditions, whereas the monomeric, 35more » kDa form predominates under the culture conditions used in this study. Antibodies to form II ribulose-1,5-bisphosphate carboxylase revealed 62 and 60 kDa forms of this protein in the alga grown as an endosymbiont and in culture, respectively. Differences in the integral membrane peridinin-chlorophyll a-c-binding proteins were also observed. These results demonstrate that there are major changes in the populations of proteins synthesized by Symbiodinium sp. in response to the conditions in hospite. Such changes may reflect a developmental switch that tailors the physiology of the alga to the conditions encountered in the endosymbiotic state. 77 refs., 6 figs.« less

Quaternary re-arrangement analysed by spectral enhancement: the interaction of a sporulation repressor with its antagonist.

PubMed

Scott, D J; Leejeerajumnean, S; Brannigan, J A; Lewis, R J; Wilkinson, A J; Hoggett, J G

1999-11-12

The protein/protein interaction between SinI and SinR has been studied by analytical ultracentrifugation and gel electrophoresis in an attempt to understand how these proteins contribute to developmental control of sporulation in Bacillus subtilis. SinR was found to be tetrameric, while SinI was found to exist as monomers and dimers in a rapidly reversible equilibrium. Labelling of SinR by incorporating the tryptophan analogue 7-azatryptophan (7AW) into the protein in place of tryptophan shifts the UV absorbance spectrum, thus allowing selective monitoring of 7AWSinR at 315 nm using the UV absorption optics of the analytical ultracentrifuge. Selective monitoring of SinR in mixtures of SinR and SinI enables the binding and stoichiometry of the interaction to be investigated quantitatively and unambiguously. We demonstrate that the oligomeric forms of SinR and SinI re-arrange to form a tight 1:1 SinR:SinI complex, with no stable intermediate species. A fragment of SinR, SinR(1-69), which contains only the DNA-binding domain, was found to be monomeric, showing that the protein appears not to oligomerise in a similar manner to the Cro repressor, a protein with which it shares a marked structural similarity. Copyright 1999 Academic Press.

Towards Understanding Plant Calcium Signaling through Calmodulin-Like Proteins: A Biochemical and Structural Perspective.

PubMed

La Verde, Valentina; Dominici, Paola; Astegno, Alessandra

2018-04-30

Ca 2+ ions play a key role in a wide variety of environmental responses and developmental processes in plants, and several protein families with Ca 2+ -binding domains have evolved to meet these needs, including calmodulin (CaM) and calmodulin-like proteins (CMLs). These proteins have no catalytic activity, but rather act as sensor relays that regulate downstream targets. While CaM is well-studied, CMLs remain poorly characterized at both the structural and functional levels, even if they are the largest class of Ca 2+ sensors in plants. The major structural theme in CMLs consists of EF-hands, and variations in these domains are predicted to significantly contribute to the functional versatility of CMLs. Herein, we focus on recent advances in understanding the features of CMLs from biochemical and structural points of view. The analysis of the metal binding and structural properties of CMLs can provide valuable insight into how such a vast array of CML proteins can coexist, with no apparent functional redundancy, and how these proteins contribute to cellular signaling while maintaining properties that are distinct from CaM and other Ca 2+ sensors. An overview of the principal techniques used to study the biochemical properties of these interesting Ca 2+ sensors is also presented.

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

Kid-1, a putative renal transcription factor: regulation during ontogeny and in response to ischemia and toxic injury.

PubMed Central

Witzgall, R; O'Leary, E; Gessner, R; Ouellette, A J; Bonventre, J V

1993-01-01

We have identified a new putative transcription factor from the rat kidney, termed Kid-1 (for kidney, ischemia and developmentally regulated gene 1). Kid-1 belongs to the C2H2 class of zinc finger genes. Its mRNA accumulates with age in postnatal renal development and is detected predominantly in the kidney. Kid-1 mRNA levels decline after renal injury secondary to ischemia or folic acid administration, two insults which result in epithelial cell dedifferentiation, followed by regenerative hyperplasia and differentiation. The low expression of Kid-1 early in postnatal development, and when renal tissue is recovering after injury, suggests that the gene product is involved in establishment of a differentiated phenotype and/or regulation of the proliferative response. The deduced protein contains 13 C2H2 zinc fingers at the COOH end in groups of 4 and 9 separated by a 32-amino-acid spacer. There are consensus sites for phosphorylation in the NH2 terminus non-zinc finger region as well as in the spacer region between zinc fingers 4 and 5. A region of the deduced protein shares extensive homology with a catalytic region of Raf kinases, a feature shared only with TFIIE among transcription factors. To determine whether Kid-1 can modulate transcription, a chimeric construct encoding the Kid-1 non-zinc finger region (sense or antisense) and the DNA-binding region of GAL4 was transfected into COS and LLC-PK1 cells together with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 binding sites, driven by either a minimal promoter or a simian virus 40 enhancer. CAT activity was markedly inhibited in cells transfected with the sense construct compared with the activity in cells transfected with the antisense construct. To our knowledge, this pattern of developmental regulation, kidney expression, and regulation of transcription is unique among the C2H2 class of zinc finger-containing DNA-binding proteins. Images PMID:8382778

Aryl hydrocarbon receptor (AHR) in the cnidarian Nematostella vectensis: comparative expression, protein interactions, and ligand binding.

PubMed

Reitzel, Adam M; Passamaneck, Yale J; Karchner, Sibel I; Franks, Diana G; Martindale, Mark Q; Tarrant, Ann M; Hahn, Mark E

2014-02-01

The aryl hydrocarbon receptor (AHR) is a member of the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcription factors and has diverse roles in development, physiology, and environmental sensing in bilaterian animals. Studying the expression of conserved genes and function of proteins in outgroups to protostomes and deuterostomes assists in understanding the antiquity of gene function and deciphering lineage-specific differences in these bilaterian clades. We describe the developmental expression of AHR from the sea anemone Nematostella vectensis and compare its expression with three other members of the bHLH-PAS family (AHR nuclear translocator (ARNT), Cycle, and a proto-Single-Minded/Trachealess). NvAHR expression was highest early in the larval stage with spatial expression in the basal portion of the ectoderm that became increasingly restricted to the oral pole with concentrated expression in tentacles of the juvenile polyp. The other bHLH-PAS genes showed a divergent expression pattern in later larval stages and polyps, in which gene expression was concentrated in the aboral end, with broader expression in the endoderm later in development. In co-immunoprecipitation assays, we found no evidence for heterodimerization of AHR with ARNT, contrary to the conservation of this specific interaction in all bilaterians studied to date. Similar to results with other invertebrate AHRs but in contrast to vertebrate AHRs, NvAHR failed to bind two prototypical xenobiotic AHR ligands (2,3,7,8-tetrachlorodibenzo-p-dioxin, β-naphthoflavone). Together, our data suggest that AHR's original function in Eumetazoa likely involved developmental patterning, potentially of neural tissue. The role of heterodimerization in the function of AHR may have arisen after the cnidarian-bilaterian ancestor. The absence of xenobiotic binding to NvAHR further supports a hypothesis for a derived role of this protein in chemical sensing within the chordates.

Aryl hydrocarbon receptor (AHR) in the cnidarian Nematostella vectensis: comparative expression, protein interactions, and ligand binding

PubMed Central

Reitzel, Adam M.; Passamaneck, Yale J.; Karchner, Sibel I.; Franks, Diana G.; Martindale, Mark Q.; Tarrant, Ann M.; Hahn, Mark E.

2014-01-01

The aryl hydrocarbon receptor (AHR) is a member of the basic-helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcription factors and has diverse roles in development, physiology, and environmental sensing in bilaterian animals. Studying the expression of conserved genes and function of proteins in outgroups to protostomes and deuterostomes assists in understanding the antiquity of gene function and deciphering lineage-specific differences in these bilaterian clades. We describe the developmental expression of AHR from the sea anemone Nematostella vectensis and compare its expression with three other members of the bHLH-PAS family (AHR nuclear translocator (ARNT), Cycle, and a proto-Single-Minded/Trachaeless). NvAHR expression was highest early in the larval stage with spatial expression in the basal portion of the ectoderm that became increasingly restricted to the oral pole with concentrated expression in tentacles of the juvenile polyp. The other bHLH-PAS genes showed a divergent expression pattern in later larval stages and polyps, in which gene expression was concentrated in the aboral end, with broader expression in the endoderm later in development. In co-immunoprecipitation assays, we found no evidence for heterodimerization of AHR with ARNT, contrary to the conservation of this specific interaction in all bilaterians studied to date. Similar to results with other invertebrate AHRs but in contrast to vertebrate AHRs, NvAHR failed to bind two prototypical xenobiotic AHR ligands (TCDD, BNF). Together, our data suggest that AHR's original function in Eumetazoa likely involved developmental patterning, potentially of neural tissue. The role of heterodimerization in the function of AHR may have arisen after the cnidarian-bilaterian ancestor. The absence of xenobiotic binding to NvAHR further supports a hypothesis for a derived role of this protein in chemical sensing within the chordates. PMID:24292160

Drosophila Polypyrimidine Tract-Binding Protein (DmPTB) Regulates Dorso-Ventral Patterning Genes in Embryos

PubMed Central

Huntley, Jim; Wesley, Cedric S.; Singh, Ravinder

2014-01-01

The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during embryogenesis. A loss of function mutation, heph03429, results in varied defects in embryonic developmental processes, leading to embryonic lethality. However, the suite of molecular functions that are disrupted in the mutant remains unknown. We have used an unbiased high throughput sequencing approach to identify transcripts that are misregulated in this mutant. Misregulated transcripts show evidence of significantly altered patterns of splicing (exon skipping, 5′ and 3′ splice site switching), alternative 5′ ends, and mRNA level changes (up and down regulation). These findings are independently supported by reverse-transcription-polymerase chain reaction (RT-PCR) analysis and in situ hybridization. We show that a group of genes, such as Zerknüllt, z600 and screw are among the most upregulated in the mutant and have been functionally linked to dorso-ventral patterning and/or dorsal closure processes. Thus, loss of dmPTB function results in specific misregulated transcripts, including those that provide the missing link between the loss of dmPTB function and observed developmental defects in embryogenesis. This study provides the first comprehensive repertoire of genes affected in vivo in the heph mutant in Drosophila and offers insight into the role of dmPTB during embryonic development. PMID:25014769

Developmental pathways from child maltreatment to adolescent marijuana dependence: Examining moderation by FK506 binding protein 5 gene (FKBP5).

PubMed

Handley, Elizabeth D; Rogosch, Fred A; Cicchetti, Dante

2015-11-01

The current study examined the prospective association between child maltreatment and the development of substance use disorder in adolescence with the aim of investigating pathways underlying this relation, as well as genetic moderation of these developmental mechanisms. Specifically, we tested whether youth who experienced maltreatment prior to age 8 were at risk for the development of marijuana dependence in adolescence by way of a childhood externalizing pathway and a childhood internalizing pathway. Moreover, we tested whether variation in FK506 binding protein 5 gene (FKBP5) CATT haplotype moderated these pathways. The participants were 326 children (n =179 maltreated; n = 147 nonmaltreated) assessed across two waves of data collection (childhood: ages 7-9 and adolescence: ages 15-18). Results indicated that higher levels of child externalizing symptoms significantly mediated the effect of child maltreatment on adolescent marijuana dependence symptoms for individuals with one or two copies of the FKBP5 CATT haplotype only. We did not find support for an internalizing pathway from child maltreatment to adolescent marijuana dependence, nor did we find evidence of moderation of the internalizing pathway by FKBP5 haplotype variation. Findings extend previous research by demonstrating that whether a maltreated child will traverse an externalizing pathway toward substance use disorder in adolescence is dependent on FKBP5 genetic variation.

The myogenic repressor gene Holes in muscles is a direct transcriptional target of Twist and Tinman in the Drosophila embryonic mesoderm.

PubMed

Elwell, Jennifer A; Lovato, TyAnna L; Adams, Melanie M; Baca, Erica M; Lee, Thai; Cripps, Richard M

2015-04-15

Understanding the regulatory circuitry controlling myogenesis is critical to understanding developmental mechanisms and developmentally-derived diseases. We analyzed the transcriptional regulation of a Drosophila myogenic repressor gene, Holes in muscles (Him). Previously, Him was shown to inhibit Myocyte enhancer factor-2 (MEF2) activity, and is expressed in myoblasts but not differentiating myotubes. We demonstrate that different phases of Him embryonic expression arises through the actions of different enhancers, and we characterize the enhancer required for its early mesoderm expression. This Him early mesoderm enhancer contains two conserved binding sites for the basic helix-loop-helix regulator Twist, and one binding site for the NK homeodomain protein Tinman. The sites for both proteins are required for enhancer activity in early embryos. Twist and Tinman activate the enhancer in tissue culture assays, and ectopic expression of either factor is sufficient to direct ectopic expression of a Him-lacZ reporter, or of the endogenous Him gene. Moreover, sustained expression of twist in the mesoderm up-regulates mesodermal Him expression in late embryos. Our findings provide a model to define mechanistically how Twist can both promotes myogenesis through direct activation of Mef2, and can place a brake on myogenesis, through direct activation of Him. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular cloning and expression profile of an ATP-binding cassette (ABC) transporter gene from the hemipteran insect Nilaparvata lugens.

PubMed

Zha, W J; Li, S H; Zhou, L; Chen, Z J; Liu, K; Yang, G C; Hu, G; He, G C; You, A Q

2015-03-30

The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. In insects, ABC transporters have important functions in the transport of molecules, and are also involved in insecticide resistance, metabolism, and development. In this study, the Nilaparvata lugens Stal (Hemiptera: Delphacidae) ABCG (NlABCG) gene was identified and characterized. The complete mRNA sequence of NlABCG was 2608-bp long, with an open reading frame of 2064 bp encoding a protein comprised of 687 amino acids. The conserved regions include three N-glycosylation and 34 phosphorylation sites, as well as seven transmembrane domains. The amino acid identity with the closely related species Acyrthosiphon pisum was 42.8%. Developmental expression analysis using quantitative real-time reverse transcriptase PCR suggested that the NlABCG transcript was expressed at all developmental stages of N. lugens. The lowest expression of NlABCG was in the 1st instar, and levels increased with larval growth. The transcript profiles of NlABCG were analyzed in various tissues from a 5th instar nymph, and the highest expression was observed in the midgut. These results suggest that the sequence, characteristics, and expression of NlABCG are highly conserved, and basic information is provided for its functional analysis.

COACTIVATOR ACTIVATOR (CoAA) PREVENTS THE TRANSCRIPTIONAL ACTIVITY OF RUNT DOMAIN TRANSCRIPTION FACTORS

PubMed Central

Li, Xiaodong; Hoeppner, Luke H.; Jensen, Eric D.; Gopalakrishnan, Rajaram; Westendorf, Jennifer J.

2013-01-01

Runx proteins are essential for a number of developmental processes and are aberrantly expressed in many human cancers. Runx factors bind DNA and co-factors to activate or repress genes crucial for bone formation, hematopoiesis, and neuronal development. Co-activator activator (CoAA) is a nuclear protein that regulates gene expression, RNA splicing and is overexpressed in many human tumors. In this study, we identified CoAA as a Runx2 binding protein. CoAA repressed Runx factor-dependent activation of reporter genes in a histone deacetylase-independent manner. CoAA also blocked Runx2-mediated repression of the Axin2 promoter, a novel Runx target gene. The carboxy-terminus of CoAA is essential for binding the Runt domains of Runx1 and Runx2. In electophoretic mobility shift assays, CoAA inhibited Runx2 interactions with DNA. These data indicate that CoAA is an inhibitor of Runx factors and can negate Runx factor regulation of gene expression. CoAA is expressed at high levels in human fetal osteoblasts and osteosarcoma cell lines. Suppression of CoAA expression by RNA interference reduced osteosarcoma cell viability in vitro, suggesting that it contributes to the proliferation and/or survival of osteoblast lineage cells. PMID:19585539

Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers

NASA Technical Reports Server (NTRS)

Poovaiah, B. W.; Xia, M.; Liu, Z.; Wang, W.; Yang, T.; Sathyanarayanan, P. V.; Franceschi, V. R.

1999-01-01

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca(2+) and Ca(2+)/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the beta-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by beta-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca(2+) concentration in target cells, thereby controlling developmental events in the anther.

Global transcriptional repression in C. elegans germline precursors by regulated sequestration of TAF-4.

PubMed

Guven-Ozkan, Tugba; Nishi, Yuichi; Robertson, Scott M; Lin, Rueyling

2008-10-03

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1-P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II after fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wild-type OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators.

Global transcriptional repression in C. elegans germline precursors by regulated sequestration of TFIID component TAF-4

PubMed Central

Guven-Ozkan, Tugba; Nishi, Yuichi; Robertson, Scott M.; Lin, Rueyling

2008-01-01

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1–P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres, but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II following fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wildtype OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators. PMID:18854162

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site.

PubMed

Mazhab-Jafari, Mohammad T; Marshall, Christopher B; Smith, Matthew J; Gasmi-Seabrook, Geneviève M C; Stathopulos, Peter B; Inagaki, Fuyuhiko; Kay, Lewis E; Neel, Benjamin G; Ikura, Mitsuhiko

2015-05-26

K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery.

Ankyrin-binding proteins related to nervous system cell adhesion molecules: candidates to provide transmembrane and intercellular connections in adult brain.

PubMed

Davis, J Q; McLaughlin, T; Bennett, V

1993-04-01

A major class of ankyrin-binding glycoproteins have been identified in adult rat brain of 186, 155, and 140 kD that are alternatively spliced products of the same pre-mRNA. Characterization of cDNAs demonstrated that ankyrin-binding glycoproteins (ABGPs) share 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides have the following features consistent with a role as ankyrin-binding proteins in vitro and in vivo: (a) ABGPs and ankyrin associate as pure proteins in a 1:1 molar stoichiometry; (b) the ankyrin-binding site is located in the COOH-terminal 21 kD of ABGP186 which contains the predicted cytoplasmic domain; (c) ABGP186 is expressed at approximately the same levels as ankyrin (15 pmoles/milligram of membrane protein); and (d) ABGP polypeptides are co-expressed with the adult form of ankyrinB late in postnatal development and are colocalized with ankyrinB by immunofluorescence. Similarity in amino acid sequence and conservation of sites of alternative splicing indicate that genes encoding ABGPs and neurofascin share a common ancestor. However, the major differences in developmental expression reported for neurofascin in embryos versus the late postnatal expression of ABGPs suggest that ABGPs and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. The predicted cytoplasmic domains of rat ABGPs and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, which includes L1, Nr-CAM, and Ng-CAM of vertebrates, and neuroglian of Drosophila. The ankyrin-binding site of rat ABGPs is localized to the C-terminal 200 residues which encompass the cytoplasmic domain, suggesting the hypothesis that ability to associate with ankyrin may be a shared feature of neurofascin and related nervous system cell adhesion molecules.

The developmental transcriptome of the bamboo snout beetle Cyrtotrachelus buqueti and insights into candidate pheromone-binding proteins

PubMed Central

Yang, Wei; Yang, Chunping; Lu, Lin; Chen, Zhangming

2017-01-01

Cyrtotrachelus buqueti is an extremely harmful bamboo borer, and the larvae of this pest attack clumping bamboo shoots. Pheromone-binding proteins (PBPs) play an important role in identifying insect sex pheromones, but the C. buqueti genome is not readily available for PBP analysis. Developmental transcriptomes of eggs, larvae from the first instar to the prepupal stage, pupae, and adults (females and males) from emergence to mating were built by RNA sequencing (RNA-Seq) in the present study to establish a sequence background of C. buqueti to help understand PBPs. Approximately 164.8 million clean reads were obtained and annotated into 108,854 transcripts. These were assembled into 24,338, 21,597, 24,798, 21,886, 24,642, and 83,115 unigenes for eggs, larvae, pupae, females, males, and the combined datasets, respectively. Unigenes were annotated against NCBI non-redundant protein sequences, NCBI non-redundant nucleotide sequences, Gene Ontology (GO), Protein family, Clusters of Orthologous Groups of Proteins/ Clusters of Eukaryotic Orthologous Groups (KOG), Swiss-Prot, and KEGG Orthology databases. A total of 17,213 unigenes were annotated into 55 sub-categories belonging to three main GO categories; 10,672 unigenes were classified into 26 functional categories by KOG classification, and 8,063 unigenes were classified into five functional KEGG categories. RSEM software for RNA sequencing showed that 4,816, 3,176, 3,661, 2,898, 4,316, 8,019, 7,273, 5,922, 5,844, and 4,570 genes were differentially expressed between larvae and males, larvae and eggs, larvae and pupae, larvae and females, males and females, males and eggs, males and pupae, females and eggs, females and pupae, and eggs and pupae, respectively. Of these, three were confirmed to be significantly differentially expressed between larvae, females, and males. Furthermore, PBP Cbuq7577_g1 was highly expressed in the antenna of males. A comprehensive sequence resource of a desirable quality was constructed from developmental transcriptomes of C. buqueti eggs, larvae, pupae, and adults. This work enriches the genomic data of C. buqueti, and facilitates our understanding of its metamorphosis, development, and response to environmental change. The identified candidate PBP Cbuq7577_g1 might play a crucial role in identifying sex pheromones, and could be used as a targeted gene to control C. buqueti numbers by disrupting sex pheromone communication. PMID:28662071

FLOWERING LOCUS C (FLC) regulates development pathways throughout the life cycle of Arabidopsis

PubMed Central

Deng, Weiwei; Ying, Hua; Helliwell, Chris A.; Taylor, Jennifer M.; Peacock, W. James; Dennis, Elizabeth S.

2011-01-01

FLOWERING LOCUS C (FLC) has a key role in the timing of the initiation of flowering in Arabidopsis. FLC binds and represses two genes that promote flowering, FT and SOC1. We show that FLC binds to many other genes, indicating that it has regulatory roles other than the repression of flowering. We identified 505 FLC binding sites, mostly located in the promoter regions of genes and containing at least one CArG box, the motif known to be associated with MADS-box proteins such as FLC. We examined 40 of the target genes, and 20 showed increased transcript levels in an flc mutant compared with the wild type. Five genes showed decreased expression in the mutant, indicating that FLC binding can result in either transcriptional repression or activation. The genes we identified as FLC targets are involved in developmental pathways throughout the life history of the plant, many of which are associated with reproductive development. FLC is also involved in vegetative development, as evidenced by its binding to SPL15, delaying the progression from juvenile to adult phase. Some of the FLC target genes are also bound by two other MADS-box proteins, AP1 and SEP3, suggesting that MADS-box genes may operate in a network of control at different stages of the life cycle, many ultimately contributing to the development of the reproductive phase of the plant. PMID:21464308

FLOWERING LOCUS C (FLC) regulates development pathways throughout the life cycle of Arabidopsis.

PubMed

Deng, Weiwei; Ying, Hua; Helliwell, Chris A; Taylor, Jennifer M; Peacock, W James; Dennis, Elizabeth S

2011-04-19

FLOWERING LOCUS C (FLC) has a key role in the timing of the initiation of flowering in Arabidopsis. FLC binds and represses two genes that promote flowering, FT and SOC1. We show that FLC binds to many other genes, indicating that it has regulatory roles other than the repression of flowering. We identified 505 FLC binding sites, mostly located in the promoter regions of genes and containing at least one CArG box, the motif known to be associated with MADS-box proteins such as FLC. We examined 40 of the target genes, and 20 showed increased transcript levels in an flc mutant compared with the wild type. Five genes showed decreased expression in the mutant, indicating that FLC binding can result in either transcriptional repression or activation. The genes we identified as FLC targets are involved in developmental pathways throughout the life history of the plant, many of which are associated with reproductive development. FLC is also involved in vegetative development, as evidenced by its binding to SPL15, delaying the progression from juvenile to adult phase. Some of the FLC target genes are also bound by two other MADS-box proteins, AP1 and SEP3, suggesting that MADS-box genes may operate in a network of control at different stages of the life cycle, many ultimately contributing to the development of the reproductive phase of the plant.

Small leucine rich proteoglycan family regulates multiple signalling pathways in neural development and maintenance.

PubMed

Dellett, Margaret; Hu, Wanzhou; Papadaki, Vasiliki; Ohnuma, Shin-ichi

2012-04-01

The small leucine-rich repeat proteoglycan (SLRPs) family of proteins currently consists of five classes, based on their structural composition and chromosomal location. As biologically active components of the extracellular matrix (ECM), SLRPs were known to bind to various collagens, having a role in regulating fibril assembly, organization and degradation. More recently, as a function of their diverse proteins cores and glycosaminoglycan side chains, SLRPs have been shown to be able to bind various cell surface receptors, growth factors, cytokines and other ECM components resulting in the ability to influence various cellular functions. Their involvement in several signaling pathways such as Wnt, transforming growth factor-β and epidermal growth factor receptor also highlights their role as matricellular proteins. SLRP family members are expressed during neural development and in adult neural tissues, including ocular tissues. This review focuses on describing SLRP family members involvement in neural development with a brief summary of their role in non-neural ocular tissues and in response to neural injury. © 2012 The Authors Development, Growth & Differentiation © 2012 Japanese Society of Developmental Biologists.

Structural and Functional Characterizations of SsgB, a Conserved Activator of Developmental Cell Division in Morphologically Complex Actinomycetes*

PubMed Central

Xu, Qingping; Traag, Bjørn A.; Willemse, Joost; McMullan, Daniel; Miller, Mitchell D.; Elsliger, Marc-André; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Bakolitsa, Constantina; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Chruszcz, Maksymilian; Clayton, Thomas; Das, Debanu; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Ernst, Dustin; Farr, Carol L.; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Grzechnik, Slawomir K.; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; Minor, Wladek; Mommaas, A. Mieke; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Puckett, Christina; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; van den Bedem, Henry; Wang, Shuren; Weekes, Dana; Hodgson, Keith O.; Wooley, John; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.; van Wezel, Gilles P.

2009-01-01

SsgA-like proteins (SALPs) are a family of homologous cell division-related proteins that occur exclusively in morphologically complex actinomycetes. We show that SsgB, a subfamily of SALPs, is the archetypal SALP that is functionally conserved in all sporulating actinomycetes. Sporulation-specific cell division of Streptomyces coelicolor ssgB mutants is restored by introduction of distant ssgB orthologues from other actinomycetes. Interestingly, the number of septa (and spores) of the complemented null mutants is dictated by the specific ssgB orthologue that is expressed. The crystal structure of the SsgB from Thermobifida fusca was determined at 2.6 Å resolution and represents the first structure for this family. The structure revealed similarities to a class of eukaryotic “whirly” single-stranded DNA/RNA-binding proteins. However, the electro-negative surface of the SALPs suggests that neither SsgB nor any of the other SALPs are likely to interact with nucleotide substrates. Instead, we show that a conserved hydrophobic surface is likely to be important for SALP function and suggest that proteins are the likely binding partners. PMID:19567872

Conserved developmental alternative splicing of muscleblind-like (MBNL) transcripts regulates MBNL localization and activity.

PubMed

Terenzi, Fulvia; Ladd, Andrea N

2010-01-01

Muscleblind-like (MBNL) proteins have been shown to regulate pre-mRNA alternative splicing, and MBNL1 has been implicated in regulating fetal-to-adult transitions in alternative splicing in the heart. MBNL1 is highly conserved, exhibiting more than 95% identity at the amino acid level between birds and mammals. To investigate MBNL1 expression during embryonic heart development, we examined MBNL1 transcript and protein expression in the embryonic chicken heart from the formation of the primitive heart tube through cardiac morphogenesis (embryonic days 1.5 through 8). MBNL1 transcript levels remained steady throughout these stages, whereas MBNL1 protein levels increased and exhibited a shift in isoforms. MBNL1 has several alternatively spliced exons. Using RT-PCR, we determined that the inclusion of one of these, exon 5, decreases dramatically during cardiac morphogenesis. This developmental transition is conserved in mice. Functional analyses of MBNL1 isoforms containing or lacking exon 5-encoded sequences revealed that exon 5 is important for the regulation of the subcellular localization, RNA binding affinity, and alternative splicing activity of MBNL1 proteins. A second MBNL protein, MBNL2, is also expressed in the embryonic heart. We found that MBNL2 exon 5, which is paralogous to MBNL1 exon 5, is similarly regulated during embryonic heart development. Analysis of MBNL1 and MBNL2 transcripts in several embryonic tissues in chicken and mouse indicate that exon 5 alternative splicing is highly conserved and tissue-specific. Thus, we propose that conserved developmental stage- and tissue-specific alternative splicing of MBNL transcripts is an important mechanism by which MBNL activity is regulated during embryonic development.

Unique and shared functions of nuclear lamina LEM domain proteins in Drosophila.

PubMed

Barton, Lacy J; Wilmington, Shameika R; Martin, Melinda J; Skopec, Hannah M; Lovander, Kaylee E; Pinto, Belinda S; Geyer, Pamela K

2014-06-01

The nuclear lamina is an extensive protein network that contributes to nuclear structure and function. LEM domain (LAP2, emerin, MAN1 domain, LEM-D) proteins are components of the nuclear lamina, identified by a shared ∼45-amino-acid motif that binds Barrier-to-autointegration factor (BAF), a chromatin-interacting protein. Drosophila melanogaster has three nuclear lamina LEM-D proteins, named Otefin (Ote), Bocksbeutel (Bocks), and dMAN1. Although these LEM-D proteins are globally expressed, loss of either Ote or dMAN1 causes tissue-specific defects in adult flies that differ from each other. The reason for such distinct tissue-restricted defects is unknown. Here, we generated null alleles of bocks, finding that loss of Bocks causes no overt adult phenotypes. Next, we defined phenotypes associated with lem-d double mutants. Although the absence of individual LEM-D proteins does not affect viability, loss of any two proteins causes lethality. Mutant phenotypes displayed by lem-d double mutants differ from baf mutants, suggesting that BAF function is retained in animals with a single nuclear lamina LEM-D protein. Interestingly, lem-d double mutants displayed distinct developmental and cellular mutant phenotypes, suggesting that Drosophila LEM-D proteins have developmental functions that are differentially shared with other LEM-D family members. This conclusion is supported by studies showing that ectopically produced LEM-D proteins have distinct capacities to rescue the tissue-specific phenotypes found in single lem-d mutants. Our findings predict that cell-specific mutant phenotypes caused by loss of LEM-D proteins reflect both the constellation of LEM-D proteins within the nuclear lamina and the capacity of functional compensation of the remaining LEM-D proteins. Copyright © 2014 by the Genetics Society of America.

Unique and Shared Functions of Nuclear Lamina LEM Domain Proteins in Drosophila

PubMed Central

Barton, Lacy J.; Wilmington, Shameika R.; Martin, Melinda J.; Skopec, Hannah M.; Lovander, Kaylee E.; Pinto, Belinda S.; Geyer, Pamela K.

2014-01-01

The nuclear lamina is an extensive protein network that contributes to nuclear structure and function. LEM domain (LAP2, emerin, MAN1 domain, LEM-D) proteins are components of the nuclear lamina, identified by a shared ∼45-amino-acid motif that binds Barrier-to-autointegration factor (BAF), a chromatin-interacting protein. Drosophila melanogaster has three nuclear lamina LEM-D proteins, named Otefin (Ote), Bocksbeutel (Bocks), and dMAN1. Although these LEM-D proteins are globally expressed, loss of either Ote or dMAN1 causes tissue-specific defects in adult flies that differ from each other. The reason for such distinct tissue-restricted defects is unknown. Here, we generated null alleles of bocks, finding that loss of Bocks causes no overt adult phenotypes. Next, we defined phenotypes associated with lem-d double mutants. Although the absence of individual LEM-D proteins does not affect viability, loss of any two proteins causes lethality. Mutant phenotypes displayed by lem-d double mutants differ from baf mutants, suggesting that BAF function is retained in animals with a single nuclear lamina LEM-D protein. Interestingly, lem-d double mutants displayed distinct developmental and cellular mutant phenotypes, suggesting that Drosophila LEM-D proteins have developmental functions that are differentially shared with other LEM-D family members. This conclusion is supported by studies showing that ectopically produced LEM-D proteins have distinct capacities to rescue the tissue-specific phenotypes found in single lem-d mutants. Our findings predict that cell-specific mutant phenotypes caused by loss of LEM-D proteins reflect both the constellation of LEM-D proteins within the nuclear lamina and the capacity of functional compensation of the remaining LEM-D proteins. PMID:24700158

Distinct Contributions of Conserved Modules to Runt Transcription Factor Activity

PubMed Central

Walrad, Pegine B.; Hang, Saiyu; Joseph, Genevieve S.; Salas, Julia

2010-01-01

Runx proteins play vital roles in regulating transcription in numerous developmental pathways throughout the animal kingdom. Two Runx protein hallmarks are the DNA-binding Runt domain and a C-terminal VWRPY motif that mediates interaction with TLE/Gro corepressor proteins. A phylogenetic analysis of Runt, the founding Runx family member, identifies four distinct regions C-terminal to the Runt domain that are conserved in Drosophila and other insects. We used a series of previously described ectopic expression assays to investigate the functions of these different conserved regions in regulating gene expression during embryogenesis and in controlling axonal projections in the developing eye. The results indicate each conserved region is required for a different subset of activities and identify distinct regions that participate in the transcriptional activation and repression of the segmentation gene sloppy-paired-1 (slp1). Interestingly, the C-terminal VWRPY-containing region is not required for repression but instead plays a role in slp1 activation. Genetic experiments indicating that Groucho (Gro) does not participate in slp1 regulation further suggest that Runt's conserved C-terminus interacts with other factors to promote transcriptional activation. These results provide a foundation for further studies on the molecular interactions that contribute to the context-dependent properties of Runx proteins as developmental regulators. PMID:20462957

Honey bee (Apis mellifera) transferrin-gene structure and the role of ecdysteroids in the developmental regulation of its expression.

PubMed

do Nascimento, Adriana Mendes; Cuvillier-Hot, Virginie; Barchuk, Angel Roberto; Simões, Zilá Luz Paulino; Hartfelder, Klaus

2004-05-01

Social life is prone to invasion by microorganisms, and binding of ferric ions by transferrin is an efficient strategy to restrict their access to iron. In this study, we isolated cDNA and genomic clones encoding an Apis mellifera transferrin (AmTRF) gene. It has an open reading frame (ORF) of 2136 bp spread over nine exons. The deduced protein sequence comprises 686 amino acid residues plus a 26 residues signal sequence, giving a predicted molecular mass of 76 kDa. Comparison of the deduced AmTRF amino acid sequence with known insect transferrins revealed significant similarity extending over the entire sequence. It clusters with monoferric transferrins, with which it shares putative iron-binding residues in the N-terminal lobe. In a functional analysis of AmTRF expression in honey bee development, we monitored its expression profile in the larval and pupal stages. The negative regulation of AmTRF by ecdysteroids deduced from the developmental expression profile was confirmed by experimental treatment of spinning-stage honey bee larvae with 20-hydroxyecdysone, and of fourth instar-larvae with juvenile hormone. A juvenile hormone application to spinning-stage larvae, in contrast, had only a minor effect on AmTRF transcript levels. This is the first study implicating ecdysteroids in the developmental regulation of transferrin expression in an insect species.

Neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) regulation by 14-3-3 protein binding at canonical serum and glucocorticoid kinase 1 (SGK1) phosphorylation sites.

PubMed

Chandran, Sindhu; Li, Hui; Dong, Wuxing; Krasinska, Karolina; Adams, Chris; Alexandrova, Ludmila; Chien, Allis; Hallows, Kenneth R; Bhalla, Vivek

2011-10-28

Regulation of epithelial Na(+) channel (ENaC)-mediated transport in the distal nephron is a critical determinant of blood pressure in humans. Aldosterone via serum and glucocorticoid kinase 1 (SGK1) stimulates ENaC by phosphorylation of the E3 ubiquitin ligase Nedd4-2, which induces interaction with 14-3-3 proteins. However, the mechanisms of SGK1- and 14-3-3-mediated regulation of Nedd4-2 are unclear. There are three canonical SGK1 target sites on Nedd4-2 that overlap phosphorylation-dependent 14-3-3 interaction motifs. Two of these are termed "minor," and one is termed "major," based on weak or strong binding to 14-3-3 proteins, respectively. By mass spectrometry, we found that aldosterone significantly stimulates phosphorylation of a minor, relative to the major, 14-3-3 binding site on Nedd4-2. Phosphorylation-deficient minor site Nedd4-2 mutants bound less 14-3-3 than did wild-type (WT) Nedd4-2, and minor site Nedd4-2 mutations were sufficient to inhibit SGK1 stimulation of ENaC cell surface expression. As measured by pulse-chase and cycloheximide chase assays, a major binding site Nedd4-2 mutant had a shorter cellular half-life than WT Nedd4-2, but this property was not dependent on binding to 14-3-3. Additionally, a dimerization-deficient 14-3-3ε mutant failed to bind Nedd4-2. We conclude that whereas phosphorylation at the Nedd4-2 major site is important for interaction with 14-3-3 dimers, minor site phosphorylation by SGK1 may be the relevant molecular switch that stabilizes Nedd4-2 interaction with 14-3-3 and thus promotes ENaC cell surface expression. We also propose that major site phosphorylation promotes cellular Nedd4-2 protein stability, which potentially represents a novel form of regulation for turnover of E3 ubiquitin ligases.

Identification of Specific DNA Binding Residues in the TCP Family of Transcription Factors in Arabidopsis[W

PubMed Central

Aggarwal, Pooja; Das Gupta, Mainak; Joseph, Agnel Praveen; Chatterjee, Nirmalya; Srinivasan, N.; Nath, Utpal

2010-01-01

The TCP transcription factors control multiple developmental traits in diverse plant species. Members of this family share an ∼60-residue-long TCP domain that binds to DNA. The TCP domain is predicted to form a basic helix-loop-helix (bHLH) structure but shares little sequence similarity with canonical bHLH domain. This classifies the TCP domain as a novel class of DNA binding domain specific to the plant kingdom. Little is known about how the TCP domain interacts with its target DNA. We report biochemical characterization and DNA binding properties of a TCP member in Arabidopsis thaliana, TCP4. We have shown that the 58-residue domain of TCP4 is essential and sufficient for binding to DNA and possesses DNA binding parameters comparable to canonical bHLH proteins. Using a yeast-based random mutagenesis screen and site-directed mutants, we identified the residues important for DNA binding and dimer formation. Mutants defective in binding and dimerization failed to rescue the phenotype of an Arabidopsis line lacking the endogenous TCP4 activity. By combining structure prediction, functional characterization of the mutants, and molecular modeling, we suggest a possible DNA binding mechanism for this class of transcription factors. PMID:20363772

The Evolutionary History and Diverse Physiological Roles of the Grapevine Calcium-Dependent Protein Kinase Gene Family

PubMed Central

Chen, Fei; Fasoli, Marianna; Tornielli, Giovanni Battista; Dal Santo, Silvia; Pezzotti, Mario; Zhang, Liangsheng; Cai, Bin; Cheng, Zong-Ming

2013-01-01

Calcium-dependent protein kinases (CDPKs) are molecular switches that bind Ca2+, ATP, and protein substrates, acting as sensor relays and responders that convert Ca2+ signals, created by developmental processes and environmental stresses, into phosphorylation events. The precise functions of the CDPKs in grapevine (Vitis vinifera) are largely unknown. We therefore investigated the phylogenetic relationships and expression profiles of the 17 CDPK genes identified in the 12x grapevine genome sequence, resolving them into four subfamilies based on phylogenetic tree topology and gene structures. The origins of the CDPKs during grapevine evolution were characterized, involving 13 expansion events. Transcriptomic analysis using 54 tissues and developmental stages revealed three types of CDPK gene expression profiles: constitutive (housekeeping CDPKs), partitioned functions, and prevalent in pollen/stamen. We identified two duplicated CDPK genes that had evolved from housekeeping to pollen-prevalent functions and whose origin correlated with that of seed plants, suggesting neofunctionalization with an important role in pollen development and also potential value in the breeding of seedless varieties. We also found that CDPKs were involved in three abiotic stress signaling pathways and could therefore be used to investigate the crosstalk between stress responses. PMID:24324631

Drosophila Fip200 is an essential regulator of autophagy that attenuates both growth and aging.

PubMed

Kim, Myungjin; Park, Hae Li; Park, Hwan-Woo; Ro, Seung-Hyun; Nam, Samuel G; Reed, John M; Guan, Jun-Lin; Lee, Jun Hee

2013-08-01

Autophagy-related 1 (Atg1)/Unc-51-like protein kinases (ULKs) are evolutionarily conserved proteins that play critical physiological roles in controlling autophagy, cell growth and neurodevelopment. RB1-inducible coiled-coil 1 (RB1CC1), also known as PTK2/FAK family-interacting protein of 200 kDa (FIP200) is a recently discovered binding partner of ULK1. Here we isolated the Drosophila RB1CC1/FIP200 homolog (Fip200/CG1347) and showed that it mediates Atg1-induced autophagy as a genetically downstream component in diverse physiological contexts. Fip200 loss-of-function mutants experienced severe mobility loss associated with neuronal autophagy defects and neurodegeneration. The Fip200 mutants were also devoid of both developmental and starvation-induced autophagy in salivary gland and fat body, while having no defects in axonal transport and projection in developing neurons. Interestingly, moderate downregulation of Fip200 accelerated both developmental growth and aging, accompanied by target of rapamycin (Tor) signaling upregulation. These results suggest that Fip200 is a critical downstream component of Atg1 and specifically mediates Atg1's autophagy-, aging- and growth-regulating functions.

Drosophila Fip200 is an essential regulator of autophagy that attenuates both growth and aging

PubMed Central

Kim, Myungjin; Park, Hae Li; Park, Hwan-Woo; Ro, Seung-Hyun; Nam, Samuel G.; Reed, John M.; Guan, Jun-Lin; Lee, Jun Hee

2013-01-01

Autophagy-related 1 (Atg1)/Unc-51-like protein kinases (ULKs) are evolutionarily conserved proteins that play critical physiological roles in controlling autophagy, cell growth and neurodevelopment. RB1-inducible coiled-coil 1 (RB1CC1), also known as PTK2/FAK family-interacting protein of 200 kDa (FIP200) is a recently discovered binding partner of ULK1. Here we isolated the Drosophila RB1CC1/FIP200 homolog (Fip200/CG1347) and showed that it mediates Atg1-induced autophagy as a genetically downstream component in diverse physiological contexts. Fip200 loss-of-function mutants experienced severe mobility loss associated with neuronal autophagy defects and neurodegeneration. The Fip200 mutants were also devoid of both developmental and starvation-induced autophagy in salivary gland and fat body, while having no defects in axonal transport and projection in developing neurons. Interestingly, moderate downregulation of Fip200 accelerated both developmental growth and aging, accompanied by target of rapamycin (Tor) signaling upregulation. These results suggest that Fip200 is a critical downstream component of Atg1 and specifically mediates Atg1’s autophagy-, aging- and growth-regulating functions. PMID:23819996

Near-atomic model of microtubule-tau interactions.

PubMed

Kellogg, Elizabeth H; Hejab, Nisreen M A; Poepsel, Simon; Downing, Kenneth H; DiMaio, Frank; Nogales, Eva

2018-06-15

Tau is a developmentally regulated axonal protein that stabilizes and bundles microtubules (MTs). Its hyperphosphorylation is thought to cause detachment from MTs and subsequent aggregation into fibrils implicated in Alzheimer's disease. It is unclear which tau residues are crucial for tau-MT interactions, where tau binds on MTs, and how it stabilizes them. We used cryo-electron microscopy to visualize different tau constructs on MTs and computational approaches to generate atomic models of tau-tubulin interactions. The conserved tubulin-binding repeats within tau adopt similar extended structures along the crest of the protofilament, stabilizing the interface between tubulin dimers. Our structures explain the effect of phosphorylation on MT affinity and lead to a model of tau repeats binding in tandem along protofilaments, tethering together tubulin dimers and stabilizing polymerization interfaces. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Diversification of HP1-like Chromo Domain Proteins in Tetrahymena thermophila.

PubMed

Wiley, Emily A; Horrell, Scott; Yoshino, Alyssa; Schornak, Cara C; Bagnani, Claire; Chalker, Douglas L

2018-01-01

Proteins that possess a chromo domain are well-known for their roles in heterochromatin assembly and maintenance. The Heterochromatin Protein 1 (HP1) family, with a chromo domain and carboxy-terminal chromo shadow domain, targets heterochromatin through interaction with histone H3 methylated on lysine 9 (H3K9me2/3). The structural and functional diversity of these proteins observed in both fission yeast and metazoans correlate with chromatin specialization. To expand these studies, we examined chromo domain proteins in the ciliate Tetrahymena thermophila, which has functionally diverse and developmentally regulated heterochromatin domains. We identified thirteen proteins similar to HP1. Together they possess only a fraction of the possible chromo domain subtypes and most lack a recognizable chromo shadow domain. Using fluorescence microscopy to track chromatin localization of tagged proteins through the life cycle, we show evidence that in T. thermophila this family has diversified with biological roles in RNAi-directed DNA elimination, germline genome structure, and somatic heterochromatin. Those proteins with H3K27me3 binding sequence characteristics localize to chromatin in mature nuclei, whereas those with H3K9me2/3 binding characteristics localize to developing nuclei undergoing DNA elimination. Findings point to an expanded and diversified family of chromo domain proteins that parallels heterochromatin diversity in ciliates. © 2017 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.

Protein targeting in the analysis of learning and memory: a potential alternative to gene targeting.

PubMed

Gerlai, R; Williams, S P; Cairns, B; Van Bruggen, N; Moran, P; Shih, A; Caras, I; Sauer, H; Phillips, H S; Winslow, J W

1998-11-01

Gene targeting using homologous recombination in embryonic stem (ES) cells offers unprecedented precision with which one may manipulate single genes and investigate the in vivo effects of defined mutations in the mouse. Geneticists argue that this technique abrogates the lack of highly specific pharmacological tools in the study of brain function and behavior. However, by now it has become clear that gene targeting has some limitations too. One problem is spatial and temporal specificity of the generated mutation, which may appear in multiple brain regions or even in other organs and may also be present throughout development, giving rise to complex, secondary phenotypical alterations. This may be a disadvantage in the functional analysis of a number of genes associated with learning and memory processes. For example, several proteins, including neurotrophins--cell-adhesion molecules--and protein kinases, that play a significant developmental role have recently been suggested to be also involved in neural and behavioral plasticity. Knocking out genes of such proteins may lead to developmental alterations or even embryonic lethality in the mouse, making it difficult to study their function in neural plasticity, learning, and memory. Therefore, alternative strategies to gene targeting may be needed. Here, we suggest a potentially useful in vivo strategy based on systemic application of immunoadhesins, genetically engineered fusion proteins possessing the Fc portion of the human IgG molecule and, for example, a binding domain of a receptor of interest. These proteins are stable in vivo and exhibit high binding specificity and affinity for the endogenous ligand of the receptor, but lack the ability to signal. Thus, if delivered to the brain, immunoadhesins may specifically block signalling of the receptor of interest. Using osmotic minipumps, the protein can be infused in a localized region of the brain for a specified period of time (days or weeks). Thus, the location and timing of delivery are controlled. Here, we present methodological details of this novel approach and argue that infusion of immunoadhesins will be useful for studying the role particular receptors play in behavioral and neural plasticity.

Exome analysis identified a novel mutation in the RBP4 gene in a consanguineous pedigree with retinal dystrophy and developmental abnormalities.

PubMed

Cukras, Catherine; Gaasterland, Terry; Lee, Pauline; Gudiseva, Harini V; Chavali, Venkata R M; Pullakhandam, Raghu; Maranhao, Bruno; Edsall, Lee; Soares, Sandra; Reddy, G Bhanuprakash; Sieving, Paul A; Ayyagari, Radha

2012-01-01

Retinitis Pigmentosa (RP) is a common form of retinal degeneration characterized by photoreceptor degeneration and retinal pigment epithelium (RPE) atrophy causing loss of visual field and acuities. Exome sequencing identified a novel homozygous splice site variant (c.111+1G>A) in the gene encoding retinol binding protein 4 (RBP4). This change segregated with early onset, progressive, and severe autosomal recessive retinitis pigmentosa (arRP) in an eight member consanguineous pedigree of European ancestry. Additionally, one patient exhibited developmental abnormalities including patent ductus arteriosus and chorioretinal and iris colobomas. The second patient developed acne from young age and extending into the 5(th) decade. Both patients had undetectable levels of RBP4 in the serum suggesting that this mutation led to either mRNA or protein instability resulting in a null phenotype. In addition, the patients exhibited severe vitamin A deficiency, and diminished serum retinol levels. Circulating transthyretin levels were normal. This study identifies the RBP4 splice site change as the cause of RP in this pedigree. The presence of developmental abnormalities and severe acne in patients with retinal degeneration may indicate the involvement of genes that regulate vitamin A absorption, transport and metabolism.

Exome Analysis Identified a Novel Mutation in the RBP4 Gene in a Consanguineous Pedigree with Retinal Dystrophy and Developmental Abnormalities

PubMed Central

Cukras, Catherine; Gaasterland, Terry; Lee, Pauline; Gudiseva, Harini V.; Chavali, Venkata R. M.; Pullakhandam, Raghu; Maranhao, Bruno; Edsall, Lee; Soares, Sandra; Reddy, G. Bhanuprakash; Sieving, Paul A.; Ayyagari, Radha

2012-01-01

Retinitis Pigmentosa (RP) is a common form of retinal degeneration characterized by photoreceptor degeneration and retinal pigment epithelium (RPE) atrophy causing loss of visual field and acuities. Exome sequencing identified a novel homozygous splice site variant (c.111+1G>A) in the gene encoding retinol binding protein 4 (RBP4). This change segregated with early onset, progressive, and severe autosomal recessive retinitis pigmentosa (arRP) in an eight member consanguineous pedigree of European ancestry. Additionally, one patient exhibited developmental abnormalities including patent ductus arteriosus and chorioretinal and iris colobomas. The second patient developed acne from young age and extending into the 5th decade. Both patients had undetectable levels of RBP4 in the serum suggesting that this mutation led to either mRNA or protein instability resulting in a null phenotype. In addition, the patients exhibited severe vitamin A deficiency, and diminished serum retinol levels. Circulating transthyretin levels were normal. This study identifies the RBP4 splice site change as the cause of RP in this pedigree. The presence of developmental abnormalities and severe acne in patients with retinal degeneration may indicate the involvement of genes that regulate vitamin A absorption, transport and metabolism. PMID:23189188

Aspp2 negatively regulates body growth but not developmental timing by modulating IRS signaling in zebrafish embryos.

PubMed

Liu, Chengdong; Luan, Jing; Bai, Yan; Li, Yun; Lu, Ling; Liu, Yunzhang; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Duan, Cunming; Zhou, Jianfeng

2014-02-01

The growth and developmental rate of developing embryos and fetus are tightly controlled and coordinated to maintain proper body shape and size. The insulin receptor substrate (IRS) proteins, key intracellular transducers of insulin and insulin-like growth factor signaling, play essential roles in the regulation of growth and development. A short isoform of apoptosis-stimulating protein of p53 2 (ASPP2) was recently identified as a binding partner of IRS-1 and IRS-2 in mammalian cells in vitro. However, it is unclear whether ASPP2 plays any role in vertebrate embryonic growth and development. Here, we show that zebrafish Aspp2a and Aspp2b negatively regulate embryonic growth without affecting developmental rate. Human ASPP2 had similar effects on body growth in zebrafish embryos. Aspp2a and 2b inhibit Akt signaling. This inhibition was reversed by coinjection of myr-Akt1, a constitutively active form of Akt1. Zebrafish Aspp2a and Aspp2b physically bound with Irs-1, and the growth inhibitory effects of ASPP2/Aspp2 depend on the presence of their ankyrin repeats and SH3 domains. These findings uncover a novel role of Aspp2 in regulating vertebrate embryonic growth. Copyright © 2013 Elsevier Inc. All rights reserved.

Autosomal recessive mutations in nuclear transport factor KPNA7 are associated with infantile spasms and cerebellar malformation.

PubMed

Paciorkowski, Alex R; Weisenberg, Judy; Kelley, Joshua B; Spencer, Adam; Tuttle, Emily; Ghoneim, Dalia; Thio, Liu Lin; Christian, Susan L; Dobyns, William B; Paschal, Bryce M

2014-05-01

Nuclear import receptors of the KPNA family recognize the nuclear localization signal in proteins and together with importin-β mediate translocation into the nucleus. Accordingly, KPNA family members have a highly conserved architecture with domains that contact the nuclear localization signal and bind to importin-β. Here, we describe autosomal recessive mutations in KPNA7 found by whole exome sequencing in a sibling pair with severe developmental disability, infantile spasms, subsequent intractable epilepsy consistent with Lennox-Gastaut syndrome, partial agenesis of the corpus callosum, and cerebellar vermis hypoplasia. The mutations mapped to exon 7 in KPNA7 result in two amino-acid substitutions, Pro339Ala and Glu344Gln. On the basis of the crystal structure of the paralog KPNA2 bound to a bipartite nuclear localization signal from the retinoblastoma protein, the amino-acid substitutions in the affected subjects were predicted to occur within the seventh armadillo repeat that forms one of the two nuclear localization signal-binding sites in KPNA family members. Glu344 is conserved in all seven KPNA proteins, and we found that the Glu354Gln mutation in KPNA2 is sufficient to reduce binding to the retinoblastoma nuclear localization signal to approximately one-half that of wild-type protein. Our data show that compound heterozygous mutations in KPNA7 are associated with a human neurodevelopmental disease, and provide the first example of a human disease associated with mutation of a nuclear transport receptor.

Autosomal recessive mutations in nuclear transport factor KPNA7 are associated with infantile spasms and cerebellar malformation

PubMed Central

Paciorkowski, Alex R; Weisenberg, Judy; Kelley, Joshua B; Spencer, Adam; Tuttle, Emily; Ghoneim, Dalia; Thio, Liu Lin; Christian, Susan L; Dobyns, William B; Paschal, Bryce M

2014-01-01

Nuclear import receptors of the KPNA family recognize the nuclear localization signal in proteins and together with importin-β mediate translocation into the nucleus. Accordingly, KPNA family members have a highly conserved architecture with domains that contact the nuclear localization signal and bind to importin-β. Here, we describe autosomal recessive mutations in KPNA7 found by whole exome sequencing in a sibling pair with severe developmental disability, infantile spasms, subsequent intractable epilepsy consistent with Lennox–Gastaut syndrome, partial agenesis of the corpus callosum, and cerebellar vermis hypoplasia. The mutations mapped to exon 7 in KPNA7 result in two amino-acid substitutions, Pro339Ala and Glu344Gln. On the basis of the crystal structure of the paralog KPNA2 bound to a bipartite nuclear localization signal from the retinoblastoma protein, the amino-acid substitutions in the affected subjects were predicted to occur within the seventh armadillo repeat that forms one of the two nuclear localization signal-binding sites in KPNA family members. Glu344 is conserved in all seven KPNA proteins, and we found that the Glu354Gln mutation in KPNA2 is sufficient to reduce binding to the retinoblastoma nuclear localization signal to approximately one-half that of wild-type protein. Our data show that compound heterozygous mutations in KPNA7 are associated with a human neurodevelopmental disease, and provide the first example of a human disease associated with mutation of a nuclear transport receptor. PMID:24045845

Functional analysis of the glycogen binding subunit CG9238/Gbs-70E of protein phosphatase 1 in Drosophila melanogaster.

PubMed

Kerekes, Éva; Kókai, Endre; Páldy, Ferenc Sándor; Dombrádi, Viktor

2014-06-01

The product of the CG9238 gene that we termed glycogen binding subunit 70E (Gbs-70E) was characterized by biochemical and molecular genetics methods. The interaction between Gbs-70E and all catalytic subunits of protein phosphatase 1 (Pp1-87B, Pp1-9C, Pp1-96A and Pp1-13C) of Drosophila melanogaster was confirmed by pairwise yeast two-hybrid tests, co-immunoprecipitation and pull down experiments. The binding of Gbs-70E to glycogen was demonstrated by sedimentation analysis. With RT-PCR we found that the mRNAs coding for the longer Gbs-70E PB/PC protein were expressed in all developmental stages of the fruit flies while the mRNA for the shorter Gbs-70E PA was restricted to the eggs and the ovaries of the adult females. The development specific expression of the shorter splice variant was not conserved in different Drosophila species. The expression level of the gene was manipulated by P-element insertions and gene deletion to analyze the functions of the gene product. A small or moderate reduction in the gene expression resulted in no significant changes, however, a deletion mutant expressing very low level of the transcript lived shorter and exhibited reduced glycogen content in the imagos. In addition, the gene deletion decreased the fertility of the fruit flies. Our results prove that Gbs-70E functions as the glycogen binding subunit of protein phosphatase 1 that regulates glycogen content and plays a role in the development of eggs in D. melanogaster. Copyright © 2014 Elsevier Ltd. All rights reserved.

70-kDa Heat Shock Cognate Protein hsc70 Mediates Calmodulin-dependent Nuclear Import of the Sex-determining Factor SRY*

PubMed Central

Kaur, Gurpreet; Lieu, Kim G.; Jans, David A.

2013-01-01

We recently showed that the developmentally important family of SOX (SRY (sex determining region on the Y chromosome)-related high mobility group (HMG) box) proteins require the calcium-binding protein calmodulin (CaM) for optimal nuclear accumulation, with clinical mutations in SRY that specifically impair nuclear accumulation via this pathway resulting in XY sex reversal. However, the mechanism by which CaM facilitates nuclear accumulation is unknown. Here, we show, for the first time, that the 70-kDa heat shock cognate protein hsc70 plays a key role in CaM-dependent nuclear import of SRY. Using a reconstituted nuclear import assay, we show that antibodies to hsc70 significantly reduce nuclear accumulation of wild type SRY and mutant derivatives thereof that retain CaM-dependent nuclear import, with an increased rate of nuclear accumulation upon addition of both CaM and hsc70, in contrast to an SRY mutant derivative with impaired CaM binding. siRNA knockdown of hsc70 in intact cells showed similar results, indicating clear dependence upon hsc70 for CaM-dependent nuclear import. Analysis using the technique of fluorescence recovery after photobleaching indicated that hsc70 is required for the maximal rate of SRY nuclear import in living cells but has no impact upon SRY nuclear retention/nuclear dynamics. Finally, we demonstrate direct binding of hsc70 to the SRY·CaM complex, with immunoprecipitation experiments from cell extracts showing association of hsc70 with wild type SRY, but not with a mutant derivative with impaired CaM binding, dependent on Ca2+. Our novel findings strongly implicate hsc70 in CaM-dependent nuclear import of SRY. PMID:23235156

EIN2 mediates direct regulation of histone acetylation in the ethylene response.

PubMed

Zhang, Fan; Wang, Likai; Qi, Bin; Zhao, Bo; Ko, Eun Esther; Riggan, Nathaniel D; Chin, Kevin; Qiao, Hong

2017-09-19

Ethylene gas is essential for developmental processes and stress responses in plants. Although the membrane-bound protein EIN2 is critical for ethylene signaling, the mechanism by which the ethylene signal is transduced remains largely unknown. Here we show the levels of H3K14Ac and H3K23Ac are correlated with the levels of EIN2 protein and demonstrate EIN2 C terminus (EIN2-C) is sufficient to rescue the levels of H3K14/23Ac of ein2 -5 at the target loci, using CRISPR/dCas9-EIN2-C. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and ChIP-reChIP-seq analyses revealed that EIN2-C associates with histone partially through an interaction with EIN2 nuclear-associated protein1 (ENAP1), which preferentially binds to the genome regions that are associated with actively expressed genes both with and without ethylene treatments. Specifically, in the presence of ethylene, ENAP1-binding regions are more accessible upon the interaction with EIN2, and more EIN3 proteins bind to the loci where ENAP1 is enriched for a quick response. Together, these results reveal EIN2-C is the key factor regulating H3K14Ac and H3K23Ac in response to ethylene and uncover a unique mechanism by which ENAP1 interacts with chromatin, potentially preserving the open chromatin regions in the absence of ethylene; in the presence of ethylene, EIN2 interacts with ENAP1, elevating the levels of H3K14Ac and H3K23Ac, promoting more EIN3 binding to the targets shared with ENAP1 and resulting in a rapid transcriptional regulation.

Germline Missense Mutations Affecting KRAS Isoform B Are Associated with a Severe Noonan Syndrome Phenotype

PubMed Central

Carta, Claudio; Pantaleoni, Francesca; Bocchinfuso, Gianfranco; Stella, Lorenzo; Vasta, Isabella; Sarkozy, Anna; Digilio, Cristina; Palleschi, Antonio; Pizzuti, Antonio; Grammatico, Paola; Zampino, Giuseppe; Dallapiccola, Bruno; Gelb, Bruce D.; Tartaglia, Marco

2006-01-01

Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart disease, and multiple skeletal and hematologic defects. NS is an autosomal dominant trait and is genetically heterogeneous. Gain of function of SHP-2, a protein tyrosine phosphatase that positively modulates RAS signaling, is observed in nearly 50% of affected individuals. Here, we report the identification of heterozygous KRAS gene mutations in two subjects exhibiting a severe NS phenotype with features overlapping those of cardiofaciocutaneous and Costello syndromes. Both mutations were de novo and affected exon 6, which encodes the C-terminal portion of KRAS isoform B but does not contribute to KRAS isoform A. Structural analysis indicated that both substitutions (Val152Gly and Asp153Val) perturb the conformation of the guanine ring–binding pocket of the protein, predicting an increase in the guanine diphosphate/guanine triphosphate (GTP) dissociation rate that would favor GTP binding to the KRASB isoform and bypass the requirement for a guanine nucleotide exchange factor. PMID:16773572

Maternal Dead-end 1 promotes translation of nanos1 by binding the eIF3 complex.

PubMed

Aguero, Tristan; Jin, Zhigang; Chorghade, Sandip; Kalsotra, Auinash; King, Mary Lou; Yang, Jing

2017-10-15

In the developing embryo, primordial germ cells (PGCs) represent the exclusive progenitors of the gametes, and their loss results in adult infertility. During early development, PGCs are exposed to numerous signals that specify somatic cell fates. To prevent somatic differentiation, PGCs must transiently silence their genome, an early developmental process that requires Nanos activity. However, it is unclear how Nanos translation is regulated in developing embryos. We report here that translation of nanos1 after fertilization requires Dead-end 1 (Dnd1), a vertebrate-specific germline RNA-binding protein. We provide evidence that Dnd1 protein, expression of which is low in oocytes, but increases dramatically after fertilization, directly interacts with, and relieves the inhibitory function of eukaryotic initiation factor 3f, a repressive component in the 43S preinitiation complex. This work uncovers a novel translational regulatory mechanism that is fundamentally important for germline development. © 2017. Published by The Company of Biologists Ltd.

Structural basis for Notch1 engagement of Delta-like 4

DOE PAGES

Luca, Vincent C.; Jude, Kevin M.; Pierce, Nathan W.; ...

2015-02-20

Notch receptors guide mammalian cell fate decisions by engaging the proteins Jagged and Delta-like (DLL). The 2.3 angstrom resolution crystal structure of the interacting regions of the Notch1-DLL4 complex reveals a two-site, antiparallel binding orientation assisted by Notch1 O-linked glycosylation. Notch1 epidermal growth factor–like repeats 11 and 12 interact with the DLL4 Delta/Serrate/Lag-2 (DSL) domain and module at the N-terminus of Notch ligands (MNNL) domains, respectively. Threonine and serine residues on Notch1 are functionalized with O-fucose and O-glucose, which act as surrogate amino acids by making specific, and essential, contacts to residues on DLL4. Lastly, the elucidation of a directmore » chemical role for O-glycans in Notch1 ligand engagement demonstrates how, by relying on posttranslational modifications of their ligand binding sites, Notch proteins have linked their functional capacity to developmentally regulated biosynthetic pathways.« less

Gas1 extends the range of Hedgehog action by facilitating its signaling

PubMed Central

Martinelli, David C.; Fan, Chen-Ming

2007-01-01

Cellular signaling initiated by Hedgehog binding to Patched1 has profound importance in mammalian embryogenesis, genetic disease, and cancer. Hedgehog acts as a morphogen to specify distinctive cell fates using different concentration thresholds, but our knowledge of how the concentration gradient is interpreted into the activity gradient is incomplete. The membrane protein Growth Arrest-Specific Gene 1 (GAS1) was thought to be a negative regulator of the Hedgehog concentration gradient. Here, we report unexpected genetic evidence that Gas1 positively regulates Hedgehog signaling in multiple developmental contexts, an effect particularly noticeable at regions where Hedgehog acts at low concentration. Using a combination of in vitro cell culture and in ovo electroporation assays, we demonstrate that GAS1 acts cooperatively with Patched1 for Hedgehog binding and enhances signaling activity in a cell-autonomous manner. Our data support a model in which GAS1 helps transform the Hedgehog protein gradient into the observed activity gradient. We propose that Gas1 is an evolutionarily novel, vertebrate-specific Hedgehog pathway regulator. PMID:17504940

Structural basis for Notch1 engagement of Delta-like 4

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luca, Vincent C.; Jude, Kevin M.; Pierce, Nathan W.

Notch receptors guide mammalian cell fate decisions by engaging the proteins Jagged and Delta-like (DLL). The 2.3 angstrom resolution crystal structure of the interacting regions of the Notch1-DLL4 complex reveals a two-site, antiparallel binding orientation assisted by Notch1 O-linked glycosylation. Notch1 epidermal growth factor–like repeats 11 and 12 interact with the DLL4 Delta/Serrate/Lag-2 (DSL) domain and module at the N-terminus of Notch ligands (MNNL) domains, respectively. Threonine and serine residues on Notch1 are functionalized with O-fucose and O-glucose, which act as surrogate amino acids by making specific, and essential, contacts to residues on DLL4. Lastly, the elucidation of a directmore » chemical role for O-glycans in Notch1 ligand engagement demonstrates how, by relying on posttranslational modifications of their ligand binding sites, Notch proteins have linked their functional capacity to developmentally regulated biosynthetic pathways.« less

High-resolution mapping of chromatin packaging in mouse embryonic stem cells and sperm.

PubMed

Carone, Benjamin R; Hung, Jui-Hung; Hainer, Sarah J; Chou, Min-Te; Carone, Dawn M; Weng, Zhiping; Fazzio, Thomas G; Rando, Oliver J

2014-07-14

Mammalian embryonic stem cells (ESCs) and sperm exhibit unusual chromatin packaging that plays important roles in cellular function. Here, we extend a recently developed technique, based on deep paired-end sequencing of lightly digested chromatin, to assess footprints of nucleosomes and other DNA-binding proteins genome-wide in murine ESCs and sperm. In ESCs, we recover well-characterized features of chromatin such as promoter nucleosome depletion and further identify widespread footprints of sequence-specific DNA-binding proteins such as CTCF, which we validate in knockdown studies. We document global differences in nuclease accessibility between ESCs and sperm, finding that the majority of histone retention in sperm preferentially occurs in large gene-poor genomic regions, with only a small subset of nucleosomes being retained over promoters of developmental regulators. Finally, we describe evidence that CTCF remains associated with the genome in mature sperm, where it could play a role in organizing the sperm genome. Copyright © 2014 Elsevier Inc. All rights reserved.

Non-homeodomain regions of Hox proteins mediate activation versus repression of Six2 via a single enhancer site in vivo

PubMed Central

Yallowitz, Alisha R.; Gong, Ke-Qin; Swinehart, Ilea T.; Nelson, Lisa T.; Wellik, Deneen M.

2009-01-01

Summary Hox genes control many developmental events along the AP axis, but few target genes have been identified. Whether target genes are activated or repressed, what enhancer elements are required for regulation, and how different domains of the Hox proteins contribute to regulatory specificity is poorly understood. Six2 is genetically downstream of both the Hox11 paralogous genes in the developing mammalian kidney and Hoxa2 in branchial arch and facial mesenchyme. Loss-of-function of Hox11 leads to loss of Six2 expression and loss-of-function of Hoxa2 leads to expanded Six2 expression. Herein we demonstrate that a single enhancer site upstream of the Six2 coding sequence is responsible for both activation by Hox11 proteins in the kidney and repression by Hoxa2 in the branchial arch and facial mesenchyme in vivo. DNA binding activity is required for both activation and repression, but differential activity is not controlled by differences in the homeodomains. Rather, protein domains N- and C-terminal to the homeodomain confer activation versus repression activity. These data support a model in which the DNA binding specificity of Hox proteins in vivo may be similar, consistent with accumulated in vitro data, and that unique functions result mainly from differential interactions mediated by non-homeodomain regions of Hox proteins. PMID:19716816

Acute tau knockdown in the hippocampus of adult mice causes learning and memory deficits.

PubMed

Velazquez, Ramon; Ferreira, Eric; Tran, An; Turner, Emily C; Belfiore, Ramona; Branca, Caterina; Oddo, Salvatore

2018-05-10

Misfolded and hyperphosphorylated tau accumulates in several neurodegenerative disorders including Alzheimer's disease, frontotemporal dementia with Parkinsonism, corticobasal degeneration, progressive supranuclear palsy, Down syndrome, and Pick's disease. Tau is a microtubule-binding protein, and its role in microtubule stabilization is well defined. In contrast, while growing evidence suggests that tau is also involved in synaptic physiology, a complete assessment of tau function in the adult brain has been hampered by robust developmental compensation of other microtubule-binding proteins in tau knockout mice. To circumvent these developmental compensations and assess the role of tau in the adult brain, we generated an adeno-associated virus (AAV) expressing a doxycycline-inducible short-hairpin (Sh) RNA targeted to tau, herein referred to as AAV-ShRNATau. We performed bilateral stereotaxic injections in 7-month-old C57Bl6/SJL wild-type mice with either the AAV-ShRNATau or a control AAV. We found that acute knockdown of tau in the adult hippocampus significantly impaired motor coordination and spatial memory. Blocking the expression of the AAV-ShRNATau, thereby allowing tau levels to return to control levels, restored motor coordination and spatial memory. Mechanistically, the reduced tau levels were associated with lower BDNF levels, reduced levels of synaptic proteins associated with learning, and decreased spine density. We provide compelling evidence that tau is necessary for motor and cognitive function in the adult brain, thereby firmly supporting that tau loss-of-function may contribute to the clinical manifestations of many tauopathies. These findings have profound clinical implications given that anti-tau therapies are in clinical trials for Alzheimer's disease. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

SETDB1 modulates PRC2 activity at developmental genes independently of H3K9 trimethylation in mouse ES cells

PubMed Central

Fei, Qi; Yang, Xiaoqin; Jiang, Hua; Wang, Qian; Yu, Yanyan; Yu, Yiling; Yi, Wei; Zhou, Shaolian; Chen, Taiping; Lu, Chris; Atadja, Peter; Liu, Xiaole Shirley; Li, En; Zhang, Yong; Shou, Jianyong

2015-01-01

SETDB1, a histone methyltransferase responsible for methylation of histone H3 lysine 9 (H3K9), is involved in maintenance of embryonic stem (ES) cells and early embryonic development of the mouse. However, how SETDB1 regulates gene expression during development is largely unknown. Here, we characterized genome-wide SETDB1 binding and H3K9 trimethylation (H3K9me3) profiles in mouse ES cells and uncovered two distinct classes of SETDB1 binding sites, termed solo and ensemble peaks. The solo peaks were devoid of H3K9me3 and enriched near developmental regulators while the ensemble peaks were associated with H3K9me3. A subset of the SETDB1 solo peaks, particularly those near neural development–related genes, was found to be associated with Polycomb Repressive Complex 2 (PRC2) as well as PRC2-interacting proteins JARID2 and MTF2. Genetic deletion of Setdb1 reduced EZH2 binding as well as histone 3 lysine 27 (H3K27) trimethylation level at SETDB1 solo peaks and facilitated neural differentiation. Furthermore, we found that H3K27me3 inhibits SETDB1 methyltransferase activity. The currently identified reciprocal action between SETDB1 and PRC2 reveals a novel mechanism underlying ES cell pluripotency and differentiation regulation. PMID:26160163

A novel paired domain DNA recognition motif can mediate Pax2 repression of gene transcription.

PubMed

Håvik, B; Ragnhildstveit, E; Lorens, J B; Saelemyr, K; Fauske, O; Knudsen, L K; Fjose, A

1999-12-20

The paired domain (PD) is an evolutionarily conserved DNA-binding domain encoded by the Pax gene family of developmental regulators. The Pax proteins are transcription factors and are involved in a variety of processes such as brain development, patterning of the central nervous system (CNS), and B-cell development. In this report we demonstrate that the zebrafish Pax2 PD can interact with a novel type of DNA sequences in vitro, the triple-A motif, consisting of a heptameric nucleotide sequence G/CAAACA/TC with an invariant core of three adjacent adenosines. This recognition sequence was found to be conserved in known natural Pax5 repressor elements involved in controlling the expression of the p53 and J-chain genes. By identifying similar high affinity binding sites in potential target genes of the Pax2 protein, including the pax2 gene itself, we obtained further evidence that the triple-A sites are biologically significant. The putative natural target sites also provide a basis for defining an extended consensus recognition sequence. In addition, we observed in transformation assays a direct correlation between Pax2 repressor activity and the presence of triple-A sites. The results suggest that a transcriptional regulatory function of Pax proteins can be modulated by PD binding to different categories of target sequences. Copyright 1999 Academic Press.

Lin-28 binds IGF-2 mRNA and participates in skeletal myogenesis by increasing translation efficiency.

PubMed

Polesskaya, Anna; Cuvellier, Sylvain; Naguibneva, Irina; Duquet, Arnaud; Moss, Eric G; Harel-Bellan, Annick

2007-05-01

Lin-28 is a highly conserved, RNA-binding, microRNA-regulated protein that is involved in regulation of developmental timing in Caenorhabditis elegans. In mammals, Lin-28 is stage-specifically expressed in embryonic muscle, neurons, and epithelia, as well as in embryonic carcinoma cells, but is suppressed in most adult tissues, with the notable exception of skeletal and cardiac muscle. The specific function and mechanism of action of Lin-28 are not well understood. Here we used loss-of-function and gain-of-function assays in cultured myoblasts to show that expression of Lin-28 is essential for skeletal muscle differentiation in mice. In order to elucidate the specific function of Lin-28, we used a combination of biochemical and functional assays, which revealed that, in differentiating myoblasts, Lin-28 binds to the polysomes and increases the efficiency of protein synthesis. An important target of Lin-28 is IGF-2, a crucial growth and differentiation factor for muscle tissue. Interaction of Lin-28 with translation initiation complexes in skeletal myoblasts and in the embryonic carcinoma cell line P19 was confirmed by localization of Lin-28 to the stress granules, temporary structures that contain stalled mRNA-protein translation complexes. Our results unravel novel mechanisms of translational regulation in skeletal muscle and suggest that Lin-28 performs the role of "translational enhancer" in embryonic and adult cells and tissues.

Paralogous SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes differentially regulate leaf initiation and reproductive phase change in petunia.

PubMed

Preston, Jill C; Jorgensen, Stacy A; Orozco, Rebecca; Hileman, Lena C

2016-02-01

Duplicated petunia clade-VI SPL genes differentially promote the timing of inflorescence and flower development, and leaf initiation rate. The timing of plant reproduction relative to favorable environmental conditions is a critical component of plant fitness, and is often associated with variation in plant architecture and habit. Recent studies have shown that overexpression of the microRNA miR156 in distantly related annual species results in plants with perennial characteristics, including late flowering, weak apical dominance, and abundant leaf production. These phenotypes are largely mediated through the negative regulation of a subset of genes belonging to the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family of transcription factors. In order to determine how and to what extent paralogous SPL genes have partitioned their roles in plant growth and development, we functionally characterized petunia clade-VI SPL genes under different environmental conditions. Our results demonstrate that PhSBP1and PhSBP2 differentially promote discrete stages of the reproductive transition, and that PhSBP1, and possibly PhCNR, accelerates leaf initiation rate. In contrast to the closest homologs in annual Arabidopsis thaliana and Mimulus guttatus, PhSBP1 and PhSBP2 transcription is not mediated by the gibberellic acid pathway, but is positively correlated with photoperiod and developmental age. The developmental functions of clade-VI SPL genes have, thus, evolved following both gene duplication and speciation within the core eudicots, likely through differential regulation and incomplete sub-functionalization.

Sfr13, a member of a large family of asymmetrically localized Sfi1-repeat proteins, is important for basal body separation and stability in Tetrahymena thermophila

PubMed Central

Stemm-Wolf, Alexander J.; Meehl, Janet B.; Winey, Mark

2013-01-01

Summary Directed fluid flow, which is achieved by the coordinated beating of motile cilia, is required for processes as diverse as cellular swimming, developmental patterning and mucus clearance. Cilia are nucleated, anchored and aligned at the plasma membrane by basal bodies, which are cylindrical microtubule-based structures with ninefold radial symmetry. In the unicellular ciliate Tetrahymena thermophila, two centrin family members associated with the basal body are important for both basal body organization and stabilization. We have identified a family of 13 proteins in Tetrahymena that contain centrin-binding repeats related to those identified in the Saccharomyces cerevisiae Sfi1 protein. We have named these proteins Sfr1–Sfr13 (for Sfi1-repeat). Nine of the Sfr proteins localize in unique polarized patterns surrounding the basal body, suggesting non-identical roles in basal body organization and association with basal body accessory structures. Furthermore, the Sfr proteins are found in distinct basal body populations in Tetrahymena cells, indicating that they are responsive to particular developmental programs. A complete genetic deletion of one of the family members, Sfr13, causes unstable basal bodies and defects in daughter basal body separation from the mother, phenotypes also observed with centrin disruption. It is likely that the other Sfr family members are involved in distinct centrin functions, providing specificity to the tasks that centrins perform at basal bodies. PMID:23426847

The Dictyostelium discoideum RACK1 orthologue has roles in growth and development

PubMed Central

2014-01-01

Background The receptor for activated C-kinase 1 (RACK1) is a conserved protein belonging to the WD40 repeat family of proteins. It folds into a beta propeller with seven blades which allow interactions with many proteins. Thus it can serve as a scaffolding protein and have roles in several cellular processes. Results We identified the product of the Dictyostelium discoideum gpbB gene as the Dictyostelium RACK1 homolog. The protein is mainly cytosolic but can also associate with cellular membranes. DdRACK1 binds to phosphoinositides (PIPs) in protein-lipid overlay and liposome-binding assays. The basis of this activity resides in a basic region located in the extended loop between blades 6 and 7 as revealed by mutational analysis. Similar to RACK1 proteins from other organisms DdRACK1 interacts with G protein subunits alpha, beta and gamma as shown by yeast two-hybrid, pulldown, and immunoprecipitation assays. Unlike the Saccharomyces cerevisiae and Cryptococcus neoformans RACK1 proteins it does not appear to take over Gβ function in D. discoideum as developmental and other defects were not rescued in Gβ null mutants overexpressing GFP-DdRACK1. Overexpression of GFP-tagged DdRACK1 and a mutant version (DdRACK1mut) which carried a charge-reversal mutation in the basic region in wild type cells led to changes during growth and development. Conclusion DdRACK1 interacts with heterotrimeric G proteins and can through these interactions impact on processes specifically regulated by these proteins. PMID:24930026

Protein-anchoring therapy to target extracellular matrix proteins to their physiological destinations.

PubMed

Ito, Mikako; Ohno, Kinji

2018-02-20

Endplate acetylcholinesterase (AChE) deficiency is a form of congenital myasthenic syndrome (CMS) caused by mutations in COLQ, which encodes collagen Q (ColQ). ColQ is an extracellular matrix (ECM) protein that anchors AChE to the synaptic basal lamina. Biglycan, encoded by BGN, is another ECM protein that binds to the dystrophin-associated protein complex (DAPC) on skeletal muscle, which links the actin cytoskeleton and ECM proteins to stabilize the sarcolemma during repeated muscle contractions. Upregulation of biglycan stabilizes the DPAC. Gene therapy can potentially ameliorate any disease that can be recapitulated in cultured cells. However, the difficulty of tissue-specific and developmental stage-specific regulated expression of transgenes, as well as the difficulty of introducing a transgene into all cells in a specific tissue, prevents us from successfully applying gene therapy to many human diseases. In contrast to intracellular proteins, an ECM protein is anchored to the target tissue via its specific binding affinity for protein(s) expressed on the cell surface within the target tissue. Exploiting this unique feature of ECM proteins, we developed protein-anchoring therapy in which a transgene product expressed even in remote tissues can be delivered and anchored to a target tissue using specific binding signals. We demonstrate the application of protein-anchoring therapy to two disease models. First, intravenous administration of adeno-associated virus (AAV) serotype 8-COLQ to Colq-deficient mice, resulting in specific anchoring of ectopically expressed ColQ-AChE at the NMJ, markedly improved motor functions, synaptic transmission, and the ultrastructure of the neuromuscular junction (NMJ). In the second example, Mdx mice, a model for Duchenne muscular dystrophy, were intravenously injected with AAV8-BGN. The treatment ameliorated motor deficits, mitigated muscle histopathologies, decreased plasma creatine kinase activities, and upregulated expression of utrophin and DAPC component proteins. We propose that protein-anchoring therapy could be applied to hereditary/acquired defects in ECM and secreted proteins, as well as therapeutic overexpression of such factors. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Deep insights into the mode of ATP-binding mechanism in Zebrafish cyclin-dependent protein kinase-like 1 (zCDKL1): A molecular dynamics approach.

PubMed

Rout, Ajaya Kumar; Dehury, Budheswar; Maharana, Jitendra; Nayak, Chirasmita; Baisvar, Vishwamitra Singh; Behera, Bijay Kumar; Das, Basanta Kumar

2018-05-01

In eukaryotes, the serine/threonine kinases (STKs) belonging to cyclin-dependent protein kinases (CDKs) play significant role in control of cell division and curb transcription in response to several extra and intra-cellular signals indispensable for enzymatic activity. The zebrafish cyclin-dependent protein kinase-like 1 protein (zCDKL1) shares a high degree of sequence and structural similarity with mammalian orthologs and express in brain, ovary, testis, and low levels in other tissues. Regardless of its importance in the developmental process, the structure, function and mode of ATP recognition have not been investigated yet due to lack of experimental data. Henceforth, to gain atomistic insights in to the structural dynamics and mode of ATP binding, a series of computational techniques involving theoretical modeling, docking, molecular dynamics (MD) simulations and MM/PBSA binding free energies were employed. The modeled bi-lobed zCDKL1 shares a high degree of secondary structure topology with human orthologs where ATP prefers to lie in the central cavity of the bi-lobed catalytic domain enclosed by strong hydrogen bonding, electrostatic and hydrophobic contacts. Long range MD simulation portrayed that catalytic domain of zCDKL1 to be highly rigid in nature as compared to the complex (zCDKL1-ATP) form. Comparative analysis with its orthologs revealed that conserved amino acids i.e., Ile10, Gly11, Glu12, Val18, Arg31, Phe80, Glu 130, Cys143 and Asp144 were crucial for ATP binding mechanism, which needs further investigation for legitimacy. MM/PBSA method revealed that van der Waals, electrostatic and polar solvation energy mostly contributes towards negative free energy. The implications of ATP binding mechanism inferred through these structural bioinformatics approaches will help in understanding the catalytic mechanisms of important STKs in eukaryotic system. Copyright © 2018. Published by Elsevier Inc.

Developmental changes in the distribution of cecal lectin-binding sites of Balb-c mice.

PubMed

Doehrn, S; Breipohl, W; Lierse, W; Romaniuk, K; Young, W

1992-01-01

The existence of lectin-binding sites was investigated in the cecum of Balb-c mice at seven developmental stages ranging from 18 days post conception (p.c.) to 8 weeks after birth. Nine horseradish-peroxidase-conjugated lectins (concanavalin A, Triticum vulgaris, Dolichus biflorus, Helix pomatia, Arachis hypogaea, Glycine maximus, Lotus tetragonolobus, Ulex europaeus, Limulus polyphemus) were applied to 5- to 7-microns thin paraffin sections of Bouin-fixed tissue. After DAB staining the sections were evaluated by light microscopy. It was shown that each lectin exhibits a unique developmental pattern. The adult binding patterns were established at the age of 3-4 weeks with only minor changes occurring thereafter. Considerable differences in binding patterns occurred not only between lectins of different groups but also between lectins with the same nominal monosaccharide specificity.

Intracellular Transport and Kinesin Superfamily Proteins: Structure, Function and Dynamics

NASA Astrophysics Data System (ADS)

Hirokawa, N.; Takemura, R.

Using various molecular cell biological and molecular genetic approaches, we identified kinesin superfamily proteins (KIFs) and characterized their significant functions in intracellular transport, which is fundamental for cellular morphogenesis, functioning, and survival. We showed that KIFs not only transport various membranous organelles, proteins complexes and mRNAs fundamental for cellular functions but also play significant roles in higher brain functions such as memory and learning, determination of important developmental processes such as left-right asymmetry formation and brain wiring. We also elucidated that KIFs recognize and bind to their specific cargoes using scaffolding or adaptor protein complexes. Concerning the mechanism of motility, we discovered the simplest unique monomeric motor KIF1A and determined by molecular biophysics, cryoelectron microscopy and X-ray crystallography that KIF1A can move on a microtubule processively as a monomer by biased Brownian motion and by hydolyzing ATP.

Information Propagation in Developmental Enhancers

NASA Astrophysics Data System (ADS)

Jena, Siddhartha; Levine, Michael

Rather than encoding information about protein sequence, certain lengths of noncoding DNA, called enhancers, interact with protein machinery such as transcription factors to precisely regulate gene expression. Enhancers have been studied extensively in the fruit fly Drosophila melanogaster, where they regulate the expression of developmental genes that establish the blueprint of the adult fly. It has been suggested that enhancer sequences possess a specific but unknown syntax with regards to the placement and strength of transcription factor binding sites. Moreover, studies in divergent fly species have shown that compensatory evolution allows for maintenance of enhancer functionality despite considerable variation in primary DNA sequence. Here, the possible role of enhancers as signal processing modules is studied as a way of explaining these two findings. We first demonstrate how this framework can be used to explain the fine-tuned spatiotemporal dynamics of gene expression. We then explore the evolutionary pressure on enhancer sequences and the resulting emergence of enhancers that are linked by compensatory mutations. This study provides a possible mechanism for the function of multiple enhancers linked to a single gene.

Modelling fragile X syndrome in the laboratory setting: A behavioral perspective.

PubMed

Melancia, Francesca; Trezza, Viviana

2018-04-25

Fragile X syndrome is the most common form of inherited mental retardation and the most frequent monogenic cause of syndromic autism spectrum disorders. The syndrome is caused by the loss of the Fragile X Mental Retardation Protein (FMRP), a key RNA-binding protein involved in synaptic plasticity and neuronal morphology. Patients show intellectual disability, social deficits, repetitive behaviors and impairments in social communication. The aim of this review is to outline the importance of behavioral phenotyping of animal models of FXS from a developmental perspective, by showing how the behavioral characteristics of FXS at the clinical level can be translated into effective, developmentally-specific and clinically meaningful behavioral readouts in the laboratory setting. After introducing the behavioral features, diagnostic criteria and off-label pharmacotherapy of FXS, we outline how FXS-relevant behavioral features can be modelled in laboratory animals in the course of development: we review the progress to date, discuss how behavioral phenotyping in animal models of FXS is essential to identify potential treatments, and discuss caveats and future directions in this research field. Copyright © 2018. Published by Elsevier B.V.

Maternal Nanos-Dependent RNA Stabilization in the Primordial Germ Cells of Drosophila Embryos.

PubMed

Sugimori, Seiko; Kumata, Yuji; Kobayashi, Satoru

2018-01-01

Nanos (Nos) is an evolutionary conserved protein expressed in the germline of various animal species. In Drosophila, maternal Nos protein is essential for germline development. In the germline progenitors, or the primordial germ cells (PGCs), Nos binds to the 3' UTR of target mRNAs to repress their translation. In contrast to this prevailing role of Nos, here we report that the 3' UTR of CG32425 mRNA mediates Nos-dependent RNA stabilization in PGCs. We found that the level of mRNA expressed from a reporter gene fused to the CG32425 3' UTR was significantly reduced in PGCs lacking maternal Nos (nos PGCs) as compared with normal PGCs. By deleting the CG32425 3' UTR, we identified the region required for mRNA stabilization, which includes Nos-binding sites. In normal embryos, CG32425 mRNA was maternally supplied into PGCs and remained in this cell type during embryogenesis. However, as expected from our reporter assay, the levels of CG32425 mRNA and its protein product expressed in nos PGCs were lower than in normal PGCs. Thus, we propose that Nos protein has dual functions in translational repression and stabilization of specific RNAs to ensure proper germline development. © 2017 Japanese Society of Developmental Biologists.

Functional Analysis of the Epidermal-Specific MYB Genes CAPRICE and WEREWOLF in Arabidopsis[W

PubMed Central

Tominaga, Rumi; Iwata, Mineko; Okada, Kiyotaka; Wada, Takuji

2007-01-01

Epidermis cell differentiation in Arabidopsis thaliana is a model system for understanding the developmental end state of plant cells. Two types of MYB transcription factors, R2R3-MYB and R3-MYB, are involved in cell fate determination. To examine the molecular basis of this process, we analyzed the functional relationship of the R2R3-type MYB gene WEREWOLF (WER) and the R3-type MYB gene CAPRICE (CPC). Chimeric constructs made from the R3 MYB regions of WER and CPC used in reciprocal complementation experiments showed that the CPC R3 region cannot functionally substitute for the WER R3 region in the differentiation of hairless cells. However, WER R3 can substantially substitute for CPC R3. There are no differences in yeast interaction assays of WER or WER chimera proteins with GLABRA3 (GL3) or ENHANCER OF GLABRA3 (EGL3). CPC and CPC chimera proteins also have similar activity in preventing GL3 WER and EGL3 WER interactions. Furthermore, we showed by gel mobility shift assays that WER chimera proteins do not bind to the GL2 promoter region. However, a CPC chimera protein, which harbors the WER R3 motif, still binds to the GL2 promoter region. PMID:17644729

Functional analysis of the epidermal-specific MYB genes CAPRICE and WEREWOLF in Arabidopsis.

PubMed

Tominaga, Rumi; Iwata, Mineko; Okada, Kiyotaka; Wada, Takuji

2007-07-01

Epidermis cell differentiation in Arabidopsis thaliana is a model system for understanding the developmental end state of plant cells. Two types of MYB transcription factors, R2R3-MYB and R3-MYB, are involved in cell fate determination. To examine the molecular basis of this process, we analyzed the functional relationship of the R2R3-type MYB gene WEREWOLF (WER) and the R3-type MYB gene CAPRICE (CPC). Chimeric constructs made from the R3 MYB regions of WER and CPC used in reciprocal complementation experiments showed that the CPC R3 region cannot functionally substitute for the WER R3 region in the differentiation of hairless cells. However, WER R3 can substantially substitute for CPC R3. There are no differences in yeast interaction assays of WER or WER chimera proteins with GLABRA3 (GL3) or ENHANCER OF GLABRA3 (EGL3). CPC and CPC chimera proteins also have similar activity in preventing GL3 WER and EGL3 WER interactions. Furthermore, we showed by gel mobility shift assays that WER chimera proteins do not bind to the GL2 promoter region. However, a CPC chimera protein, which harbors the WER R3 motif, still binds to the GL2 promoter region.

Rice Dwarf Virus P2 Protein Hijacks Auxin Signaling by Directly Targeting the Rice OsIAA10 Protein, Enhancing Viral Infection and Disease Development

PubMed Central

Jin, Lian; Qin, Qingqing; Wang, Yu; Pu, Yingying; Liu, Lifang; Wen, Xing; Ji, Shaoyi; Wu, Jianguo; Wei, Chunhong; Li, Yi

2016-01-01

The phytohormone auxin plays critical roles in regulating myriads of plant growth and developmental processes. Microbe infection can disturb auxin signaling resulting in defects in these processes, but the underlying mechanisms are poorly understood. Auxin signaling begins with perception of auxin by a transient co-receptor complex consisting of an F-box transport inhibitor response 1/auxin signaling F-box (TIR1/AFB) protein and an auxin/indole-3-acetic acid (Aux/IAA) protein. Auxin binding to the co-receptor triggers ubiquitination and 26S proteasome degradation of the Aux/IAA proteins, leading to subsequent events, including expression of auxin-responsive genes. Here we report that Rice dwarf virus (RDV), a devastating pathogen of rice, causes disease symptoms including dwarfing, increased tiller number and short crown roots in infected rice as a result of reduced sensitivity to auxin signaling. The RDV capsid protein P2 binds OsIAA10, blocking the interaction between OsIAA10 and OsTIR1 and inhibiting 26S proteasome-mediated OsIAA10 degradation. Transgenic rice plants overexpressing wild-type or a dominant-negative (degradation-resistant) mutant of OsIAA10 phenocopy RDV symptoms are more susceptible to RDV infection; however, knockdown of OsIAA10 enhances the resistance of rice to RDV infection. Our findings reveal a previously unknown mechanism of viral protein reprogramming of a key step in auxin signaling initiation that enhances viral infection and pathogenesis. PMID:27606959

Prospects in the use of aptamers for characterizing the structure and stability of bioactive proteins and peptides in food.

PubMed

Agyei, Dominic; Acquah, Caleb; Tan, Kei Xian; Hii, Hieng Kok; Rajendran, Subin R C K; Udenigwe, Chibuike C; Danquah, Michael K

2018-01-01

Food-derived bioactive proteins and peptides have gained acceptance among researchers, food manufacturers and consumers as health-enhancing functional food components that also serve as natural alternatives for disease prevention and/or management. Bioactivity in food proteins and peptides is determined by their conformations and binding characteristics, which in turn depend on their primary and secondary structures. To maintain their bioactivities, the molecular integrity of bioactive peptides must remain intact, and this warrants the study of peptide form and structure, ideally with robust, highly specific and sensitive techniques. Short single-stranded nucleic acids (i.e. aptamers) are known to have high affinity for cognate targets such as proteins and peptides. Aptamers can be produced cost-effectively and chemically derivatized to increase their stability and shelf life. Their improved binding characteristics and minimal modification of the target molecular signature suggests their suitability for real-time detection of conformational changes in both proteins and peptides. This review discusses the developmental progress of systematic evolution of ligands by exponential enrichment (SELEX), an iterative technology for generating cost-effective aptamers with low dissociation constants (K d ) for monitoring the form and structure of bioactive proteins and peptides. The review also presents case studies of this technique in monitoring the structural stability of bioactive peptide formulations to encourage applications in functional foods. The challenges and potential of aptamers in this research field are also discussed. Graphical abstract Advancing bioactive proteins and peptide functionality via aptameric ligands.

Core Histones and HIRIP3, a Novel Histone-Binding Protein, Directly Interact with WD Repeat Protein HIRA

PubMed Central

Lorain, Stéphanie; Quivy, Jean-Pierre; Monier-Gavelle, Frédérique; Scamps, Christine; Lécluse, Yann; Almouzni, Geneviève; Lipinski, Marc

1998-01-01

The human HIRA gene has been named after Hir1p and Hir2p, two corepressors which together appear to act on chromatin structure to control gene transcription in Saccharomyces cerevisiae. HIRA homologs are expressed in a regulated fashion during mouse and chicken embryogenesis, and the human gene is a major candidate for the DiGeorge syndrome and related developmental disorders caused by a reduction to single dose of a fragment of chromosome 22q. Western blot analysis and double-immunofluorescence experiments using a specific antiserum revealed a primary nuclear localization of HIRA. Similar to Hir1p, HIRA contains seven amino-terminal WD repeats and probably functions as part of a multiprotein complex. HIRA and core histone H2B were found to physically interact in a yeast double-hybrid protein interaction trap, in GST pull-down assays, and in coimmunoprecipitation experiments performed from cellular extracts. In vitro, HIRA also interacted with core histone H4. H2B- and H4-binding domains were overlapping but distinguishable in the carboxy-terminal region of HIRA, and the region for HIRA interaction was mapped to the amino-terminal tail of H2B and the second α helix of H4. HIRIP3 (HIRA-interacting protein 3) is a novel gene product that was identified from its HIRA-binding properties in the yeast protein interaction trap. In vitro, HIRIP3 directly interacted with HIRA but also with core histones H2B and H3, suggesting that a HIRA-HIRIP3-containing complex could function in some aspects of chromatin and histone metabolism. Insufficient production of HIRA, which we report elsewhere interacts with homeodomain-containing DNA-binding factors during mammalian embryogenesis, could perturb the stoichiometric assembly of multimolecular complexes required for normal embryonic development. PMID:9710638

Selection of homeotic proteins for binding to a human DNA replication origin.

PubMed

de Stanchina, E; Gabellini, D; Norio, P; Giacca, M; Peverali, F A; Riva, S; Falaschi, A; Biamonti, G

2000-06-09

We have previously shown that a cell cycle-dependent nucleoprotein complex assembles in vivo on a 74 bp sequence within the human DNA replication origin associated to the Lamin B2 gene. Here, we report the identification, using a one-hybrid screen in yeast, of three proteins interacting with the 74 bp sequence. All of them, namely HOXA13, HOXC10 and HOXC13, are orthologues of the Abdominal-B gene of Drosophila melanogaster and are members of the homeogene family of developmental regulators. We describe the complete open reading frame sequence of HOXC10 and HOXC13 along with the structure of the HoxC13 gene. The specificity of binding of these two proteins to the Lamin B2 origin is confirmed by both band-shift and in vitro footprinting assays. In addition, the ability of HOXC10 and HOXC13 to increase the activity of a promoter containing the 74 bp sequence, as assayed by CAT-assay experiments, demonstrates a direct interaction of these homeoproteins with the origin sequence in mammalian cells. We also show that HOXC10 expression is cell-type-dependent and positively correlates with cell proliferation. Copyright 2000 Academic Press.

Combinatorial control of messenger RNAs by Pumilio, Nanos and Brain Tumor Proteins

PubMed Central

Arvola, René M.

2017-01-01

ABSTRACT Eukaryotes possess a vast array of RNA-binding proteins (RBPs) that affect mRNAs in diverse ways to control protein expression. Combinatorial regulation of mRNAs by RBPs is emerging as the rule. No example illustrates this as vividly as the partnership of 3 Drosophila RBPs, Pumilio, Nanos and Brain Tumor, which have overlapping functions in development, stem cell maintenance and differentiation, fertility and neurologic processes. Here we synthesize 30 y of research with new insights into their molecular functions and mechanisms of action. First, we provide an overview of the key properties of each RBP. Next, we present a detailed analysis of their collaborative regulatory mechanism using a classic example of the developmental morphogen, hunchback, which is spatially and temporally regulated by the trio during embryogenesis. New biochemical, structural and functional analyses provide insights into RNA recognition, cooperativity, and regulatory mechanisms. We integrate these data into a model of combinatorial RNA binding and regulation of translation and mRNA decay. We then use this information, transcriptome wide analyses and bioinformatics predictions to assess the global impact of Pumilio, Nanos and Brain Tumor on gene regulation. Together, the results support pervasive, dynamic post-transcriptional control. PMID:28318367

Combinatorial control of messenger RNAs by Pumilio, Nanos and Brain Tumor Proteins.

PubMed

Arvola, René M; Weidmann, Chase A; Tanaka Hall, Traci M; Goldstrohm, Aaron C

2017-11-02

Eukaryotes possess a vast array of RNA-binding proteins (RBPs) that affect mRNAs in diverse ways to control protein expression. Combinatorial regulation of mRNAs by RBPs is emerging as the rule. No example illustrates this as vividly as the partnership of 3 Drosophila RBPs, Pumilio, Nanos and Brain Tumor, which have overlapping functions in development, stem cell maintenance and differentiation, fertility and neurologic processes. Here we synthesize 30 y of research with new insights into their molecular functions and mechanisms of action. First, we provide an overview of the key properties of each RBP. Next, we present a detailed analysis of their collaborative regulatory mechanism using a classic example of the developmental morphogen, hunchback, which is spatially and temporally regulated by the trio during embryogenesis. New biochemical, structural and functional analyses provide insights into RNA recognition, cooperativity, and regulatory mechanisms. We integrate these data into a model of combinatorial RNA binding and regulation of translation and mRNA decay. We then use this information, transcriptome wide analyses and bioinformatics predictions to assess the global impact of Pumilio, Nanos and Brain Tumor on gene regulation. Together, the results support pervasive, dynamic post-transcriptional control.

The Cotton Kinesin-Like Calmodulin-Binding Protein Associates with Cortical Microtubles in Cotton Fibers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Preuss, Mary L.; Delmar, Deborah P.; Liu, Bo

Microtubules in interphase plant cells form a cortical array, which is critical for plant cell morphogenesis. Genetic studies imply that the minus end-directed microtubule motor kinesin-like calmodulin-binding protein (KCBP) plays a role in trichome morphogenesis in Arabidopsis. However, it was not clear whether this motor interacted with interphase microtubules. In cotton (Gossypium hirsutum) fibers, cortical microtubules undergo dramatic reorganization during fiber development. In this study, cDNA clones of the cotton KCBP homolog GhKCBP were isolated from a cotton fiber-specific cDNA library. During cotton fiber development from 10 to 21 DPA, the GhKCBP protein level gradually decreases. By immunofluorescence, GhKCBP wasmore » detected as puncta along cortical microtubules in fiber cells of different developmental stages. Thus the results provide evidence that GhKCBP plays a role in interphase cell growth likely by interacting with cortical microtubules. In contrast to fibers, in dividing cells of cotton, GhKCBP localized to the nucleus, the microtubule preprophase band, mitotic spindle, and the phragmoplast. Therefore KCBP likely exerts multiple roles in cell division and cell growth in flowering plants.« less

Tuning the chemosensory window

PubMed Central

Zhou, Shanshan; Mackay, Trudy FC

2010-01-01

Accurate perception of chemical signals from the environment is critical for the fitness of most animals. Drosophila melanogaster experiences its chemical environment through families of chemoreceptors that include olfactory receptors, gustatory receptors and odorant binding proteins. Its chemical environment, however, changes during its life cycle and the interpretation of chemical signals is dependent on dynamic social and physical surroundings. Phenotypic plasticity of gene expression of the chemoreceptor repertoire allows flies to adjust the chemosensory window through which they “view” their world and to modify the ensemble of expressed chemoreceptor proteins in line with their developmental and physiological state and according to their needs to locate food and oviposition sites under different social and physical environmental conditions. Furthermore, males and females differ in their expression profiles of chemoreceptor genes. Thus, each sex experiences its chemical environment via combinatorial activation of distinct chemoreceptor ensembles. The remarkable phenotypic plasticity of the chemoreceptor repertoire raises several fundamental questions. What are the mechanisms that translate environmental cues into regulation of chemoreceptor gene expression? How are gustatory and olfactory cues integrated perceptually? What is the relationship between ensembles of odorant binding proteins and odorant receptors? And, what is the significance of co-regulated chemoreceptor transcriptional networks? PMID:20305396

Structure–function studies of STAR family Quaking proteins bound to their in vivo RNA target sites

PubMed Central

Teplova, Marianna; Hafner, Markus; Teplov, Dmitri; Essig, Katharina; Tuschl, Thomas; Patel, Dinshaw J.

2013-01-01

Mammalian Quaking (QKI) and its Caenorhabditis elegans homolog, GLD-1 (defective in germ line development), are evolutionarily conserved RNA-binding proteins, which post-transcriptionally regulate target genes essential for developmental processes and myelination. We present X-ray structures of the STAR (signal transduction and activation of RNA) domain, composed of Qua1, K homology (KH), and Qua2 motifs of QKI and GLD-1 bound to high-affinity in vivo RNA targets containing YUAAY RNA recognition elements (RREs). The KH and Qua2 motifs of the STAR domain synergize to specifically interact with bases and sugar-phosphate backbones of the bound RRE. Qua1-mediated homodimerization generates a scaffold that enables concurrent recognition of two RREs, thereby plausibly targeting tandem RREs present in many QKI-targeted transcripts. Structure-guided mutations reduced QKI RNA-binding affinity in vitro and in vivo, and expression of QKI mutants in human embryonic kidney cells (HEK293) significantly decreased the abundance of QKI target mRNAs. Overall, our studies define principles underlying RNA target selection by STAR homodimers and provide insights into the post-transcriptional regulatory function of mammalian QKI proteins. PMID:23630077

Synthetic activation of caspases: Artificial death switches

PubMed Central

MacCorkle, Rebecca A.; Freeman, Kevin W.; Spencer, David M.

1998-01-01

The development of safe vectors for gene therapy requires fail-safe mechanisms to terminate therapy or remove genetically altered cells. The ideal “suicide switch” would be nonimmunogenic and nontoxic when uninduced and able to trigger cell death independent of tissue type or cell cycle stage. By using chemically induced dimerization, we have developed powerful death switches based on the cysteine proteases, caspase-1 ICE (interleukin-1β converting enzyme) and caspase-3 YAMA. In both cases, aggregation of the target protein is achieved by a nontoxic lipid-permeable dimeric FK506 analog that binds to the attached FK506-binding proteins, FKBPs. We find that intracellular cross-linking of caspase-1 or caspase-3 is sufficient to trigger rapid apoptosis in a Bcl-xL-independent manner, suggesting that these conditional proapoptotic molecules can bypass intracellular checkpoint genes, such as Bcl-xL, that limit apoptosis. Because these chimeric molecules are derived from autologous proteins, they should be nonimmunogenic and thus ideal for long-lived gene therapy vectors. These properties should also make chemically induced apoptosis useful for developmental studies, for treating hyperproliferative disorders, and for developing animal models to a wide variety of diseases. PMID:9520421

Feedback control of mammalian Hedgehog signaling by the Hedgehog-binding protein, Hip1, modulates Fgf signaling during branching morphogenesis of the lung

PubMed Central

Chuang, Pao-Tien; Kawcak, T'Nay; McMahon, Andrew P.

2003-01-01

Hedgehog (Hh) signaling plays a major role in multiple aspects of embryonic development. A key issue is how negative regulation of Hh signaling might contribute to generating differential responses over tens of cell diameters. In cells that respond to Hh, two proteins that are up-regulated are Patched1 (Ptch1), the Hh receptor, a general target in both invertebrate and vertebrate organisms, and Hip1, a Hh-binding protein that is vertebrate specific. To address the developmental role of Hip1 in the context of Hh signaling, we generated Hip1 mutants in the mouse. Loss of Hip1 function results in specific defects in two Hh target issues, the lung, a target of Sonic hedgehog (Shh) signaling, and the endochondral skeleton, a target of Indian hedgehog (Ihh) signaling. Hh signaling was up-regulated in Hip1 mutants, substantiating Hip1's general role in negatively regulating Hh signaling. Our studies focused on Hip1 in the lung. Here, a dynamic interaction between Hh and fibroblast growth factor (Fgf) signaling, modulated at least in part by Hip1, controls early lung branching. PMID:12569124

Feedback control of mammalian Hedgehog signaling by the Hedgehog-binding protein, Hip1, modulates Fgf signaling during branching morphogenesis of the lung.

PubMed

Chuang, Pao-Tien; Kawcak, T'Nay; McMahon, Andrew P

2003-02-01

Hedgehog (Hh) signaling plays a major role in multiple aspects of embryonic development. A key issue is how negative regulation of Hh signaling might contribute to generating differential responses over tens of cell diameters. In cells that respond to Hh, two proteins that are up-regulated are Patched1 (Ptch1), the Hh receptor, a general target in both invertebrate and vertebrate organisms, and Hip1, a Hh-binding protein that is vertebrate specific. To address the developmental role of Hip1 in the context of Hh signaling, we generated Hip1 mutants in the mouse. Loss of Hip1 function results in specific defects in two Hh target issues, the lung, a target of Sonic hedgehog (Shh) signaling, and the endochondral skeleton, a target of Indian hedgehog (Ihh) signaling. Hh signaling was up-regulated in Hip1 mutants, substantiating Hip1's general role in negatively regulating Hh signaling. Our studies focused on Hip1 in the lung. Here, a dynamic interaction between Hh and fibroblast growth factor (Fgf) signaling, modulated at least in part by Hip1, controls early lung branching.

Functional conservation of MBD proteins: MeCP2 and Drosophila MBD proteins alter sleep.

PubMed

Gupta, T; Morgan, H R; Bailey, J A; Certel, S J

2016-11-01

Proteins containing a methyl-CpG-binding domain (MBD) bind 5mC and convert the methylation pattern information into appropriate functional cellular states. The correct readout of epigenetic marks is of particular importance in the nervous system where abnormal expression or compromised MBD protein function, can lead to disease and developmental disorders. Recent evidence indicates that the genome of Drosophila melanogaster is methylated and two MBD proteins, dMBD2/3 and dMBD-R2, are present. Are Drosophila MBD proteins required for neuronal function, and as MBD-containing proteins have diverged and evolved, does the MBD domain retain the molecular properties required for conserved cellular function across species? To address these questions, we expressed the human MBD-containing protein, hMeCP2, in distinct amine neurons and quantified functional changes in sleep circuitry output using a high throughput assay in Drosophila. hMeCP2 expression resulted in phase-specific sleep loss and sleep fragmentation with the hMeCP2-mediated sleep deficits requiring an intact MBD domain. Reducing endogenous dMBD2/3 and dMBD-R2 levels also generated sleep fragmentation, with an increase in sleep occurring upon dMBD-R2 reduction. To examine if hMeCP2 and dMBD-R2 are targeting common neuronal functions, we reduced dMBD-R2 levels in combination with hMeCP2 expression and observed a complete rescue of sleep deficits. Furthermore, chromosomal binding experiments indicate MBD-R2 and MeCP2 associate on shared genomic loci. Our results provide the first demonstration that Drosophila MBD-containing family members are required for neuronal function and suggest that the MBD domain retains considerable functional conservation at the whole organism level across species. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Splicing regulation and dysregulation of cholinergic genes expressed at the neuromuscular junction.

PubMed

Ohno, Kinji; Rahman, Mohammad Alinoor; Nazim, Mohammad; Nasrin, Farhana; Lin, Yingni; Takeda, Jun-Ichi; Masuda, Akio

2017-08-01

We humans have evolved by acquiring diversity of alternative RNA metabolisms including alternative means of splicing and transcribing non-coding genes, and not by acquiring new coding genes. Tissue-specific and developmental stage-specific alternative RNA splicing is achieved by tightly regulated spatiotemporal regulation of expressions and activations of RNA-binding proteins that recognize their cognate splicing cis-elements on nascent RNA transcripts. Genes expressed at the neuromuscular junction are also alternatively spliced. In addition, germline mutations provoke aberrant splicing by compromising binding of RNA-binding proteins, and cause congenital myasthenic syndromes (CMS). We present physiological splicing mechanisms of genes for agrin (AGRN), acetylcholinesterase (ACHE), MuSK (MUSK), acetylcholine receptor (AChR) α1 subunit (CHRNA1), and collagen Q (COLQ) in human, and their aberration in diseases. Splicing isoforms of AChE T , AChE H , and AChE R are generated by hnRNP H/F. Skipping of MUSK exon 10 makes a Wnt-insensitive MuSK isoform, which is unique to human. Skipping of exon 10 is achieved by coordinated binding of hnRNP C, YB-1, and hnRNP L to exon 10. Exon P3A of CHRNA1 is alternatively included to generate a non-functional AChR α1 subunit in human. Molecular dissection of splicing mutations in patients with CMS reveals that exon P3A is alternatively skipped by hnRNP H, polypyrimidine tract-binding protein 1, and hnRNP L. Similarly, analysis of an exonic mutation in COLQ exon 16 in a CMS patient discloses that constitutive splicing of exon 16 requires binding of serine arginine-rich splicing factor 1. Intronic and exonic splicing mutations in CMS enable us to dissect molecular mechanisms underlying alternative and constitutive splicing of genes expressed at the neuromuscular junction. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms. © 2017 International Society for Neurochemistry.

MURC, a Muscle-Restricted Coiled-Coil Protein That Modulates the Rho/ROCK Pathway, Induces Cardiac Dysfunction and Conduction Disturbance▿

PubMed Central

Ogata, Takehiro; Ueyama, Tomomi; Isodono, Koji; Tagawa, Masashi; Takehara, Naofumi; Kawashima, Tsuneaki; Harada, Koichiro; Takahashi, Tomosaburo; Shioi, Tetsuo; Matsubara, Hiroaki; Oh, Hidemasa

2008-01-01

We identified a novel muscle-restricted putative coiled-coil protein, MURC, which is evolutionarily conserved from frog to human. MURC was localized to the cytoplasm with accumulation in the Z-line of the sarcomere in the murine adult heart. MURC mRNA expression in the heart increased during the developmental process from the embryonic stage to adulthood. In response to pressure overload, MURC mRNA expression increased in the hypertrophied heart. Using the yeast two-hybrid system, we identified the serum deprivation response (SDPR) protein, a phosphatidylserine-binding protein, as a MURC-binding protein. MURC induced activation of the RhoA/ROCK pathway, which modulated serum response factor-mediated atrial natriuretic peptide (ANP) expression and myofibrillar organization. SDPR augmented MURC-induced transactivation of the ANP promoter in cardiomyocytes, and RNA interference of SDPR attenuated the action of MURC on the ANP promoter. Transgenic mice expressing cardiac-specific MURC (Tg-MURC) exhibited cardiac contractile dysfunction and atrioventricular (AV) conduction disturbances with atrial chamber enlargement, reduced thickness of the ventricular wall, and interstitial fibrosis. Spontaneous episodes of atrial fibrillation and AV block were observed in Tg-MURC mice. These findings indicate that MURC modulates RhoA signaling and that MURC plays an important role in the development of cardiac dysfunction and conduction disturbance with increased vulnerability to atrial arrhythmias. PMID:18332105

MURC, a muscle-restricted coiled-coil protein that modulates the Rho/ROCK pathway, induces cardiac dysfunction and conduction disturbance.

PubMed

Ogata, Takehiro; Ueyama, Tomomi; Isodono, Koji; Tagawa, Masashi; Takehara, Naofumi; Kawashima, Tsuneaki; Harada, Koichiro; Takahashi, Tomosaburo; Shioi, Tetsuo; Matsubara, Hiroaki; Oh, Hidemasa

2008-05-01

We identified a novel muscle-restricted putative coiled-coil protein, MURC, which is evolutionarily conserved from frog to human. MURC was localized to the cytoplasm with accumulation in the Z-line of the sarcomere in the murine adult heart. MURC mRNA expression in the heart increased during the developmental process from the embryonic stage to adulthood. In response to pressure overload, MURC mRNA expression increased in the hypertrophied heart. Using the yeast two-hybrid system, we identified the serum deprivation response (SDPR) protein, a phosphatidylserine-binding protein, as a MURC-binding protein. MURC induced activation of the RhoA/ROCK pathway, which modulated serum response factor-mediated atrial natriuretic peptide (ANP) expression and myofibrillar organization. SDPR augmented MURC-induced transactivation of the ANP promoter in cardiomyocytes, and RNA interference of SDPR attenuated the action of MURC on the ANP promoter. Transgenic mice expressing cardiac-specific MURC (Tg-MURC) exhibited cardiac contractile dysfunction and atrioventricular (AV) conduction disturbances with atrial chamber enlargement, reduced thickness of the ventricular wall, and interstitial fibrosis. Spontaneous episodes of atrial fibrillation and AV block were observed in Tg-MURC mice. These findings indicate that MURC modulates RhoA signaling and that MURC plays an important role in the development of cardiac dysfunction and conduction disturbance with increased vulnerability to atrial arrhythmias.

Multiple Targets of Salicylic Acid and Its Derivatives in Plants and Animals

PubMed Central

Klessig, Daniel F.; Tian, Miaoying; Choi, Hyong Woo

2016-01-01

Salicylic acid (SA) is a critical plant hormone that is involved in many processes, including seed germination, root initiation, stomatal closure, floral induction, thermogenesis, and response to abiotic and biotic stresses. Its central role in plant immunity, although extensively studied, is still only partially understood. Classical biochemical approaches and, more recently, genome-wide high-throughput screens have identified more than two dozen plant SA-binding proteins (SABPs), as well as multiple candidates that have yet to be characterized. Some of these proteins bind SA with high affinity, while the affinity of others exhibit is low. Given that SA levels vary greatly even within a particular plant species depending on subcellular location, tissue type, developmental stage, and with respect to both time and location after an environmental stimulus such as infection, the presence of SABPs exhibiting a wide range of affinities for SA may provide great flexibility and multiple mechanisms through which SA can act. SA and its derivatives, both natural and synthetic, also have multiple targets in animals/humans. Interestingly, many of these proteins, like their plant counterparts, are associated with immunity or disease development. Two recently identified SABPs, high mobility group box protein and glyceraldehyde 3-phosphate dehydrogenase, are critical proteins that not only serve key structural or metabolic functions but also play prominent roles in disease responses in both kingdoms. PMID:27303403

Proteomic analysis of JAZ interacting proteins under methyl jasmonate treatment in finger millet.

PubMed

Sen, Saswati; Kundu, Sangeeta; Dutta, Samir Kr

2016-11-01

Jasmonic acid (JA) signaling pathway in plants is activated against various developmental processes as well as biotic and abiotic stresses. The Jasmonate ZIM-domain (JAZ) protein family, the key regulator of plant JA signaling pathway, also participates in phytohormone crosstalk. This is the first study revealing the in vivo interactions of finger millet (Eleusine coracana (L.) Gaertn.) JAZ protein (EcJAZ) under methyl jasmonate (MJ) treatment. The aim of the study was to explore not only the JA signaling pathway but also the phytohormone signaling crosstalk of finger millet, a highly important future crop. From the MJ-treated finger millet seedlings, the EcJAZ interacting proteins were purified by affinity chromatography with the EcJAZ-matrix. Twenty-one proteins of varying functionalities were successfully identified by MALDI-TOF-TOF Mass spectrometry. Apart from the previously identified JAZ binding proteins, most prominently, EcJAZ was found to interact with transcription factors like NAC, GATA and also with Cold responsive protein (COR), etc. that might have extended the range of functionalities of JAZ proteins. Moreover, to evaluate the interactions of EcJAZ in the JA-co-receptor complex, we generated ten in-silico models containing the EcJAZ degron and the COI1-SKP1 of five monocot cereals viz., rice, wheat, maize, Sorghum and Setaria with JA-Ile or coronatine. Our results indicated that the EcJAZ protein of finger millet could act as the signaling hub for the JA and other phytohormone signaling pathways, in response to a diverse set of stressors and developmental cues to provide survival fitness to the plant. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Developmental Considerations of Sperm Protein 17 Gene Expression in Rheumatoid Arthritis Synoviocytes

PubMed Central

Takeoka, Yuichi; Kenny, Thomas P.; Yago, Hisashi; Naiki, Mitsuru; Gershwin, M. Eric; Robbins, Dick L.

2002-01-01

Rheumatoid arthritis (RA) is an autoimmune disease characterized by proliferative synovial tissue. We used mRNA differential display and library subtraction to compare mRNA expression in RA and osteoarthritis (OA) synoviocytes. We initially compared the mRNA expression patterns in 1 female RA and 1 OA synovia and found a differentially expressed 350 bp transcript in the RA synoviocytes which was, by sequence analysis, 100% homologous to sperm protein 17 (Sp17). Moreover, the Sp17 transcript was found differentially expressed in a RA synovial library that was subtracted with an OA synovial library. Using specific primers for full length Sp17, a 1.1 kb transcript was amplified from the synoviocytes of 7 additional female RA patients, sequenced and found to 100% homologous to Sp17. Thus, we found the unexpected expression of Sp17, a thought to be gamete-specific protein, in the synoviocytes of 8/8 female RA patients in contrast to control OA synoviocytes. Interestingly, Sp17's structural relationship with cell-binding and recognition proteins, suggests that Sp17 may function in cell-cell recognition and signaling in the RA synoviocyte. Further, Sp17 could have a significant regulatory role in RA synoviocyte gene transcription and/or signal transduction. Thus, Sp17 could have an important role in RA synoviocyte proliferation or defective apoptosis. Finally, the presence of Sp17 in synoviocytes has interesting developmental considerations. PMID:12739786

Post-synaptic BDNF-TrkB Signaling in Synapse Maturation, Plasticity and Disease

PubMed Central

Yoshii, Akira; Constantine-Paton, Martha

2010-01-01

Brain-derived neurotrophic factor (BDNF) is a prototypic neurotrophin that regulates diverse developmental events from the selection of neural progenitors to the terminal dendritic differentiation and connectivity of neurons. We focus here on activity-dependent synaptic regulation by BDNF and its receptor, full length TrkB. BDNF-TrkB signaling is involved in transcription, translation, and trafficking of proteins during various phases of synaptic development and has been implicated in several forms of synaptic plasticity. These functions are carried out by a combination of the three signaling cascades triggered when BDNF binds TrkB: the mitogen-activated protein kinase (MAPK), the phospholipase Cγ (PLC PLCγ), and the phosphatidylinositol 3-kinase (PI3K) pathways. MAPK and PI3K play crucial roles in both translation and/or trafficking of proteins induced by synaptic activity while PLCγ regulates intracellular Ca2+ that can drive transcription via cyclic AMP and a Protein Kinase C. Conversely, the abnormal regulation of BDNF is implicated in various developmental and neurodegenerative diseases that perturb neural development and function. We will discuss the current state of understanding BDNF signaling in the context of synaptic development and plasticity with a focus on the post-synaptic cell and close with the evidence that basic mechanisms of BDNF function still need to be understood in order to effectively treat genetic disruptions of these pathways that cause devastating neurodevelopmental diseases. PMID:20186705

Local requirement of the Drosophila insulin binding protein imp-L2 in coordinating developmental progression with nutritional conditions.

PubMed

Sarraf-Zadeh, Ladan; Christen, Stefan; Sauer, Uwe; Cognigni, Paola; Miguel-Aliaga, Irene; Stocker, Hugo; Köhler, Katja; Hafen, Ernst

2013-09-01

In Drosophila, growth takes place during the larval stages until the formation of the pupa. Starvation delays pupariation to allow prolonged feeding, ensuring that the animal reaches an appropriate size to form a fertile adult. Pupariation is induced by a peak of the steroid hormone ecdysone produced by the prothoracic gland (PG) after larvae have reached a certain body mass. Local downregulation of the insulin/insulin-like growth factor signaling (IIS) activity in the PG interferes with ecdysone production, indicating that IIS activity in the PG couples the nutritional state to development. However, the underlying mechanism is not well understood. In this study we show that the secreted Imaginal morphogenesis protein-Late 2 (Imp-L2), a growth inhibitor in Drosophila, is involved in this process. Imp-L2 inhibits the activity of the Drosophila insulin-like peptides by direct binding and is expressed by specific cells in the brain, the ring gland, the gut and the fat body. We demonstrate that Imp-L2 is required to regulate and adapt developmental timing to nutritional conditions by regulating IIS activity in the PG. Increasing Imp-L2 expression at its endogenous sites using an Imp-L2-Gal4 driver delays pupariation, while Imp-L2 mutants exhibit a slight acceleration of development. These effects are strongly enhanced by starvation and are accompanied by massive alterations of ecdysone production resulting most likely from increased Imp-L2 production by neurons directly contacting the PG and not from elevated Imp-L2 levels in the hemolymph. Taken together our results suggest that Imp-L2-expressing neurons sense the nutritional state of Drosophila larvae and coordinate dietary information and ecdysone production to adjust developmental timing under starvation conditions. Copyright © 2013 Elsevier Inc. All rights reserved.

The sarcomeric Z-disc component myopodin is a multiadapter protein that interacts with filamin and alpha-actinin.

PubMed

Linnemann, Anja; van der Ven, Peter F M; Vakeel, Padmanabhan; Albinus, Britta; Simonis, Dirk; Bendas, Gerd; Schenk, Jörg A; Micheel, Burkhard; Kley, Rudolf A; Fürst, Dieter O

2010-09-01

Here we introduce myopodin as a novel filamin C binding partner. Corroborative yeast two-hybrid and biochemical analyses indicate that the central part of myopodin that shows high homology to the closely related protein synaptopodin and that is common to all its currently known or predicted variants interacts with filamin C immunoglobulin-like domains 20-21. A detailed characterization of the previously described interaction between myopodin and alpha-actinin demonstrates for the first time that myopodin contains three independent alpha-actinin-binding sites. Newly developed myopodin-specific antibodies reveal expression at the earliest stages of in vitro differentiation of human skeletal muscle cells preceding the expression of sarcomeric alpha-actinin. Myopodin colocalizes with filamin and alpha-actinin during all stages of muscle development. By contrast, colocalization with its previously identified binding partner zyxin is restricted to early developmental stages. Genetic and cellular analyses of skeletal muscle provided direct evidence for an alternative transcriptional start site in exon three, corroborating the expression of a myopodin variant lacking the PDZ domain encoded by exons 1 and 2 in skeletal muscle. We conclude that myopodin is a multiadapter protein of the sarcomeric Z-disc that links nascent myofibrils to the sarcolemma via zyxin, and might play a role in early assembly and stabilization of the Z-disc. Mutations in FLNC, ACTN2 and several other genes encoding Z-disc-related proteins cause myopathy and cardiomyopathy. Its localization and its association with the myopathy-associated proteins filamin C and alpha-actinin make myopodin an interesting candidate for a muscle disease gene. Copyright 2010 Elsevier GmbH. All rights reserved.

SLXL1, a novel acrosomal protein, interacts with DKKL1 and is involved in fertilization in mice.

PubMed

Zhuang, Xin-jie; Hou, Xiao-jun; Liao, Shang-Ying; Wang, Xiu-Xia; Cooke, Howard J; Zhang, Ming; Han, Chunsheng

2011-01-01

Spermatogenesis is a complex cellular developmental process which involves diverse families of genes. The Xlr (X-linked, lymphocyte regulated) family includes multiple members, only a few of which have reported functions in meiosis, post-meiotic maturation, and fertilization of germ cells. Slx-like1 (Slxl1) is a member of the Xlr family, whose expression and function in spermatogenesis need to be elucidated. The mRNA and protein expression and localization of Slxl1 were investigated by RT-PCR, Western blotting and immunohistochemistry in different tissues and at different stages of spermatogenesis. The interacting partner of SLXL1 was examined by co-immunoprecipitation and co-localization. Assessment of the role of SLXL1 in capacitation, acrosome reaction, zona pellucida binding/penetration, and fertilization was carried out in vitro using blocking antisera. The results showed that Slxl1 mRNA and protein were specifically expressed in the testis. SLXL1 was exclusively located in the acrosome of post-meiotic germ cells and interacts with DKKL1 (Dickkopf-like1), which is an acrosome-associated protein and plays an important role in fertilization. The rates of zona pellucida binding/penetration and fertilization were significantly reduced by the anti-SLXL1 polyclonal antiserum. SLXL1 is the first identified member of the XLR family that is associated with acrosome and is involved in zona pellucid binding/penetration and subsequent fertilization. These results, together with previous studies, suggest that Xlr family members participate in diverse processes from meiosis to fertilization during spermatogenesis.

Regulatory elements in vivo in the promoter of the abscisic acid responsive gene rab17 from maize.

PubMed

Busk, P K; Jensen, A B; Pagès, M

1997-06-01

The rab17 gene from maize is transcribed in late embryonic development and is responsive to abscisic acid and water stress in embryo and vegetative tissues. In vivo footprinting and transient transformation of rab17 were performed in embryos and vegetative tissues to characterize the cis-elements involved in regulation of the gene. By in vivo footprinting, protein binding was observed to nine elements in the promoter, which correspond to five putative ABREs (abscisic acid responsive elements) and four other sequences. The footprints indicated that distinct proteins interact with these elements in the two developmental stages. In transient transformation, six of the elements were important for high level expression of the rab17 promoter in embryos, whereas only three elements were important in leaves. The cis-acting sequences can be divided in embryo-specific, ABA-specific and leaf-specific elements on the basis of protein binding and the ability to confer expression of rab17. We found one positive, new element, called GRA, with the sequence CACTGGCCGCCC. This element was important for transcription in leaves but not in embryos. Two other non-ABRE elements that stimulated transcription from the rab17 promoter resemble previously described abscisic acid and drought-inducible elements. There were differences in protein binding and function of the five ABREs in the rab17 promoter. The possible reasons for these differences are discussed. The in vivo data obtained suggest that an embryo-specific pathway regulates transcription of the rab genes during development, whereas another pathway is responsible for induction in response to ABA and drought in vegetative tissues.

A Systemic Small RNA Signaling System in Plants

PubMed Central

Yoo, Byung-Chun; Kragler, Friedrich; Varkonyi-Gasic, Erika; Haywood, Valerie; Archer-Evans, Sarah; Lee, Young Moo; Lough, Tony J.; Lucas, William J.

2004-01-01

Systemic translocation of RNA exerts non-cell-autonomous control over plant development and defense. Long-distance delivery of mRNA has been proven, but transport of small interfering RNA and microRNA remains to be demonstrated. Analyses performed on phloem sap collected from a range of plants identified populations of small RNA species. The dynamic nature of this population was reflected in its response to growth conditions and viral infection. The authenticity of these phloem small RNA molecules was confirmed by bioinformatic analysis; potential targets for a set of phloem small RNA species were identified. Heterografting studies, using spontaneously silencing coat protein (CP) plant lines, also established that transgene-derived siRNA move in the long-distance phloem and initiate CP gene silencing in the scion. Biochemical analysis of pumpkin (Cucurbita maxima) phloem sap led to the characterization of C. maxima Phloem SMALL RNA BINDING PROTEIN1 (CmPSRP1), a unique component of the protein machinery probably involved in small RNA trafficking. Equivalently sized small RNA binding proteins were detected in phloem sap from cucumber (Cucumis sativus) and lupin (Lupinus albus). PSRP1 binds selectively to 25-nucleotide single-stranded RNA species. Microinjection studies provided direct evidence that PSRP1 could mediate the cell-to-cell trafficking of 25-nucleotide single-stranded, but not double-stranded, RNA molecules. The potential role played by PSRP1 in long-distance transmission of silencing signals is discussed with respect to the pathways and mechanisms used by plants to exert systemic control over developmental and physiological processes. PMID:15258266

Distribution of monoamine oxidase proteins in human brain: implications for brain imaging studies

PubMed Central

Tong, Junchao; Meyer, Jeffrey H; Furukawa, Yoshiaki; Boileau, Isabelle; Chang, Li-Jan; Wilson, Alan A; Houle, Sylvain; Kish, Stephen J

2013-01-01

Positron emission tomography (PET) imaging of monoamine oxidases (MAO-A: [11C]harmine, [11C]clorgyline, and [11C]befloxatone; MAO-B: [11C]deprenyl-D2) has been actively pursued given clinical importance of MAOs in human neuropsychiatric disorders. However, it is unknown how well PET outcome measures for the different radiotracers are quantitatively related to actual MAO protein levels. We measured regional distribution (n=38) and developmental/aging changes (21 hours to 99 years) of both MAOs by quantitative immunoblotting in autopsied normal human brain. MAO-A was more abundant than MAO-B in infants, which was reversed as MAO-B levels increased faster before 1 year and, unlike MAO-A, kept increasing steadily to senescence. In adults, regional protein levels of both MAOs were positively and proportionally correlated with literature postmortem data of MAO activities and binding densities. With the exception of [11C]befloxatone (binding potential (BP), r=0.61, P=0.15), correlations between regional PET outcome measures of binding in the literature and MAO protein levels were good (P<0.01) for [11C]harmine (distribution volume, r=0.86), [11C]clorgyline (λk3, r=0.82), and [11C]deprenyl-D2 (λk3 or modified Patlak slope, r=0.78 to 0.87), supporting validity of the latter imaging measures. However, compared with in vitro data, the latter PET measures underestimated regional contrast by ∼2-fold. Further studies are needed to address cause of the in vivo vs. in vitro nonproportionality. PMID:23403377

F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases.

PubMed

Liu, Suxuan; Xiong, Xinyu; Zhao, Xianxian; Yang, Xiaofeng; Wang, Hong

2015-05-09

Eukaryotic cell membrane dynamics change in curvature during physiological and pathological processes. In the past ten years, a novel protein family, Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain proteins, has been identified to be the most important coordinators in membrane curvature regulation. The F-BAR domain family is a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily that is associated with dynamic changes in cell membrane. However, the molecular basis in membrane structure regulation and the biological functions of F-BAR protein are unclear. The pathophysiological role of F-BAR protein is unknown. This review summarizes the current understanding of structure and function in the BAR domain superfamily, classifies F-BAR family proteins into nine subfamilies based on domain structure, and characterizes F-BAR protein structure, domain interaction, and functional relevance. In general, F-BAR protein binds to cell membrane via F-BAR domain association with membrane phospholipids and initiates membrane curvature and scission via Src homology-3 (SH3) domain interaction with its partner proteins. This process causes membrane dynamic changes and leads to seven important cellular biological functions, which include endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, via distinct signaling pathways determined by specific domain-binding partners. These cellular functions play important roles in many physiological and pathophysiological processes. We further summarize F-BAR protein expression and mutation changes observed in various diseases and developmental disorders. Considering the structure feature and functional implication of F-BAR proteins, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating extracellular matrix degradation, and transmitting signaling for cell proliferation.

A homeodomain transcription factor gene, PfMSX, activates expression of Pif gene in the pearl oyster Pinctada fucata.

PubMed

Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi

2014-01-01

We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5' flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster.

A Homeodomain Transcription Factor Gene, PfMSX, Activates Expression of Pif Gene in the Pearl Oyster Pinctada fucata

PubMed Central

Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi

2014-01-01

We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5′ flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster. PMID:25099698

A Functional Study of AUXILIN-LIKE1 and 2, Two Putative Clathrin Uncoating Factors in Arabidopsis[OPEN

PubMed Central

Adamowski, Maciek; Kania, Urszula

2018-01-01

Clathrin-mediated endocytosis (CME) is a cellular trafficking process in which cargoes and lipids are internalized from the plasma membrane into vesicles coated with clathrin and adaptor proteins. CME is essential for many developmental and physiological processes in plants, but its underlying mechanism is not well characterized compared with that in yeast and animal systems. Here, we searched for new factors involved in CME in Arabidopsis thaliana by performing tandem affinity purification of proteins that interact with clathrin light chain, a principal component of the clathrin coat. Among the confirmed interactors, we found two putative homologs of the clathrin-coat uncoating factor auxilin previously described in non-plant systems. Overexpression of AUXILIN-LIKE1 and AUXILIN-LIKE2 in Arabidopsis caused an arrest of seedling growth and development. This was concomitant with inhibited endocytosis due to blocking of clathrin recruitment after the initial step of adaptor protein binding to the plasma membrane. By contrast, auxilin-like1/2 loss-of-function lines did not present endocytosis-related developmental or cellular phenotypes under normal growth conditions. This work contributes to the ongoing characterization of the endocytotic machinery in plants and provides a robust tool for conditionally and specifically interfering with CME in Arabidopsis. PMID:29511054

Receptor-mediated signalling in plants: molecular patterns and programmes

PubMed Central

Tör, Mahmut; Lotze, Michael T.; Holton, Nicholas

2009-01-01

A highly evolved surveillance system in plants is able to detect a broad range of signals originating from pathogens, damaged tissues, or altered developmental processes, initiating sophisticated molecular mechanisms that result in defence, wound healing, and development. Microbe-associated molecular pattern molecules (MAMPs), damage-associated molecular pattern molecules (DAMPs), virulence factors, secreted proteins, and processed peptides can be recognized directly or indirectly by this surveillance system. Nucleotide binding-leucine rich repeat proteins (NB-LRR) are intracellular receptors and have been targeted by breeders for decades to elicit resistance to crop pathogens in the field. Receptor-like kinases (RLKs) or receptor like proteins (RLPs) are membrane bound signalling molecules with an extracellular receptor domain. They provide an early warning system for the presence of potential pathogens and activate protective immune signalling in plants. In addition, they act as a signal amplifier in the case of tissue damage, establishing symbiotic relationships and effecting developmental processes. The identification of several important ligands for the RLK-type receptors provided an opportunity to understand how plants differentiate, how they distinguish beneficial and detrimental stimuli, and how they co-ordinate the role of various types of receptors under varying environmental conditions. The diverse roles of extra-and intracellular plant receptors are examined here and the recent findings on how they promote defence and development is reviewed. PMID:19628572

Detection and Quantification of the Fragile X Mental Retardation Protein 1 (FMRP).

PubMed

LaFauci, Giuseppe; Adayev, Tatyana; Kascsak, Richard; Brown, W Ted

2016-12-09

The final product of FMR1 gene transcription, Fragile X Mental Retardation Protein 1 (FMRP), is an RNA binding protein that acts as a repressor of translation. FMRP is expressed in several tissues and plays important roles in neurogenesis, synaptic plasticity, and ovarian functions and has been implicated in a number of neuropsychological disorders. The loss of FMRP causes Fragile X Syndrome (FXS). In most cases, FXS is due to large expansions of a CGG repeat in FMR1 -normally containing 6-54 repeats-to over 200 CGGs and identified as full mutation (FM). Hypermethylation of the repeat induces FMR1 silencing and lack of FMRP expression in FM male. Mosaic FM males express low levels of FMRP and present a less severe phenotype that inversely correlates with FMRP levels. Carriers of pre-mutations (55-200 CGG) show increased mRNA, and normal to reduced FMRP levels. Alternative splicing of FMR1 mRNA results in 24 FMRP predicted isoforms whose expression are tissues and developmentally regulated. Here, we summarize the approaches used by several laboratories including our own to (a) detect and estimate the amount of FMRP in different tissues, developmental stages and various pathologies; and (b) to accurately quantifying FMRP for a direct diagnosis of FXS in adults and newborns.

Detection and Quantification of the Fragile X Mental Retardation Protein 1 (FMRP)

PubMed Central

LaFauci, Giuseppe; Adayev, Tatyana; Kascsak, Richard; Brown, W. Ted

2016-01-01

The final product of FMR1 gene transcription, Fragile X Mental Retardation Protein 1 (FMRP), is an RNA binding protein that acts as a repressor of translation. FMRP is expressed in several tissues and plays important roles in neurogenesis, synaptic plasticity, and ovarian functions and has been implicated in a number of neuropsychological disorders. The loss of FMRP causes Fragile X Syndrome (FXS). In most cases, FXS is due to large expansions of a CGG repeat in FMR1—normally containing 6–54 repeats—to over 200 CGGs and identified as full mutation (FM). Hypermethylation of the repeat induces FMR1 silencing and lack of FMRP expression in FM male. Mosaic FM males express low levels of FMRP and present a less severe phenotype that inversely correlates with FMRP levels. Carriers of pre-mutations (55–200 CGG) show increased mRNA, and normal to reduced FMRP levels. Alternative splicing of FMR1 mRNA results in 24 FMRP predicted isoforms whose expression are tissues and developmentally regulated. Here, we summarize the approaches used by several laboratories including our own to (a) detect and estimate the amount of FMRP in different tissues, developmental stages and various pathologies; and (b) to accurately quantifying FMRP for a direct diagnosis of FXS in adults and newborns. PMID:27941672

Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish.

PubMed

Callard, G V; Tchoudakova, A V; Kishida, M; Wood, E

2001-12-01

Teleost fish are characterized by exceptionally high levels of brain estrogen biosynthesis when compared to the brains of other vertebrates or to the ovaries of the same fish. Goldfish (Carassius auratus) and zebrafish (Danio rerio) have utility as complementary models for understanding the molecular basis and functional significance of exaggerated neural estrogen biosynthesis. Multiple cytochrome P450 aromatase (P450arom) cDNAs that derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (P450aromB>A) and ovary (P450aromA>B) and have a different developmental program (B>A) and response to estrogen upregulation (B only). As measured by increased P450aromB mRNA, a functional estrogen response system is first detected 24-48 h post-fertilization (hpf), consistent with the onset of estrogen receptor (ER) expression (alpha, beta, and gamma). The 5'-flanking region of the cyp19b gene has a TATA box, two estrogen response elements (EREs), an ERE half-site (ERE1/2), a nerve growth factor inducible-B protein (NGFI-B)/Nur77 responsive element (NBRE) binding site, and a sequence identical to the zebrafish GATA-2 gene neural specific enhancer. The cyp19a promoter region has TATA and CAAT boxes, a steroidogenic factor-1 (SF-1) binding site, and two aryl hydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) binding motifs. Both genes have multiple potential SRY/SOX binding sites (16 and 8 in cyp19b and cyp19a, respectively). Luciferase reporters have basal promoter activity in GH3 cells, but differences (a>b) are opposite to fish pituitary (b>a). When microinjected into fertilized zebrafish eggs, a cyp19b promoter-driven green fluorescent protein (GFP) reporter (but not cyp19a) is expressed in neurons of 30-48 hpf embryos, most prominently in retinal ganglion cells (RGCs) and their projections to optic tectum. Further studies are required to identify functionally relevant cis-elements and cellular factors, and to determine the regulatory role of estrogen in neurodevelopment.

Proteomes of hard and soft near-isogenic wheat lines reveal that kernel hardness is related to the amplification of a stress response during endosperm development.

PubMed

Lesage, Véronique S; Merlino, Marielle; Chambon, Christophe; Bouchet, Brigitte; Marion, Didier; Branlard, Gérard

2012-01-01

Wheat kernel texture, a major trait determining the end-use quality of wheat flour, is mainly influenced by puroindolines. These small basic proteins display in vitro lipid binding and antimicrobial properties, but their cellular functions during grain development remain unknown. To gain an insight into their biological function, a comparative proteome analysis of two near-isogenic lines (NILs) of bread wheat Triticum aestivum L. cv. Falcon differing in the presence or absence of the puroindoline-a gene (Pina) and kernel hardness, was performed. Proteomes of the two NILs were compared at four developmental stages of the grain for the metabolic albumin/globulin fraction and the Triton-extracted amphiphilic fraction. Proteome variations showed that, during grain development, folding proteins and stress-related proteins were more abundant in the hard line compared with the soft one. These results, taken together with ultrastructural observations showing that the formation of the protein matrix occurred earlier in the hard line, suggested that a stress response, possibly the unfolded protein response, is induced earlier in the hard NIL than in the soft one leading to earlier endosperm cell death. Quantification of the albumin/globulin fraction and amphiphilic proteins at each developmental stage strengthened this hypothesis as a plateau was revealed from the 500 °Cd stage in the hard NIL whereas synthesis continued in the soft one. These results open new avenues concerning the function of puroindolines which could be involved in the storage protein folding machinery, consequently affecting the development of wheat endosperm and the formation of the protein matrix.

Pbx3 Deficiency Results in Central Hypoventilation

PubMed Central

Rhee, Joon Whan; Arata, Akiko; Selleri, Licia; Jacobs, Yakop; Arata, Satoru; Onimaru, Hiroshi; Cleary, Michael L.

2004-01-01

Pbx proteins comprise a family of TALE (three amino acid loop extension) class homeodomain transcription factors that are implicated in developmental gene expression through their abilities to form hetero-oligomeric DNA-binding complexes and function as transcriptional regulators in numerous cell types. We demonstrate here that one member of this family, Pbx3, is expressed at high levels predominantly in the developing central nervous system, including a region of the medulla oblongata that is implicated in the control of respiration. Pbx3-deficient mice develop to term but die within a few hours of birth from central respiratory failure due to abnormal activity of inspiratory neurons in the medulla. This partially phenocopies the defect in mice deficient for Rnx, a metaHox homeodomain transcription factor, that we demonstrate here is capable of forming a DNA-binding complex with Pbx3. Rnx expression is unperturbed in Pbx3-deficient mice, but its ability to enhance transcription in vitro as a complex with TALE proteins is compromised in the absence of Pbx3. Thus, Pbx3 is essential for respiration and, like its DNA-binding partner Rnx, is critical for proper development of medullary respiratory control mechanisms. Pbx3-deficient mice provide a model for congenital central hypoventilation syndrome and suggest that Pbx3 mutations may promote the pathogenesis of this disorder. PMID:15466398

Drosophila tubulin-binding cofactor B is required for microtubule network formation and for cell polarity

PubMed Central

Baffet, Alexandre D.; Benoit, Béatrice; Januschke, Jens; Audo, Jennifer; Gourhand, Vanessa; Roth, Siegfried; Guichet, Antoine

2012-01-01

Microtubules (MTs) are essential for cell division, shape, intracellular transport, and polarity. MT stability is regulated by many factors, including MT-associated proteins and proteins controlling the amount of free tubulin heterodimers available for polymerization. Tubulin-binding cofactors are potential key regulators of free tubulin concentration, since they are required for α-β–tubulin dimerization in vitro. In this paper, we show that mutation of the Drosophila tubulin-binding cofactor B (dTBCB) affects the levels of both α- and β-tubulins and dramatically destabilizes the MT network in different fly tissues. However, we find that dTBCB is dispensable for the early MT-dependent steps of oogenesis, including cell division, and that dTBCB is not required for mitosis in several tissues. In striking contrast, the absence of dTBCB during later stages of oogenesis causes major defects in cell polarity. We show that dTBCB is required for the polarized localization of the axis-determining mRNAs within the oocyte and for the apico-basal polarity of the surrounding follicle cells. These results establish a developmental function for the dTBCB gene that is essential for viability and MT-dependent cell polarity, but not cell division. PMID:22855530

Longitudinal Investigation of Source Memory Reveals Different Developmental Trajectories for Item Memory and Binding

ERIC Educational Resources Information Center

Riggins, Tracy

2014-01-01

The present study used a cohort-sequential design to examine developmental changes in children's ability to bind items in memory during early and middle childhood. Three cohorts of children (aged 4, 6, or 8 years) were followed longitudinally for 3 years. Each year, children completed a source memory paradigm assessing memory for items and…

Specific Eph receptor-cytoplasmic effector signaling mediated by SAM-SAM domain interactions.

PubMed

Wang, Yue; Shang, Yuan; Li, Jianchao; Chen, Weidi; Li, Gang; Wan, Jun; Liu, Wei; Zhang, Mingjie

2018-05-11

The Eph receptor tyrosine kinase (RTK) family is the largest subfamily of RTKs playing critical roles in many developmental processes such as tissue patterning, neurogenesis and neuronal circuit formation, angiogenesis, etc. How the 14 Eph proteins, via their highly similar cytoplasmic domains, can transmit diverse and sometimes opposite cellular signals upon engaging ephrins is a major unresolved question. Here we systematically investigated the bindings of each SAM domain of Eph receptors to the SAM domains from SHIP2 and Odin, and uncover a highly specific SAM-SAM interaction-mediated cytoplasmic Eph-effector binding pattern. Comparative X-ray crystallographic studies of several SAM-SAM heterodimer complexes, together with biochemical and cell biology experiments, not only revealed the exquisite specificity code governing Eph/effector interactions but also allowed us to identify SAMD5 as a new Eph binding partner. Finally, these Eph/effector SAM heterodimer structures can explain many Eph SAM mutations identified in patients suffering from cancers and other diseases. © 2018, Wang et al.

The mechanism of dioxin toxicity: relationship to risk assessment.

PubMed Central

Birnbaum, L S

1994-01-01

Risk characterization involves hazard identification, determination of dose-response relationships, and exposure assessment. Improvement of the risk assessment process requires inclusion of the best available science. Recent findings in the area of dioxin toxicity have led to a major effort to reassess its risk. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), commonly referred to as "dioxin," is the most toxic member of a class of related chemicals including the polyhalogenated dibenzo-p-dioxins, dibenzofurans, biphenyls, naphthalenes, azo- and azoxy-benzenes, whose toxicities can be expressed as fractional equivalencies of TCDD. These chemicals exert their effects through interaction with a specific intracellular protein, the Ah receptor. While binding to the receptor is necessary, it is not sufficient to bring about a chain of events leading to various responses including enzyme induction, immunotoxicity, reproductive and endocrine effects, developmental toxicity, chloracne, tumor promotion, etc. Some of these responses appear to be linear at low doses. Immunotoxicity and effects on the reproductive system appear to be among the most sensitive responses. The Ah receptor functions as a transcriptional enhancer, interacting with a number of other regulatory proteins (heat shock proteins, kinases, translocases, DNA binding species). Interaction with specific base sequences in the DNA appear to be modulated by the presence of other growth factors, hormones and their receptors as well as other regulatory proteins. Thus, dioxin appears to function as a hormone, initiating a cascade of events that is dependent upon the environment of each cell and tissue. While Ah receptor variants exist, all vertebrates examined have demonstrated such a protein with similar numbers of receptors and binding affinity for TCDD. Most species respond similarly to dioxin and related compounds. While a given species may be an outlier for a given response, it will behave like other animals for other responses. For both in vivo and in vitro end points where animal and human data exist, such as enzyme induction, chloracne, immunotoxicity, developmental toxicity, and cancer, the sensitivity of humans appears similar to that of experimental animals. Current levels of environmental exposure to this class of chemicals may be resulting in subtle responses in populations at special risk such as subsistence fisherman and the developing infant, as well as in the general population. Increased understanding of the mechanism of dioxin's effects as well as elucidation of exposure-dose relationships is leading to the development of a biologically based dose-response model in the ongoing process of incorporating the best science into the risk assessment of TCDD and related compounds. PMID:7698077

Chemical synthesis of a Zwitterhormon, insulaxin, and of a relaxin-like bombyxin derivative.

PubMed

Büllesbach, E E; Steinetz, B G; Schwabe, C

1996-07-30

The structural motif of insulin and relaxin is frequently seen in molecules of divergent functions and origins. The insect developmental factor bombyxin, the relaxin-like factor from Leydig cells, and the insulin-like factor 4 (INSL4) all are made of two disulfide-linked chains and have one disulfide bond within the A-chain. The polyclonal antibody R6, which was raised against porcine relaxin, reacts with a wide variety of naturally occurring relaxins from primates, marine and terrestrial mammals, and elasmobranchs but does not recognize insulin. The antibody binds mainly to the arginines that occur in the N, N+4 positions in the B-chains of all relaxins which also constitute the receptor-binding site. The receptor-binding haptens were incorporated by total synthesis into human despentapeptide insulin and bombyxin II, a developmental factor from the silk moth Bombyx mori. In the process the insect factor became a perfect antigen for the anti-relaxin antibody, whereas the human insulin was transformed into a bona fide relaxin. The conversion was affected by changing four critical residues so that the insulin activity was retained to the extent of 10% of the original level. This, to the best of our knowledge, is the first designer protein to incorporate two unrelated biological functions in one primary sequence, and we are therefore proposing that, analogous to zwitterion, the generic name "Zwitterhormon" (German spelling) be used for this type of construct.

Characterization of two mosquito STATs, AaSTAT and CtSTAT. Differential regulation of tyrosine phosphorylation and DNA binding activity by lipopolysaccharide treatment and by Japanese encephalitis virus infection.

PubMed

Lin, Chang-Chi; Chou, Chih-Ming; Hsu, Ya-Li; Lien, Jih-Ching; Wang, Yu-Ming; Chen, Shui-Tsung; Tsai, Shu-Chuan; Hsiao, Pei-Wen; Huang, Chang-Jen

2004-01-30

Two mosquito STATs, AaSTAT and CtSTAT, have been cloned from Aedes albopictus and Culex tritaeniorhynchus mosquitoes, respectively. These two STATs are more similar to those of Drosophila, Anopheles, and mammalian STAT5 in the DNA binding and Src homology 2 domains. The mRNA transcripts are expressed at all developmental stages, and the proteins are present predominantly at the pupal and adult stages in both mosquitoes. Stimulation with lipopolysaccharide resulted in an increase of tyrosine phosphorylation and DNA binding activity of AaSTAT and CtSTAT as well as an increase of luciferase activity of a reporter gene containing Drosophila STAT binding motif in mosquito C6/36 cells. After being infected with Japanese encephalitis virus, nuclear extracts of C6/36 cells revealed a decrease of tyrosine phosphorylation and DNA binding activity of AaSTAT which could be restored by sodium orthovanadate treatment. Taking all of the data together, this is the first report to clone and characterize two mosquito STATs with 81% identity and to demonstrate a different response of tyrosine phosphorylation and DNA binding of these two STATs by lipopolysaccharide treatment and by Japanese encephalitis virus infection.

Widespread evidence of cooperative DNA binding by transcription factors in Drosophila development

PubMed Central

Kazemian, Majid; Pham, Hannah; Wolfe, Scot A.; Brodsky, Michael H.; Sinha, Saurabh

2013-01-01

Regulation of eukaryotic gene transcription is often combinatorial in nature, with multiple transcription factors (TFs) regulating common target genes, often through direct or indirect mutual interactions. Many individual examples of cooperative binding by directly interacting TFs have been identified, but it remains unclear how pervasive this mechanism is during animal development. Cooperative TF binding should be manifest in genomic sequences as biased arrangements of TF-binding sites. Here, we explore the extent and diversity of such arrangements related to gene regulation during Drosophila embryogenesis. We used the DNA-binding specificities of 322 TFs along with chromatin accessibility information to identify enriched spacing and orientation patterns of TF-binding site pairs. We developed a new statistical approach for this task, specifically designed to accurately assess inter-site spacing biases while accounting for the phenomenon of homotypic site clustering commonly observed in developmental regulatory regions. We observed a large number of short-range distance preferences between TF-binding site pairs, including examples where the preference depends on the relative orientation of the binding sites. To test whether these binding site patterns reflect physical interactions between the corresponding TFs, we analyzed 27 TF pairs whose binding sites exhibited short distance preferences. In vitro protein–protein binding experiments revealed that >65% of these TF pairs can directly interact with each other. For five pairs, we further demonstrate that they bind cooperatively to DNA if both sites are present with the preferred spacing. This study demonstrates how DNA-binding motifs can be used to produce a comprehensive map of sequence signatures for different mechanisms of combinatorial TF action. PMID:23847101

The Nedd4 binding protein 3 is required for anterior neural development in Xenopus laevis.

PubMed

Kiem, Lena-Maria; Dietmann, Petra; Linnemann, Alexander; Schmeisser, Michael J; Kühl, Susanne J

2017-03-01

The Fezzin family member Nedd4-binding protein 3 (N4BP3) is known to regulate axonal and dendritic branching. Here, we show that n4bp3 is expressed in the neural tissue of the early Xenopus laevis embryo including the eye, the brain and neural crest cells. Knockdown of N4bp3 in the Xenopus anterior neural tissue results in severe developmental impairment of the eye, the brain and neural crest derived cranial cartilage structures. Moreover, we demonstrate that N4bp3 depletion leads to a significant reduction of both eye and brain specific marker genes and reduced neural crest cell migration. Finally, we demonstrate an impact of N4bp3 deficiency on cell apoptosis and proliferation. Our studies indicate that N4bp3 is required for early anterior neural development of vertebrates. This is in line with a study implicating that genetic disruption of N4BP3 in humans might be related to neurodevelopmental disease. Copyright © 2017 Elsevier Inc. All rights reserved.

A conserved phosphorylation switch controls the interaction between cadherin and β-catenin in vitro and in vivo

DOE PAGES

Choi, Hee -Jung; Loveless, Timothy; Lynch, Allison M.; ...

2015-04-06

In metazoan adherens junctions, β-catenin links the cytoplasmic tail of classical cadherins to the F-actin-binding protein α-catenin. Phosphorylation of a Ser/Thr-rich region in the cadherin tail dramatically enhances affinity for β-catenin and promotes cell-cell adhesion in cell culture systems, but its importance has not been demonstrated in vivo. In this paper, we identify a critical phosphorylated serine in the C. elegans cadherin HMR-1 required for strong binding to the β-catenin homolog HMP-2. Ablation of this phosphoserine interaction produces developmental defects that resemble full loss-of-function (Hammerhead and Humpback) phenotypes. Most metazoans possess a single gene for β-catenin, which is also amore » transcriptional coactivator in Wnt signaling. Nematodes and planaria, however, have a set of paralogous β-catenins; for example, C. elegans HMP-2 functions only in cell-cell adhesion, whereas SYS-1 mediates transcriptional activation through interactions with POP-1/Tcf. Finally, our structural data define critical sequence differences responsible for the unique ligand specificities of these two proteins.« less

Rett Syndrome Mutation MeCP2 T158A Disrupts DNA Binding, Protein Stability and ERP Responses

PubMed Central

Goffin, Darren; Allen, Megan; Zhang, Le; Amorim, Maria; Wang, I-Ting Judy; Reyes, Arith-Ruth S.; Mercado-Berton, Amy; Ong, Caroline; Cohen, Sonia; Hu, Linda; Blendy, Julie A.; Carlson, Gregory C.; Siegel, Steve J.; Greenberg, Michael E.; Zhou, Zhaolan (Joe)

2011-01-01

Mutations in the MECP2 gene cause the autism spectrum disorder Rett Syndrome (RTT). One of the most common mutations associated with RTT occurs at MeCP2 Threonine 158 converting it to Methionine (T158M) or Alanine (T158A). To understand the role of T158 mutation in the pathogenesis of RTT, we generated knockin mice recapitulating MeCP2 T158A mutation. Here we show a causal role for T158A mutation in the development of RTT-like phenotypes including developmental regression, motor dysfunction, and learning and memory deficits. These phenotypes resemble those in Mecp2-null mice and manifest through a reduction in MeCP2 binding to methylated DNA and a decrease in MeCP2 protein stability. Importantly, the age-dependent development of event-related neuronal responses are disrupted by MeCP2 mutation, suggesting that impaired neuronal circuitry underlies the pathogenesis of RTT and that assessment of event-related potentials may serve as a biomarker for RTT and treatment evaluation. PMID:22119903

The Role of Dimerization in Raf Signaling | Center for Cancer Research

Cancer.gov

One frequently mutated pathway in a variety of cancers and developmental disorders is the Ras-Raf-MEK-ERK cascade. Normally, binding of a growth factor to its receptor switches on Ras, which, in turn, activates one or more of the Raf kinase family members, A-Raf, B-Raf, and C-Raf. Rafs perpetuate the signal by phosphorylating and activating MEK, another kinase that phosphorylates a third kinase, ERK. ERK then phosphorylates a number of key growth-, survival-, or differentiation-promoting targets. Of the proteins in the cascade, Rafs have the most complex regulatory mechanisms, including the ability to form dimers. Because the role that dimerization plays in Raf function has been unclear, researchers working with Deborah Morrison, Ph.D., Chief of CCR’s Laboratory of Cell and Developmental Signaling, decided to investigate its significance in normal and disease-associated Raf signaling.

LncRNA, a new component of expanding RNA-protein regulatory network important for animal sperm development.

PubMed

Zhang, Chenwang; Gao, Liuze; Xu, Eugene Yujun

2016-11-01

Spermatogenesis is one of the fundamental processes of sexual reproduction, present in almost all metazoan animals. Like many other reproductive traits, developmental features and traits of spermatogenesis are under strong selective pressure to change, both at morphological and underlying molecular levels. Yet evidence suggests that some fundamental features of spermatogenesis may be ancient and conserved among metazoan species. Identifying the underlying conserved molecular mechanisms could reveal core components of metazoan spermatogenic machinery and provide novel insight into causes of human infertility. Conserved RNA-binding proteins and their interacting RNA network emerge to be a common theme important for animal sperm development. We review research on the recent addition to the RNA family - Long non-coding RNA (lncRNA) and its roles in spermatogenesis in the context of the expanding RNA-protein network. Copyright © 2016 Elsevier Ltd. All rights reserved.

Stage-specific excretory/secretory small heat shock proteins from the parasitic nematode Strongyloides ratti: putative links to host’s intestinal mucosal defense system

PubMed Central

Younis, Abuelhassan Elshazly; Geisinger, Frank; Ajonina-Ekoti, Irene; Soblik, Hanns; Steen, Hanno; Mitreva, Makedonka; Erttmann, Klaus D.; Perbandt, Markus; Liebau, Eva; Brattig, Norbert W.

2013-01-01

SUMMARY In search of molecules involved in the interaction of intestinal nematodes and mammalian mucosal host cells, we performed mass spectrometry to identify excretory/secretory proteins (ESP) from Strongyloides ratti. In addition to other peptides, we detected in the ESP of parasitic female stage peptides homologous to the Caenorhabditis elegans heat shock protein-17, named Sra-HSP-17.1 (~19 kDa) and Sra-HSP-17.2 (~ 18 kDa) with 49% amino acid identity. The full-length cDNAs (483 bp and 474 bp, respectively) were identified and the genomic organization analyzed. To allow further characterization, the proteins were recombinantly expressed and purified. Profiling of transcription by qRT-PCR and of protein by ELISA in various developmental stages revealed parasitic female-specific expression. The sequence analysis of both DNA and amino acid sequence showed two genes share a conserved alpha-crystallin domain and variable N-terminals. The Sra-HSP-17 proteins showed the highest homology to the deduced small heat-shock protein sequence of the human pathogen S. stercoralis. We observed strong immunogenicity of both proteins, leading to high IgG responses following infection of rats. Flow cytometric analysis indicated the binding of Sra-HSP-17s to the monocytes/macrophage lineage but not to peripheral lymphocytes or neutrophils. A rat intestinal epithelial cell line showed dose dependent binding to Sra-HSP-17.1, but not to Sra-HSP-17.2. Exposed monocytes released IL-10 but not TNF-alpha in response to Sra-HSP-17s, suggesting a possible involvement of secreted female proteins in host immune responses. PMID:21762402

The presence of an insulin-like androgenic gland factor (IAG) and insulin-like peptide binding protein (ILPBP) in the ovary of the blue crab, Callinectes sapidus and their roles in ovarian development.

PubMed

Huang, Xiaoshuai; Ye, Haihui; Chung, J Sook

2017-08-01

Insulin-like androgenic gland factor (IAG) that is produced by the male androgenic gland (AG), plays a role in sexual differentiation and maintenance of male secondary sex characteristics in decapod crustaceans. With an earlier finding of IAG expression in a female Callinectes sapidus ovary, we aimed to examine a putative role of IAG during the ovarian development of this species. To this end, the full-length cDNA sequence of the ovarian CasIAG (termed CasIAG-ova) has been isolated. The predicted mature peptide sequence of CasIAG-ova is identical to that of the IAG from the AG, except in their signal peptide regions. The CasIAG-ova contains an alternative initiation codon (UUG) as the start codon, which suggests that the translational regulation of CasIAG-ova may differ from that of the IAG from AG. To define the function of CasIAG-ova, the expressions of CasIAG-ova as well as its putative binding protein, insulin-like peptide binding protein (ILPBP), are measured in the ovaries at various developmental stages obtained from different seasons. Season affects both CasIAG and ILPBP expression in the ovary. Overall, summer females at earlier ovarian stages contain high levels of CasIAG and ILPBP than spring or fall females. These findings indicate that CasIAG-ova and CasILPBP may be involved in the ovarian development. When comparing the levels of CasIAG and CasILPBP in the ovary, the latter are much higher (∼10-10000 fold) than the former. Expression patterns of CasILPBP differ from those of CasIAG-ova during ovarian development and by season, suggesting that ILPBP may have an additional role in ovarian development rather than a function of a putative binding protein of IAG. Copyright © 2017 Elsevier Inc. All rights reserved.

Colorful protein-based fluorescent probes for collagen imaging.

PubMed

Aper, Stijn J A; van Spreeuwel, Ariane C C; van Turnhout, Mark C; van der Linden, Ardjan J; Pieters, Pascal A; van der Zon, Nick L L; de la Rambelje, Sander L; Bouten, Carlijn V C; Merkx, Maarten

2014-01-01

Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.

Drosophila COP9 signalosome subunit 7 interacts with multiple genomic loci to regulate development

PubMed Central

Singer, Ruth; Atar, Shimshi; Atias, Osnat; Oron, Efrat; Segal, Daniel; Hirsch, Joel A.; Tuller, Tamir; Orian, Amir; Chamovitz, Daniel A.

2014-01-01

The COP9 signalosome protein complex has a central role in the regulation of development of multicellular organisms. While the function of this complex in ubiquitin-mediated protein degradation is well established, results over the past few years have hinted that the COP9 signalosome may function more broadly in the regulation of gene expression. Here, using DamID technology, we show that COP9 signalosome subunit 7 functionally associates with a large number of genomic loci in the Drosophila genome, and show that the expression of many genes within these loci is COP9 signalosome-dependent. This association is likely direct as we show CSN7 binds DNA in vitro. The genes targeted by CSN7 are preferentially enriched for transcriptionally active regions of the genome, and are involved in the regulation of distinct gene ontology groupings including imaginal disc development and cell-cycle control. In accord, loss of CSN7 function leads to cell-cycle delay and altered wing development. These results indicate that CSN7, and by extension the entire COP9 signalosome, functions directly in transcriptional control. While the COP9 signalosome protein complex has long been known to regulate protein degradation, here we expand the role of this complex by showing that subunit 7 binds DNA in vitro and functions directly in vivo in transcriptional control of developmentally important pathways that are relevant for human health. PMID:25106867

Impact of bilirubin-induced neurologic dysfunction on neurodevelopmental outcomes

PubMed Central

Loe, Irene M.

2015-01-01

Bilirubin-induced neurologic dysfunction (BIND) is the constellation of neurologic sequelae following milder degrees of neonatal hyperbilirubinemia than are associated with kernicterus. Clinically, BIND may manifest after the neonatal period as developmental delay, cognitive impairment, disordered executive function, and behavioral and psychiatric disorders. However, there is controversy regarding the relative contribution of neonatal hyperbilirubinemia versus other risk factors to the development of later neurodevelopmental disorders in children with BIND. In this review, we focus on the empiric data from the past 25 years regarding neurodevelopmental outcomes and BIND, including specific effects on developmental delay, cognition, speech and language development, executive function, and th neurobehavioral disorders, such as attention deficit/hyperactivity disorder and autism. PMID:25585889

Co-Localization of the Oncogenic Transcription Factor MYCN and the DNA Methyl Binding Protein MeCP2 at Genomic Sites in Neuroblastoma

PubMed Central

Murphy, Derek M.; Buckley, Patrick G.; Das, Sudipto; Watters, Karen M.; Bryan, Kenneth; Stallings, Raymond L.

2011-01-01

Background MYCN is a transcription factor that is expressed during the development of the neural crest and its dysregulation plays a major role in the pathogenesis of pediatric cancers such as neuroblastoma, medulloblastoma and rhabdomyosarcoma. MeCP2 is a CpG methyl binding protein which has been associated with a number of cancers and developmental disorders, particularly Rett syndrome. Methods and Findings Using an integrative global genomics approach involving chromatin immunoprecipitation applied to microarrays, we have determined that MYCN and MeCP2 co-localize to gene promoter regions, as well as inter/intragenic sites, within the neuroblastoma genome (MYCN amplified Kelly cells) at high frequency (70.2% of MYCN sites were also positive for MeCP2). Intriguingly, the frequency of co-localization was significantly less at promoter regions exhibiting substantial hypermethylation (8.7%), as determined by methylated DNA immunoprecipitation (MeDIP) applied to the same microarrays. Co-immunoprecipitation of MYCN using an anti-MeCP2 antibody indicated that a MYCN/MeCP2 interaction occurs at protein level. mRNA expression profiling revealed that the median expression of genes with promoters bound by MYCN was significantly higher than for genes bound by MeCP2, and that genes bound by both proteins had intermediate expression. Pathway analysis was carried out for genes bound by MYCN, MeCP2 or MYCN/MeCP2, revealing higher order functions. Conclusions Our results indicate that MYCN and MeCP2 protein interact and co-localize to similar genomic sites at very high frequency, and that the patterns of binding of these proteins can be associated with significant differences in transcriptional activity. Although it is not yet known if this interaction contributes to neuroblastoma disease pathogenesis, it is intriguing that the interaction occurs at the promoter regions of several genes important for the development of neuroblastoma, including ALK, AURKA and BDNF. PMID:21731748

Co-localization of the oncogenic transcription factor MYCN and the DNA methyl binding protein MeCP2 at genomic sites in neuroblastoma.

PubMed

Murphy, Derek M; Buckley, Patrick G; Das, Sudipto; Watters, Karen M; Bryan, Kenneth; Stallings, Raymond L

2011-01-01

MYCN is a transcription factor that is expressed during the development of the neural crest and its dysregulation plays a major role in the pathogenesis of pediatric cancers such as neuroblastoma, medulloblastoma and rhabdomyosarcoma. MeCP2 is a CpG methyl binding protein which has been associated with a number of cancers and developmental disorders, particularly Rett syndrome. Using an integrative global genomics approach involving chromatin immunoprecipitation applied to microarrays, we have determined that MYCN and MeCP2 co-localize to gene promoter regions, as well as inter/intragenic sites, within the neuroblastoma genome (MYCN amplified Kelly cells) at high frequency (70.2% of MYCN sites were also positive for MeCP2). Intriguingly, the frequency of co-localization was significantly less at promoter regions exhibiting substantial hypermethylation (8.7%), as determined by methylated DNA immunoprecipitation (MeDIP) applied to the same microarrays. Co-immunoprecipitation of MYCN using an anti-MeCP2 antibody indicated that a MYCN/MeCP2 interaction occurs at protein level. mRNA expression profiling revealed that the median expression of genes with promoters bound by MYCN was significantly higher than for genes bound by MeCP2, and that genes bound by both proteins had intermediate expression. Pathway analysis was carried out for genes bound by MYCN, MeCP2 or MYCN/MeCP2, revealing higher order functions. Our results indicate that MYCN and MeCP2 protein interact and co-localize to similar genomic sites at very high frequency, and that the patterns of binding of these proteins can be associated with significant differences in transcriptional activity. Although it is not yet known if this interaction contributes to neuroblastoma disease pathogenesis, it is intriguing that the interaction occurs at the promoter regions of several genes important for the development of neuroblastoma, including ALK, AURKA and BDNF.

Transcriptional activation by MEIS1A in response to protein kinase A signaling requires the transducers of regulated CREB family of CREB co-activators.

PubMed

Goh, Siew-Lee; Looi, Yvonne; Shen, Hui; Fang, Jun; Bodner, Caroline; Houle, Martin; Ng, Andy Cheuk-Him; Screaton, Robert A; Featherstone, Mark

2009-07-10

The transcription factor encoded by the murine ecotropic integration site 1 gene (MEIS1) is a partner of HOX and PBX proteins. It has been implicated in embryonic patterning and leukemia, and causally linked to restless legs syndrome. The MEIS1A C terminus harbors a transcriptional activation domain that is stimulated by protein kinase A (PKA) in a manner dependent on the co-activator of cAMP response element-binding protein (CREB), CREB-binding protein (CBP). We explored the involvement of another mediator of PKA-inducible transcription, namely the CREB co-activators transducers of regulated CREB activity (TORCs). Overexpression of TORC1 or TORC2 bypassed PKA for activation by MEIS1A. Co-immunoprecipitation experiments demonstrated a physical interaction between MEIS1 and TORC2 that is dependent on the MEIS1A C terminus, whereas chromatin immunoprecipitation revealed PKA-inducible recruitment of MEIS1, PBX1, and TORC2 on the MEIS1 target genes Hoxb2 and Meis1. The MEIS1 interaction domain on TORC1 was mapped to the N-terminal coiled-coil region, and TORC1 mutants lacking this domain attenuated the response to PKA on a natural MEIS1A target enhancer. Thus, TORCs physically cooperate with MEIS1 to achieve PKA-inducible transactivation through the MEIS1A C terminus, suggesting a concerted action in developmental and oncogenic processes.

Transcriptional Activation by MEIS1A in Response to Protein Kinase A Signaling Requires the Transducers of Regulated CREB Family of CREB Co-activators*

PubMed Central

Goh, Siew-Lee; Looi, Yvonne; Shen, Hui; Fang, Jun; Bodner, Caroline; Houle, Martin; Ng, Andy Cheuk-Him; Screaton, Robert A.; Featherstone, Mark

2009-01-01

The transcription factor encoded by the murine ecotropic integration site 1 gene (MEIS1) is a partner of HOX and PBX proteins. It has been implicated in embryonic patterning and leukemia, and causally linked to restless legs syndrome. The MEIS1A C terminus harbors a transcriptional activation domain that is stimulated by protein kinase A (PKA) in a manner dependent on the co-activator of cAMP response element-binding protein (CREB), CREB-binding protein (CBP). We explored the involvement of another mediator of PKA-inducible transcription, namely the CREB co-activators transducers of regulated CREB activity (TORCs). Overexpression of TORC1 or TORC2 bypassed PKA for activation by MEIS1A. Co-immunoprecipitation experiments demonstrated a physical interaction between MEIS1 and TORC2 that is dependent on the MEIS1A C terminus, whereas chromatin immunoprecipitation revealed PKA-inducible recruitment of MEIS1, PBX1, and TORC2 on the MEIS1 target genes Hoxb2 and Meis1. The MEIS1 interaction domain on TORC1 was mapped to the N-terminal coiled-coil region, and TORC1 mutants lacking this domain attenuated the response to PKA on a natural MEIS1A target enhancer. Thus, TORCs physically cooperate with MEIS1 to achieve PKA-inducible transactivation through the MEIS1A C terminus, suggesting a concerted action in developmental and oncogenic processes. PMID:19473990

An m6A-YTH Module Controls Developmental Timing and Morphogenesis in Arabidopsis.

PubMed

Arribas-Hernández, Laura; Bressendorff, Simon; Hansen, Mathias Henning; Poulsen, Christian; Erdmann, Susanne; Brodersen, Peter

2018-04-11

Methylation of N6-adenosine (m6A) in mRNA is an important post-transcriptional gene regulatory mechanism in eukaryotes. m6A provides a binding site for effector proteins ("readers") that influence pre-mRNA splicing, mRNA degradation or translational efficiency. YT521-B homology (YTH) domain proteins are important m6A readers with established functions in animals. Plants contain more YTH domain proteins than other eukaryotes, but their biological importance remains unknown. Here, we show that the cytoplasmic Arabidopsis thaliana YTH domain proteins EVOLUTIONARILY CONSERVED C-TERMINAL REGION2/3 (ECT2/3) are required for the correct timing of leaf formation and for normal leaf morphology. These functions depend fully on intact m6A binding sites of ECT2 and ECT3, indicating that they function as m6A readers. Mutation of the close ECT2 homolog, ECT4, enhances the delayed leaf emergence and leaf morphology defects of ect2/ect3 mutants, and all three ECT proteins are expressed at leaf formation sites in the shoot apex of young seedlings and in the division zone of developing leaves. ECT2 and ECT3 are also highly expressed at early stages of trichome development and are required for trichome morphology, as previously reported for m6A itself. Overall, our study establishes the relevance of a cytoplasmic m6A-YTH regulatory module in the timing and execution of plant organogenesis. © 2018 American Society of Plant Biologists. All rights reserved.

Mutations in the SPARC-related modular calcium-binding protein 1 gene, SMOC1, cause waardenburg anophthalmia syndrome.

PubMed

Abouzeid, Hana; Boisset, Gaëlle; Favez, Tatiana; Youssef, Mohamed; Marzouk, Iman; Shakankiry, Nihal; Bayoumi, Nader; Descombes, Patrick; Agosti, Céline; Munier, Francis L; Schorderet, Daniel F

2011-01-07

Waardenburg anophthalmia syndrome, also known as microphthalmia with limb anomalies, ophthalmoacromelic syndrome, and anophthalmia-syndactyly, is a rare autosomal-recessive developmental disorder that has been mapped to 10p11.23. Here we show that this disease is heterogeneous by reporting on a consanguineous family, not linked to the 10p11.23 locus, whose two affected children have a homozygous mutation in SMOC1. Knockdown experiments of the zebrafish smoc1 revealed that smoc1 is important in eye development and that it is expressed in many organs, including brain and somites.

Bombyx mori cyclin-dependent kinase inhibitor is involved in regulation of the silkworm cell cycle.

PubMed

Tang, X-F; Zhou, X-L; Zhang, Q; Chen, P; Lu, C; Pan, M-H

2018-06-01

Cyclin-dependent kinase inhibitors (CKIs) are negative regulators of the cell cycle. They can bind to cyclin-dependent kinase (CDK)-cyclin complexes and inhibit CDK activities. We identified a single homologous gene of the CDK interacting protein/kinase inhibitory protein (Cip/Kip) family, BmCKI, in the silkworm, Bombyx mori. The gene transcribes two splice variants: a 654-bp-long BmCKI-L (the longer splice variant) encoding a protein with 217 amino acids and a 579-bp-long BmCKI-S (the shorter splice variant) encoding a protein with 192 amino acids. BmCKI-L and BmCKI-S contain the Cip/Kip family conserved cyclin-binding domain and the CDK-binding domain. They are localized in the nucleus and have an unconventional bipartite nuclear localization signal at amino acid residues 181-210. Overexpression of BmCKI-L or BmCKI-S affected cell cycle progression; the cell cycle was arrested in the first gap phase of cell cycle (G1). RNA interference of BmCKI-L or BmCKI-S led to cells accumulating in the second gap phase and the mitotic phase of cell cycle (G2/M). Both BmCKI-L and BmCKI-S are involved in cell cycle regulation and probably have similar effects. The transgenic silkworm with BmCKI-L overexpression (BmCKI-L-OE), exhibited embryonic lethal, larva developmental retardation and lethal phenotypes. These results suggest that BmCKI-L might regulate the growth and development of silkworm. These findings clarify the function of CKIs and increase our understanding of cell cycle regulation in the silkworm. © 2018 The Royal Entomological Society.

JMJ27, an Arabidopsis H3K9 histone demethylase, modulates defense against Pseudomonas syringae and flowering time.

PubMed

Dutta, Aditya; Choudhary, Pratibha; Caruana, Julie; Raina, Ramesh

2017-09-01

Histone methylation is known to dynamically regulate diverse developmental and physiological processes. Histone methyl marks are written by methyltransferases and erased by demethylases, and result in modification of chromatin structure to repress or activate transcription. However, little is known about how histone methylation may regulate defense mechanisms and flowering time in plants. Here we report characterization of JmjC DOMAIN-CONTAINING PROTEIN 27 (JMJ27), an Arabidopsis JHDM2 (JmjC domain-containing histone demethylase 2) family protein, which modulates defense against pathogens and flowering time. JMJ27 is a nuclear protein containing a zinc-finger motif and a catalytic JmjC domain with conserved Fe(II) and α-ketoglutarate binding sites, and displays H3K9me1/2 demethylase activity both in vitro and in vivo. JMJ27 is induced in response to virulent Pseudomonas syringae pathogens and is required for resistance against these pathogens. JMJ27 is a negative modulator of WRKY25 (a repressor of defense) and a positive modulator of several pathogenesis-related (PR) proteins. Additionally, loss of JMJ27 function leads to early flowering. JMJ27 negatively modulates the major flowering regulator CONSTANS (CO) and positively modulates FLOWERING LOCUS C (FLC). Taken together, our results indicate that JMJ27 functions as a histone demethylase to modulate both physiological (defense) and developmental (flowering time) processes in Arabidopsis. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Tropomodulins: pointed-end capping proteins that regulate actin filament architecture in diverse cell types

PubMed Central

Yamashiro, Sawako; Gokhin, David S.; Kimura, Sumiko; Nowak, Roberta B.; Fowler, Velia M.

2012-01-01

Tropomodulins are a family of four proteins (Tmods 1–4) that cap the pointed ends of actin filaments in actin cytoskeletal structures in a developmentally regulated and tissue-specific manner. Unique among capping proteins, Tmods also bind tropomyosins (TMs), which greatly enhance the actin filament pointed-end capping activity of Tmods. Tmods are defined by a tropomyosin (TM)-regulated/Pointed-End Actin Capping (TM-Cap) domain in their unstructured N-terminal portion, followed by a compact, folded Leucine-Rich Repeat/Pointed-End Actin Capping (LRR-Cap) domain. By inhibiting actin monomer association and dissociation from pointed ends, Tmods regulate regulate actin dynamics and turnover, stabilizing actin filament lengths and cytoskeletal architecture. In this review, we summarize the genes, structural features, molecular and biochemical properties, actin regulatory mechanisms, expression patterns, and cell and tissue functions of Tmods. By understanding Tmods’ functions in the context of their molecular structure, actin regulation, binding partners, and related variants (leiomodins 1–3), we can draw broad conclusions that can explain the diverse morphological and functional phenotypes that arise from Tmod perturbation experiments in vitro and in vivo. Tmod-based stabilization and organization of intracellular actin filament networks provide key insights into how the emergent properties of the actin cytoskeleton drive tissue morphogenesis and physiology. PMID:22488942

Phospholipase C and D regulation of Src, calcium release and membrane fusion during Xenopus laevis development

PubMed Central

Stith, Bradley J.

2015-01-01

This review emphasizes how lipids regulate membrane fusion and the proteins involved in three developmental stages: oocyte maturation to the fertilizable egg, fertilization and during first cleavage. Decades of work show that phosphatidic acid (PA) releases intracellular calcium, and recent work shows that the lipid can activate Src tyrosine kinase or phospholipase C during Xenopus fertilization. Numerous reports are summarized to show three levels of increase in lipid second messengers inositol 1,4,5-trisphosphate and sn 1,2-diacylglycerol (DAG) during the three different developmental stages. In addition, possible roles for PA, ceramide, lysophosphatidylcholine, plasmalogens, phosphatidylinositol 4-phosphate, phosphatidylinositol 5-phosphate, phosphatidylinositol 4,5-bisphosphate, membrane microdomains (rafts) and phosphatidylinositol 3,4,5-trisphosphate in regulation of membrane fusion (acrosome reaction, sperm-egg fusion, cortical granule exocytosis), inositol 1,4,5-trisphosphate receptors, and calcium release are discussed. The role of six lipases involved in generating putative lipid second messengers during fertilization is also discussed: phospholipase D, autotaxin, lipin1, sphingomyelinase, phospholipase C, and phospholipase A2. More specifically, proteins involved in developmental events and their regulation through lipid binding to SH3, SH4, PH, PX, or C2 protein domains is emphasized. New models are presented for PA activation of Src (through SH3, SH4 and a unique domain), that this may be why the SH2 domain of PLCγ is not required for Xenopus fertilization, PA activation of phospholipase C, a role for PA during the calcium wave after fertilization, and that calcium/calmodulin may be responsible for the loss of Src from rafts after fertilization. Also discussed is that the large DAG increase during fertilization derives from phospholipase D production of PA and lipin dephosphorylation to DAG. PMID:25748412

Developmental exposure to lead (Pb) alters the expression of the human tau gene and its products in a transgenic animal model

PubMed Central

Dash, M.; Eid, A.; Subaiea, G.; Chang, J.; Deeb, R.; Masoud, A.; Renehan, W.E.; Adem, A.; Zawia, N.H.

2016-01-01

Tauopathies are a class of neurodegenerative diseases associated with the pathological aggregationof the tau protein in the human brain. The best known of these illnesses is Alzheimer's disease (AD); a disease where the microtubule associated protein tau (MAPT) becomes hyperphosphorylated (lowering its binding affinity to microtubules) and aggregates within neurons in the form of neurofibrillary tangles (NFTs). In this paper we examine whether environmental factors play a significant role in tau pathogenesis. Our studies were conducted in a double mutant mouse model that expressed the human tau gene and lacked the gene for murine tau. The human tau mouse model was tested for the transgene's ability to respond to an environmental toxicant. Pups were developmentally exposed to lead (Pb) from postnatal day (PND) 1-20 with 0.2% Pb acetate. Mice were then sacrificed at PND 20, 30, 40 and 60. Protein and mRNA levels for tau and CDK5 as well as tau phosphorylation at Ser396 were determined. In addition, the potential role of miRNA in tau expression was investigated by measuring levels of miR-34c, a miRNA that targets the mRNA for human tau, at PND20 and 50. The expression of the human tau transgene was altered by developmental exposure to Pb. This exposure also altered the expression of miR-34c. Our findings are the first of their kind to test the responsiveness of the human tau gene to an environmental toxicant and to examine an epigenetic mechanism that may be involved in the regulation of this gene's expression. PMID:27293183

Molecular and cellular aspects of calcium action in plants

NASA Technical Reports Server (NTRS)

Poovaiah, B. W.

1988-01-01

Calcium is known to be a second messenger in many developmental processes in animal systems, but it has only recently become evident that Ca is an important intracellular messenger in plants as well. The level of free Ca concentration in the cytoplasm is extremely low, and it is influenced by extracellular signals such as light, gravity, and hormones. Investigations from our laboratory indicated that Ca and its binding protein, calmodulin, play an important role in stimulus-response coupling by regulating enzyme activities, especially through protein phosphorylation. In vivo and in vitro protein phosphorylation studies have revealed Ca-dependent changes in various plant tissues. We have also been able to influence various physiological processes such as cell elongation, abscission, senescence, and tuberization by altering extracellular and intracellular Ca levels. Other examples of Ca-mediated processes in plants are as follows: a) cell division, b) geotropism, c) protoplasmic streaming, d) stomatal control, e) chloroplast movement, f) secretion, g) hormone-dependent changes, h) enzyme activation, and i) protein phosphorylation.

The DAN family: modulators of TGF-β signaling and beyond.

PubMed

Nolan, Kristof; Thompson, Thomas B

2014-08-01

Extracellular binding proteins or antagonists are important factors that modulate ligands in the transforming growth factor (TGF-β) family. While the interplay between antagonists and ligands are essential for developmental and normal cellular processes, their imbalance can lead to the pathology of several disease states. In particular, recent studies have implicated members of the differential screening-selected gene in neuroblastoma (DAN) family in disease such as renal fibrosis, pulmonary arterial hypertension, and reactivation of metastatic cancer stem cells. DAN family members are known to inhibit the bone morphogenetic proteins (BMP) of the TGF-β family. However, unlike other TGF-β antagonist families, DAN family members have roles beyond ligand inhibition and can modulate Wnt and vascular endothelial growth factor (VEGF) signaling pathways. This review describes recent structural and functional advances that have expanded our understanding of DAN family proteins with regards to BMP inhibition and also highlights their emerging roles in the modulation of Wnt and VEGF signaling pathways. © 2014 The Protein Society.

Production of a reference transcriptome and transcriptomic database (EdwardsiellaBase) for the lined sea anemone, Edwardsiella lineata, a parasitic cnidarian

PubMed Central

2014-01-01

Background The lined sea anemone Edwardsiella lineata is an informative model system for evolutionary-developmental studies of parasitism. In this species, it is possible to compare alternate developmental pathways leading from a larva to either a free-living polyp or a vermiform parasite that inhabits the mesoglea of a ctenophore host. Additionally, E. lineata is confamilial with the model cnidarian Nematostella vectensis, providing an opportunity for comparative genomic, molecular and organismal studies. Description We generated a reference transcriptome for E. lineata via high-throughput sequencing of RNA isolated from five developmental stages (parasite; parasite-to-larva transition; larva; larva-to-adult transition; adult). The transcriptome comprises 90,440 contigs assembled from >15 billion nucleotides of DNA sequence. Using a molecular clock approach, we estimated the divergence between E. lineata and N. vectensis at 215–364 million years ago. Based on gene ontology and metabolic pathway analyses and gene family surveys (bHLH-PAS, deiodinases, Fox genes, LIM homeodomains, minicollagens, nuclear receptors, Sox genes, and Wnts), the transcriptome of E. lineata is comparable in depth and completeness to N. vectensis. Analyses of protein motifs and revealed extensive conservation between the proteins of these two edwardsiid anemones, although we show the NF-κB protein of E. lineata reflects the ancestral structure, while the NF-κB protein of N. vectensis has undergone a split that separates the DNA-binding domain from the inhibitory domain. All contigs have been deposited in a public database (EdwardsiellaBase), where they may be searched according to contig ID, gene ontology, protein family motif (Pfam), enzyme commission number, and BLAST. The alignment of the raw reads to the contigs can also be visualized via JBrowse. Conclusions The transcriptomic data and database described here provide a platform for studying the evolutionary developmental genomics of a derived parasitic life cycle. In addition, these data from E. lineata will aid in the interpretation of evolutionary novelties in gene sequence or structure that have been reported for the model cnidarian N. vectensis (e.g., the split NF-κB locus). Finally, we include custom computational tools to facilitate the annotation of a transcriptome based on high-throughput sequencing data obtained from a “non-model system.” PMID:24467778

Production of a reference transcriptome and transcriptomic database (EdwardsiellaBase) for the lined sea anemone, Edwardsiella lineata, a parasitic cnidarian.

PubMed

Stefanik, Derek J; Lubinski, Tristan J; Granger, Brian R; Byrd, Allyson L; Reitzel, Adam M; DeFilippo, Lukas; Lorenc, Allison; Finnerty, John R

2014-01-28

The lined sea anemone Edwardsiella lineata is an informative model system for evolutionary-developmental studies of parasitism. In this species, it is possible to compare alternate developmental pathways leading from a larva to either a free-living polyp or a vermiform parasite that inhabits the mesoglea of a ctenophore host. Additionally, E. lineata is confamilial with the model cnidarian Nematostella vectensis, providing an opportunity for comparative genomic, molecular and organismal studies. We generated a reference transcriptome for E. lineata via high-throughput sequencing of RNA isolated from five developmental stages (parasite; parasite-to-larva transition; larva; larva-to-adult transition; adult). The transcriptome comprises 90,440 contigs assembled from >15 billion nucleotides of DNA sequence. Using a molecular clock approach, we estimated the divergence between E. lineata and N. vectensis at 215-364 million years ago. Based on gene ontology and metabolic pathway analyses and gene family surveys (bHLH-PAS, deiodinases, Fox genes, LIM homeodomains, minicollagens, nuclear receptors, Sox genes, and Wnts), the transcriptome of E. lineata is comparable in depth and completeness to N. vectensis. Analyses of protein motifs and revealed extensive conservation between the proteins of these two edwardsiid anemones, although we show the NF-κB protein of E. lineata reflects the ancestral structure, while the NF-κB protein of N. vectensis has undergone a split that separates the DNA-binding domain from the inhibitory domain. All contigs have been deposited in a public database (EdwardsiellaBase), where they may be searched according to contig ID, gene ontology, protein family motif (Pfam), enzyme commission number, and BLAST. The alignment of the raw reads to the contigs can also be visualized via JBrowse. The transcriptomic data and database described here provide a platform for studying the evolutionary developmental genomics of a derived parasitic life cycle. In addition, these data from E. lineata will aid in the interpretation of evolutionary novelties in gene sequence or structure that have been reported for the model cnidarian N. vectensis (e.g., the split NF-κB locus). Finally, we include custom computational tools to facilitate the annotation of a transcriptome based on high-throughput sequencing data obtained from a "non-model system."

Development of peptide and protein based radiopharmaceuticals.

PubMed

Wynendaele, Evelien; Bracke, Nathalie; Stalmans, Sofie; De Spiegeleer, Bart

2014-01-01

Radiolabelled peptides and proteins have recently gained great interest as theranostics, due to their numerous and considerable advantages over small (organic) molecules. Developmental procedures of these radiolabelled biomolecules start with the radiolabelling process, greatly defined by the amino acid composition of the molecule and the radionuclide used. Depending on the radionuclide selection, radiolabelling starting materials are whether or not essential for efficient radiolabelling, resulting in direct or indirect radioiodination, radiometal-chelate coupling, indirect radiofluorination or (3)H/(14)C-labelling. Before preclinical investigations are performed, quality control analyses of the synthesized radiopharmaceutical are recommended to eliminate false positive or negative functionality results, e.g. changed receptor binding properties due to (radiolabelled) impurities. Therefore, radionuclidic, radiochemical and chemical purity are investigated, next to the general peptide attributes as described in the European and the United States Pharmacopeia. Moreover, in vitro and in vivo stability characteristics of the peptides and proteins also need to be explored, seen their strong sensitivity to proteinases and peptidases, together with radiolysis and trans-chelation phenomena of the radiopharmaceuticals. In vitro biomedical characterization of the radiolabelled peptides and proteins is performed by saturation, kinetic and competition binding assays, analyzing KD, Bmax, kon, koff and internalization properties, taking into account the chemical and metabolic stability and adsorption events inherent to peptides and proteins. In vivo biodistribution can be adapted by linker, chelate or radionuclide modifications, minimizing normal tissue (e.g. kidney and liver) radiation, and resulting in favorable dosimetry analyses. Finally, clinical trials are initiated, eventually leading to the marketing of radiolabelled peptides and proteins for PET/SPECT-imaging and therapy of different clinical diseases.

SPOROCYTELESS is a novel embryophyte-specific transcription repressor that interacts with TPL and TCP proteins in Arabidopsis.

PubMed

Chen, Guang-Hui; Sun, Jia-Ying; Liu, Man; Liu, Jie; Yang, Wei-Cai

2014-12-20

Germlines in plants are formed de novo during post-embryonic development, while little is known about the mechanism that controls this process. In Arabidopsis, the earliest gene controlling this process is SPOROCYTELESS (SPL). A decade ago, we showed that loss of SPL function abolished sporogenesis in both male and female organs of Arabidopsis. However, its function is unclear up to now. In this study, we showed that SPL belongs to a novel transcription repressor family specific in embryophyte, which consists of 173 members in the land plants so far. All of them contain a conserved SPL-motif in their N-terminal and an ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif in the C-terminal, therefore designated as SPL-like, EAR-containing proteins (SPEARs). Consistently, SPL acts as a transcriptional repressor in yeast and tobacco cells, and SPEAR proteins are able to form homodimer and/or heterodimer with each other in vitro. Furthermore, SPEARs interact with the TOPLESS (TPL) co-repressors via the EAR motif and TCP family transcription factors in yeast cells. Together, we propose that SPL and SPEARs most likely belong to a novel transcription repressor family in land plants which may play a variety of developmental roles in plants. Copyright © 2014 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

l-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome

PubMed Central

Xu, Baoshan; Sowa, Nenja; Cardenas, Maria E.; Gerton, Jennifer L.

2015-01-01

Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (α-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite α-KIC can partially rescue development in zebrafish models for CdLS. PMID:25378554

Gene Families of Cuticular Proteins Analogous to Peritrophins (CPAPs) in Tribolium castaneum Have Diverse Functions

PubMed Central

Jasrapuria, Sinu; Specht, Charles A.; Kramer, Karl J.; Beeman, Richard W.; Muthukrishnan, Subbaratnam

2012-01-01

The functional characterization of an entire class of 17 genes from the red flour beetle, Tribolium castaneum, which encode two families of Cuticular Proteins Analogous to Peritrophins (CPAPs) has been carried out. CPAP genes in T. castaneum are expressed exclusively in cuticle-forming tissues and have been classified into two families, CPAP1 and CPAP3, based on whether the proteins contain either one (CPAP1), or three copies (CPAP3) of the chitin-binding domain, ChtBD2, with its six characteristically spaced cysteine residues. Individual members of the TcCPAP1 and TcCPAP3 gene families have distinct developmental patterns of expression. Many of these proteins serve essential and non-redundant functions in maintaining the structural integrity of the cuticle in different parts of the insect anatomy. Three genes of the TcCPAP1 family and five genes of the TcCPAP3 family are essential for insect development, molting, cuticle integrity, proper locomotion or fecundity. RNA interference (RNAi) targeting TcCPAP1-C, TcCPAP1-H, TcCPAP1-J or TcCPAP3-C transcripts resulted in death at the pharate adult stage of development. RNAi for TcCPAP3-A1, TcCPAP3-B, TcCPAP3-D1 or TcCPAP3-D2 genes resulted in different developmental defects, including adult/embryonic mortality, abnormal elytra or hindwings, or an abnormal ‘stiff-jointed’ gait. These results provide experimental support for specialization in the functions of CPAP proteins in T. castaneum and a biological rationale for the conservation of CPAP orthologs in other orders of insects. This is the first comprehensive functional analysis of an entire class of cuticular proteins with one or more ChtBD2 domains in any insect species. PMID:23185457

Gene families of cuticular proteins analogous to peritrophins (CPAPs) in Tribolium castaneum have diverse functions.

PubMed

Jasrapuria, Sinu; Specht, Charles A; Kramer, Karl J; Beeman, Richard W; Muthukrishnan, Subbaratnam

2012-01-01

The functional characterization of an entire class of 17 genes from the red flour beetle, Tribolium castaneum, which encode two families of Cuticular Proteins Analogous to Peritrophins (CPAPs) has been carried out. CPAP genes in T. castaneum are expressed exclusively in cuticle-forming tissues and have been classified into two families, CPAP1 and CPAP3, based on whether the proteins contain either one (CPAP1), or three copies (CPAP3) of the chitin-binding domain, ChtBD2, with its six characteristically spaced cysteine residues. Individual members of the TcCPAP1 and TcCPAP3 gene families have distinct developmental patterns of expression. Many of these proteins serve essential and non-redundant functions in maintaining the structural integrity of the cuticle in different parts of the insect anatomy. Three genes of the TcCPAP1 family and five genes of the TcCPAP3 family are essential for insect development, molting, cuticle integrity, proper locomotion or fecundity. RNA interference (RNAi) targeting TcCPAP1-C, TcCPAP1-H, TcCPAP1-J or TcCPAP3-C transcripts resulted in death at the pharate adult stage of development. RNAi for TcCPAP3-A1, TcCPAP3-B, TcCPAP3-D1 or TcCPAP3-D2 genes resulted in different developmental defects, including adult/embryonic mortality, abnormal elytra or hindwings, or an abnormal 'stiff-jointed' gait. These results provide experimental support for specialization in the functions of CPAP proteins in T. castaneum and a biological rationale for the conservation of CPAP orthologs in other orders of insects. This is the first comprehensive functional analysis of an entire class of cuticular proteins with one or more ChtBD2 domains in any insect species.

L-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome.

PubMed

Xu, Baoshan; Sowa, Nenja; Cardenas, Maria E; Gerton, Jennifer L

2015-03-15

Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (α-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite α-KIC can partially rescue development in zebrafish models for CdLS. © The Author 2014. Published by Oxford University Press.

Mutations in the Paxillin-binding Site of Integrin-linked Kinase (ILK) Destabilize the Pseudokinase Domain and Cause Embryonic Lethality in Mice*

PubMed Central

Moik, Daniel; Böttcher, Anika; Makhina, Tatiana; Grashoff, Carsten; Bulus, Nada; Zent, Roy; Fässler, Reinhard

2013-01-01

Integrin-linked kinase (ILK) localizes to focal adhesions (FAs) where it regulates cell spreading, migration, and growth factor receptor signaling. Previous reports showed that overexpressed ILK in which Val386 and Thr387 were substituted with glycine residues (ILK-VT/GG) could neither interact with paxillin nor localize to FA in cells expressing endogenous wild-type ILK, implying that paxillin binding to ILK is required for its localization to FAs. Here, we show that introducing this mutation into the germ line of mice (ILK-VT/GG) caused vasculogenesis defects, resulting in a general developmental delay and death at around embryonic day 12.5. Fibroblasts isolated from ILK-VT/GG mice contained mutant ILK in FAs, showed normal adhesion to and spreading on extracellular matrix substrates but displayed impaired migration. Biochemical analysis revealed that VT/GG substitutions decreased ILK protein stability leading to decreased ILK levels and reduced binding to paxillin and α-parvin. Because paxillin depletion did not affect ILK localization to FAs, the embryonic lethality and the in vitro migration defects are likely due to the reduced levels of ILK-VT/GG and diminished binding to parvins. PMID:23658024

Impact of bilirubin-induced neurologic dysfunction on neurodevelopmental outcomes.

PubMed

Wusthoff, Courtney J; Loe, Irene M

2015-02-01

Bilirubin-induced neurologic dysfunction (BIND) is the constellation of neurologic sequelae following milder degrees of neonatal hyperbilirubinemia than are associated with kernicterus. Clinically, BIND may manifest after the neonatal period as developmental delay, cognitive impairment, disordered executive function, and behavioral and psychiatric disorders. However, there is controversy regarding the relative contribution of neonatal hyperbilirubinemia versus other risk factors to the development of later neurodevelopmental disorders in children with BIND. In this review, we focus on the empiric data from the past 25 years regarding neurodevelopmental outcomes and BIND, including specific effects on developmental delay, cognition, speech and language development, executive function, and the neurobehavioral disorders, such as attention deficit/hyperactivity disorder and autism. Copyright © 2014 Elsevier Ltd. All rights reserved.

Three RNA recognition motifs participate in RNA recognition and structural organization by the pro-apoptotic factor TIA-1

PubMed Central

Bauer, William J.; Heath, Jason; Jenkins, Jermaine L.; Kielkopf, Clara L.

2012-01-01

T-cell intracellular antigen-1 (TIA-1) regulates developmental and stress-responsive pathways through distinct activities at the levels of alternative pre-mRNA splicing and mRNA translation. The TIA-1 polypeptide contains three RNA recognition motifs (RRMs). The central RRM2 and C-terminal RRM3 associate with cellular mRNAs. The N-terminal RRM1 enhances interactions of a C-terminal Q-rich domain of TIA-1 with the U1-C splicing factor, despite linear separation of the domains in the TIA-1 sequence. Given the expanded functional repertoire of the RRM family, it was unknown whether TIA-1 RRM1 contributes to RNA binding as well as documented protein interactions. To address this question, we used isothermal titration calorimetry and small-angle X-ray scattering (SAXS) to dissect the roles of the TIA-1 RRMs in RNA recognition. Notably, the fas RNA exhibited two binding sites with indistinguishable affinities for TIA-1. Analyses of TIA-1 variants established that RRM1 was dispensable for binding AU-rich fas sites, yet all three RRMs were required to bind a polyU RNA with high affinity. SAXS analyses demonstrated a `V' shape for a TIA-1 construct comprising the three RRMs, and revealed that its dimensions became more compact in the RNA-bound state. The sequence-selective involvement of TIA-1 RRM1 in RNA recognition suggests a possible role for RNA sequences in regulating the distinct functions of TIA-1. Further implications for U1-C recruitment by the adjacent TIA-1 binding sites of the fas pre-mRNA and the bent TIA-1 shape, which organizes the N- and C-termini on the same side of the protein, are discussed. PMID:22154808

A secreted WNT-ligand-binding domain of FZD5 generated by a frameshift mutation causes autosomal dominant coloboma

PubMed Central

Liu, Chunqiao; Widen, Sonya A.; Williamson, Kathleen A.; Ratnapriya, Rinki; Gerth-Kahlert, Christina; Rainger, Joe; Alur, Ramakrishna P.; Strachan, Erin; Manjunath, Souparnika H.; Balakrishnan, Archana; Floyd, James A.; Li, Tiansen; Waskiewicz, Andrew; Brooks, Brian P.; Lehmann, Ordan J.; FitzPatrick, David R.; Swaroop, Anand

2016-01-01

Ocular coloboma is a common eye malformation resulting from incomplete fusion of the optic fissure during development. Coloboma is often associated with microphthalmia and/or contralateral anophthalmia. Coloboma shows extensive locus heterogeneity associated with causative mutations identified in genes encoding developmental transcription factors or components of signaling pathways. We report an ultra-rare, heterozygous frameshift mutation in FZD5 (p.Ala219Glufs*49) that was identified independently in two branches of a large family with autosomal dominant non-syndromic coloboma. FZD5 has a single-coding exon and consequently a transcript with this frameshift variant is not a canonical substrate for nonsense-mediated decay. FZD5 encodes a transmembrane receptor with a conserved extracellular cysteine rich domain for ligand binding. The frameshift mutation results in the production of a truncated protein, which retains the Wingless-type MMTV integration site family member-ligand-binding domain, but lacks the transmembrane domain. The truncated protein was secreted from cells, and behaved as a dominant-negative FZD5 receptor, antagonizing both canonical and non-canonical WNT signaling. Expression of the resultant mutant protein caused coloboma and microphthalmia in zebrafish, and disruption of the apical junction of the retinal neural epithelium in mouse, mimicking the phenotype of Fz5/Fz8 compound conditional knockout mutants. Our studies have revealed a conserved role of Wnt–Frizzled (FZD) signaling in ocular development and directly implicate WNT–FZD signaling both in normal closure of the human optic fissure and pathogenesis of coloboma. PMID:26908622

ISOLATION AND CHARACTERIZATION OF AXOLOTL NPDC-1 AND ITS EFFECTS ON RETINOIC ACID RECEPTOR SIGNALING

PubMed Central

Theodosiou, Maria; Monaghan, James R; Spencer, Michael L; Voss, S Randal; Noonan, Daniel J

2009-01-01

Retinoic acid, a key morphogen in early vertebrate development and tissue regeneration, mediates its effects through the binding of receptors that act as ligand-induced transcription factors. These binding events function to recruit an array of transcription co-regulatory proteins to specific gene promoters. One such co-regulatory protein, neuronal proliferation and differentiation control-1 (NPDC-1), is broadly expressed during mammalian development and functions as an in vitro repressor of retinoic acid receptor (RAR)-mediated transcription. To obtain comparative and developmental insights about NPDC-1 function, we cloned the axolotl (Ambystoma mexicanum) orthologue and measured transcript abundances among tissues sampled during the embryonic and juvenile phases of development, and also during spinal cord regeneration. Structurally, the axolotl orthologue of NPDC-1 retained sequence identity to mammalian sequences in all functional domains. Functionally, we observed that axolotl NPDC-1 mRNA expression peaked late in embryogenesis, with highest levels of expression occurring during the time of limb development, a process regulated by retinoic acid signaling. Also similar to what has been observed in mammals, axolotl NPDC-1 directly interacts with axolotl RAR, modulates axolotl RAR DNA binding, and represses cell proliferation and axolotl RAR-mediated gene transcription. These data justify axolotl as a model to further investigate NPDC-1 and its role in regulating retinoic acid signaling. PMID:17331771

Regulation of DNA Replication Timing on Human Chromosome by a Cell-Type Specific DNA Binding Protein SATB1

PubMed Central

Oda, Masako; Kanoh, Yutaka; Watanabe, Yoshihisa; Masai, Hisao

2012-01-01

Background Replication timing of metazoan DNA during S-phase may be determined by many factors including chromosome structures, nuclear positioning, patterns of histone modifications, and transcriptional activity. It may be determined by Mb-domain structures, termed as “replication domains”, and recent findings indicate that replication timing is under developmental and cell type-specific regulation. Methodology/Principal Findings We examined replication timing on the human 5q23/31 3.5-Mb segment in T cells and non-T cells. We used two independent methods to determine replication timing. One is quantification of nascent replicating DNA in cell cycle-fractionated stage-specific S phase populations. The other is FISH analyses of replication foci. Although the locations of early- and late-replicating domains were common between the two cell lines, the timing transition region (TTR) between early and late domains were offset by 200-kb. We show that Special AT-rich sequence Binding protein 1 (SATB1), specifically expressed in T-cells, binds to the early domain immediately adjacent to TTR and delays the replication timing of the TTR. Measurement of the chromosome copy number along the TTR during synchronized S phase suggests that the fork movement may be slowed down by SATB1. Conclusions Our results reveal a novel role of SATB1 in cell type-specific regulation of replication timing along the chromosome. PMID:22879953

Regulation of DNA replication timing on human chromosome by a cell-type specific DNA binding protein SATB1.

PubMed

Oda, Masako; Kanoh, Yutaka; Watanabe, Yoshihisa; Masai, Hisao

2012-01-01

Replication timing of metazoan DNA during S-phase may be determined by many factors including chromosome structures, nuclear positioning, patterns of histone modifications, and transcriptional activity. It may be determined by Mb-domain structures, termed as "replication domains", and recent findings indicate that replication timing is under developmental and cell type-specific regulation. We examined replication timing on the human 5q23/31 3.5-Mb segment in T cells and non-T cells. We used two independent methods to determine replication timing. One is quantification of nascent replicating DNA in cell cycle-fractionated stage-specific S phase populations. The other is FISH analyses of replication foci. Although the locations of early- and late-replicating domains were common between the two cell lines, the timing transition region (TTR) between early and late domains were offset by 200-kb. We show that Special AT-rich sequence Binding protein 1 (SATB1), specifically expressed in T-cells, binds to the early domain immediately adjacent to TTR and delays the replication timing of the TTR. Measurement of the chromosome copy number along the TTR during synchronized S phase suggests that the fork movement may be slowed down by SATB1. Our results reveal a novel role of SATB1 in cell type-specific regulation of replication timing along the chromosome.

High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis.

PubMed

Sridharan, Vinod; Heimiller, Joseph; Robida, Mark D; Singh, Ravinder

2016-01-01

The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during spermatogenesis. The heph2 mutation in this gene results in a specific defect in spermatogenesis, causing aberrant spermatid individualization and male sterility. However, the array of molecular defects in the mutant remains uncharacterized. Using an unbiased high throughput sequencing approach, we have identified transcripts that are misregulated in this mutant. Aberrant transcripts show altered expression levels, exon skipping, and alternative 5' ends. We independently verified these findings by reverse-transcription and polymerase chain reaction (RT-PCR) analysis. Our analysis shows misregulation of transcripts that have been connected to spermatogenesis, including components of the actomyosin cytoskeletal apparatus. We show, for example, that the Myosin light chain 1 (Mlc1) transcript is aberrantly spliced. Furthermore, bioinformatics analysis reveals that Mlc1 contains a high affinity binding site(s) for dmPTB and that the site is conserved in many Drosophila species. We discuss that Mlc1 and other components of the actomyosin cytoskeletal apparatus offer important molecular links between the loss of dmPTB function and the observed developmental defect in spermatogenesis. This study provides the first comprehensive list of genes misregulated in vivo in the heph2 mutant in Drosophila and offers insight into the role of dmPTB during spermatogenesis.

RNA-binding proteins in eye development and disease: implication of conserved RNA granule components.

PubMed

Dash, Soma; Siddam, Archana D; Barnum, Carrie E; Janga, Sarath Chandra; Lachke, Salil A

2016-07-01

The molecular biology of metazoan eye development is an area of intense investigation. These efforts have led to the surprising recognition that although insect and vertebrate eyes have dramatically different structures, the orthologs or family members of several conserved transcription and signaling regulators such as Pax6, Six3, Prox1, and Bmp4 are commonly required for their development. In contrast, our understanding of posttranscriptional regulation in eye development and disease, particularly regarding the function of RNA-binding proteins (RBPs), is limited. We examine the present knowledge of RBPs in eye development in the insect model Drosophila as well as several vertebrate models such as fish, frog, chicken, and mouse. Interestingly, of the 42 RBPs that have been investigated for their expression or function in vertebrate eye development, 24 (~60%) are recognized in eukaryotic cells as components of RNA granules such as processing bodies, stress granules, or other specialized ribonucleoprotein (RNP) complexes. We discuss the distinct developmental and cellular events that may necessitate potential RBP/RNA granule-associated RNA regulon models to facilitate posttranscriptional control of gene expression in eye morphogenesis. In support of these hypotheses, three RBPs and RNP/RNA granule components Tdrd7, Caprin2, and Stau2 are linked to ocular developmental defects such as congenital cataract, Peters anomaly, and microphthalmia in human patients or animal models. We conclude by discussing the utility of interdisciplinary approaches such as the bioinformatics tool iSyTE (integrated Systems Tool for Eye gene discovery) to prioritize RBPs for deriving posttranscriptional regulatory networks in eye development and disease. WIREs RNA 2016, 7:527-557. doi: 10.1002/wrna.1355 For further resources related to this article, please visit the WIREs website. © 2016 Wiley Periodicals, Inc.

Mutations in the BAF-Complex Subunit DPF2 Are Associated with Coffin-Siris Syndrome.

PubMed

Vasileiou, Georgia; Vergarajauregui, Silvia; Endele, Sabine; Popp, Bernt; Büttner, Christian; Ekici, Arif B; Gerard, Marion; Bramswig, Nuria C; Albrecht, Beate; Clayton-Smith, Jill; Morton, Jenny; Tomkins, Susan; Low, Karen; Weber, Astrid; Wenzel, Maren; Altmüller, Janine; Li, Yun; Wollnik, Bernd; Hoganson, George; Plona, Maria-Renée; Cho, Megan T; Thiel, Christian T; Lüdecke, Hermann-Josef; Strom, Tim M; Calpena, Eduardo; Wilkie, Andrew O M; Wieczorek, Dagmar; Engel, Felix B; Reis, André

2018-03-01

Variants affecting the function of different subunits of the BAF chromatin-remodelling complex lead to various neurodevelopmental syndromes, including Coffin-Siris syndrome. Furthermore, variants in proteins containing PHD fingers, motifs recognizing specific histone tail modifications, have been associated with several neurological and developmental-delay disorders. Here, we report eight heterozygous de novo variants (one frameshift, two splice site, and five missense) in the gene encoding the BAF complex subunit double plant homeodomain finger 2 (DPF2). Affected individuals share common clinical features described in individuals with Coffin-Siris syndrome, including coarse facial features, global developmental delay, intellectual disability, speech impairment, and hypoplasia of fingernails and toenails. All variants occur within the highly conserved PHD1 and PHD2 motifs. Moreover, missense variants are situated close to zinc binding sites and are predicted to disrupt these sites. Pull-down assays of recombinant proteins and histone peptides revealed that a subset of the identified missense variants abolish or impaire DPF2 binding to unmodified and modified H3 histone tails. These results suggest an impairment of PHD finger structural integrity and cohesion and most likely an aberrant recognition of histone modifications. Furthermore, the overexpression of these variants in HEK293 and COS7 cell lines was associated with the formation of nuclear aggregates and the recruitment of both wild-type DPF2 and BRG1 to these aggregates. Expression analysis of truncating variants found in the affected individuals indicated that the aberrant transcripts escape nonsense-mediated decay. Altogether, we provide compelling evidence that de novo variants in DPF2 cause Coffin-Siris syndrome and propose a dominant-negative mechanism of pathogenicity. Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Heterozygous missense variants of LMX1A lead to nonsyndromic hearing impairment and vestibular dysfunction.

PubMed

Wesdorp, Mieke; de Koning Gans, Pia A M; Schraders, Margit; Oostrik, Jaap; Huynen, Martijn A; Venselaar, Hanka; Beynon, Andy J; van Gaalen, Judith; Piai, Vitória; Voermans, Nicol; van Rossum, Michelle M; Hartel, Bas P; Lelieveld, Stefan H; Wiel, Laurens; Verbist, Berit; Rotteveel, Liselotte J; van Dooren, Marieke F; Lichtner, Peter; Kunst, Henricus P M; Feenstra, Ilse; Admiraal, Ronald J C; Yntema, Helger G; Hoefsloot, Lies H; Pennings, Ronald J E; Kremer, Hannie

2018-05-12

Unraveling the causes and pathomechanisms of progressive disorders is essential for the development of therapeutic strategies. Here, we identified heterozygous pathogenic missense variants of LMX1A in two families of Dutch origin with progressive nonsyndromic hearing impairment (HI), using whole exome sequencing. One variant, c.721G > C (p.Val241Leu), occurred de novo and is predicted to affect the homeodomain of LMX1A, which is essential for DNA binding. The second variant, c.290G > C (p.Cys97Ser), predicted to affect a zinc-binding residue of the second LIM domain that is involved in protein-protein interactions. Bi-allelic deleterious variants of Lmx1a are associated with a complex phenotype in mice, including deafness and vestibular defects, due to arrest of inner ear development. Although Lmx1a mouse mutants demonstrate neurological, skeletal, pigmentation and reproductive system abnormalities, no syndromic features were present in the participating subjects of either family. LMX1A has previously been suggested as a candidate gene for intellectual disability, but our data do not support this, as affected subjects displayed normal cognition. Large variability was observed in the age of onset (a)symmetry, severity and progression rate of HI. About half of the affected individuals displayed vestibular dysfunction and experienced symptoms thereof. The late-onset progressive phenotype and the absence of cochleovestibular malformations on computed tomography scans indicate that heterozygous defects of LMX1A do not result in severe developmental abnormalities in humans. We propose that a single LMX1A wild-type copy is sufficient for normal development but insufficient for maintenance of cochleovestibular function. Alternatively, minor cochleovestibular developmental abnormalities could eventually lead to the progressive phenotype seen in the families.

RNA Binding Proteins in Eye Development and Disease: Implication of Conserved RNA Granule Components

PubMed Central

Dash, Soma; Siddam, Archana D.; Barnum, Carrie E.; Janga, Sarath Chandra

2016-01-01

The molecular biology of metazoan eye development is an area of intense investigation. These efforts have led to the surprising recognition that although insect and vertebrate eyes have dramatically different structures, the orthologs or family members of several conserved transcription and signaling regulators such as Pax6, Six3, Prox1 and Bmp4 are commonly required for their development. In contrast, our understanding of post-transcriptional regulation in eye development and disease, particularly regarding the function of RNA binding proteins (RBPs), is limited. We examine the present knowledge of RBPs in eye development in the insect model Drosophila, as well as several vertebrate models such as fish, frog, chicken and mouse. Interestingly, of the 42 RBPs that have been investigated with for their expression or function in vertebrate eye development, 24 (~60%) are recognized in eukaryotic cells as components of RNA granules such as Processing bodies (P-bodies), Stress granules, or other specialized ribonucleoprotein (RNP) complexes. We discuss the distinct developmental and cellular events that may necessitate potential RBP/RNA granule-associated RNA regulon models to facilitate post-transcriptional control of gene expression in eye morphogenesis. In support of these hypotheses, three RBPs and RNP/RNA granule components Tdrd7, Caprin2 and Stau2 are linked to ocular developmental defects such as congenital cataract, Peters anomaly and microphthalmia in human patients or animal models. We conclude by discussing the utility of interdisciplinary approaches such as the bioinformatics tool iSyTE (integrated Systems Tool for Eye gene discovery) to prioritize RBPs for deriving post-transcriptional regulatory networks in eye development and disease. PMID:27133484

Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus.

PubMed

Belak, Zachery R; Ovsenek, Nicholas; Eskiw, Christopher H

2018-05-23

Yin-Yang 1 (YY1) is a highly conserved transcription factor possessing RNA-binding activity. A putative YY1 homologue was previously identified in the developmental model organism Strongylocentrotus purpuratus (the purple sea urchin) by genomic sequencing. We identified a high degree of sequence similarity with YY1 homologues of vertebrate origin which shared 100% protein sequence identity over the DNA- and RNA-binding zinc-finger region with high similarity in the N-terminal transcriptional activation domain. SpYY1 demonstrated identical DNA- and RNA-binding characteristics between Xenopus laevis and S. purpuratus indicating that it maintains similar functional and biochemical properties across widely divergent deuterostome species. SpYY1 binds to the consensus YY1 DNA element, and also to U-rich RNA sequences. Although we detected SpYY1 RNA-binding activity in ova lysates and observed cytoplasmic localization, SpYY1 was not associated with maternal mRNA in ova. SpYY1 expressed in Xenopus oocytes was excluded from the nucleus and associated with maternally expressed cytoplasmic mRNA molecules. These data demonstrate the existence of an YY1 homologue in S. purpuratus with similar structural and biochemical features to those of the well-studied vertebrate YY1; however, the data reveal major differences in the biological role of YY1 in the regulation of maternally expressed mRNA in the two species.

Localization of palmitoylated and activated G protein α-subunit in Dictyostelium discoideum.

PubMed

Alamer, Sarah; Kageyama, Yusuke; Gundersen, Robert E

2018-06-01

Guanine nucleotide-binding proteins (G proteins) act as molecular switches to regulate many fundamental cellular processes. The lipid modification, palmitoylation, can be considered as a key factor for proper G protein function and plasma membrane localization. In Dictyostelium discoidum, Gα2 is essential for the chemotactic response to cAMP in their developmental life cycle. However, the regulation of Gα2 with respect to palmitoylation, activation and Gβγ association is less clear. In this study, Gα2 is shown to be palmitoylated on Cys-4 by [ 3 H]palmitate labeling. Loss of this palmitoylation site results in redistribution of Gα2 within the cell and poor D. discoideum development. Cellular re-localization is also observed for activated Gα2. In the membrane fraction, Gα2-wt (YFP) is highly enriched in a low-density membrane fraction, which is palmitoylation-dependent. Activated Gα2 monomer and heterotrimer are shifted to two different higher-density fractions. These results broaden our understanding of how G protein localization and function are regulated inside the cells. © 2018 Wiley Periodicals, Inc.

AES/GRG5: more than just a dominant-negative TLE/GRG family member.

PubMed

Beagle, Brandon; Johnson, Gail V W

2010-11-01

The human Transducin-like Enhancer of Split (TLE) and mouse homologue, Groucho gene-related protein (GRG), represent a family of conserved non-DNA binding transcriptional modulatory proteins divided into two subgroups based upon size. The long TLE/GRGs consist of four pentadomain proteins that are dedicated co-repressors for multiple transcription factors (TF). The second TLE/GRG subgroup is composed of the Amino-terminal Enhancer of Split (AES) in humans and its mouse homolog GRG5 (AES/GRG5). In contrast to the dedicated co-repressor function of long TLE/GRGs, AES/GRG5 can both positively or negatively modulate various TF as well as non-TF proteins in a long TLE/GRG-dependent or -independent manner. Therefore, AES/GRG5 is a functionally dynamic protein that is not exclusively defined by its role as a long TLE/GRG antagonist. AES/GRG5 may function in various developmental and pathological processes but the functional characteristics of endogenous AES/GRG5 in a physiologically relevant context remains to be determined. © 2010 Wiley-Liss, Inc.

Structural basis of glycan specificity in neonate-specific bovine-human reassortant rotavirus

DOE PAGES

Hu, Liya; Ramani, Sasirekha; Czako, Rita; ...

2015-09-30

We report that strain-dependent variation of glycan recognition during initial cell attachment of viruses is a critical determinant of host specificity, tissue-tropism and zoonosis. Rotaviruses (RVs), which cause life-threatening gastroenteritis in infants and children, display significant genotype-dependent variations in glycan recognition resulting from sequence alterations in the VP8* domain of the spike protein VP4. The structural basis of this genotype-dependent glycan specificity, particularly in human RVs, remains poorly understood. Here, from crystallographic studies, we show how genotypic variations configure a novel binding site in the VP8* of a neonate-specific bovine-human reassortant to uniquely recognize either type I or type IImore » precursor glycans, and to restrict type II glycan binding in the bovine counterpart. In conclusion, such a distinct glycan-binding site that allows differential recognition of the precursor glycans, which are developmentally regulated in the neonate gut and abundant in bovine and human milk provides a basis for age-restricted tropism and zoonotic transmission of G10P[11] rotaviruses.« less

Structural basis of glycan specificity in neonate-specific bovine-human reassortant rotavirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Liya; Ramani, Sasirekha; Czako, Rita

We report that strain-dependent variation of glycan recognition during initial cell attachment of viruses is a critical determinant of host specificity, tissue-tropism and zoonosis. Rotaviruses (RVs), which cause life-threatening gastroenteritis in infants and children, display significant genotype-dependent variations in glycan recognition resulting from sequence alterations in the VP8* domain of the spike protein VP4. The structural basis of this genotype-dependent glycan specificity, particularly in human RVs, remains poorly understood. Here, from crystallographic studies, we show how genotypic variations configure a novel binding site in the VP8* of a neonate-specific bovine-human reassortant to uniquely recognize either type I or type IImore » precursor glycans, and to restrict type II glycan binding in the bovine counterpart. In conclusion, such a distinct glycan-binding site that allows differential recognition of the precursor glycans, which are developmentally regulated in the neonate gut and abundant in bovine and human milk provides a basis for age-restricted tropism and zoonotic transmission of G10P[11] rotaviruses.« less

Changing Faces of Transcriptional Regulation Reflected by Zic3

PubMed Central

Winata, Cecilia Lanny; Kondrychyn, Igor; Deddens, J.C.; Korzh, Vladimir

2015-01-01

The advent of genomics in the study of developmental mechanisms has brought a trove of information on gene datasets and regulation during development, where the Zic family of zinc-finger proteins plays an important role. Genomic analysis of the modes of action of Zic3 in pluripotent cells demonstrated its requirement for maintenance of stem cells pluripotency upon binding to the proximal regulatory regions (promoters) of genes associated with cell pluripotency (Nanog, Sox2, Oct4, etc.) as well as cell cycle, proliferation, oncogenesis and early embryogenesis. In contrast, during gastrulation and neurulation Zic3 acts by binding the distal regulatory regions (enhancers, etc) associated with control of gene transcription in the Nodal and Wnt signaling pathways, including genes that act to break body symmetry. This illustrates a general role of Zic3 as a transcriptional regulator that acts not only alone, but in many instances in conjunction with other transcription factors. The latter is done by binding to adjacent sites in the context of multi-transcription factor complexes associated with regulatory elements. PMID:26085810

Control of seed dormancy and germination by DOG1-AHG1 PP2C phosphatase complex via binding to heme.

PubMed

Nishimura, Noriyuki; Tsuchiya, Wataru; Moresco, James J; Hayashi, Yuki; Satoh, Kouji; Kaiwa, Nahomi; Irisa, Tomoko; Kinoshita, Toshinori; Schroeder, Julian I; Yates, John R; Hirayama, Takashi; Yamazaki, Toshimasa

2018-06-06

Abscisic acid (ABA) regulates abiotic stress and developmental responses including regulation of seed dormancy to prevent seeds from germinating under unfavorable environmental conditions. ABA HYPERSENSITIVE GERMINATION1 (AHG1) encoding a type 2C protein phosphatase (PP2C) is a central negative regulator of ABA response in germination; however, the molecular function and regulation of AHG1 remain elusive. Here we report that AHG1 interacts with DELAY OF GERMINATION1 (DOG1), which is a pivotal positive regulator in seed dormancy. DOG1 acts upstream of AHG1 and impairs the PP2C activity of AHG1 in vitro. Furthermore, DOG1 has the ability to bind heme. Binding of DOG1 to AHG1 and heme are independent processes, but both are essential for DOG1 function in vivo. Our study demonstrates that AHG1 and DOG1 constitute an important regulatory system for seed dormancy and germination by integrating multiple environmental signals, in parallel with the PYL/RCAR ABA receptor-mediated regulatory system.

A novel DCX missense mutation in a family with X-linked lissencephaly and subcortical band heterotopia syndrome inherited from a low-level somatic mosaic mother: Genetic and functional studies.

PubMed

Tsai, Meng-Han; Kuo, Pei-Wen; Myers, Candace T; Li, Shih-Wen; Lin, Wei-Che; Fu, Ting-Ying; Chang, Hsin-Yun; Mefford, Heather C; Chang, Yao-Chung; Tsai, Jin-Wu

2016-09-01

To study the genetics and functional alteration of a family with X-linked lissencephaly and subcortical band heterotopia. Five affected patients (one male with lissencephaly, four female with subcortical band heterotopia) and their relatives were studied. Sanger sequencing of DCX gene, allele specific PCR and molecular inversion probe technique were performed. Mutant and wild type of the gene products, namely doublecortin, were expressed in cells followed by immunostaining to explore the localization of doublecortin and microtubules in cells. In vitro microtubule-binding protein spin-down assay was performed to quantify the binding ability of doublecortin to microtubules. We identified a novel DCX mutation c.785A > G, p.Asp262Gly that segregated with the affected members of the family. Allele specific PCR and molecular inversion probe technique demonstrated that the asymptomatic female carrier had an 8% mutant allele fraction in DNA derived from peripheral leukocytes. This mother had 7 children, 4 of whom were affected and all four affected siblings carried the mutation. Functional study showed that the mutant doublecortin protein had a significant reduction of its ability to bind microtubules. Low level mosaicism could be a cause of inherited risk from asymptomatic parents for DCX related lissencephaly-subcortical band heterotopia spectrum. This is particularly important in terms of genetic counselling for recurrent risk of future pregnancies. The reduced binding affinity of mutant doublecortin may contribute to developmental malformation of the cerebral cortex. Copyright © 2016 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

A positive feedback mechanism that regulates expression of miR-9 during neurogenesis.

PubMed

Davila, Jonathan L; Goff, Loyal A; Ricupero, Christopher L; Camarillo, Cynthia; Oni, Eileen N; Swerdel, Mavis R; Toro-Ramos, Alana J; Li, Jiali; Hart, Ronald P

2014-01-01

MiR-9, a neuron-specific miRNA, is an important regulator of neurogenesis. In this study we identify how miR-9 is regulated during early differentiation from a neural stem-like cell. We utilized two immortalized rat precursor clones, one committed to neurogenesis (L2.2) and another capable of producing both neurons and non-neuronal cells (L2.3), to reproducibly study early neurogenesis. Exogenous miR-9 is capable of increasing neurogenesis from L2.3 cells. Only one of three genomic loci capable of encoding miR-9 was regulated during neurogenesis and the promoter region of this locus contains sufficient functional elements to drive expression of a luciferase reporter in a developmentally regulated pattern. Furthermore, among a large number of potential regulatory sites encoded in this sequence, Mef2 stood out because of its known pro-neuronal role. Of four Mef2 paralogs, we found only Mef2C mRNA was regulated during neurogenesis. Removal of predicted Mef2 binding sites or knockdown of Mef2C expression reduced miR-9-2 promoter activity. Finally, the mRNA encoding the Mef2C binding partner HDAC4 was shown to be targeted by miR-9. Since HDAC4 protein could be co-immunoprecipitated with Mef2C protein or with genomic Mef2 binding sequences, we conclude that miR-9 regulation is mediated, at least in part, by Mef2C binding but that expressed miR-9 has the capacity to reduce inhibitory HDAC4, stabilizing its own expression in a positive feedback mechanism.

Characterization of Three Novel Fatty Acid- and Retinoid-Binding Protein Genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the Cereal Cyst Nematodes Heterodera avenae and H. filipjevi.

PubMed

Qiao, Fen; Luo, Lilian; Peng, Huan; Luo, Shujie; Huang, Wenkun; Cui, Jiangkuan; Li, Xin; Kong, Lingan; Jiang, Daohong; Chitwood, David J; Peng, Deliang

2016-01-01

Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 (KU877266), Ha-far-2 (KU877267), Hf-far-1 (KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in all stages. The highest expression level of Ha-far-1 was observed in fourth-stage juvenile (J4), whereas the highest expression level of Ha-far-2 occurred in second-stage juvenile (J2). In conclusion, we have identified two novel far genes from H. avenae and one from H. filipjevi and have provided further indication that nematode far genes are present in a variety of nematode species, where the FAR proteins share similar basic structure, expression pattern and biochemical activities.

Engineering of a Histone-Recognition Domain in Dnmt3a Alters the Epigenetic Landscape and Phenotypic Features of Mouse ESCs.

PubMed

Noh, Kyung-Min; Wang, Haibo; Kim, Hyunjae R; Wenderski, Wendy; Fang, Fang; Li, Charles H; Dewell, Scott; Hughes, Stephen H; Melnick, Ari M; Patel, Dinshaw J; Li, Haitao; Allis, C David

2015-07-02

Histone modification and DNA methylation are associated with varying epigenetic "landscapes," but detailed mechanistic and functional links between the two remain unclear. Using the ATRX-DNMT3-DNMT3L (ADD) domain of the DNA methyltransferase Dnmt3a as a paradigm, we apply protein engineering to dissect the molecular interactions underlying the recruitment of this enzyme to specific regions of chromatin in mouse embryonic stem cells (ESCs). By rendering the ADD domain insensitive to histone modification, specifically H3K4 methylation or H3T3 phosphorylation, we demonstrate the consequence of dysregulated Dnmt3a binding and activity. Targeting of a Dnmt3a mutant to H3K4me3 promoters decreases gene expression in a subset of developmental genes and alters ESC differentiation, whereas aberrant binding of another mutant to H3T3ph during mitosis promotes chromosome instability. Our studies support the general view that histone modification "reading" and DNA methylation are closely coupled in mammalian cells, and suggest an avenue for the functional assessment of chromatin-associated proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Hormones and Human and Nonhuman Primate Growth.

PubMed

Bernstein, Robin Miriam

2017-01-01

The aim of this paper was to review information pertaining to the hormonal regulation of nonhuman primate growth, with specific focus on the growth hormone (GH)-insulin-like growth factor (IGF) axis and adrenal androgens. Hormones of the GH-IGF axis are consistently associated with measures of growth - linear, weight, or both - during the growth period; in adulthood, concentrations of IGF-I, IGF-binding protein-3, and GH-binding protein are not associated with any measures of size. Comparing patterns of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) may be especially relevant for understanding whether the childhood stage of growth and development is unique to humans and perhaps other apes. Genetic, hormonal, and morphological data on adrenarche in other nonhuman primate species suggest that this endocrine transition is delayed in humans, chimpanzees, and possibly gorillas, while present very early in postnatal life in macaques. This suggests that although perhaps permitted by an extension of the pre-adolescent growth period, childhood builds upon existing developmental substrates rather than having been inserted de novo into an ancestral growth trajectory. Hormones can provide insight regarding the evolution of the human growth trajectory. © 2017 S. Karger AG, Basel.

Deciphering the roles of acyl-CoA-binding proteins in plant cells.

PubMed

Lung, Shiu-Cheung; Chye, Mee-Len

2016-09-01

Lipid trafficking is vital for metabolite exchange and signal communications between organelles and endomembranes. Acyl-CoA-binding proteins (ACBPs) are involved in the intracellular transport, protection, and pool formation of acyl-CoA esters, which are important intermediates and regulators in lipid metabolism and cellular signaling. In this review, we highlight recent advances in our understanding of plant ACBP families from a cellular and developmental perspective. Plant ACBPs have been extensively studied in Arabidopsis thaliana (a dicot) and to a lesser extent in Oryza sativa (a monocot). Thus far, they have been detected in the plasma membrane, vesicles, endoplasmic reticulum, Golgi apparatus, apoplast, cytosol, nuclear periphery, and peroxisomes. In combination with biochemical and molecular genetic tools, the widespread subcellular distribution of respective ACBP members has been explicitly linked to their functions in lipid metabolism during development and in response to stresses. At the cellular level, strong expression of specific ACBP homologs in specialized cells, such as embryos, stem epidermis, guard cells, male gametophytes, and phloem sap, is of relevance to their corresponding distinct roles in organ development and stress responses. Other interesting patterns in their subcellular localization and spatial expression that prompt new directions in future investigations are discussed.

ATP-Binding Cassette Efflux Transporters in Human Placenta

PubMed Central

Ni, Zhanglin; Mao, Qingcheng

2010-01-01

Pregnant women are often complicated with diseases including viral or bacterial infections, epilepsy, hypertension, or pregnancy-induced conditions such as depression and gestational diabetes that require treatment with medication. In addition, substance abuse during pregnancy remains a major public health problem. Many drugs used by pregnant women are off label without the necessary dose, efficacy, and safety data required for rational dosing regimens of these drugs. Thus, a major concern arising from the widespread use of drugs by pregnant women is the transfer of drugs across the placental barrier, leading to potential toxicity to the developing fetus. Knowledge regarding the ATP-binding cassette (ABC) efflux transporters, which play an important role in drug transfer across the placental barrier, is absolutely critical for optimizing the therapeutic strategy to treat the mother while protecting the fetus during pregnancy. Such transporters include P-glycoprotein (P-gp, gene symbol ABCB1), the breast cancer resistance protein (BCRP, gene symbol ABCG2), and the multidrug resistance proteins (MRPs, gene symbol ABCCs). In this review, we summarize the current knowledge with respect to developmental expression and regulation, membrane localization, functional significance, and genetic polymorphisms of these ABC transporters in the placenta and their relevance to fetal drug exposure and toxicity. PMID:21118087

Two chemoreceptors mediate developmental effects of dauer pheromone in C. elegans.

PubMed

Kim, Kyuhyung; Sato, Koji; Shibuya, Mayumi; Zeiger, Danna M; Butcher, Rebecca A; Ragains, Justin R; Clardy, Jon; Touhara, Kazushige; Sengupta, Piali

2009-11-13

Intraspecific chemical communication is mediated by signals called pheromones. Caenorhabditis elegans secretes a mixture of small molecules (collectively termed dauer pheromone) that regulates entry into the alternate dauer larval stage and also modulates adult behavior via as yet unknown receptors. Here, we identify two heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) that mediate dauer formation in response to a subset of dauer pheromone components. The SRBC-64 and SRBC-66 GPCRs are members of the large Caenorhabditis-specific SRBC subfamily and are expressed in the ASK chemosensory neurons, which are required for pheromone-induced dauer formation. Expression of both, but not each receptor alone, confers pheromone-mediated effects on heterologous cells. Identification of dauer pheromone receptors will allow a better understanding of the signaling cascades that transduce the context-dependent effects of ecologically important chemical signals.

Embryogenesis and laboratory maintenance of the foam-nesting túngara frogs, genus Engystomops (= Physalaemus)

PubMed Central

Romero-Carvajal, Andrés; Sáenz-Ponce, Natalia; Venegas-Ferrín, Michael; Almeida-Reinoso, Diego; Lee, Chanjae; Bond, Jennifer; Ryan, Michael J.; Wallingford, John B.; del Pino, Eugenia M.

2010-01-01

The vast majority of embryological research on amphibians focuses on just a single genus of frogs, Xenopus. To attain a more comprehensive understanding of amphibian development, experimentation on non-model frogs will be essential. Here, we report on the early development, rearing, and embryological analysis of túngara frogs (genus Engystomops, also called Physaleamus). The frogs Engystomops pustulosus, Engystomops coloradorum and Engystomops randi construct floating foam-nests with small eggs. We define a table of 23 stages for the developmental period in the foam-nest. Embryos were immunostained against Lim1, neural, and somite-specific proteins and the expression pattern of RetinoBlastoma Binding Protein 6 (RBBP6) was analyzed by in situ hybridization. Due to their brief life-cycle, frogs belonging to the genus Engystomops are attractive for comparative and genetic studies of development. PMID:19384855

Osmotin: a plant sentinel and a possible agonist of mammalian adiponectin

PubMed Central

Anil Kumar, S.; Hima Kumari, P.; Shravan Kumar, G.; Mohanalatha, C.; Kavi Kishor, P. B.

2015-01-01

Osmotin is a stress responsive antifungal protein belonging to the pathogenesis-related (PR)-5 family that confers tolerance to both biotic and abiotic stresses in plants. Protective efforts of osmotin in plants range from high temperature to cold and salt to drought. It lyses the plasma membrane of the pathogens. It is widely distributed in fruits and vegetables. It is a differentially expressed and developmentally regulated protein that protects the cells from osmotic stress and invading pathogens as well, by structural or metabolic alterations. During stress conditions, osmotin helps in the accumulation of the osmolyte proline, which quenches reactive oxygen species and free radicals. Osmotin expression results in the accumulation of storage reserves and increases the shelf-life of fruits. It binds to a seven-transmembrane-domain receptor-like protein and induces programmed cell death in Saccharomyces cerevisiae through RAS2/cAMP signaling pathway. Adiponectin, produced in adipose tissues of mammals, is an insulin-sensitizing hormone. Strangely, osmotin acts like the mammalian hormone adiponectin in various in vitro and in vivo models. Adiponectin and osmotin, the two receptor binding proteins do not share sequence similarity at the amino acid level, but interestingly they have a similar structural and functional properties. In experimental mice, adiponectin inhibits endothelial cell proliferation and migration, primary tumor growth, and reduces atherosclerosis. This retrospective work examines the vital role of osmotin in plant defense and as a potential targeted therapeutic drug for humans. PMID:25852715

Osmotin: a plant sentinel and a possible agonist of mammalian adiponectin.

PubMed

Anil Kumar, S; Hima Kumari, P; Shravan Kumar, G; Mohanalatha, C; Kavi Kishor, P B

2015-01-01

Osmotin is a stress responsive antifungal protein belonging to the pathogenesis-related (PR)-5 family that confers tolerance to both biotic and abiotic stresses in plants. Protective efforts of osmotin in plants range from high temperature to cold and salt to drought. It lyses the plasma membrane of the pathogens. It is widely distributed in fruits and vegetables. It is a differentially expressed and developmentally regulated protein that protects the cells from osmotic stress and invading pathogens as well, by structural or metabolic alterations. During stress conditions, osmotin helps in the accumulation of the osmolyte proline, which quenches reactive oxygen species and free radicals. Osmotin expression results in the accumulation of storage reserves and increases the shelf-life of fruits. It binds to a seven-transmembrane-domain receptor-like protein and induces programmed cell death in Saccharomyces cerevisiae through RAS2/cAMP signaling pathway. Adiponectin, produced in adipose tissues of mammals, is an insulin-sensitizing hormone. Strangely, osmotin acts like the mammalian hormone adiponectin in various in vitro and in vivo models. Adiponectin and osmotin, the two receptor binding proteins do not share sequence similarity at the amino acid level, but interestingly they have a similar structural and functional properties. In experimental mice, adiponectin inhibits endothelial cell proliferation and migration, primary tumor growth, and reduces atherosclerosis. This retrospective work examines the vital role of osmotin in plant defense and as a potential targeted therapeutic drug for humans.

Function and evolution of a gene family encoding odorant binding-like proteins in a social insect, the honey bee (Apis mellifera).

PubMed

Forêt, Sylvain; Maleszka, Ryszard

2006-11-01

The remarkable olfactory power of insect species is thought to be generated by a combinatorial action of two large protein families, G protein-coupled olfactory receptors (ORs) and odorant binding proteins (OBPs). In olfactory sensilla, OBPs deliver hydrophobic airborne molecules to ORs, but their expression in nonolfactory tissues suggests that they also may function as general carriers in other developmental and physiological processes. Here we used bioinformatic and experimental approaches to characterize the OBP-like gene family in a highly social insect, the Western honey bee. Comparison with other insects shows that the honey bee has the smallest set of these genes, consisting of only 21 OBPs. This number stands in stark contrast to the more than 70 OBPs in Anopheles gambiae and 51 in Drosophila melanogaster. In the honey bee as in the two dipterans, these genes are organized in clusters. We show that the evolution of their structure involved frequent intron losses. We describe a monophyletic subfamily of OBPs where the diversification of some amino acids appears to have been accelerated by positive selection. Expression profiling under a wide range of conditions shows that in the honey bee only nine OBPs are antenna-specific. The remaining genes are expressed either ubiquitously or are tightly regulated in specialized tissues or during development. These findings support the view that OBPs are not restricted to olfaction and are likely to be involved in broader physiological functions.

Drosophila COP9 signalosome subunit 7 interacts with multiple genomic loci to regulate development.

PubMed

Singer, Ruth; Atar, Shimshi; Atias, Osnat; Oron, Efrat; Segal, Daniel; Hirsch, Joel A; Tuller, Tamir; Orian, Amir; Chamovitz, Daniel A

2014-09-01

The COP9 signalosome protein complex has a central role in the regulation of development of multicellular organisms. While the function of this complex in ubiquitin-mediated protein degradation is well established, results over the past few years have hinted that the COP9 signalosome may function more broadly in the regulation of gene expression. Here, using DamID technology, we show that COP9 signalosome subunit 7 functionally associates with a large number of genomic loci in the Drosophila genome, and show that the expression of many genes within these loci is COP9 signalosome-dependent. This association is likely direct as we show CSN7 binds DNA in vitro. The genes targeted by CSN7 are preferentially enriched for transcriptionally active regions of the genome, and are involved in the regulation of distinct gene ontology groupings including imaginal disc development and cell-cycle control. In accord, loss of CSN7 function leads to cell-cycle delay and altered wing development. These results indicate that CSN7, and by extension the entire COP9 signalosome, functions directly in transcriptional control. While the COP9 signalosome protein complex has long been known to regulate protein degradation, here we expand the role of this complex by showing that subunit 7 binds DNA in vitro and functions directly in vivo in transcriptional control of developmentally important pathways that are relevant for human health. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis

PubMed Central

Chau, Johnnie; Kulnane, Laura Shapiro; Salz, Helen K.

2012-01-01

Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. We address the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. We find that this dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3′ untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior. PMID:22645327

Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis.

PubMed

Chau, Johnnie; Kulnane, Laura Shapiro; Salz, Helen K

2012-06-12

Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. We address the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. We find that this dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3' untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior.

ATM Substrate Chk2-interacting Zn2+ Finger (ASCIZ) Is a Bi-functional Transcriptional Activator and Feedback Sensor in the Regulation of Dynein Light Chain (DYNLL1) Expression*

PubMed Central

Jurado, Sabine; Conlan, Lindus A.; Baker, Emma K.; Ng, Jane-Lee; Tenis, Nora; Hoch, Nicolas C.; Gleeson, Kimberly; Smeets, Monique; Izon, David; Heierhorst, Jörg

2012-01-01

The highly conserved DYNLL1 (LC8) protein was originally discovered as a light chain of the dynein motor complex, but is increasingly emerging as a sequence-specific regulator of protein dimerization with hundreds of targets and wide-ranging cellular functions. Despite its important roles, DYNLL1's own regulation remains poorly understood. Here we identify ASCIZ (ATMIN/ZNF822), an essential Zn2+ finger protein with dual roles in the DNA base damage response and as a developmental transcription factor, as a conserved regulator of Dynll1 gene expression. DYNLL1 levels are reduced by ∼10-fold in the absence of ASCIZ in human, mouse and chicken cells. ASCIZ binds directly to the Dynll1 promoter and regulates its activity in a Zn2+ finger-dependent manner. DYNLL1 protein in turn interacts with ten binding sites in the ASCIZ transcription activation domain, and high DYNLL1 levels inhibit the transcriptional activity of ASCIZ. In addition, DYNLL1 was also required for DNA damage-induced ASCIZ focus formation. The dual ability of ASCIZ to activate Dynll1 gene expression and to sense free DYNLL1 protein levels enables a simple dynamic feedback loop to adjust DYNLL1 levels to cellular needs. The ASCIZ-DYNLL1 feedback loop represents a novel mechanism for auto-regulation of gene expression, where the gene product directly inhibits the transcriptional activator while bound at its own promoter. PMID:22167198

Identification, cloning and characterization of the tomato TCP transcription factor family.

PubMed

Parapunova, Violeta; Busscher, Marco; Busscher-Lange, Jacqueline; Lammers, Michiel; Karlova, Rumyana; Bovy, Arnaud G; Angenent, Gerco C; de Maagd, Ruud A

2014-06-06

TCP proteins are plant-specific transcription factors, which are known to have a wide range of functions in different plant species such as in leaf development, flower symmetry, shoot branching, and senescence. Only a small number of TCP genes has been characterised from tomato (Solanum lycopersicum). Here we report several functional features of the members of the entire family present in the tomato genome. We have identified 30 Solanum lycopersicum SlTCP genes, most of which have not been described before. Phylogenetic analysis clearly distinguishes two homology classes of the SlTCP transcription factor family - class I and class II. Class II differentiates in two subclasses, the CIN-TCP subclass and the CYC/TB1 subclass, involved in leaf development and axillary shoots formation, respectively. The expression patterns of all members were determined by quantitative PCR. Several SlTCP genes, like SlTCP12, SlTCP15 and SlTCP18 are preferentially expressed in the tomato fruit, suggesting a role during fruit development or ripening. These genes are regulated by RIN (RIPENING INHIBITOR), CNR (COLORLESS NON-RIPENING) and SlAP2a (APETALA2a) proteins, which are transcription factors with key roles in ripening. With a yeast one-hybrid assay we demonstrated that RIN binds the promoter fragments of SlTCP12, SlTCP15 and SlTCP18, and that CNR binds the SlTCP18 promoter. This data strongly suggests that these class I SlTCP proteins are involved in ripening. Furthermore, we demonstrate that SlTCPs bind the promoter fragments of members of their own family, indicating that they regulate each other. Additional yeast one-hybrid studies performed with Arabidopsis transcription factors revealed binding of the promoter fragments by proteins involved in the ethylene signal transduction pathway, contributing to the idea that these SlTCP genes are involved in the ripening process. Yeast two-hybrid data shows that SlTCP proteins can form homo and heterodimers, suggesting that they act together in order to form functional protein complexes and together regulate developmental processes in tomato. The comprehensive analysis we performed, like phylogenetic analysis, expression studies, identification of the upstream regulators and the dimerization specificity of the tomato TCP transcription factor family provides the basis for functional studies to reveal the role of this family in tomato development.

Identification, cloning and characterization of the tomato TCP transcription factor family

PubMed Central

2014-01-01

Background TCP proteins are plant-specific transcription factors, which are known to have a wide range of functions in different plant species such as in leaf development, flower symmetry, shoot branching, and senescence. Only a small number of TCP genes has been characterised from tomato (Solanum lycopersicum). Here we report several functional features of the members of the entire family present in the tomato genome. Results We have identified 30 Solanum lycopersicum SlTCP genes, most of which have not been described before. Phylogenetic analysis clearly distinguishes two homology classes of the SlTCP transcription factor family - class I and class II. Class II differentiates in two subclasses, the CIN-TCP subclass and the CYC/TB1 subclass, involved in leaf development and axillary shoots formation, respectively. The expression patterns of all members were determined by quantitative PCR. Several SlTCP genes, like SlTCP12, SlTCP15 and SlTCP18 are preferentially expressed in the tomato fruit, suggesting a role during fruit development or ripening. These genes are regulated by RIN (RIPENING INHIBITOR), CNR (COLORLESS NON-RIPENING) and SlAP2a (APETALA2a) proteins, which are transcription factors with key roles in ripening. With a yeast one-hybrid assay we demonstrated that RIN binds the promoter fragments of SlTCP12, SlTCP15 and SlTCP18, and that CNR binds the SlTCP18 promoter. This data strongly suggests that these class I SlTCP proteins are involved in ripening. Furthermore, we demonstrate that SlTCPs bind the promoter fragments of members of their own family, indicating that they regulate each other. Additional yeast one-hybrid studies performed with Arabidopsis transcription factors revealed binding of the promoter fragments by proteins involved in the ethylene signal transduction pathway, contributing to the idea that these SlTCP genes are involved in the ripening process. Yeast two-hybrid data shows that SlTCP proteins can form homo and heterodimers, suggesting that they act together in order to form functional protein complexes and together regulate developmental processes in tomato. Conclusions The comprehensive analysis we performed, like phylogenetic analysis, expression studies, identification of the upstream regulators and the dimerization specificity of the tomato TCP transcription factor family provides the basis for functional studies to reveal the role of this family in tomato development. PMID:24903607

BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

PubMed

Wei, Qing; La, David; Kihara, Daisuke

2017-01-01

Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

PubMed

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

ELAV Links Paused Pol II to Alternative Polyadenylation in the Drosophila Nervous System

PubMed Central

Oktaba, Katarzyna; Zhang, Wei; Lotz, Thea Sabrina; Jun, David Jayhyun; Lemke, Sandra Beatrice; Ng, Samuel Pak; Esposito, Emilia; Levine, Michael; Hilgers, Valérie

2014-01-01

SUMMARY Alternative polyadenylation (APA) has been implicated in a variety of developmental and disease processes. A particularly dramatic form of APA occurs in the developing nervous system of flies and mammals, whereby various developmental genes undergo coordinate 3′ UTR extension. In Drosophila, the RNA-binding protein ELAV inhibits RNA processing at proximal polyadenylation sites, thereby fostering the formation of exceptionally long 3′ UTRs. Here, we present evidence that paused Pol II promotes recruitment of ELAV to extended genes. Replacing promoters of extended genes with heterologous promoters blocks normal 3′ extension in the nervous system, while extension-associated promoters can induce 3′ extension in ectopic tissues expressing ELAV. Computational analyses suggest that promoter regions of extended genes tend to contain paused Pol II and associated cis-regulatory elements such as GAGA. ChIP-Seq assays identify ELAV in the promoter regions of extended genes. Our study provides evidence for a regulatory link between promoter-proximal pausing and APA. PMID:25544561

Plasticity-related genes in brain development and amygdala-dependent learning.

PubMed

Ehrlich, D E; Josselyn, S A

2016-01-01

Learning about motivationally important stimuli involves plasticity in the amygdala, a temporal lobe structure. Amygdala-dependent learning involves a growing number of plasticity-related signaling pathways also implicated in brain development, suggesting that learning-related signaling in juveniles may simultaneously influence development. Here, we review the pleiotropic functions in nervous system development and amygdala-dependent learning of a signaling pathway that includes brain-derived neurotrophic factor (BDNF), extracellular signaling-related kinases (ERKs) and cyclic AMP-response element binding protein (CREB). Using these canonical, plasticity-related genes as an example, we discuss the intersection of learning-related and developmental plasticity in the immature amygdala, when aversive and appetitive learning may influence the developmental trajectory of amygdala function. We propose that learning-dependent activation of BDNF, ERK and CREB signaling in the immature amygdala exaggerates and accelerates neural development, promoting amygdala excitability and environmental sensitivity later in life. © 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Regulation of the Myxococcus xanthus C-Signal-Dependent Ω4400 Promoter by the Essential Developmental Protein FruA

PubMed Central

Yoder-Himes, Deborah R.; Kroos, Lee

2006-01-01

The bacterium Myxococcus xanthus employs extracellular signals to coordinate aggregation and sporulation during multicellular development. Extracellular, contact-dependent signaling that involves the CsgA protein (called C-signaling) activates FruA, a putative response regulator that governs a branched signaling pathway inside cells. One branch regulates cell movement, leading to aggregation. The other branch regulates gene expression, leading to sporulation. C-signaling is required for full expression of most genes induced after 6 h into development, including the gene identified by Tn5 lac insertion Ω4400. To determine if FruA is a direct regulator of Ω4400 transcription, a combination of in vivo and in vitro experiments was performed. Ω4400 expression was abolished in a fruA mutant. The DNA-binding domain of FruA bound specifically to DNA upstream of the promoter −35 region in vitro. Mutations between bp −86 and −77 greatly reduced binding. One of these mutations had been shown previously to reduce Ω4400 expression in vivo and make it independent of C-signaling. For the first time, chromatin immunoprecipitation (ChIP) experiments were performed on M. xanthus. The ChIP experiments demonstrated that FruA is associated with the Ω4400 promoter region late in development, even in the absence of C-signaling. Based on these results, we propose that FruA directly activates Ω4400 transcription to a moderate level prior to C-signaling and, in response to C-signaling, binds near bp −80 and activates transcription to a higher level. Also, the highly localized effects of mutations between bp −86 and −77 on DNA binding in vitro, together with recently published footprints, allow us to predict a consensus binding site of GTCG/CGA/G for the FruA DNA-binding domain. PMID:16816188

Functional assignment to JEV proteins using SVM.

PubMed

Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

2008-01-01

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP).

Functional assignment to JEV proteins using SVM

PubMed Central

Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

2008-01-01

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP). PMID:19052658

The Arabidopsis Polycomb Repressive Complex 1 (PRC1) Components AtBMI1A, B, and C Impact Gene Networks throughout All Stages of Plant Development1[OPEN

PubMed Central

Zhou, Yue

2017-01-01

Polycomb Group regulation in Arabidopsis (Arabidopsis thaliana) is required to maintain cell differentiation and allow developmental phase transitions. This is achieved by the activity of three PcG repressive complex 2s (PRC2s) and the participation of a yet poorly defined PRC1. Previous results showed that apparent PRC1 components perform discrete roles during plant development, suggesting the existence of PRC1 variants; however, it is not clear in how many processes these components participate. We show that AtBMI1 proteins are required to promote all developmental phase transitions and to control cell proliferation during organ growth and development, expanding their proposed range of action. While AtBMI1 function during germination is closely linked to B3 domain transcription factors VAL1/2 possibly in combination with GT-box binding factors, other AtBMI1 regulatory networks require participation of different factor combinations. Conversely, EMF1 and LHP1 bind many H3K27me3 positive genes up-regulated in atbmi1a/b/c mutants; however, loss of their function affects expression of a different subset, suggesting that even if EMF1, LHP1, and AtBMI1 exist in a common PRC1 variant, their role in repression depends on the functional context. PMID:27837089

Regulation of bone morphogenetic proteins in early embryonic development

NASA Astrophysics Data System (ADS)

Yamamoto, Yukiyo; Oelgeschläger, Michael

2004-11-01

Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

Polypyrimidine Tract Binding Protein Homologs from Arabidopsis Are Key Regulators of Alternative Splicing with Implications in Fundamental Developmental Processes[W

PubMed Central

Rühl, Christina; Stauffer, Eva; Kahles, André; Wagner, Gabriele; Drechsel, Gabriele; Rätsch, Gunnar; Wachter, Andreas

2012-01-01

Alternative splicing (AS) generates transcript variants by variable exon/intron definition and massively expands transcriptome diversity. Changes in AS patterns have been found to be linked to manifold biological processes, yet fundamental aspects, such as the regulation of AS and its functional implications, largely remain to be addressed. In this work, widespread AS regulation by Arabidopsis thaliana Polypyrimidine tract binding protein homologs (PTBs) was revealed. In total, 452 AS events derived from 307 distinct genes were found to be responsive to the levels of the splicing factors PTB1 and PTB2, which predominantly triggered splicing of regulated introns, inclusion of cassette exons, and usage of upstream 5′ splice sites. By contrast, no major AS regulatory function of the distantly related PTB3 was found. Dependent on their position within the mRNA, PTB-regulated events can both modify the untranslated regions and give rise to alternative protein products. We find that PTB-mediated AS events are connected to diverse biological processes, and the functional implications of selected instances were further elucidated. Specifically, PTB misexpression changes AS of PHYTOCHROME INTERACTING FACTOR6, coinciding with altered rates of abscisic acid–dependent seed germination. Furthermore, AS patterns as well as the expression of key flowering regulators were massively changed in a PTB1/2 level-dependent manner. PMID:23192226

Transcriptional deregulation of oncogenic myocyte enhancer factor 2C in T-cell acute lymphoblastic leukemia.

PubMed

Nagel, Stefan; Venturini, Letizia; Meyer, Corinna; Kaufmann, Maren; Scherr, Michaela; Drexler, Hans G; Macleod, Roderick A F

2011-02-01

Myocyte enhancer factor 2C (MEF2C) encodes a transcription factor which is ectopically expressed in T-cell acute lymphoblastic leukemia (T-ALL) cell lines, deregulated directly by ectopically expressed homeodomain protein NKX2-5 or by loss of promoter regions via del(5)(q14). Here, we analyzed the MEF2C 5'-region, thus identifying potential regulatory binding sites for GFI1B, basic helix-loop-helix proteins, STAT5, and HOXA9/HOXA10. Chromatin immunoprecipitation and overexpression analyses demonstrated direct activation by GFI1B and LYL1 and inhibition by STAT5. HOXA9/HOXA10 activated expression of NMYC which in turn mediated MEF2C repression, indicating an indirect mode of regulation via NMYC interactor (NMI) and STAT5. Lacking comma: Chromosomal deletion of the STAT5 binding site in LOUCY cells reduced protein levels of STAT5 in some MEF2C-positve T-ALL cell lines, and the presence of inhibitory IL7-JAK-STAT5 signaling highlighted the repressive impact of this factor in MEF2C regulation. Taken together, our results indicate that the expression of MEF2C in T-ALL cells is principally deregulated via activating leukemic transcription factors GFI1B or NKX2-5 and by escaping inhibitory developmental STAT5 signaling.

Abrogation of both short and long forms of latent transforming growth factor-β binding protein-1 causes defective cardiovascular development and is perinatally lethal.

PubMed

Horiguchi, Masahito; Todorovic, Vesna; Hadjiolova, Krassimira; Weiskirchen, Ralf; Rifkin, Daniel B

2015-04-01

Latent transforming growth factor-β binding protein-1 (LTBP-1) is an extracellular protein that is structurally similar to fibrillin and has an important role in controlling transforming growth factor-β (TGF-β) signaling by storing the cytokine in the extracellular matrix and by being involved in the conversion of the latent growth factor to its active form. LTBP-1 is found as both short (LTBP-1S) and long (LTBP-1L) forms, which are derived through the use of separate promoters. There is controversy regarding the importance of LTBP-1L, as Ltbp1L knockout mice showed multiple cardiovascular defects but the complete null mice did not. Here, we describe a third line of Ltbp1 knockout mice generated utilizing a conditional knockout strategy that ablated expression of both L and S forms of LTBP-1. These mice show severe developmental cardiovascular abnormalities and die perinatally; thus these animals display a phenotype similar to previously reported Ltbp1L knockout mice. We reinvestigated the other "complete" knockout line and found that these mice express a splice variant of LTBP-1L and, therefore, are not complete Ltbp1 knockouts. Our results clarify the phenotypes of Ltbp1 null mice and re-emphasize the importance of LTBP-1 in vivo. Copyright © 2015. Published by Elsevier B.V.

Differential RPA-1 and RAD-51 recruitment in vivo throughout the C. elegans germline, as revealed by laser microirradiation.

PubMed

Koury, Emily; Harrell, Kailey; Smolikove, Sarit

2018-01-25

Studies of the repair pathways associated with DNA double strand breaks (DSBs) are numerous, and provide evidence for cell-cycle specific regulation of homologous recombination (HR) by the regulation of its associated proteins. Laser microirradiation is a well-established method to examine in vitro kinetics of repair and allows for live-imaging of DSB repair from the moment of induction. Here we apply this method to whole, live organisms, introducing an effective system to analyze exogenous, microirradiation-induced breaks in the Caenorhabditis elegans germline. Through this method we observed the sequential kinetics of the recruitment of ssDNA binding proteins RPA-1 and RAD-51 in vivo. We analyze these kinetics throughout different regions of the germline, and thus throughout a range of developmental stages of mitotic and meiotic nuclei. Our analysis demonstrates a largely conserved timing of recruitment of ssDNA binding proteins to DSBs throughout the germline, with a delay of RAD-51 recruitment at mid-pachytene nuclei. Microirradiated nuclei are viable and undergo a slow kinetics of resolution. We observe RPA-1 and RAD-51 colocalization for hours post-microirradiation throughout the germline, suggesting that there are mixed RPA-1/RAD-51 filaments. Finally, through live imaging analysis we observed RAD-51 foci movement with low frequency of coalescence. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Differential RPA-1 and RAD-51 recruitment in vivo throughout the C. elegans germline, as revealed by laser microirradiation

PubMed Central

Koury, Emily; Harrell, Kailey

2018-01-01

Abstract Studies of the repair pathways associated with DNA double strand breaks (DSBs) are numerous, and provide evidence for cell-cycle specific regulation of homologous recombination (HR) by the regulation of its associated proteins. Laser microirradiation is a well-established method to examine in vitro kinetics of repair and allows for live-imaging of DSB repair from the moment of induction. Here we apply this method to whole, live organisms, introducing an effective system to analyze exogenous, microirradiation-induced breaks in the Caenorhabditis elegans germline. Through this method we observed the sequential kinetics of the recruitment of ssDNA binding proteins RPA-1 and RAD-51 in vivo. We analyze these kinetics throughout different regions of the germline, and thus throughout a range of developmental stages of mitotic and meiotic nuclei. Our analysis demonstrates a largely conserved timing of recruitment of ssDNA binding proteins to DSBs throughout the germline, with a delay of RAD-51 recruitment at mid-pachytene nuclei. Microirradiated nuclei are viable and undergo a slow kinetics of resolution. We observe RPA-1 and RAD-51 colocalization for hours post-microirradiation throughout the germline, suggesting that there are mixed RPA-1/RAD-51 filaments. Finally, through live imaging analysis we observed RAD-51 foci movement with low frequency of coalescence. PMID:29244155

Different Expression Profiles Suggest Functional Differentiation Among Chemosensory Proteins in Nilaparvata lugens (Hemiptera: Delphacidae)

PubMed Central

Yang, Ke; He, Peng; Dong, Shuang-Lin

2014-01-01

Abstract Chemosensory proteins (CSPs) play various roles in insect physiology including olfaction and development. The brown planthopper, Nilaparvata lugens Stål , is one of the most notorious rice pests worldwide. The wing-from variation and annually long distance migration imply that olfaction would play a key role in N. lugens behavior. In this study, full-length cDNAs of nine CSPs were cloned by the rapid amplification of cDNA ends procedure, and their expression profiles were determined by the quantitative real-time Polymerase Chain Reaction (qPCR), with regard to developmental stage, wing-form, gender, and tissues of short-wing adult. These NlugCSP genes showed distinct expression patterns, indicating different roles they play. In particular, NlugCSP5 was long wing form biased and highly expressed in female wings among tissues; NlugCSP1 was mainly expressed in male adults and abdomen; NlugCSP7 was widely expressed in chemosensory tissues but little in the nonchemosensory abdomen. The function of NlugCSP7 in olfaction was further explored by the competitive fluorescence binding assay using the recombinant protein. However, the recombinant NlugCSP7 showed no obvious binding with all tested volatile compounds, suggesting that it may participate in physiological processes other than olfaction. Our results provide bases and some important clues for the function of NlugCSPs . PMID:25527582

Long-Range Control of Gene Expression: Emerging Mechanisms and Disruption in Disease

PubMed Central

Kleinjan, Dirk A.; van Heyningen, Veronica

2005-01-01

Transcriptional control is a major mechanism for regulating gene expression. The complex machinery required to effect this control is still emerging from functional and evolutionary analysis of genomic architecture. In addition to the promoter, many other regulatory elements are required for spatiotemporally and quantitatively correct gene expression. Enhancer and repressor elements may reside in introns or up- and downstream of the transcription unit. For some genes with highly complex expression patterns—often those that function as key developmental control genes—the cis-regulatory domain can extend long distances outside the transcription unit. Some of the earliest hints of this came from disease-associated chromosomal breaks positioned well outside the relevant gene. With the availability of wide-ranging genome sequence comparisons, strong conservation of many noncoding regions became obvious. Functional studies have shown many of these conserved sites to be transcriptional regulatory elements that sometimes reside inside unrelated neighboring genes. Such sequence-conserved elements generally harbor sites for tissue-specific DNA-binding proteins. Developmentally variable chromatin conformation can control protein access to these sites and can regulate transcription. Disruption of these finely tuned mechanisms can cause disease. Some regulatory element mutations will be associated with phenotypes distinct from any identified for coding-region mutations. PMID:15549674

Immune-mediated steroid-responsive epileptic spasms and epileptic encephalopathy associated with VGKC-complex antibodies.

PubMed

Suleiman, Jehan; Brenner, Tanja; Gill, Deepak; Troedson, Christopher; Sinclair, Adriane J; Brilot, Fabienne; Vincent, Angela; Lang, Bethan; Dale, Russell C

2011-11-01

Autoantibodies that bind to voltage-gated potassium-channel complex proteins (VGKC-complex antibodies) occur frequently in adults with limbic encephalitis presenting with cognitive impairment and seizures. Recently, VGKC-complex antibodies have been described in a few children with limbic encephalitis, and children with unexplained encephalitis presenting with status epilepticus. We report a case of infantile-onset epileptic spasms and developmental delay compatible with epileptic encephalopathy. Our patient was a female infant, aged 4 months at presentation. She had evidence of immune activation in the central nervous system with elevated cerebrospinal fluid neopterin and mirrored oligoclonal bands, which prompted testing for autoantibodies. VGKC-complex antibodies were elevated (201 pmol/L, normal<100), but extended antibody testing, including leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein 2 (CASPR2), was negative. The patient showed a partial response to steroid treatment, which was started late in the disease course. On review at 13 months of age, her development was consistent with an age of 5 to 6 months. These results suggest that VGKC-complex antibodies might represent a marker of immune therapy responsiveness in a subgroup of patients with infantile epileptic encephalopathy. © The Authors. Developmental Medicine & Child Neurology © 2011 Mac Keith Press.

Developmentally regulated, alternative splicing of the Rpn10 gene generates multiple forms of 26S proteasomes

PubMed Central

Kawahara, Hiroyuki; Kasahara, Masanori; Nishiyama, Atsuya; Ohsumi, Keita; Goto, Tetsuya; Kishimoto, Takeo; Saeki, Yasushi; Yokosawa, Hideyoshi; Shimbara, Naoki; Murata, Shigeo; Chiba, Tomoki; Suzuki, Koichi; Tanaka, Keiji

2000-01-01

The 26S proteasome is a multisubunit protein- destroying machinery that degrades ubiquitin-tagged proteins. To date only a single species of Rpn10, which possibly functions as a multiubiquitin chain-binding subunit, has been identified in various organisms. Here we report that mouse Rpn10 mRNAs occur in at least five distinct forms, named Rpn10a to Rpn10e, and that they are generated from a single gene by developmentally regulated, alternative splicing. Rpn10a is ubiquitously expressed, whereas Rpn10e is expressed only in embryos, with the highest levels of expression in the brain. Both forms of Rpn10 are components of the 26S proteasome, with an apparently similar affinity for multiubiquitylated [125I]lysozyme in vitro. However, they exert markedly divergent effects on the destruction of B-type cyclin in Xenopus egg extracts. Thus, the 26S proteasome occurs in at least two functionally distinct forms: one containing a ubiquitously expressed Rpn10a and the other a newly identified, embryo-specific Rpn10e. While the former is thought to perform proteolysis constitutively in a wide variety of cells, the latter may play a specialized role in early embryonic development. PMID:10921894

Evaluation of a developmental hierarchy for breast cancer cells to assess risk-based patient selection for targeted treatment.

PubMed

Bliss, Sarah A; Paul, Sunirmal; Pobiarzyn, Piotr W; Ayer, Seda; Sinha, Garima; Pant, Saumya; Hilton, Holly; Sharma, Neha; Cunha, Maria F; Engelberth, Daniel J; Greco, Steven J; Bryan, Margarette; Kucia, Magdalena J; Kakar, Sham S; Ratajczak, Mariusz Z; Rameshwar, Pranela

2018-01-10

This study proposes that a novel developmental hierarchy of breast cancer (BC) cells (BCCs) could predict treatment response and outcome. The continued challenge to treat BC requires stratification of BCCs into distinct subsets. This would provide insights on how BCCs evade treatment and adapt dormancy for decades. We selected three subsets, based on the relative expression of octamer-binding transcription factor 4 A (Oct4A) and then analysed each with Affymetrix gene chip. Oct4A is a stem cell gene and would separate subsets based on maturation. Data analyses and gene validation identified three membrane proteins, TMEM98, GPR64 and FAT4. BCCs from cell lines and blood from BC patients were analysed for these three membrane proteins by flow cytometry, along with known markers of cancer stem cells (CSCs), CD44, CD24 and Oct4, aldehyde dehydrogenase 1 (ALDH1) activity and telomere length. A novel working hierarchy of BCCs was established with the most immature subset as CSCs. This group was further subdivided into long- and short-term CSCs. Analyses of 20 post-treatment blood indicated that circulating CSCs and early BC progenitors may be associated with recurrence or early death. These results suggest that the novel hierarchy may predict treatment response and prognosis.

Non-coding RNAs—Novel targets in neurotoxicity

PubMed Central

Tal, Tamara L.; Tanguay, Robert L.

2012-01-01

Over the past ten years non-coding RNAs (ncRNAs) have emerged as pivotal players in fundamental physiological and cellular processes and have been increasingly implicated in cancer, immune disorders, and cardiovascular, neurodegenerative, and metabolic diseases. MicroRNAs (miRNAs) represent a class of ncRNA molecules that function as negative regulators of post-transcriptional gene expression. miRNAs are predicted to regulate 60% of all human protein-coding genes and as such, play key roles in cellular and developmental processes, human health, and disease. Relative to counterparts that lack bindings sites for miRNAs, genes encoding proteins that are post-transcriptionally regulated by miRNAs are twice as likely to be sensitive to environmental chemical exposure. Not surprisingly, miRNAs have been recognized as targets or effectors of nervous system, developmental, hepatic, and carcinogenic toxicants, and have been identified as putative regulators of phase I xenobiotic-metabolizing enzymes. In this review, we give an overview of the types of ncRNAs and highlight their roles in neurodevelopment, neurological disease, activity-dependent signaling, and drug metabolism. We then delve into specific examples that illustrate their importance as mediators, effectors, or adaptive agents of neurotoxicants or neuroactive pharmaceutical compounds. Finally, we identify a number of outstanding questions regarding ncRNAs and neurotoxicity. PMID:22394481

Fragile X Mental Retardation Protein is Required for Programmed Cell Death and Clearance of Developmentally-Transient Peptidergic Neurons

PubMed Central

Gatto, Cheryl L.; Broadie, Kendal

2011-01-01

Fragile X syndrome (FXS), caused by loss of fragile X mental retardation 1 (FMR1) gene function, is the most common heritable cause of intellectual disability and autism spectrum disorders. The FMR1 product (FMRP) is an RNA-binding protein best established to function in activity-dependent modulation of synaptic connections. In the Drosophila FXS disease model, loss of functionally-conserved dFMRP causes synaptic overgrowth and overelaboration in pigment dispersing factor (PDF) peptidergic neurons in the adult brain. Here, we identify a very different component of PDF neuron misregulation in dfmr1 mutants: the aberrant retention of normally developmentally-transient PDF tritocerebral (PDF-TRI) neurons. In wild-type animals, PDF-TRI neurons in the central brain undergo programmed cell death and complete, processive clearance within days of eclosion. In the absence of dFMRP, a defective apoptotic program leads to constitutive maintenance of these peptidergic neurons. We tested whether this apoptotic defect is circuit-specific by examining crustacean cardioactive peptide (CCAP) and bursicon circuits, which are similarly developmentally-transient and normally eliminated immediately post-eclosion. In dfmr1 null mutants, CCAP/bursicon neurons also exhibit significantly delayed clearance dynamics, but are subsequently eliminated from the nervous system, in contrast to the fully persistent PDF-TRI neurons. Thus, the requirement of dFMRP for the retention of transitory peptidergic neurons shows evident circuit specificity. The novel defect of impaired apoptosis and aberrant neuron persistence in the Drosophila FXS model suggests an entirely new level of “pruning” dysfunction may contribute to the FXS disease state. PMID:21596027

Dysferlin is essential for endocytosis in the sea star oocyte.

PubMed

Oulhen, Nathalie; Onorato, Thomas M; Ramos, Isabela; Wessel, Gary M

2014-04-01

Dysferlin is a calcium-binding transmembrane protein involved in membrane fusion and membrane repair. In humans, mutations in the dysferlin gene are associated with muscular dystrophy. In this study, we isolated plasma membrane-enriched fractions from full-grown immature oocytes of the sea star, and identified dysferlin by mass spectrometry analysis. The full-length dysferlin sequence is highly conserved between human and the sea star. We learned that in the sea star Patiria miniata, dysferlin RNA and protein are expressed from oogenesis to gastrulation. Interestingly, the protein is highly enriched in the plasma membrane of oocytes. Injection of a morpholino against dysferlin leads to a decrease of endocytosis in oocytes, and to a developmental arrest during gastrulation. These results suggest that dysferlin is critical for normal endocytosis during oogenesis and for embryogenesis in the sea star and that this animal may be a useful model for studying the relationship of dysferlin structure as it relates to its function. Copyright © 2014 Elsevier Inc. All rights reserved.

Dysferlin is essential for endocytosis in the sea star oocyte

PubMed Central

Oulhen, Nathalie; Onorato, Thomas M.; Ramos, Isabela; Wessel, Gary M.

2014-01-01

Dysferlin is a calcium-binding transmembrane protein involved in membrane fusion and membrane repair. In humans, mutations in the dysferlin gene are associated with muscular dystrophy. In this study, we isolated plasma membrane-enriched fractions from full-grown immature oocytes of the sea star, and identified dysferlin by mass spectrometry analysis. The full-length dysferlin sequence is highly conserved between human and the sea star. We learned that in the sea star Patiria miniata, dysferlin RNA and protein are expressed from oogenesis to gastrulation. Interestingly, the protein is highly enriched in the plasma membrane of oocytes. Injection of a morpholino against dysferlin leads to a decrease of endocytosis in oocytes, and to a developmental arrest during gastrulation. These results suggest that dysferlin is critical for normal endocytosis during oogenesis and for embryogenesis in the sea star and that this animal may be a useful model for studying the relationship of dysferlin structure as it relates to its function. PMID:24368072

A Homozygous Nme7 Mutation Is Associated with Situs Inversus Totalis.

PubMed

Reish, Orit; Aspit, Liam; Zouella, Arielle; Roth, Yehudah; Polak-Charcon, Sylvie; Baboushkin, Tatiana; Benyamini, Lilach; Scheetz, Todd E; Mussaffi, Huda; Sheffield, Val C; Parvari, Ruti

2016-08-01

We investigated the cause of situs inversus totalis (SIT) in two siblings from a consanguineous family. Genotyping and whole-exome analysis revealed a homozygous change in NME7, resulting in deletion of an exon causing an in-frame deletion of 34 amino acids located in the second NDK domain of the protein and segregated with the defective lateralization in the family. NME7 is an important developmental gene, and NME7 protein is a component of the γ-tubulin ring complex. This mutation is predicted to affect the interaction of NME7 protein with this complex as it deletes the amino acids crucial for the binding. SIT associated with homozygous deletion in our patients is in line with Nme7(-/-) mutant mice phenotypes consisting of congenital hydrocephalus and SIT, indicating a novel human laterality patterning role for NME7. Further cases are required to elaborate the full human phenotype associated with NME7 mutations. © 2016 WILEY PERIODICALS, INC.

Control of developmentally primed erythroid genes by combinatorial co-repressor actions

PubMed Central

Stadhouders, Ralph; Cico, Alba; Stephen, Tharshana; Thongjuea, Supat; Kolovos, Petros; Baymaz, H. Irem; Yu, Xiao; Demmers, Jeroen; Bezstarosti, Karel; Maas, Alex; Barroca, Vilma; Kockx, Christel; Ozgur, Zeliha; van Ijcken, Wilfred; Arcangeli, Marie-Laure; Andrieu-Soler, Charlotte; Lenhard, Boris; Grosveld, Frank; Soler, Eric

2015-01-01

How transcription factors (TFs) cooperate within large protein complexes to allow rapid modulation of gene expression during development is still largely unknown. Here we show that the key haematopoietic LIM-domain-binding protein-1 (LDB1) TF complex contains several activator and repressor components that together maintain an erythroid-specific gene expression programme primed for rapid activation until differentiation is induced. A combination of proteomics, functional genomics and in vivo studies presented here identifies known and novel co-repressors, most notably the ETO2 and IRF2BP2 proteins, involved in maintaining this primed state. The ETO2–IRF2BP2 axis, interacting with the NCOR1/SMRT co-repressor complex, suppresses the expression of the vast majority of archetypical erythroid genes and pathways until its decommissioning at the onset of terminal erythroid differentiation. Our experiments demonstrate that multimeric regulatory complexes feature a dynamic interplay between activating and repressing components that determines lineage-specific gene expression and cellular differentiation. PMID:26593974

Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4.

PubMed

Boulanger, Marie-Chloé; Miranda, Tina Branscombe; Clarke, Steven; Di Fruscio, Marco; Suter, Beat; Lasko, Paul; Richard, Stéphane

2004-04-15

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 ( Drosophila arginine methyltransferases 1-9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation.

Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4.

PubMed Central

Boulanger, Marie-Chloé; Miranda, Tina Branscombe; Clarke, Steven; Di Fruscio, Marco; Suter, Beat; Lasko, Paul; Richard, Stéphane

2004-01-01

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 ( Drosophila arginine methyltransferases 1-9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation. PMID:14705965

Mutations in STAMBP, encoding a deubiquitinating enzyme, cause Microcephaly-Capillary Malformation syndrome

PubMed Central

McDonell, Laura M.; Mirzaa, Ghayda M.; Alcantara, Diana; Schwartzentruber, Jeremy; Carter, Melissa T.; Lee, Leo J.; Clericuzio, Carol L.; Graham, John M.; Morris-Rosendahl, Deborah J.; Polster, Tilman; Acsadi, Gyula; Townshend, Sharron; Williams, Simon; Halbert, Anne; Isidor, Bertrand; Smyser, Christopher D.; Paciorkowski, Alex R.; Willing, Marcia; Woulfe, John; Das, Soma; Beaulieu, Chandree L.; Marcadier, Janet; Geraghty, Michael T.; Frey, Brendan J.; Majewski, Jacek; Bulman, Dennis E.; Dobyns, William B.; O’Driscoll, Mark; Boycott, Kym M.

2014-01-01

Microcephaly-capillary malformation (MIC-CAP) syndrome exhibits severe microcephaly with progressive cortical atrophy, intractable epilepsy, profound developmental delay and multiple small capillary malformations on the skin. We employed whole-exome sequencing of five patients with MIC-CAP syndrome and identified novel recessive mutations in STAMBP, a gene encoding the deubiquitinating (DUB) isopeptidase STAMBP (STAM-binding protein)/AMSH (Associated Molecule with the SH3 domain of STAM), that plays a key role in cell surface receptor-mediated endocytosis and sorting. Patient cell lines showed reduced STAMBP expression associated with accumulation of ubiquitin-conjugated protein aggregates, elevated apoptosis and insensitive activation of the RAS-MAPK and PI3K-AKT-mTOR pathways. The latter cellular phenotype is significant considering the established connection between these pathways and their association with vascular and capillary malformations. Furthermore, our findings of a congenital human disorder caused by a defective DUB protein that functions in endocytosis, implicates ubiquitin-conjugate aggregation and elevated apoptosis as factors potentially influencing the progressive neuronal loss underlying MIC-CAP. PMID:23542699

21 CFR 866.5765 - Retinol-binding protein immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement of...

21 CFR 866.5765 - Retinol-binding protein immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement of...

21 CFR 866.5765 - Retinol-binding protein immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement of...

21 CFR 866.5765 - Retinol-binding protein immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement of...

Mechanisms of epigenetic and cell-type specific regulation of Hey target genes in ES cells and cardiomyocytes.

PubMed

Weber, David; Heisig, Julia; Kneitz, Susanne; Wolf, Elmar; Eilers, Martin; Gessler, Manfred

2015-02-01

Hey bHLH transcription factors are critical effectors of Notch signaling. During mammalian heart development they are expressed in atrial and ventricular cardiomyocytes and in the developing endocardium. Hey knockout mice suffer from lethal cardiac defects, such as ventricular septum defects, valve defects and cardiomyopathy. Despite this functional relevance, little is known about the regulation of downstream targets in relevant cell types. The objective of this study was to elucidate the regulatory mechanisms by which Hey proteins affect gene expression in a cell type specific manner. We used an in vitro cardiomyocyte differentiation system with inducible Hey1 or Hey2 expression to study target gene regulation in cardiomyocytes (CM) generated from murine embryonic stem cells (ESC). The effects of Hey1 and Hey2 are largely redundant, but cell type specific. The number of regulated genes is comparable between ESC and CM, but the total number of binding sites is much higher, especially in ESC, targeting mainly genes involved in transcriptional regulation and developmental processes. Repression by Hey proteins generally correlates with the extent of Hey-binding to target promoters, Hdac recruitment and lower histone acetylation. Functionally, treatment with the Hdac inhibitor TSA abolished Hey target gene regulation. However, in CM the repressive effect of Hey-binding is lost for a subset of genes. These also lack Hey-dependent histone deacetylation in CM and are enriched for binding sites of cardiac specific activators like Srf, Nkx2-5, and Gata4. Ectopic Nkx2-5 overexpression in ESC blocks Hey-mediated repression of these genes. Thus, Hey proteins mechanistically repress target genes via Hdac recruitment and histone deacetylation. In CM Hey-repression is counteracted by cardiac activators, which recruit histone acetylases and prevent Hey mediated deacetylation and subsequent repression for a subset of genes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dynamic CRM occupancy reflects a temporal map of developmental progression.

PubMed

Wilczyński, Bartek; Furlong, Eileen E M

2010-06-22

Development is driven by tightly coordinated spatio-temporal patterns of gene expression, which are initiated through the action of transcription factors (TFs) binding to cis-regulatory modules (CRMs). Although many studies have investigated how spatial patterns arise, precise temporal control of gene expression is less well understood. Here, we show that dynamic changes in the timing of CRM occupancy is a prevalent feature common to all TFs examined in a developmental ChIP time course to date. CRMs exhibit complex binding patterns that cannot be explained by the sequence motifs or expression of the TFs themselves. The temporal changes in TF binding are highly correlated with dynamic patterns of target gene expression, which in turn reflect transitions in cellular function during different stages of development. Thus, it is not only the timing of a TF's expression, but also its temporal occupancy in refined time windows, which determines temporal gene expression. Systematic measurement of dynamic CRM occupancy may therefore serve as a powerful method to decode dynamic changes in gene expression driving developmental progression.

Identification of compound-protein interactions through the analysis of gene ontology, KEGG enrichment for proteins and molecular fragments of compounds.

PubMed

Chen, Lei; Zhang, Yu-Hang; Zheng, Mingyue; Huang, Tao; Cai, Yu-Dong

2016-12-01

Compound-protein interactions play important roles in every cell via the recognition and regulation of specific functional proteins. The correct identification of compound-protein interactions can lead to a good comprehension of this complicated system and provide useful input for the investigation of various attributes of compounds and proteins. In this study, we attempted to understand this system by extracting properties from both proteins and compounds, in which proteins were represented by gene ontology and KEGG pathway enrichment scores and compounds were represented by molecular fragments. Advanced feature selection methods, including minimum redundancy maximum relevance, incremental feature selection, and the basic machine learning algorithm random forest, were used to analyze these properties and extract core factors for the determination of actual compound-protein interactions. Compound-protein interactions reported in The Binding Databases were used as positive samples. To improve the reliability of the results, the analytic procedure was executed five times using different negative samples. Simultaneously, five optimal prediction methods based on a random forest and yielding maximum MCCs of approximately 77.55 % were constructed and may be useful tools for the prediction of compound-protein interactions. This work provides new clues to understanding the system of compound-protein interactions by analyzing extracted core features. Our results indicate that compound-protein interactions are related to biological processes involving immune, developmental and hormone-associated pathways.

Genome-wide identification and characterization of TCP genes involved in ovule development of Phalaenopsis equestris.

PubMed

Lin, Yu-Fu; Chen, You-Yi; Hsiao, Yu-Yun; Shen, Ching-Yu; Hsu, Jui-Ling; Yeh, Chuan-Ming; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Liu, Zhong-Jian; Tsai, Wen-Chieh

2016-09-01

TEOSINTE-BRANCHED/CYCLOIDEA/PCF (TCP) proteins are plant-specific transcription factors known to have a role in multiple aspects of plant growth and development at the cellular, organ and tissue levels. However, there has been no related study of TCPs in orchids. Here we identified 23 TCP genes from the genome sequence of Phalaenopsis equestris Phylogenetic analysis distinguished two homology classes of PeTCP transcription factor families: classes I and II. Class II was further divided into two subclasses, CIN and CYC/TB1. Spatial and temporal expression analysis showed that PePCF10 was predominantly expressed in ovules at early developmental stages and PeCIN8 had high expression at late developmental stages in ovules, with overlapping expression at day 16 after pollination. Subcellular localization and protein-protein interaction analyses revealed that PePCF10 and PeCIN8 could form homodimers and localize in the nucleus. However, PePCF10 and PeCIN8 could not form heterodimers. In transgenic Arabidopsis thaliana plants (overexpression and SRDX, a super repression motif derived from the EAR-motif of the repression domain of tobacco ETHYLENE-RESPONSIVE ELEMENT-BINDING FACTOR 3 and SUPERMAN, dominantly repressed), the two genes helped regulate cell proliferation. Together, these results suggest that PePCF10 and PeCIN8 play important roles in orchid ovule development by modulating cell division. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

In vitro reconstitution of T cell receptor-mediated segregation of the CD45 phosphatase

PubMed Central

Carbone, Catherine B.; Fernandes, Ricardo A.; Hui, Enfu; Su, Xiaolei; Garcia, K. Christopher; Vale, Ronald D.

2017-01-01

T cell signaling initiates upon the binding of peptide-loaded MHC (pMHC) on an antigen-presenting cell to the T cell receptor (TCR) on a T cell. TCR phosphorylation in response to pMHC binding is accompanied by segregation of the transmembrane phosphatase CD45 away from TCR–pMHC complexes. The kinetic segregation hypothesis proposes that CD45 exclusion shifts the local kinase–phosphatase balance to favor TCR phosphorylation. Spatial partitioning may arise from the size difference between the large CD45 extracellular domain and the smaller TCR–pMHC complex, although parsing potential contributions of extracellular protein size, actin activity, and lipid domains is difficult in living cells. Here, we reconstitute segregation of CD45 from bound receptor–ligand pairs using purified proteins on model membranes. Using a model receptor–ligand pair (FRB–FKBP), we first test physical and computational predictions for protein organization at membrane interfaces. We then show that the TCR–pMHC interaction causes partial exclusion of CD45. Comparing two developmentally regulated isoforms of CD45, the larger RABC variant is excluded more rapidly and efficiently (∼50%) than the smaller R0 isoform (∼20%), suggesting that CD45 isotypes could regulate signaling thresholds in different T cell subtypes. Similar to the sensitivity of T cell signaling, TCR–pMHC interactions with Kds of ≤15 µM were needed to exclude CD45. We further show that the coreceptor PD-1 with its ligand PD-L1, immunotherapy targets that inhibit T cell signaling, also exclude CD45. These results demonstrate that the binding energies of physiological receptor–ligand pairs on the T cell are sufficient to create spatial organization at membrane–membrane interfaces. PMID:29042512

Secreted Phosphoprotein 1 Is a Determinant of Lung Function Development in Mice

PubMed Central

Martin, Timothy M.; Concel, Vincent J.; Upadhyay, Swapna; Bein, Kiflai; Brant, Kelly A.; George, Leema; Mitra, Ankita; Thimraj, Tania A.; Fabisiak, James P.; Vuga, Louis J.; Fattman, Cheryl; Kaminski, Naftali; Schulz, Holger; Leikauf, George D.

2014-01-01

Secreted phosphoprotein 1 (Spp1) is located within quantitative trait loci associated with lung function that was previously identified by contrasting C3H/HeJ and JF1/Msf mouse strains that have extremely divergent lung function. JF1/Msf mice with diminished lung function had reduced lung SPP1 transcript and protein during the peak stage of alveologenesis (postnatal day [P]14–P28) as compared with C3H/HeJ mice. In addition to a previously identified genetic variant that altered runt-related transcription factor 2 (RUNX2) binding in the Spp1 promoter, we identified another promoter variant in a putative RUNX2 binding site that increased the DNA protein binding. SPP1 induced dose-dependent mouse lung epithelial-15 cell proliferation. Spp1(−/−) mice have decreased specific total lung capacity/body weight, higher specific compliance, and increased mean airspace chord length (Lm) compared with Spp1(+/+) mice. Microarray analysis revealed enriched gene ontogeny categories, with numerous genes associated with lung development and/or respiratory disease. Insulin-like growth factor 1, Hedgehog-interacting protein, wingless-related mouse mammary tumor virus integration site 5A, and NOTCH1 transcripts decreased in the lung of P14 Spp1(−/−) mice as determined by quantitative RT-PCR analysis. SPP1 promotes pneumocyte growth, and mice lacking SPP1 have smaller, more compliant lungs with enlarged airspace (i.e., increased Lm). Microarray analysis suggests a dysregulation of key lung developmental transcripts in gene-targeted Spp1(−/−) mice, particularly during the peak phase of alveologenesis. In addition to its known roles in lung disease, this study supports SPP1 as a determinant of lung development in mice. PMID:24816281

Control of dopaminergic neuron survival by the unfolded protein response transcription factor XBP1

PubMed Central

Valdés, Pamela; Mercado, Gabriela; Vidal, Rene L.; Molina, Claudia; Parsons, Geoffrey; Court, Felipe A.; Martinez, Alexis; Galleguillos, Danny; Armentano, Donna; Schneider, Bernard L.; Hetz, Claudio

2014-01-01

Parkinson disease (PD) is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta (SNpc). Although growing evidence indicates that endoplasmic reticulum (ER) stress is a hallmark of PD, its exact contribution to the disease process is not well understood. Here we report that developmental ablation of X-Box binding protein 1 (XBP1) in the nervous system, a key regulator of the unfolded protein response (UPR), protects dopaminergic neurons against a PD-inducing neurotoxin. This survival effect was associated with a preconditioning condition that resulted from induction of an adaptive ER stress response in dopaminergic neurons of the SNpc, but not in other brain regions. In contrast, silencing XBP1 in adult animals triggered chronic ER stress and dopaminergic neuron degeneration. Supporting this finding, gene therapy to deliver an active form of XBP1 provided neuroprotection and reduced striatal denervation in animals injected with 6-hydroxydopamine. Our results reveal a physiological role of the UPR in the maintenance of protein homeostasis in dopaminergic neurons that may help explain the differential neuronal vulnerability observed in PD. PMID:24753614

BASIC PENTACYSTEINE Proteins Mediate MADS Domain Complex Binding to the DNA for Tissue-Specific Expression of Target Genes in Arabidopsis[W

PubMed Central

Simonini, Sara; Roig-Villanova, Irma; Gregis, Veronica; Colombo, Bilitis; Colombo, Lucia; Kater, Martin M.

2012-01-01

BASIC PENTACYSTEINE (BPC) transcription factors have been identified in a large variety of plant species. In Arabidopsis thaliana there are seven BPC genes, which, except for BPC5, are expressed ubiquitously. BPC genes are functionally redundant in a wide range of developmental processes. Recently, we reported that BPC1 binds to guanine and adenine (GA)–rich consensus sequences in the SEEDSTICK (STK) promoter in vitro and induces conformational changes. Here we show by chromatin immunoprecipitation experiments that in vivo BPCs also bind to the consensus boxes, and when these were mutated, expression from the STK promoter was derepressed, resulting in ectopic expression in the inflorescence. We also reveal that SHORT VEGETATIVE PHASE (SVP) is a direct regulator of STK. SVP is a floral meristem identity gene belonging to the MADS box gene family. The SVP-APETALA1 (AP1) dimer recruits the SEUSS (SEU)-LEUNIG (LUG) transcriptional cosuppressor to repress floral homeotic gene expression in the floral meristem. Interestingly, we found that GA consensus sequences in the STK promoter to which BPCs bind are essential for recruitment of the corepressor complex to this promoter. Our data suggest that we have identified a new regulatory mechanism controlling plant gene expression that is probably generally used, when considering BPCs’ wide expression profile and the frequent presence of consensus binding sites in plant promoters. PMID:23054472

Thioredoxin binding protein (TBP)-2/Txnip and α-arrestin proteins in cancer and diabetes mellitus.

PubMed

Masutani, Hiroshi; Yoshihara, Eiji; Masaki, So; Chen, Zhe; Yodoi, Junji

2012-01-01

Thioredoxin binding protein -2/ thioredoxin interacting protein is an α-arrestin protein that has attracted much attention as a multifunctional regulator. Thioredoxin binding protein -2 expression is downregulated in tumor cells and the level of thioredoxin binding protein is correlated with clinical stage of cancer. Mice with mutations or knockout of the thioredoxin binding protein -2 gene are much more susceptible to carcinogenesis than wild-type mice, indicating a role for thioredoxin binding protein -2 in cancer suppression. Studies have also revealed roles for thioredoxin binding protein -2 in metabolic control. Enhancement of thioredoxin binding protein -2 expression causes impairment of insulin sensitivity and glucose-induced insulin secretion, and β-cell apoptosis. These changes are important characteristics of type 2 diabetes mellitus. Thioredoxin binding protein -2 regulates transcription of metabolic regulating genes. Thioredoxin binding protein -2-like inducible membrane protein/ arrestin domain containing 3 regulates endocytosis of receptors such as the β(2)-adrenergic receptor. The α-arrestin family possesses PPXY motifs and may function as an adaptor/scaffold for NEDD family ubiquitin ligases. Elucidation of the molecular mechanisms of α-arrestin proteins would provide a new pharmacological basis for developing approaches against cancer and type 2 diabetes mellitus.

A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

PubMed Central

Greibe, Eva; Fedosov, Sergey; Sorensen, Boe S.; Højrup, Peter; Poulsen, Steen S.; Nexo, Ebba

2012-01-01

Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin-binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases, it appeared to be the same protein based on analysis of partial sequences and immunological responses. The trout cobalamin-binding protein was purified from roe fluid, sequenced, and further characterized. Like haptocorrin, the trout cobalamin-binding protein was stable at low pH and had a high binding affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation by trypsin and chymotrypsin. It also resembled intrinsic factor in the composition of conserved residues in the primary cobalamin-binding site in the C terminus. The trout cobalamin-binding protein was glycosylated and displayed spectral properties comparable with those of haptocorrin and intrinsic factor. In conclusion, only one soluble cobalamin-binding protein was identified in the rainbow trout, a protein that structurally behaves like an intermediate between the three human cobalamin-binding proteins. PMID:22872637

A novel RNA binding protein affects rbcL gene expression and is specific to bundle sheath chloroplasts in C4 plants

PubMed Central

2013-01-01

Background Plants that utilize the highly efficient C4 pathway of photosynthesis typically possess kranz-type leaf anatomy that consists of two morphologically and functionally distinct photosynthetic cell types, the bundle sheath (BS) and mesophyll (M) cells. These two cell types differentially express many genes that are required for C4 capability and function. In mature C4 leaves, the plastidic rbcL gene, encoding the large subunit of the primary CO2 fixation enzyme Rubisco, is expressed specifically within BS cells. Numerous studies have demonstrated that BS-specific rbcL gene expression is regulated predominantly at post-transcriptional levels, through the control of translation and mRNA stability. The identification of regulatory factors associated with C4 patterns of rbcL gene expression has been an elusive goal for many years. Results RLSB, encoded by the nuclear RLSB gene, is an S1-domain RNA binding protein purified from C4 chloroplasts based on its specific binding to plastid-encoded rbcL mRNA in vitro. Co-localized with LSU to chloroplasts, RLSB is highly conserved across many plant species. Most significantly, RLSB localizes specifically to leaf bundle sheath (BS) cells in C4 plants. Comparative analysis using maize (C4) and Arabidopsis (C3) reveals its tight association with rbcL gene expression in both plants. Reduced RLSB expression (through insertion mutation or RNA silencing, respectively) led to reductions in rbcL mRNA accumulation and LSU production. Additional developmental effects, such as virescent/yellow leaves, were likely associated with decreased photosynthetic function and disruption of associated signaling networks. Conclusions Reductions in RLSB expression, due to insertion mutation or gene silencing, are strictly correlated with reductions in rbcL gene expression in both maize and Arabidopsis. In both plants, accumulation of rbcL mRNA as well as synthesis of LSU protein were affected. These findings suggest that specific accumulation and binding of the RLSB binding protein to rbcL mRNA within BS chloroplasts may be one determinant leading to the characteristic cell type-specific localization of Rubisco in C4 plants. Evolutionary modification of RLSB expression, from a C3 “default” state to BS cell-specificity, could represent one mechanism by which rbcL expression has become restricted to only one cell type in C4 plants. PMID:24053212

Sprouty proteins are in vivo targets of Corkscrew/SHP-2 tyrosine phosphatases.

PubMed

Jarvis, Lesley A; Toering, Stephanie J; Simon, Michael A; Krasnow, Mark A; Smith-Bolton, Rachel K

2006-03-01

Drosophila Corkscrew protein and its vertebrate ortholog SHP-2 (now known as Ptpn11) positively modulate receptor tyrosine kinase (RTK) signaling during development, but how these tyrosine phosphatases promote tyrosine kinase signaling is not well understood. Sprouty proteins are tyrosine-phosphorylated RTK feedback inhibitors, but their regulation and mechanism of action are also poorly understood. Here, we show that Corkscrew/SHP-2 proteins control Sprouty phosphorylation and function. Genetic experiments demonstrate that Corkscrew/SHP-2 and Sprouty proteins have opposite effects on RTK-mediated developmental events in Drosophila and an RTK signaling process in cultured mammalian cells, and the genes display dose-sensitive genetic interactions. In cultured cells, inactivation of SHP-2 increases phosphorylation on the critical tyrosine of Sprouty 1. SHP-2 associates in a complex with Sprouty 1 in cultured cells and in vitro, and a purified SHP-2 protein dephosphorylates the critical tyrosine of Sprouty 1. Substrate-trapping forms of Corkscrew bind Sprouty in cultured Drosophila cells and the developing eye. These results identify Sprouty proteins as in vivo targets of Corkscrew/SHP-2 tyrosine phosphatases and show how Corkscrew/SHP-2 proteins can promote RTK signaling by inactivating a feedback inhibitor. We propose that this double-negative feedback circuit shapes the output profile of RTK signaling events.

PTPRT regulates the interaction of Syntaxin-binding protein 1 with Syntaxin 1 through dephosphorylation of specific tyrosine residue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, So-Hee; Moon, Jeonghee; Lee, Myungkyu

2013-09-13

Highlights: •PTPRT is a brain-specific, expressed, protein tyrosine phosphatase. •PTPRT regulated the interaction of Syntaxin-binding protein 1 with Syntaxin 1. •PTPRT dephosphorylated the specific tyrosine residue of Syntaxin-binding protein 1. •Dephosphorylation of Syntaxin-binding protein 1 enhanced the interaction with Syntaxin 1. •PTPRT appears to regulate the fusion of synaptic vesicle through dephosphorylation. -- Abstract: PTPRT (protein tyrosine phosphatase receptor T), a brain-specific tyrosine phosphatase, has been found to regulate synaptic formation and development of hippocampal neurons, but its regulation mechanism is not yet fully understood. Here, Syntaxin-binding protein 1, a key component of synaptic vesicle fusion machinery, was identified asmore » a possible interaction partner and an endogenous substrate of PTPRT. PTPRT interacted with Syntaxin-binding protein 1 in rat synaptosome, and co-localized with Syntaxin-binding protein 1 in cultured hippocampal neurons. PTPRT dephosphorylated tyrosine 145 located around the linker between domain 1 and 2 of Syntaxin-binding protein 1. Syntaxin-binding protein 1 directly binds to Syntaxin 1, a t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein, and plays a role as catalysts of SNARE complex formation. Syntaxin-binding protein 1 mutant mimicking non-phosphorylation (Y145F) enhanced the interaction with Syntaxin 1 compared to wild type, and therefore, dephosphorylation of Syntaxin-binding protein 1 appeared to be important for SNARE-complex formation. In conclusion, PTPRT could regulate the interaction of Syntaxin-binding protein 1 with Syntaxin 1, and as a result, the synaptic vesicle fusion appeared to be controlled through dephosphorylation of Syntaxin-binding protein 1.« less

Antagonism of LIN-17/Frizzled and LIN-18/Ryk in nematode vulva induction reveals evolutionary alterations in core developmental pathways.

PubMed

Wang, Xiaoyue; Sommer, Ralf J

2011-07-01

Most diversity in animals and plants results from the modification of already existing structures. Many organ systems, for example, are permanently modified during evolution to create developmental and morphological diversity, but little is known about the evolution of the underlying developmental mechanisms. The theory of developmental systems drift proposes that the development of conserved morphological structures can involve large-scale modifications in their regulatory mechanisms. We test this hypothesis by comparing vulva induction in two genetically tractable nematodes, Caenorhabditis elegans and Pristionchus pacificus. Previous work indicated that the vulva is induced by epidermal growth factor (EGF)/RAS and WNT signaling in Caenorhabditis and Pristionchus, respectively. Here, we show that the evolution of vulva induction involves major molecular alterations and that this shift of signaling pathways involves a novel wiring of WNT signaling and the acquisition of novel domains in otherwise conserved receptors in Pristionchus vulva induction. First, Ppa-LIN-17/Frizzled acts as an antagonist of WNT signaling and suppresses the ligand Ppa-EGL-20 by ligand sequestration. Second, Ppa-LIN-18/Ryk transmits WNT signaling and requires inhibitory SH3 domain binding motifs, unknown from Cel-LIN-18/Ryk. Third, Ppa-LIN-18/Ryk signaling involves Axin and β-catenin and Ppa-axl-1/Axin is epistatic to Ppa-lin-18/Ryk. These results confirm developmental system drift as an important theory for the evolution of organ systems and they highlight the significance of protein modularity in signal transduction and the dynamics of signaling networks.

Antagonism of LIN-17/Frizzled and LIN-18/Ryk in Nematode Vulva Induction Reveals Evolutionary Alterations in Core Developmental Pathways

PubMed Central

Wang, Xiaoyue; Sommer, Ralf J.

2011-01-01

Most diversity in animals and plants results from the modification of already existing structures. Many organ systems, for example, are permanently modified during evolution to create developmental and morphological diversity, but little is known about the evolution of the underlying developmental mechanisms. The theory of developmental systems drift proposes that the development of conserved morphological structures can involve large-scale modifications in their regulatory mechanisms. We test this hypothesis by comparing vulva induction in two genetically tractable nematodes, Caenorhabditis elegans and Pristionchus pacificus. Previous work indicated that the vulva is induced by epidermal growth factor (EGF)/RAS and WNT signaling in Caenorhabditis and Pristionchus, respectively. Here, we show that the evolution of vulva induction involves major molecular alterations and that this shift of signaling pathways involves a novel wiring of WNT signaling and the acquisition of novel domains in otherwise conserved receptors in Pristionchus vulva induction. First, Ppa-LIN-17/Frizzled acts as an antagonist of WNT signaling and suppresses the ligand Ppa-EGL-20 by ligand sequestration. Second, Ppa-LIN-18/Ryk transmits WNT signaling and requires inhibitory SH3 domain binding motifs, unknown from Cel-LIN-18/Ryk. Third, Ppa-LIN-18/Ryk signaling involves Axin and β-catenin and Ppa-axl-1/Axin is epistatic to Ppa-lin-18/Ryk. These results confirm developmental system drift as an important theory for the evolution of organ systems and they highlight the significance of protein modularity in signal transduction and the dynamics of signaling networks. PMID:21814488

Genome-Wide Mapping of Collier In Vivo Binding Sites Highlights Its Hierarchical Position in Different Transcription Regulatory Networks

PubMed Central

Dubois, Laurence; Bataillé, Laetitia; Painset, Anaïs; Le Gras, Stéphanie; Jost, Bernard; Crozatier, Michèle; Vincent, Alain

2015-01-01

Collier, the single Drosophila COE (Collier/EBF/Olf-1) transcription factor, is required in several developmental processes, including head patterning and specification of muscle and neuron identity during embryogenesis. To identify direct Collier (Col) targets in different cell types, we used ChIP-seq to map Col binding sites throughout the genome, at mid-embryogenesis. In vivo Col binding peaks were associated to 415 potential direct target genes. Gene Ontology analysis revealed a strong enrichment in proteins with DNA binding and/or transcription-regulatory properties. Characterization of a selection of candidates, using transgenic CRM-reporter assays, identified direct Col targets in dorso-lateral somatic muscles and specific neuron types in the central nervous system. These data brought new evidence that Col direct control of the expression of the transcription regulators apterous and eyes-absent (eya) is critical to specifying neuronal identities. They also showed that cross-regulation between col and eya in muscle progenitor cells is required for specification of muscle identity, revealing a new parallel between the myogenic regulatory networks operating in Drosophila and vertebrates. Col regulation of eya, both in specific muscle and neuronal lineages, may illustrate one mechanism behind the evolutionary diversification of Col biological roles. PMID:26204530

Identification and characterization of a histamine-binding lipocalin-like molecule from the relapsing fever tick Ornithodoros turicata.

PubMed

Neelakanta, G; Sultana, H; Sonenshine, D E; Andersen, J F

2018-04-01

Lipocalins are low molecular weight membrane transporters that are abundantly expressed in the salivary glands and other tissues of ticks. In this study, we identified a lipocalin-like molecule, designated as otlip, from the soft ticks Ornithodoros turicata, the vector for the relapsing fever causing spirochete Borrelia turicatae. We noted that the expression of otlip was developmentally regulated, with adult ticks expressing significantly higher levels in comparison to the larvae or nymphal ticks. Expression of otlip was evident in both fed and unfed O. turicata ticks, with significantly increased expression in the salivary glands in comparison to the midgut or ovary tissues. High conservation of the biogenic amine-binding motif was evident in the deduced primary amino acid sequence of Otlip. Protein modelling of Otlip revealed conservation of most of the residues involved in binding histamine or serotonin ligand. In vitro assays demonstrated binding of recombinant Otlip with histamine. Furthermore, prediction of post-translational modifications revealed that Otlip contained phosphorylation and myristoylation sites. Taken together, our study not only provides evidence for the presence of a lipocalin-like molecule in O. turicata ticks but also suggests a role for this molecule in the salivary glands of this medically important vector. © 2017 The Royal Entomological Society.

Dysbindin-1, WAVE2 and Abi-1 form a complex that regulates dendritic spine formation.

PubMed

Ito, H; Morishita, R; Shinoda, T; Iwamoto, I; Sudo, K; Okamoto, K; Nagata, K

2010-10-01

Genetic variations in dysbindin-1 (dystrobrevin-binding protein-1) are one of the most commonly reported variations associated with schizophrenia. As schizophrenia could be regarded as a neurodevelopmental disorder resulting from abnormalities of synaptic connectivity, we attempted to clarify the function of dysbindin-1 in neuronal development. We examined the developmental change of dysbindin-1 in rat brain by western blotting and found that a 50 kDa isoform is highly expressed during the embryonic stage, whereas a 40 kDa one is detected at postnatal day 11 and increased thereafter. Immunofluorescent analyses revealed that dysbindin-1 is enriched at the spine-like structure of primary cultured rat hippocampal neurons. We identified WAVE2, but not N-WASP, as a binding partner for dysbindin-1. We also found that Abi-1, a binding molecule for WAVE2 involved in spine morphogenesis, interacts with dysbindin-1. Although dysbindin-1, WAVE2 and Abi-1 form a ternary complex, dysbindin-1 promoted the binding of WAVE2 to Abi-1. RNA interference-mediated knockdown of dysbindin-1 led to the generation of abnormally elongated immature dendritic protrusions. The present results indicate possible functions of dysbindin-1 at the postsynapse in the regulation of dendritic spine morphogenesis through the interaction with WAVE2 and Abi-1.

Monoclonal antibodies to human vitamin D-binding protein.

PubMed Central

Pierce, E A; Dame, M C; Bouillon, R; Van Baelen, H; DeLuca, H F

1985-01-01

Monoclonal antibodies to vitamin D-binding protein isolated from human serum have been produced. The antibodies obtained have been shown to be specific for human vitamin D-binding protein by three independent assays. The antibodies recognize human vitamin D-binding protein specifically in an enzyme-linked immunosorbent assay. Human vitamin D-binding protein is detected specifically in both pure and crude samples by a radiometric immunosorbent assay (RISA) and by an immunoprecipitation assay. The anti-human vitamin D-binding protein antibodies cross-react with monkey and pig vitamin D-binding protein, but not with vitamin D-binding protein from rat, mouse, or chicken, as determined by the RISA and immunoprecipitation assays. Images PMID:3936035

Complexes of D-type cyclins with CDKs during maize germination

PubMed Central

Vázquez-Ramos, Jorge M.

2013-01-01

The importance of cell proliferation in plant growth and development has been well documented. The majority of studies on basic cell cycle mechanisms in plants have been at the level of gene expression and much less knowledge has accumulated in terms of protein interactions and activation. Two key proteins, cyclins and cyclin-dependent kinases (CDKs) are fundamental for cell cycle regulation and advancement. Our aim has been to understand the role of D-type cyclins and type A and B CDKs in the cell cycle taking place during a developmental process such as maize seed germination. Results indicate that three maize D-type cyclins—D2;2, D4;2, and D5;3—(G1-S cyclins by definition) bind and activate two different types of CDK—A and B1;1—in a differential way during germination. Whereas CDKA–D-type cyclin complexes are more active at early germination times than at later times, it was surprising to observe that CDKB1;1, a supposedly G2-M kinase, bound in a differential way to all D-type cyclins tested during germination. Binding to cyclin D2;2 was detectable at all germination times, forming a complex with kinase activity, whereas binding to D4;2 and D5;3 was more variable; in particular, D5;3 was only detected at late germination times. Results are discussed in terms of cell cycle advancement and its importance for seed germination. PMID:24127516

Phosphatidic acid binding proteins display differential binding as a function of membrane curvature stress and chemical properties.

PubMed

Putta, Priya; Rankenberg, Johanna; Korver, Ruud A; van Wijk, Ringo; Munnik, Teun; Testerink, Christa; Kooijman, Edgar E

2016-11-01

Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins. Copyright © 2016. Published by Elsevier B.V.

Thioredoxin binding protein (TBP)-2/Txnip and α-arrestin proteins in cancer and diabetes mellitus

PubMed Central

Masutani, Hiroshi; Yoshihara, Eiji; Masaki, So; Chen, Zhe; Yodoi, Junji

2012-01-01

Thioredoxin binding protein −2/ thioredoxin interacting protein is an α-arrestin protein that has attracted much attention as a multifunctional regulator. Thioredoxin binding protein −2 expression is downregulated in tumor cells and the level of thioredoxin binding protein is correlated with clinical stage of cancer. Mice with mutations or knockout of the thioredoxin binding protein −2 gene are much more susceptible to carcinogenesis than wild-type mice, indicating a role for thioredoxin binding protein −2 in cancer suppression. Studies have also revealed roles for thioredoxin binding protein −2 in metabolic control. Enhancement of thioredoxin binding protein −2 expression causes impairment of insulin sensitivity and glucose-induced insulin secretion, and β-cell apoptosis. These changes are important characteristics of type 2 diabetes mellitus. Thioredoxin binding protein −2 regulates transcription of metabolic regulating genes. Thioredoxin binding protein −2-like inducible membrane protein/ arrestin domain containing 3 regulates endocytosis of receptors such as the β2-adrenergic receptor. The α-arrestin family possesses PPXY motifs and may function as an adaptor/scaffold for NEDD family ubiquitin ligases. Elucidation of the molecular mechanisms of α-arrestin proteins would provide a new pharmacological basis for developing approaches against cancer and type 2 diabetes mellitus. PMID:22247597

Discovery of binding proteins for a protein target using protein-protein docking-based virtual screening.

PubMed

Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua

2014-10-01

Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets. © 2014 Wiley Periodicals, Inc.

A Positive Feedback Mechanism That Regulates Expression of miR-9 during Neurogenesis

PubMed Central

Oni, Eileen N.; Swerdel, Mavis R.; Toro-Ramos, Alana J.; Li, Jiali; Hart, Ronald P.

2014-01-01

MiR-9, a neuron-specific miRNA, is an important regulator of neurogenesis. In this study we identify how miR-9 is regulated during early differentiation from a neural stem-like cell. We utilized two immortalized rat precursor clones, one committed to neurogenesis (L2.2) and another capable of producing both neurons and non-neuronal cells (L2.3), to reproducibly study early neurogenesis. Exogenous miR-9 is capable of increasing neurogenesis from L2.3 cells. Only one of three genomic loci capable of encoding miR-9 was regulated during neurogenesis and the promoter region of this locus contains sufficient functional elements to drive expression of a luciferase reporter in a developmentally regulated pattern. Furthermore, among a large number of potential regulatory sites encoded in this sequence, Mef2 stood out because of its known pro-neuronal role. Of four Mef2 paralogs, we found only Mef2C mRNA was regulated during neurogenesis. Removal of predicted Mef2 binding sites or knockdown of Mef2C expression reduced miR-9-2 promoter activity. Finally, the mRNA encoding the Mef2C binding partner HDAC4 was shown to be targeted by miR-9. Since HDAC4 protein could be co-immunoprecipitated with Mef2C protein or with genomic Mef2 binding sequences, we conclude that miR-9 regulation is mediated, at least in part, by Mef2C binding but that expressed miR-9 has the capacity to reduce inhibitory HDAC4, stabilizing its own expression in a positive feedback mechanism. PMID:24714615

Novel marker for the onset of frontotemporal dementia: early increase in activity-dependent neuroprotective protein (ADNP) in the face of Tau mutation.

PubMed

Schirer, Yulie; Malishkevich, Anna; Ophir, Yotam; Lewis, Jada; Giladi, Eliezer; Gozes, Illana

2014-01-01

Tauopathy, a major pathology in Alzheimer's disease, is also found in ~50% of frontotemporal dementias (FTDs). Tau transcript, a product of a single gene, undergoes alternative splicing to yield 6 protein species, each with either 3 or 4 microtubule binding repeat domains (tau 3R or 4R, associated with dynamic and stable microtubules, respectively). While the healthy human brain shows a 1/1 ratio of tau 3R/4R, this ratio may be dramatically changed in the FTD brain. We have previously discovered that activity-dependent neuroprotective protein (ADNP) is essential for brain formation in the mouse, with ADNP+/- mice exhibiting tauopathy, age-driven neurodegeneration and behavioral deficits. Here, in transgenic mice overexpressing a mutated tau 4R species, in the cerebral cortex but not in the cerebellum, we showed significantly increased ADNP expression (~3-fold transcripts) in the cerebral cortex of young transgenic mice (~disease onset), but not in the cerebellum, as compared to control littermates. The transgene-age-related increased ADNP expression paralleled augmented dynamic tau 3R transcript level compared to control littermates. Blocking mutated tau 4R transgene expression resulted in normalization of ADNP and tau 3R expression. ADNP was previously shown to be a member of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex. Here, Brahma (Brm), a component of the SWI/SNF complex regulating alternative splicing, showed a similar developmental expression pattern to ADNP. Immunoprecipitations further suggested Brm-ADNP interaction coupled to ADNP - polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF)-binding, with PSF being a direct regulator of tau transcript splicing. It should be noted that although we have shown a correlation between levels of ADNP and tau isoform expression three months of age, we are not presenting evidence of a direct link between the two. Future research into ADNP/tau relations is warranted.

Genome wide analysis reveals Zic3 interaction with distal regulatory elements of stage specific developmental genes in zebrafish.

PubMed

Winata, Cecilia L; Kondrychyn, Igor; Kumar, Vibhor; Srinivasan, Kandhadayar G; Orlov, Yuriy; Ravishankar, Ashwini; Prabhakar, Shyam; Stanton, Lawrence W; Korzh, Vladimir; Mathavan, Sinnakaruppan

2013-10-01

Zic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches--ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in zic3-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using in vivo enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.

In Vivo Chromatin Targets of the Transcription Factor Yin Yang 2 in Trophoblast Stem Cells

PubMed Central

Pérez-Palacios, Raquel; Macías-Redondo, Sofía; Climent, María; Contreras-Moreira, Bruno; Muniesa, Pedro; Schoorlemmer, Jon

2016-01-01

Background Yin Yang 2 (YY2) is a zinc finger protein closely related to the well-characterized Yin Yang 1 (YY1). YY1 is a DNA-binding transcription factor, with defined functions in multiple developmental processes, such as implantation, cell differentiation, X inactivation, imprinting and organogenesis. Yy2 has been treated as a largely immaterial duplication of Yy1, as they share high homology in the Zinc Finger-region and similar if not identical in vitro binding sites. In contrast to these similarities, gene expression alterations in HeLa cells with attenuated levels of either Yy1 or Yy2 were to some extent gene-specific. Moreover, the chromatin binding sites for YY2, except for its association with transposable retroviral elements (RE) and Endogenous Retroviral Elements (ERVs), remain to be identified. As a first step towards defining potential Yy2 functions matching or complementary to Yy1, we considered in vivo DNA binding sites of YY2 in trophoblast stem (TS) cells. Results We report the presence of YY2 protein in mouse-derived embryonic stem (ES) and TS cell lines. Following up on our previous report on ERV binding by YY2 in TS cells, we investigated the tissue-specificity of REX1 and YY2 binding and confirm binding to RE/ERV targets in both ES cells and TS cells. Because of the higher levels of expression, we chose TS cells to understand the role of Yy2 in gene and chromatin regulation. We used in vivo YY2 association as a measure to identify potential target genes. Sequencing of chromatin obtained in chromatin-immunoprecipitation (ChIP) assays carried out with αYY2 serum allowed us to identify a limited number of chromatin targets for YY2. Some putative binding sites were validated in regular ChIP assays and gene expression of genes nearby was altered in the absence of Yy2. Conclusions YY2 binding to ERVs is not confined to TS cells. In vivo binding sites share the presence of a consensus binding motif. Selected sites were uniquely bound by YY2 as opposed to YY1, suggesting that YY2 exerts unique contributions to gene regulation. YY2 binding was not generally associated with gene promoters. However, several YY2 binding sites are linked to long noncoding RNA (lncRNA) genes and we show that the expression levels of a few of those are Yy2-dependent. PMID:27191592

Functional assignment of solute-binding proteins of ABC transporters using a fluorescence-based thermal shift assay.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giulliani, S. E.; Frank, A. E.; Collart, F. R.

2008-12-08

We have used a fluorescence-based thermal shift (FTS) assay to identify amino acids that bind to solute-binding proteins in the bacterial ABC transporter family. The assay was validated with a set of six proteins with known binding specificity and was consistently able to map proteins with their known binding ligands. The assay also identified additional candidate binding ligands for several of the amino acid-binding proteins in the validation set. We extended this approach to additional targets and demonstrated the ability of the FTS assay to unambiguously identify preferential binding for several homologues of amino acid-binding proteins with known specificity andmore » to functionally annotate proteins of unknown binding specificity. The assay is implemented in a microwell plate format and provides a rapid approach to validate an anticipated function or to screen proteins of unknown function. The ABC-type transporter family is ubiquitous and transports a variety of biological compounds, but the current annotation of the ligand-binding proteins is limited to mostly generic descriptions of function. The results illustrate the feasibility of the FTS assay to improve the functional annotation of binding proteins associated with ABC-type transporters and suggest this approach that can also be extended to other protein families.« less

Glutathione S-transferase pi isoform (GSTP1) expression in murine retina increases with developmental maturity.

PubMed

Lee, Wen-Hsiang; Joshi, Pratibha; Wen, Rong

2014-01-01

Glutathione S-transferase pi isoform (GSTP1) is an intracellular detoxification enzyme that catalyzes reduction of chemically reactive electrophiles and is a zeaxanthin-binding protein in the human macula. We have previously demonstrated that GSTP1 levels are decreased in human age-related macular degeneration (AMD) retina compared to normal controls (Joshi et al., Invest Ophthalmol Vis Sci, e-abstract, 2009). We also showed that GSTP1 levels parallel survival of human retinal pigment epithelial (RPE) cells exposed to ultraviolet (UV) light, and GSTP1 over-expression protects them against UV light damage (Joshi et al., Invest Ophthalmol Vis Sci, e-abstract, 2010). In the present work, we determined the developmental time course of GSTP1 expression in murine retina and in response to light challenge. Eyes from BALB/c mice at postnatal day 20, 1 month, and 2 months of age were prepared for retinal protein extraction and cryo sectioning, and GSTP1 levels in the retina were analyzed by Western blot and immunohistochemistry (IHC). Another group of BALB/c mice with the same age ranges was exposed to 1000 lx of white fluorescent light for 24 h, and their retinas were analyzed for GSTP1 expression by Western blot and IHC in a similar manner. GSTP1 levels in the murine retina increased in ascending order from postnatal day 20, 1 month, and 2 months of age. Moreover, GSTP1 expression in murine retina at postnatal day 20, 1 month, and 2 months of age increased in response to brief light exposure compared to age-matched controls under normal condition. GSTP1 expression in retina increases with developmental age in mice and accompanies murine retinal maturation. Brief exposure to light induces GSTP1 expression in the murine retina across various developmental ages. GSTP1 induction may be a protective response to light-induced oxidative damage in the murine retina.

Glutathione S-Transferase Pi Isoform (GSTP1) Expression in Murine Retina Increases with Developmental Maturity

PubMed Central

Lee, Wen-Hsiang; Joshi, Pratibha; Wen, Rong

2014-01-01

Background and Aims Glutathione S-transferase pi isoform (GSTP1) is an intracellular detoxification enzyme that catalyzes reduction of chemically reactive electrophiles and is a zeaxanthin-binding protein in the human macula. We have previously demonstrated that GSTP1 levels are decreased in human age-related macular degeneration (AMD) retina compared to normal controls [1]. We also showed that GSTP1 levels parallel survival of human retinal pigment epithelial (RPE) cells exposed to UV light, and GSTP1 over-expression protects them against UV light damage [2]. In the present work, we determined the developmental time course of GSTP1 expression in murine retina and in response to light challenge. Methods Eyes from BALB/c mice at post-natal day 20, 1 month, and 2 months of age were prepared for retinal protein extraction and cryo sectioning, and GSTP1 levels in the retina were analyzed by Western blot and immunohistochemistry (IHC). Another group of BALB/c mice with the same age ranges was exposed to 1000 lux of white fluorescent light for 24 hours, and their retinas were analyzed for GSTP1 expression by Western blot and IHC in a similar manner. Results GSTP1 levels in the murine retina increased in ascending order from post-natal day 20, 1 month, and 2 months of age. Moreover, GSTP1 expression in murine retina at post-natal day 20, 1 month, and 2 months of age increased in response to brief light exposure compared to age-matched controls under normal condition. Conclusions GSTP1 expression in retina increases with developmental age in mice and accompanies murine retinal maturation. Brief exposure to light induces GSTP1 expression in the murine retina across various developmental ages. GSTP1 induction may be a protective response to light-induced oxidative damage in the murine retina. PMID:24664677

Analysis of Cytoskeletal and Motility Proteins in the Sea Urchin Genome Assembly

PubMed Central

RL, Morris; MP, Hoffman; RA, Obar; SS, McCafferty; IR, Gibbons; AD, Leone; J, Cool; EL, Allgood; AM, Musante; KM, Judkins; BJ, Rossetti; AP, Rawson; DR, Burgess

2007-01-01

The sea urchin embryo is a classical model system for studying the role of the cytoskeleton in such events as fertilization, mitosis, cleavage, cell migration and gastrulation. We have conducted an analysis of gene models derived from the Strongylocentrotus purpuratus genome assembly and have gathered strong evidence for the existence of multiple gene families encoding cytoskeletal proteins and their regulators in sea urchin. While many cytoskeletal genes have been cloned from sea urchin with sequences already existing in public databases, genome analysis reveals a significantly higher degree of diversity within certain gene families. Furthermore, genes are described corresponding to homologs of cytoskeletal proteins not previously documented in sea urchins. To illustrate the varying degree of sequence diversity that exists within cytoskeletal gene families, we conducted an analysis of genes encoding actins, specific actin-binding proteins, myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins, and intermediate filaments. We conducted ontological analysis of select genes to better understand the relatedness of urchin cytoskeletal genes to those of other deuterostomes. We analyzed developmental expression (EST) data to confirm the existence of select gene models and to understand their differential expression during various stages of early development. PMID:17027957

Double-bromo and extraterminal (BET) domain proteins regulate dendrite morphology and mechanosensory function

PubMed Central

Bagley, Joshua A.; Yan, Zhiqiang; Zhang, Wei; Wildonger, Jill

2014-01-01

A complex array of genetic factors regulates neuronal dendrite morphology. Epigenetic regulation of gene expression represents a plausible mechanism to control pathways responsible for specific dendritic arbor shapes. By studying the Drosophila dendritic arborization (da) neurons, we discovered a role of the double-bromodomain and extraterminal (BET) family proteins in regulating dendrite arbor complexity. A loss-of-function mutation in the single Drosophila BET protein encoded by female sterile 1 homeotic [fs(1)h] causes loss of fine, terminal dendritic branches. Moreover, fs(1)h is necessary for the induction of branching caused by a previously identified transcription factor, Cut (Ct), which regulates subtype-specific dendrite morphology. Finally, disrupting fs(1)h function impairs the mechanosensory response of class III da sensory neurons without compromising the expression of the ion channel NompC, which mediates the mechanosensitive response. Thus, our results identify a novel role for BET family proteins in regulating dendrite morphology and a possible separation of developmental pathways specifying neural cell morphology and ion channel expression. Since the BET proteins are known to bind acetylated histone tails, these results also suggest a role of epigenetic histone modifications and the “histone code,” in regulating dendrite morphology. PMID:25184680

Differential conservation of transcriptional domains of mammalian Prophet of Pit-1 proteins revealed by structural studies of the bovine gene and comparative functional analysis of the protein.

PubMed

Showalter, Aaron D; Smith, Timothy P L; Bennett, Gary L; Sloop, Kyle W; Whitsett, Julie A; Rhodes, Simon J

2002-05-29

The Prophet of Pit-1 (PROP1) gene encodes a paired class homeodomain transcription factor that is exclusively expressed in the developing mammalian pituitary gland. PROP1 function is essential for anterior pituitary organogenesis, and heritable mutations in the gene are associated with combined pituitary hormone deficiency in human patients and animals. By cloning the bovine PROP1 gene and by comparative analysis, we demonstrate that the homeodomains and carboxyl termini of mammalian PROP1 proteins are highly conserved while the amino termini are diverged. Whereas the carboxyl termini of the human and bovine PROP1 proteins contain potent transcriptional activation domains, the amino termini and homeodomains have repressive activities. The bovine PROP1 gene has four exons and three introns and maps to a region of chromosome seven carrying a quantitative trait locus affecting ovulation rate. Two alleles of the bovine gene were found that encode distinct protein products with different DNA binding and transcriptional activities. These experiments demonstrate that mammalian PROP1 genes encode proteins with complex regulatory capacities and that modest changes in protein sequence can significantly alter the activity of this pituitary developmental transcription factor.

Signal recognition particle assembly in relation to the function of amplified nucleoli of Xenopus oocytes.

PubMed

Sommerville, John; Brumwell, Craig L; Politz, Joan C Ritland; Pederson, Thoru

2005-03-15

The signal recognition particle (SRP) is a ribonucleoprotein machine that controls the translation and intracellular sorting of membrane and secreted proteins. The SRP contains a core RNA subunit with which six proteins are assembled. Recent work in both yeast and mammalian cells has identified the nucleolus as a possible initial site of SRP assembly. In the present study, SRP RNA and protein components were identified in the extrachromosomal, amplified nucleoli of Xenopus laevis oocytes. Fluorescent SRP RNA microinjected into the oocyte nucleus became specifically localized in the nucleoli, and endogenous SRP RNA was also detected in oocyte nucleoli by RNA in situ hybridization. An initial step in the assembly of SRP involves the binding of the SRP19 protein to SRP RNA. When green fluorescent protein (GFP)-tagged SRP19 protein was injected into the oocyte cytoplasm it was imported into the nucleus and became concentrated in the amplified nucleoli. After visiting the amplified nucleoli, GFP-tagged SRP19 protein was detected in the cytoplasm in a ribonucleoprotein complex, having a sedimentation coefficient characteristic of the SRP. These results suggest that the amplified nucleoli of Xenopus oocytes produce maternal stores not only of ribosomes, the classical product of nucleoli, but also of SRP, presumably as a global developmental strategy for stockpiling translational machinery for early embryogenesis.

Food proteins and maturation of small intestinal microvillus membranes (MVM). I. Binding characteristics of cow's milk proteins and concanavalin A to MVM from newborn and adult rats.

PubMed

Stern, M; Gellermann, B

1988-01-01

To study maturational changes of food protein and lectin binding to rat small intestinal microvillus membranes (MVM), MVM were prepared from newborn and adult animals by a modified CaCl2 precipitation technique. Radiolabeled cow's milk proteins [alpha-lactalbumin, alpha-casein, beta-lactoglobulin, bovine serum albumin (BSA)] and the lectin concanavalin A (Con A) were used for incubations. Binding assays were done using miniature ultracentrifugation for separation of unbound material. Binding of Con A to MVM from newborn and adult rats was strong, specific, and saturable. Binding of Con A was inhibited by cold Con A and by the sugar ligand polymer mannan. Adult MVM bound more Con A than newborn preparations. Unlike Con A, binding of cow's milk proteins by MVM was weak, nonspecific, and noninhibitable. Newborn MVM bound more cow's milk proteins than adult controls. This was true for all the proteins tested (p less than 0.001). Binding rose with decreased molecular weight of cow's milk proteins, but molecular weight was not the only determining factor for binding. Trypsin treatment of MVM caused a marked increase of BSA binding in adult but not in newborn preparations. This finding indicated the importance of protein components of MVM for cow's milk protein binding. Maturational changes in protein-lipid interactions and membrane fluidity possibly influence nonspecific cow's milk protein binding to MVM. Differences in binding between newborns and adults were not directly related to maturational shifts in membrane glycosylation that are indicated by differential Con A binding. Increased cow's milk protein binding in newborn individuals might increase the potential risk to develop an adverse reaction to food proteins.

Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

PubMed

Miao, Yinglong; McCammon, J Andrew

2018-03-20

Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

Cell-type specific role of the RNA-binding protein, NONO, in the DNA double-strand break response in the mouse testes.

PubMed

Li, Shuyi; Shu, Feng-Jue; Li, Zhentian; Jaafar, Lahcen; Zhao, Shourong; Dynan, William S

2017-03-01

The tandem RNA recognition motif protein, NONO, was previously identified as a candidate DNA double-strand break (DSB) repair factor in a biochemical screen for proteins with end-joining stimulatory activity. Subsequent work showed that NONO and its binding partner, SFPQ, have many of the properties expected for bona fide repair factors in cell-based assays. Their contribution to the DNA damage response in intact tissue in vivo has not, however, been demonstrated. Here we compare DNA damage sensitivity in the testes of wild-type mice versus mice bearing a null allele of the NONO homologue (Nono gt ). In wild-type mice, NONO protein was present in Sertoli, peritubular myoid, and interstitial cells, with an increase in expression following induction of DNA damage. As expected for the product of an X-linked gene, NONO was not detected in germ cells. The Nono gt/0 mice had at most a mild testis developmental phenotype in the absence of genotoxic stress. However, following irradiation at sublethal, 2-4 Gy doses, Nono gt/0 mice displayed a number of indicators of radiosensitivity as compared to their wild-type counterparts. These included higher levels of persistent DSB repair foci, increased numbers of apoptotic cells in the seminiferous tubules, and partial degeneration of the blood-testis barrier. There was also an almost complete loss of germ cells at later times following irradiation, evidently arising as an indirect effect reflecting loss of stromal support. Results demonstrate a role for NONO protein in protection against direct and indirect biological effects of ionizing radiation in the whole animal. Copyright © 2017 Elsevier B.V. All rights reserved.

Evolution of SH2 domains and phosphotyrosine signalling networks

PubMed Central

Liu, Bernard A.; Nash, Piers D.

2012-01-01

Src homology 2 (SH2) domains mediate selective protein–protein interactions with tyrosine phosphorylated proteins, and in doing so define specificity of phosphotyrosine (pTyr) signalling networks. SH2 domains and protein-tyrosine phosphatases expand alongside protein-tyrosine kinases (PTKs) to coordinate cellular and organismal complexity in the evolution of the unikont branch of the eukaryotes. Examination of conserved families of PTKs and SH2 domain proteins provides fiduciary marks that trace the evolutionary landscape for the development of complex cellular systems in the proto-metazoan and metazoan lineages. The evolutionary provenance of conserved SH2 and PTK families reveals the mechanisms by which diversity is achieved through adaptations in tissue-specific gene transcription, altered ligand binding, insertions of linear motifs and the gain or loss of domains following gene duplication. We discuss mechanisms by which pTyr-mediated signalling networks evolve through the development of novel and expanded families of SH2 domain proteins and the elaboration of connections between pTyr-signalling proteins. These changes underlie the variety of general and specific signalling networks that give rise to tissue-specific functions and increasingly complex developmental programmes. Examination of SH2 domains from an evolutionary perspective provides insight into the process by which evolutionary expansion and modification of molecular protein interaction domain proteins permits the development of novel protein-interaction networks and accommodates adaptation of signalling networks. PMID:22889907

A tool for calculating binding-site residues on proteins from PDB structures.

PubMed

Hu, Jing; Yan, Changhui

2009-08-03

In the research on protein functional sites, researchers often need to identify binding-site residues on a protein. A commonly used strategy is to find a complex structure from the Protein Data Bank (PDB) that consists of the protein of interest and its interacting partner(s) and calculate binding-site residues based on the complex structure. However, since a protein may participate in multiple interactions, the binding-site residues calculated based on one complex structure usually do not reveal all binding sites on a protein. Thus, this requires researchers to find all PDB complexes that contain the protein of interest and combine the binding-site information gleaned from them. This process is very time-consuming. Especially, combing binding-site information obtained from different PDB structures requires tedious work to align protein sequences. The process becomes overwhelmingly difficult when researchers have a large set of proteins to analyze, which is usually the case in practice. In this study, we have developed a tool for calculating binding-site residues on proteins, TCBRP http://yanbioinformatics.cs.usu.edu:8080/ppbindingsubmit. For an input protein, TCBRP can quickly find all binding-site residues on the protein by automatically combining the information obtained from all PDB structures that consist of the protein of interest. Additionally, TCBRP presents the binding-site residues in different categories according to the interaction type. TCBRP also allows researchers to set the definition of binding-site residues. The developed tool is very useful for the research on protein binding site analysis and prediction.

Interaction entropy for protein-protein binding

NASA Astrophysics Data System (ADS)

Sun, Zhaoxi; Yan, Yu N.; Yang, Maoyou; Zhang, John Z. H.

2017-03-01

Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interaction entropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interaction entropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

Interaction entropy for protein-protein binding.

PubMed

Sun, Zhaoxi; Yan, Yu N; Yang, Maoyou; Zhang, John Z H

2017-03-28

Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interactionentropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interactionentropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

Block of ATP-Binding Cassette B19 Ion Channel Activity by 5-Nitro-2-(3-Phenylpropylamino)-Benzoic Acid Impairs Polar Auxin Transport and Root Gravitropism1[OPEN

PubMed Central

Cho, Misuk; Henry, Elizabeth M.; Lewis, Daniel R.; Wu, Guosheng; Muday, Gloria K.

2014-01-01

Polar transport of the hormone auxin through tissues and organs depends on membrane proteins, including some B-subgroup members of the ATP-binding cassette (ABC) transporter family. The messenger RNA level of at least one B-subgroup ABCB gene in Arabidopsis (Arabidopsis thaliana), ABCB19, increases upon treatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), possibly to compensate for an inhibitory effect of the drug on ABCB19 activity. Consistent with this hypothesis, NPPB blocked ion channel activity associated with ABCB19 expressed in human embryonic kidney cells as measured by patch-clamp electrophysiology. NPPB inhibited polar auxin transport through Arabidopsis seedling roots similarly to abcb19 mutations. NPPB also inhibited shootward auxin transport, which depends on the related ABCB4 protein. NPPB substantially decreased ABCB4 and ABCB19 protein levels when cycloheximide concomitantly inhibited new protein synthesis, indicating that blockage by NPPB enhances the degradation of ABCB transporters. Impairing the principal auxin transport streams in roots with NPPB caused aberrant patterns of auxin signaling reporters in root apices. Formation of the auxin-signaling gradient across the tips of gravity-stimulated roots, and its developmental consequence (gravitropism), were inhibited by micromolar concentrations of NPPB that did not affect growth rate. These results identify ion channel activity of ABCB19 that is blocked by NPPB, a compound that can now be considered an inhibitor of polar auxin transport with a defined molecular target. PMID:25324509

Microarray analysis of gene expression patterns in the leaf during potato tuberization in the potato somatic hybrid Solanum tuberosum and Solanum etuberosum.

PubMed

Tiwari, Jagesh Kumar; Devi, Sapna; Sundaresha, S; Chandel, Poonam; Ali, Nilofer; Singh, Brajesh; Bhardwaj, Vinay; Singh, Bir Pal

2015-06-01

Genes involved in photoassimilate partitioning and changes in hormonal balance are important for potato tuberization. In the present study, we investigated gene expression patterns in the tuber-bearing potato somatic hybrid (E1-3) and control non-tuberous wild species Solanum etuberosum (Etb) by microarray. Plants were grown under controlled conditions and leaves were collected at eight tuber developmental stages for microarray analysis. A t-test analysis identified a total of 468 genes (94 up-regulated and 374 down-regulated) that were statistically significant (p ≤ 0.05) and differentially expressed in E1-3 and Etb. Gene Ontology (GO) characterization of the 468 genes revealed that 145 were annotated and 323 were of unknown function. Further, these 145 genes were grouped based on GO biological processes followed by molecular function and (or) PGSC description into 15 gene sets, namely (1) transport, (2) metabolic process, (3) biological process, (4) photosynthesis, (5) oxidation-reduction, (6) transcription, (7) translation, (8) binding, (9) protein phosphorylation, (10) protein folding, (11) ubiquitin-dependent protein catabolic process, (12) RNA processing, (13) negative regulation of protein, (14) methylation, and (15) mitosis. RT-PCR analysis of 10 selected highly significant genes (p ≤ 0.01) confirmed the microarray results. Overall, we show that candidate genes induced in leaves of E1-3 were implicated in tuberization processes such as transport, carbohydrate metabolism, phytohormones, and transcription/translation/binding functions. Hence, our results provide an insight into the candidate genes induced in leaf tissues during tuberization in E1-3.

Block of ATP-binding cassette B19 ion channel activity by 5-nitro-2-(3-phenylpropylamino)-benzoic acid impairs polar auxin transport and root gravitropism.

PubMed

Cho, Misuk; Henry, Elizabeth M; Lewis, Daniel R; Wu, Guosheng; Muday, Gloria K; Spalding, Edgar P

2014-12-01

Polar transport of the hormone auxin through tissues and organs depends on membrane proteins, including some B-subgroup members of the ATP-binding cassette (ABC) transporter family. The messenger RNA level of at least one B-subgroup ABCB gene in Arabidopsis (Arabidopsis thaliana), ABCB19, increases upon treatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), possibly to compensate for an inhibitory effect of the drug on ABCB19 activity. Consistent with this hypothesis, NPPB blocked ion channel activity associated with ABCB19 expressed in human embryonic kidney cells as measured by patch-clamp electrophysiology. NPPB inhibited polar auxin transport through Arabidopsis seedling roots similarly to abcb19 mutations. NPPB also inhibited shootward auxin transport, which depends on the related ABCB4 protein. NPPB substantially decreased ABCB4 and ABCB19 protein levels when cycloheximide concomitantly inhibited new protein synthesis, indicating that blockage by NPPB enhances the degradation of ABCB transporters. Impairing the principal auxin transport streams in roots with NPPB caused aberrant patterns of auxin signaling reporters in root apices. Formation of the auxin-signaling gradient across the tips of gravity-stimulated roots, and its developmental consequence (gravitropism), were inhibited by micromolar concentrations of NPPB that did not affect growth rate. These results identify ion channel activity of ABCB19 that is blocked by NPPB, a compound that can now be considered an inhibitor of polar auxin transport with a defined molecular target. © 2014 American Society of Plant Biologists. All Rights Reserved.

Characterization of Three Novel Fatty Acid- and Retinoid-Binding Protein Genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the Cereal Cyst Nematodes Heterodera avenae and H. filipjevi

PubMed Central

Peng, Huan; Luo, Shujie; Huang, Wenkun; Cui, Jiangkuan; Li, Xin; Kong, Lingan; Jiang, Daohong; Chitwood, David J.; Peng, Deliang

2016-01-01

Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 (KU877266), Ha-far-2 (KU877267), Hf-far-1 (KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in all stages. The highest expression level of Ha-far-1 was observed in fourth-stage juvenile (J4), whereas the highest expression level of Ha-far-2 occurred in second-stage juvenile (J2). In conclusion, we have identified two novel far genes from H. avenae and one from H. filipjevi and have provided further indication that nematode far genes are present in a variety of nematode species, where the FAR proteins share similar basic structure, expression pattern and biochemical activities. PMID:27479008

Paxillin associates with poly(A)-binding protein 1 at the dense endoplasmic reticulum and the leading edge of migrating cells.

PubMed

Woods, Alison J; Roberts, Marnie S; Choudhary, Jyoti; Barry, Simon T; Mazaki, Yuichi; Sabe, Hisataka; Morley, Simon J; Critchley, David R; Norman, Jim C

2002-02-22

Using mass spectrometry we have identified proteins which co-immunoprecipitate with paxillin, an adaptor protein implicated in the integrin-mediated signaling pathways of cell motility. A major component of paxillin immunoprecipitates was poly(A)-binding protein 1, a 70-kDa mRNA-binding protein. Poly(A)-binding protein 1 associated with both the alpha and beta isoforms of paxillin, and this was unaffected by RNase treatment consistent with a protein-protein interaction. The NH(2)-terminal region of paxillin (residues 54-313) associated directly with poly(A)-binding protein 1 in cell lysates, and with His-poly(A)-binding protein 1 immobilized in microtiter wells. Binding was specific, saturable and of high affinity (K(d) of approximately 10 nm). Cell fractionation studies showed that at steady state, the bulk of paxillin and poly(A)-binding protein 1 was present in the "dense" polyribosome-associated endoplasmic reticulum. However, inhibition of nuclear export with leptomycin B caused paxillin and poly(A)-binding protein 1 to accumulate in the nucleus, indicating that they shuttle between the nuclear and cytoplasmic compartments. When cells migrate, poly(A)-binding protein 1 colocalized with paxillin-beta at the tips of lamellipodia. Our results suggest a new mechanism whereby a paxillin x poly(A)-binding protein 1 complex facilitates transport of mRNA from the nucleus to sites of protein synthesis at the endoplasmic reticulum and the leading lamella during cell migration.

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...

Effects of salts on protein-surface interactions: applications for column chromatography.

PubMed

Tsumoto, Kouhei; Ejima, Daisuke; Senczuk, Anna M; Kita, Yoshiko; Arakawa, Tsutomu

2007-07-01

Development of protein pharmaceuticals depends on the availability of high quality proteins. Various column chromatographies are used to purify proteins and characterize the purity and properties of the proteins. Most column chromatographies require salts, whether inorganic or organic, for binding, elution or simply better recovery and resolution. The salts modulate affinity of the proteins for particular columns and nonspecific protein-protein or protein-surface interactions, depending on the type and concentration of the salts, in both specific and nonspecific manners. Salts also affect the binding capacity of the column, which determines the size of the column to be used. Binding capacity, whether equilibrium or dynamic (under an approximation of a slow flow rate), depends on the binding constant, protein concentration and the number of the binding site on the column as well as nonspecific binding. This review attempts to summarize the mechanism of the salt effects on binding affinity and capacity for various column chromatographies and on nonspecific protein-protein or protein-surface interactions. Understanding such salt effects should also be useful in preventing nonspecific protein binding to various containers. Copyright 2007 Wiley-Liss, Inc.

Loss of Sfpq Causes Long-Gene Transcriptopathy in the Brain.

PubMed

Takeuchi, Akihide; Iida, Kei; Tsubota, Toshiaki; Hosokawa, Motoyasu; Denawa, Masatsugu; Brown, J B; Ninomiya, Kensuke; Ito, Mikako; Kimura, Hiroshi; Abe, Takaya; Kiyonari, Hiroshi; Ohno, Kinji; Hagiwara, Masatoshi

2018-05-01

Genes specifically expressed in neurons contain members with extended long introns. Longer genes present a problem with respect to fulfilment of gene length transcription, and evidence suggests that dysregulation of long genes is a mechanism underlying neurodegenerative and psychiatric disorders. Here, we report the discovery that RNA-binding protein Sfpq is a critical factor for maintaining transcriptional elongation of long genes. We demonstrate that Sfpq co-transcriptionally binds to long introns and is required for sustaining long-gene transcription by RNA polymerase II through mediating the interaction of cyclin-dependent kinase 9 with the elongation complex. Phenotypically, Sfpq disruption caused neuronal apoptosis in developing mouse brains. Expression analysis of Sfpq-regulated genes revealed specific downregulation of developmentally essential neuronal genes longer than 100 kb in Sfpq-disrupted brains; those genes are enriched in associations with neurodegenerative and psychiatric diseases. The identified molecular machinery yields directions for targeted investigations of the association between long-gene transcriptopathy and neuronal diseases. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Chromatibody, a novel non-invasive molecular tool to explore and manipulate chromatin in living cells

PubMed Central

Jullien, Denis; Vignard, Julien; Fedor, Yoann; Béry, Nicolas; Olichon, Aurélien; Crozatier, Michèle; Erard, Monique; Cassard, Hervé; Ducommun, Bernard; Salles, Bernard

2016-01-01

ABSTRACT Chromatin function is involved in many cellular processes, its visualization or modification being essential in many developmental or cellular studies. Here, we present the characterization of chromatibody, a chromatin-binding single-domain, and explore its use in living cells. This non-intercalating tool specifically binds the heterodimer of H2A–H2B histones and displays a versatile reactivity, specifically labeling chromatin from yeast to mammals. We show that this genetically encoded probe, when fused to fluorescent proteins, allows non-invasive real-time chromatin imaging. Chromatibody is a dynamic chromatin probe that can be modulated. Finally, chromatibody is an efficient tool to target an enzymatic activity to the nucleosome, such as the DNA damage-dependent H2A ubiquitylation, which can modify this epigenetic mark at the scale of the genome and result in DNA damage signaling and repair defects. Taken together, these results identify chromatibody as a universal non-invasive tool for either in vivo chromatin imaging or to manipulate the chromatin landscape. PMID:27206857

De novo mutations in MED13, a component of the Mediator complex, are associated with a novel neurodevelopmental disorder.

PubMed

Snijders Blok, Lot; Hiatt, Susan M; Bowling, Kevin M; Prokop, Jeremy W; Engel, Krysta L; Cochran, J Nicholas; Bebin, E Martina; Bijlsma, Emilia K; Ruivenkamp, Claudia A L; Terhal, Paulien; Simon, Marleen E H; Smith, Rosemarie; Hurst, Jane A; McLaughlin, Heather; Person, Richard; Crunk, Amy; Wangler, Michael F; Streff, Haley; Symonds, Joseph D; Zuberi, Sameer M; Elliott, Katherine S; Sanders, Victoria R; Masunga, Abigail; Hopkin, Robert J; Dubbs, Holly A; Ortiz-Gonzalez, Xilma R; Pfundt, Rolph; Brunner, Han G; Fisher, Simon E; Kleefstra, Tjitske; Cooper, Gregory M

2018-05-08

Many genetic causes of developmental delay and/or intellectual disability (DD/ID) are extremely rare, and robust discovery of these requires both large-scale DNA sequencing and data sharing. Here we describe a GeneMatcher collaboration which led to a cohort of 13 affected individuals harboring protein-altering variants, 11 of which are de novo, in MED13; the only inherited variant was transmitted to an affected child from an affected mother. All patients had intellectual disability and/or developmental delays, including speech delays or disorders. Other features that were reported in two or more patients include autism spectrum disorder, attention deficit hyperactivity disorder, optic nerve abnormalities, Duane anomaly, hypotonia, mild congenital heart abnormalities, and dysmorphisms. Six affected individuals had mutations that are predicted to truncate the MED13 protein, six had missense mutations, and one had an in-frame-deletion of one amino acid. Out of the seven non-truncating mutations, six clustered in two specific locations of the MED13 protein: an N-terminal and C-terminal region. The four N-terminal clustering mutations affect two adjacent amino acids that are known to be involved in MED13 ubiquitination and degradation, p.Thr326 and p.Pro327. MED13 is a component of the CDK8-kinase module that can reversibly bind Mediator, a multi-protein complex that is required for Polymerase II transcription initiation. Mutations in several other genes encoding subunits of Mediator have been previously shown to associate with DD/ID, including MED13L, a paralog of MED13. Thus, our findings add MED13 to the group of CDK8-kinase module-associated disease genes.

Prenatal Dexamethasone Augments the Sex-Selective Developmental Neurotoxicity of Chlorpyrifos: Implications for Vulnerability after Pharmacotherapy for Preterm Labor

PubMed Central

Slotkin, Theodore A.; Card, Jennifer; Infante, Alice; Seidler, Frederic J.

2013-01-01

Glucocorticoids are routinely given in preterm labor and are also elevated by maternal stress; organophosphate exposures are virtually ubiquitous, so coexposures to these two agents are pervasive. We administered dexamethasone to pregnant rats on gestational days 17–19 at a standard therapeutic dose (0.2 mg/kg); offspring were then given chlorpyrifos on postnatal days 1–4, at a dose (1 mg/kg) that produces barely-detectable (<10%) inhibition of brain cholinesterase activity. We evaluated indices for acetylcholine (ACh) synaptic function throughout adolescence, young adulthood and later adulthood, in brain regions possessing the majority of ACh projections and cell bodies; we measured nicotinic ACh receptor binding, hemicholinium-3 binding to the presynaptic choline transporter and choline acetyltransferase activity, all known targets for the adverse developmental effects of dexamethasone and chlorpyrifos given individually. Dexamethasone did not enhance the systemic toxicity of chlorpyrifos, as evidenced by weight gain and measurements of cholinesterase inhibition during chlorpyrifos treatment. Nevertheless, it enhanced the loss of presynaptic ACh function selectively in females, who ordinarily show sparing of organophosphate developmental neurotoxicity relative to males. Females receiving the combined treatment showed decrements in choline transporter binding and choline acetyltransferase activity that were unique (not found with either treatment alone), as well as additive decrements in nicotinic receptor binding. On the other hand, males given dexamethasone showed no augmentation of the effects of chlorpyrifos. Our findings indicate that prior dexamethasone exposure could create a subpopulation that is especially vulnerable to the adverse effects of organophosphates or other developmental neurotoxicants. PMID:23416428

Selective enrichment of metal-binding proteins based on magnetic core/shell microspheres functionalized with metal cations.

PubMed

Fang, Caiyun; Zhang, Lei; Zhang, Xiaoqin; Lu, Haojie

2015-06-21

Metal binding proteins play many important roles in a broad range of biological processes. Characterization of metal binding proteins is important for understanding their structure and biological functions, thus leading to a clear understanding of metal associated diseases. The present study is the first to investigate the effectiveness of magnetic microspheres functionalized with metal cations (Ca(2+), Cu(2+), Zn(2+) and Fe(3+)) as the absorbent matrix in IMAC technology to enrich metal containing/binding proteins. The putative metal binding proteins in rat liver were then globally characterized by using this strategy which is very easy to handle and can capture a number of metal binding proteins effectively. In total, 185 putative metal binding proteins were identified from rat liver including some known less abundant and membrane-bound metal binding proteins such as Plcg1, Acsl5, etc. The identified proteins are involved in many important processes including binding, catalytic activity, translation elongation factor activity, electron carrier activity, and so on.

Fragile X Mental Retardation Protein Regulates Activity-Dependent Membrane Trafficking and Trans-Synaptic Signaling Mediating Synaptic Remodeling

PubMed Central

Sears, James C.; Broadie, Kendal

2018-01-01

Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. The disease arises through loss of fragile X mental retardation protein (FMRP), which normally exhibits peak expression levels in early-use critical periods, and is required for activity-dependent synaptic remodeling during this transient developmental window. FMRP canonically binds mRNA to repress protein translation, with targets that regulate cytoskeleton dynamics, membrane trafficking, and trans-synaptic signaling. We focus here on recent advances emerging in these three areas from the Drosophila disease model. In the well-characterized central brain mushroom body (MB) olfactory learning/memory circuit, FMRP is required for activity-dependent synaptic remodeling of projection neurons innervating the MB calyx, with function tightly restricted to an early-use critical period. FMRP loss is phenocopied by conditional removal of FMRP only during this critical period, and rescued by FMRP conditional expression only during this critical period. Consistent with FXS hyperexcitation, FMRP loss defects are phenocopied by heightened sensory experience and targeted optogenetic hyperexcitation during this critical period. FMRP binds mRNA encoding Drosophila ESCRTIII core component Shrub (human CHMP4 homolog) to restrict Shrub translation in an activity-dependent mechanism only during this same critical period. Shrub mediates endosomal membrane trafficking, and perturbing Shrub expression is known to interfere with neuronal process pruning. Consistently, FMRP loss and Shrub overexpression targeted to projection neurons similarly causes endosomal membrane trafficking defects within synaptic boutons, and genetic reduction of Shrub strikingly rescues Drosophila FXS model defects. In parallel work on the well-characterized giant fiber (GF) circuit, FMRP limits iontophoretic dye loading into central interneurons, demonstrating an FMRP role controlling core neuronal properties through the activity-dependent repression of translation. In the well-characterized Drosophila neuromuscular junction (NMJ) model, developmental synaptogenesis and activity-dependent synaptic remodeling both require extracellular matrix metalloproteinase (MMP) enzymes interacting with the heparan sulfate proteoglycan (HSPG) glypican dally-like protein (Dlp) to restrict trans-synaptic Wnt signaling, with FXS synaptogenic defects alleviated by both MMP and HSPG reduction. This new mechanistic axis spanning from activity to FMRP to HSPG-dependent MMP regulation modulates activity-dependent synaptogenesis. We discuss future directions for these mechanisms, and intersecting research priorities for FMRP in glial and signaling interactions. PMID:29375303

Tighter Ligand Binding Can Compensate for Impaired Stability of an RNA-Binding Protein.

PubMed

Wallis, Christopher P; Richman, Tara R; Filipovska, Aleksandra; Rackham, Oliver

2018-06-15

It has been widely shown that ligand-binding residues, by virtue of their orientation, charge, and solvent exposure, often have a net destabilizing effect on proteins that is offset by stability conferring residues elsewhere in the protein. This structure-function trade-off can constrain possible adaptive evolutionary changes of function and may hamper protein engineering efforts to design proteins with new functions. Here, we present evidence from a large randomized mutant library screen that, in the case of PUF RNA-binding proteins, this structural relationship may be inverted and that active-site mutations that increase protein activity are also able to compensate for impaired stability. We show that certain mutations in RNA-protein binding residues are not necessarily destabilizing and that increased ligand-binding can rescue an insoluble, unstable PUF protein. We hypothesize that these mutations restabilize the protein via thermodynamic coupling of protein folding and RNA binding.

Telobox motifs recruit CLF/SWN-PRC2 for H3K27me3 deposition via TRB factors in Arabidopsis.

PubMed

Zhou, Yue; Wang, Yuejun; Krause, Kristin; Yang, Tingting; Dongus, Joram A; Zhang, Yijing; Turck, Franziska

2018-05-01

Polycomb repressive complexes (PRCs) control organismic development in higher eukaryotes through epigenetic gene repression 1-4 . PRC proteins do not contain DNA-binding domains, thus prompting questions regarding how PRCs find their target loci 5 . Here we present genome-wide evidence of PRC2 recruitment by telomere-repeat-binding factors (TRBs) through telobox-related motifs in Arabidopsis. A triple trb1-2, trb2-1, and trb3-2 (trb1/2/3) mutant with a developmental phenotype and a transcriptome strikingly similar to those of strong PRC2 mutants showed redistribution of trimethyl histone H3 Lys27 (H3K27me3) marks and lower H3K27me3 levels, which were correlated with derepression of TRB1-target genes. TRB1-3 physically interacted with the PRC2 proteins CLF and SWN. A SEP3 reporter gene with a telobox mutation showed ectopic expression, which was correlated with H3K27me3 depletion, whereas tethering TRB1 to the mutated cis element partially restored repression. We propose that telobox-related motifs recruit PRC2 through the interaction between TRBs and CLF/SWN, a mechanism essential for H3K27me3 deposition at a subset of target genes.

Transcription factor GATA-1 regulates human HOXB2 gene expression in erythroid cells.

PubMed

Vieille-Grosjean, I; Huber, P

1995-03-03

The human HOXB2 gene is a member of the vertebrate Hox gene family that contains genes coding for specific developmental stage DNA-binding proteins. Remarkably, within the hematopoietic compartment, genes of the HOXB complex are expressed specifically in erythromegakaryocytic cell lines and, for some of them, in hematopoietic progenitors. Here, we report the study of HOXB2 gene transcriptional regulation in hematopoietic cells, an initial step in understanding the lineage-specific expression of the whole HOXB complex in these cells. We have isolated the HOXB2 5'-flanking sequence and have characterized a promoter fragment extending 323 base pairs upstream from the transcriptional start site, which, in transfection experiments, was sufficient to direct the tissue-specific expression of HOXB2 in the erythroid cell line K562. In this fragment, we have identified a potential GATA-binding site that is essential to the promoter activity as demonstrated by point mutation experiments. Gel shift analysis revealed the formation of a specific complex in both erythroleukemic lines K562 and HEL that could be prevented by the addition of a specific antiserum raised against GATA-1 protein. These findings suggest a regulatory hierarchy in which GATA-1 is upstream of the HOXB2 gene in erythroid cells.

On the nature of in vivo requirements for rde-4 in RNAi and developmental pathways in C. elegans

PubMed Central

Lopez-Molina, Javier; Gent, Jonathan; Saynuk, Jamie Fleenor

2011-01-01

C. elegans RDE-4 is a double-stranded RNA binding protein that has been shown to play a key role in response to foreign double-stranded RNA (dsRNA). We have used diverse tools for analysis of gene function to characterize the domain and organismal foci of RDE-4 action in C. elegans. First, we examined the focus of activity within the RDE-4 protein, by testing a series of RDE-4 deletion constructs for their ability to support dsRNA-triggered gene silencing. These assays indicated a molecular requirement for a linker region and the second dsRNA-binding domain of RDE-4, with ancillary contributions to function from the C and N terminal domains. Second, we used mosaic analysis to explore the cellular focus of action of RDE-4. These experiments indicated an ability of RDE-4 to function non-autonomously in foreign RNA responses. Third, we used growth under stressful conditions to search for evidence of an organismal focus of action for RDE-4 distinct from its role in response to foreign dsRNA. Propagation at high temperatures exposed a conditional requirement for RDE-4 for optimal growth and fertility, indicating at least under these conditions that RDE-4 can serve an essential role in C. elegans. PMID:21519199

Nuclear DNA polymerase beta from Leishmania infantum. Cloning, molecular analysis and developmental regulation

PubMed Central

Taladriz, Soraya; Hanke, Tobias; Ramiro, María J.; García-Díaz, Miguel; Lacoba, Mario García de; Blanco, Luis; Larraga, Vicente

2001-01-01

We have identified a novel polymerase beta (Pol β)-like enzyme from Leishmania infantum, a parasite protozoon causing disease in humans. This protein, named Li Pol β, shows a nuclear localization that contrasts with the mitochondrial localization of Pol β from Crithidia fasciculata, a closely related parasite, the only polymerase β described so far in Trypanosomatidae. Li Pol β, that belongs to the DNA polymerase X family, displays an evolutionarily conserved Pol β-type DNA polymerase core, in which most of the key residues involved in DNA binding, nucleotide binding, dRPase and polymerization catalysis are conserved. In agreement with this, Li Pol β, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity. Cell synchronization experiments showed a correlation between both Li Pol β mRNA and protein levels along the parasite cell cycle. Analysis of these parameters at the different growth phases of the parasite, from the proliferative (non-infective) logarithmic phase to the non-dividing (highly infectious) stationary phase, showed high levels of Li Pol β at the infective phase of the parasite. The data suggest a role of Li Pol β in base excision repair in L.infantum, a parasite usually affected by oxygen stress environments into the macrophage host cells. PMID:11557814

Surfactant Protein-C Promoter Variants Associated with Neonatal Respiratory Distress Syndrome Reduce Transcription

PubMed Central

Wambach, Jennifer A.; Yang, Ping; Wegner, Daniel J.; An, Ping; Hackett, Brian P.; Cole, F. S.; Hamvas, Aaron

2010-01-01

Dominant mutations in coding regions of the surfactant protein-C gene (SFTPC) cause respiratory distress syndrome (RDS) in infants. However, the contribution of variants in noncoding regions of SFTPC to pulmonary phenotypes is unknown. Using a case-control group of infants ≥34 weeks gestation (n=538), we used complete resequencing of SFTPC and its promoter, genotyping, and logistic regression to identify 80 single nucleotide polymorphisms (SNPs). Three promoter SNPs were statistically associated with neonatal RDS among European descent infants. To assess the transcriptional effects of these three promoter SNPs, we selectively mutated the SFTPC promoter and performed transient transfection using MLE-15 cells and a firefly luciferase reporter vector. Each promoter SNP decreased SFTPC transcription. The combination of two variants in high linkage dysequilibrium also decreased SFTPC transcription. In silico evaluation of transcription factor binding demonstrated that the rare allele at g.-1167 disrupts a SOX (SRY-related high mobility group box) consensus motif and introduces a GATA-1 site, at g.-2385 removes a MZF-1 (myeloid zinc finger) binding site, and at g.-1647 removes a potential methylation site. This combined statistical, in vitro, and in silico approach suggests that reduced SFTPC transcription contributes to the genetic risk for neonatal RDS in developmentally susceptible infants. PMID:20539253

Sequence-Based Prediction of RNA-Binding Residues in Proteins.

PubMed

Walia, Rasna R; El-Manzalawy, Yasser; Honavar, Vasant G; Dobbs, Drena

2017-01-01

Identifying individual residues in the interfaces of protein-RNA complexes is important for understanding the molecular determinants of protein-RNA recognition and has many potential applications. Recent technical advances have led to several high-throughput experimental methods for identifying partners in protein-RNA complexes, but determining RNA-binding residues in proteins is still expensive and time-consuming. This chapter focuses on available computational methods for identifying which amino acids in an RNA-binding protein participate directly in contacting RNA. Step-by-step protocols for using three different web-based servers to predict RNA-binding residues are described. In addition, currently available web servers and software tools for predicting RNA-binding sites, as well as databases that contain valuable information about known protein-RNA complexes, RNA-binding motifs in proteins, and protein-binding recognition sites in RNA are provided. We emphasize sequence-based methods that can reliably identify interfacial residues without the requirement for structural information regarding either the RNA-binding protein or its RNA partner.

An oocyte-specific ELAVL2 isoform is a translational repressor ablated from meiotically competent antral oocytes

PubMed Central

Chalupnikova, Katerina; Solc, Petr; Sulimenko, Vadym; Sedlacek, Radislav; Svoboda, Petr

2014-01-01

At the end of the growth phase, mouse antral follicle oocytes acquire full developmental competence. In the mouse, this event is marked by the transition from the so-called non-surrounded nucleolus (NSN) chromatin configuration into the transcriptionally quiescent surrounded nucleolus (SN) configuration, which is named after a prominent perinucleolar condensed chromatin ring. However, the SN chromatin configuration alone is not sufficient for determining the developmental competence of the SN oocyte. There are additional nuclear and cytoplamic factors involved, while a little is known about the changes occurring in the cytoplasm during the NSN/SN transition. Here, we report functional analysis of maternal ELAVL2 an AU-rich element binding protein. Elavl2 gene encodes an oocyte-specific protein isoform (denoted ELAVL2°), which acts as a translational repressor. ELAVL2° is abundant in fully grown NSN oocytes, is ablated during the NSN/SN transition and remains low during the oocyte-to-embryo transition (OET). ELAVL2° overexpression during meiotic maturation causes errors in chromosome segregation, indicating the significance of naturally reduced ELAVL2° levels in SN oocytes. On the other hand, during oocyte growth, prematurely reduced Elavl2 expression results in lower yields of fully grown and meiotically matured oocytes, suggesting that Elavl2 is necessary for proper oocyte maturation. Moreover, Elavl2 knockdown showed stimulating effects on translation in fully grown oocytes. We propose that ELAVL2 has an ambivalent role in oocytes: it functions as a pleiotropic translational repressor in efficient production of fully grown oocytes, while its disposal during the NSN/SN transition contributes to the acquisition of full developmental competence. PMID:24553115

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family

PubMed Central

2010-01-01

Background Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. Findings De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Conclusions Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein. PMID:20979600

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family.

PubMed

Antonets, Denis V; Nepomnyashchikh, Tatyana S; Shchelkunov, Sergei N

2010-10-27

Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.

PubMed

Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur

2016-04-06

Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites.

Developmental characterization and environmental stress responses of Y-box binding protein 1 gene (AccYB-1) from Apis cerana cerana.

PubMed

Li, Guilin; Wang, Lijun; Wang, Ying; Li, Han; Liu, Zhenguo; Wang, Hongfang; Xu, Baohua; Guo, Xingqi

2018-06-22

Y-box binding protein 1 (YB-1) is a member of the cold shock domain protein superfamily and is involved in development, environmental stresses and DNA oxidative damage in many organisms. However, the precise functions of YB-1 are still not well understood in various insects, including bees. In the current study, we identified a YB-1 gene in Apis cerana cerana (AccYB-1). The predicted cis-acting elements in the promoter sequence of AccYB-1 indicated its possible roles in development and stress responses. AccYB-1 expression was higher in one-day-old larvae and dark-eyed pupae than in other development stages. Tissue-specific expression analysis showed that the mRNA level of AccYB-1 was higher in the thorax and midgut than in other tissues. The results from real-time PCR showed that AccYB-1 was induced by many environmental stresses. Silencing AccYB-1 downregulated the transcriptional level of some growth- and development-related genes and antioxidant genes and decreased the enzyme activities of several antioxidant-related enzymes, further indicating a possible function of AccYB-1 in growth, development and stress responses. Taken together, our findings suggest that AccYB-1 may play an indispensable role in growth and development and environmental stress responses in Apis cerana cerana. To our knowledge, this is the first paper to explore the role of YB-1 in bees. Copyright © 2018. Published by Elsevier B.V.

The membrane trafficking and functionality of the K+-Cl- co-transporter KCC2 is regulated by TGF-β2.

PubMed

Roussa, Eleni; Speer, Jan Manuel; Chudotvorova, Ilona; Khakipoor, Shokoufeh; Smirnov, Sergei; Rivera, Claudio; Krieglstein, Kerstin

2016-09-15

Functional activation of the neuronal K(+)-Cl(-) co-transporter KCC2 (also known as SLC12A5) is a prerequisite for shifting GABAA responses from depolarizing to hyperpolarizing during development. Here, we introduce transforming growth factor β2 (TGF-β2) as a new regulator of KCC2 membrane trafficking and functional activation. TGF-β2 controls membrane trafficking, surface expression and activity of KCC2 in developing and mature mouse primary hippocampal neurons, as determined by immunoblotting, immunofluorescence, biotinylation of surface proteins and KCC2-mediated Cl(-) extrusion. We also identify the signaling pathway from TGF-β2 to cAMP-response-element-binding protein (CREB) and Ras-associated binding protein 11b (Rab11b) as the underlying mechanism for TGF-β2-mediated KCC2 trafficking and functional activation. TGF-β2 increases colocalization and interaction of KCC2 with Rab11b, as determined by 3D stimulated emission depletion (STED) microscopy and co-immunoprecipitation, respectively, induces CREB phosphorylation, and enhances Rab11b gene expression. Loss of function of either CREB1 or Rab11b suppressed TGF-β2-dependent KCC2 trafficking, surface expression and functionality. Thus, TGF-β2 is a new regulatory factor for KCC2 functional activation and membrane trafficking, and a putative indispensable molecular determinant for the developmental shift of GABAergic transmission. © 2016. Published by The Company of Biologists Ltd.

Proteome-wide Identification of Novel Ceramide-binding Proteins by Yeast Surface cDNA Display and Deep Sequencing.

PubMed

Bidlingmaier, Scott; Ha, Kevin; Lee, Nam-Kyung; Su, Yang; Liu, Bin

2016-04-01

Although the bioactive sphingolipid ceramide is an important cell signaling molecule, relatively few direct ceramide-interacting proteins are known. We used an approach combining yeast surface cDNA display and deep sequencing technology to identify novel proteins binding directly to ceramide. We identified 234 candidate ceramide-binding protein fragments and validated binding for 20. Most (17) bound selectively to ceramide, although a few (3) bound to other lipids as well. Several novel ceramide-binding domains were discovered, including the EF-hand calcium-binding motif, the heat shock chaperonin-binding motif STI1, the SCP2 sterol-binding domain, and the tetratricopeptide repeat region motif. Interestingly, four of the verified ceramide-binding proteins (HPCA, HPCAL1, NCS1, and VSNL1) and an additional three candidate ceramide-binding proteins (NCALD, HPCAL4, and KCNIP3) belong to the neuronal calcium sensor family of EF hand-containing proteins. We used mutagenesis to map the ceramide-binding site in HPCA and to create a mutant HPCA that does not bind to ceramide. We demonstrated selective binding to ceramide by mammalian cell-produced wild type but not mutant HPCA. Intriguingly, we also identified a fragment from prostaglandin D2synthase that binds preferentially to ceramide 1-phosphate. The wide variety of proteins and domains capable of binding to ceramide suggests that many of the signaling functions of ceramide may be regulated by direct binding to these proteins. Based on the deep sequencing data, we estimate that our yeast surface cDNA display library covers ∼60% of the human proteome and our selection/deep sequencing protocol can identify target-interacting protein fragments that are present at extremely low frequency in the starting library. Thus, the yeast surface cDNA display/deep sequencing approach is a rapid, comprehensive, and flexible method for the analysis of protein-ligand interactions, particularly for the study of non-protein ligands. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

[Determination of plasma protein binding rate of arctiin and arctigenin with ultrafiltration].

PubMed

Han, Xue-Ying; Wang, Wei; Tan, Ri-Qiu; Dou, De-Qiang

2013-02-01

To determine the plasma protein binding rate of arctiin and arctigenin. The ultrafiltration combined with HPLC was employed to determine the plasma protein binding rate of arctiin and arctigenin as well as rat plasma and healthy human plasma proteins. The plasma protein binding rate of arctiin with rat plasma at the concentrations of 64. 29, 32.14, 16.07 mg x L(-1) were (71.2 +/- 2.0)%, (73.4 +/- 0.61)%, (78.2 +/- 1.9)%, respectively; while the plasma protein binding rate of arctiin with healthy human plasma at the above concentrations were (64.8 +/- 3.1)%, (64.5 +/- 2.5)%, (77.5 +/- 1.7)%, respectively. The plasma protein binding rate of arctigenin with rat plasma at the concentrations of 77.42, 38.71, 19.36 mg x L(-1) were (96.7 +/- 0.41)%, (96.8 +/- 1.6)%, (97.3 +/- 0.46)%, respectively; while the plasma protein binding rate of arctigenin with normal human plasma at the above concentrations were (94.7 +/- 3.1)%, (96.8 +/- 1.6)%, (97.9 +/- 1.3)%, respectively. The binding rate of arctiin with rat plasma protein was moderate, which is slightly higher than the binding rate of arctiin with healthy human plasma protein. The plasma protein binding rates of arctigenin with both rat plasma and healthy human plasma are very high.

Binding, relational memory, and recall of naturalistic events: a developmental perspective.

PubMed

Sluzenski, Julia; Newcombe, Nora S; Kovacs, Stacie L

2006-01-01

This research was an investigation of children's performance on a task that requires memory binding. In Experiments 1 and 2, 4-year-olds, 6-year-olds, and adults viewed complex pictures and were tested on memory for isolated parts in the pictures and on the part combinations (combination condition). The results suggested improvement in memory for the combinations between the ages of 4 and 6 years but not in memory for the isolated parts. In Experiments 2 and 3, the authors also examined the developmental relationship between performance in the combination condition and free recall of a naturalistic event, finding preliminary evidence that performance on a memory task that requires binding is positively related to performance in episodic memory. ((c) 2006 APA, all rights reserved).

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caberoy, Nora B.; Zhou, Yixiong; Alvarado, Gabriela

To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in {approx}300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressedmore » for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.« less

Isolation and characterizations of oxalate-binding proteins in the kidney

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roop-ngam, Piyachat; Chaiyarit, Sakdithep; Pongsakul, Nutkridta

Highlights: Black-Right-Pointing-Pointer The first large-scale characterizations of oxalate-binding kidney proteins. Black-Right-Pointing-Pointer The recently developed oxalate-conjugated EAH Sepharose 4B beads were applied. Black-Right-Pointing-Pointer 38 forms of 26 unique oxalate-binding kidney proteins were identified. Black-Right-Pointing-Pointer 25/26 (96%) of identified proteins had 'L-x(3,5)-R-x(2)-[AGILPV]' domain. -- Abstract: Oxalate-binding proteins are thought to serve as potential modulators of kidney stone formation. However, only few oxalate-binding proteins have been identified from previous studies. Our present study, therefore, aimed for large-scale identification of oxalate-binding proteins in porcine kidney using an oxalate-affinity column containing oxalate-conjugated EAH Sepharose 4B beads for purification followed by two-dimensional gel electrophoresis (2-DE) tomore » resolve the recovered proteins. Comparing with those obtained from the controlled column containing uncoupled EAH-Sepharose 4B (to subtract the background of non-specific bindings), a total of 38 protein spots were defined as oxalate-binding proteins. These protein spots were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS) as 26 unique proteins, including several nuclear proteins, mitochondrial proteins, oxidative stress regulatory proteins, metabolic enzymes and others. Identification of oxalate-binding domain using the PRATT tool revealed 'L-x(3,5)-R-x(2)-[AGILPV]' as a functional domain responsible for oxalate-binding in 25 of 26 (96%) unique identified proteins. We report herein, for the first time, large-scale identification and characterizations of oxalate-binding proteins in the kidney. The presence of positively charged arginine residue in the middle of this functional domain suggested its significance for binding to the negatively charged oxalate. These data will enhance future stone research, particularly on stone modulators.« less

Structural Analysis of Semi-specific Oligosaccharide Recognition by a Cellulose-binding Protein of Thermotoga maritima Reveals Adaptations for Functional Diversification of the Oligopeptide Periplasmic Binding Protein Fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cuneo, Matthew J.; Beese, Lorena S.; Hellinga, Homme W.

Periplasmic binding proteins (PBPs) constitute a protein superfamily that binds a wide variety of ligands. In prokaryotes, PBPs function as receptors for ATP-binding cassette or tripartite ATP-independent transporters and chemotaxis systems. In many instances, PBPs bind their cognate ligands with exquisite specificity, distinguishing, for example, between sugar epimers or structurally similar anions. By contrast, oligopeptide-binding proteins bind their ligands through interactions with the peptide backbone but do not distinguish between different side chains. The extremophile Thermotoga maritima possesses a remarkable array of carbohydrate-processing metabolic systems, including the hydrolysis of cellulosic polymers. Here, we present the crystal structure of a T.more » maritima cellobiose-binding protein (tm0031) that is homologous to oligopeptide-binding proteins. T. maritima cellobiose-binding protein binds a variety of lengths of {beta}(1 {yields} 4)-linked glucose oligomers, ranging from two rings (cellobiose) to five (cellopentaose). The structure reveals that binding is semi-specific. The disaccharide at the nonreducing end binds specifically; the other rings are located in a large solvent-filled groove, where the reducing end makes several contacts with the protein, thereby imposing an upper limit of the oligosaccharides that are recognized. Semi-specific recognition, in which a molecular class rather than individual species is selected, provides an efficient solution for the uptake of complex mixtures.« less

Genome-wide identification and characterization of TCP genes involved in ovule development of Phalaenopsis equestris

PubMed Central

Lin, Yu-Fu; Chen, You-Yi; Hsiao, Yu-Yun; Shen, Ching-Yu; Hsu, Jui-Ling; Yeh, Chuan-Ming; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Liu, Zhong-Jian; Tsai, Wen-Chieh

2016-01-01

TEOSINTE-BRANCHED/CYCLOIDEA/PCF (TCP) proteins are plant-specific transcription factors known to have a role in multiple aspects of plant growth and development at the cellular, organ and tissue levels. However, there has been no related study of TCPs in orchids. Here we identified 23 TCP genes from the genome sequence of Phalaenopsis equestris. Phylogenetic analysis distinguished two homology classes of PeTCP transcription factor families: classes I and II. Class II was further divided into two subclasses, CIN and CYC/TB1. Spatial and temporal expression analysis showed that PePCF10 was predominantly expressed in ovules at early developmental stages and PeCIN8 had high expression at late developmental stages in ovules, with overlapping expression at day 16 after pollination. Subcellular localization and protein–protein interaction analyses revealed that PePCF10 and PeCIN8 could form homodimers and localize in the nucleus. However, PePCF10 and PeCIN8 could not form heterodimers. In transgenic Arabidopsis thaliana plants (overexpression and SRDX, a super repression motif derived from the EAR-motif of the repression domain of tobacco ETHYLENE-RESPONSIVE ELEMENT-BINDING FACTOR 3 and SUPERMAN, dominantly repressed), the two genes helped regulate cell proliferation. Together, these results suggest that PePCF10 and PeCIN8 play important roles in orchid ovule development by modulating cell division. PMID:27543606

Skeletal Muscle Satellite Cells Are Committed to Myogenesis and Do Not Spontaneously Adopt Nonmyogenic Fates

PubMed Central

Starkey, Jessica D.; Yamamoto, Masakazu; Yamamoto, Shoko; Goldhamer, David J.

2011-01-01

The developmental potential of skeletal muscle stem cells (satellite cells) remains controversial. The authors investigated satellite cell developmental potential in single fiber and clonal cultures derived from MyoDiCre/+;R26REYFP/+ muscle, in which essentially all satellite cells are permanently labeled. Approximately 60% of the clones derived from cells that co-purified with muscle fibers spontaneously underwent adipogenic differentiation. These adipocytes stained with Oil-Red-O and expressed the terminal differentiation markers, adipsin and fatty acid binding protein 4, but did not express EYFP and were therefore not of satellite cell origin. Satellite cells mutant for either MyoD or Myf-5 also maintained myogenic programming in culture and did not adopt an adipogenic fate. Incorporation of additional wash steps prior to muscle fiber plating virtually eliminated the non-myogenic cells but did not reduce the number of adherent Pax7+ satellite cells. More than half of the adipocytes observed in cultures from Tie2-Cre mice were recombined, further demonstrating a non-satellite cell origin. Under adipogenesis-inducing conditions, satellite cells accumulated cytoplasmic lipid but maintained myogenic protein expression and did not fully execute the adipogenic differentiation program, distinguishing them from adipocytes observed in muscle fiber cultures. The authors conclude that skeletal muscle satellite cells are committed to myogenesis and do not spontaneously adopt an adipogenic fate. PMID:21339173

Mutations in MYB3R1 and MYB3R4 Cause Pleiotropic Developmental Defects and Preferential Down-Regulation of Multiple G2/M-Specific Genes in Arabidopsis1[C][W

PubMed Central

Haga, Nozomi; Kobayashi, Kosuke; Suzuki, Takamasa; Maeo, Kenichiro; Kubo, Minoru; Ohtani, Misato; Mitsuda, Nobutaka; Demura, Taku; Nakamura, Kenzo; Jürgens, Gerd; Ito, Masaki

2011-01-01

R1R2R3-Myb proteins represent an evolutionarily conserved class of Myb family proteins important for cell cycle regulation and differentiation in eukaryotic cells. In plants, this class of Myb proteins are believed to regulate the transcription of G2/M phase-specific genes by binding to common cis-elements, called mitosis-specific activator (MSA) elements. In Arabidopsis (Arabidopsis thaliana), MYB3R1 and MYB3R4 act as transcriptional activators and positively regulate cytokinesis by activating the transcription of KNOLLE, which encodes a cytokinesis-specific syntaxin. Here, we show that the double mutation myb3r1 myb3r4 causes pleiotropic developmental defects, some of which are due to deficiency of KNOLLE whereas other are not, suggesting that multiple target genes are involved. Consistently, microarray analysis of the double mutant revealed altered expression of many genes, among which G2/M-specific genes showed significant overrepresentation of the MSA motif and a strong tendency to be down-regulated by the double mutation. Our results demonstrate, on a genome-wide level, the importance of the MYB3R-MSA pathway for regulating G2/M-specific transcription. In addition, MYB3R1 and MYB3R4 may have diverse roles during plant development by regulating G2/M-specific genes with various functions as well as genes possibly unrelated to the cell cycle. PMID:21862669

Mutations in the Na+/Citrate Cotransporter NaCT (SLC13A5) in Pediatric Patients with Epilepsy and Developmental Delay

PubMed Central

Klotz, Jenna; Porter, Brenda E; Colas, Claire; Schlessinger, Avner; Pajor, Ana M

2016-01-01

Mutations in the SLC13A5 gene that codes for the Na+/citrate cotransporter, NaCT, are associated with early onset epilepsy, developmental delay and tooth dysplasia in children. In this study, we identify additional SLC13A5 mutations in nine epilepsy patients from six families. To better characterize the syndrome, families with affected children answered questions about the scope of illness and the treatment strategies. Currently, there are no effective treatments, but some antiepileptic drugs targeting the γ-aminobutyric acid system reduce seizure frequency. Acetazolamide, a carbonic anhydrase inhibitor and atypical antiseizure medication, decreases seizures in four patients. In contrast to previous reports, the ketogenic diet and fasting resulted in worsening of symptoms. The effects of the mutations on NaCT transport function and protein expression were examined by transient transfections of COS-7 cells. There was no transport activity from any of the mutant transporters, although some of the mutant transporter proteins were present on the plasma membrane. The structural model of NaCT suggests that these mutations can affect helix packing or substrate binding. We tested various treatments, including chemical chaperones and low temperatures, but none improved transport function in the NaCT mutants. Interestingly, coexpression of NaCT and the mutants results in decreased protein expression and activity of the wild-type transporter, indicating functional interaction. In conclusion, this study has identified additional SLC13A5 mutations in patients with chronic epilepsy starting in the neonatal period, with the mutations producing inactive Na+/citrate transporters. PMID:27261973

SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.

PubMed

Brannan, Kristopher W; Jin, Wenhao; Huelga, Stephanie C; Banks, Charles A S; Gilmore, Joshua M; Florens, Laurence; Washburn, Michael P; Van Nostrand, Eric L; Pratt, Gabriel A; Schwinn, Marie K; Daniels, Danette L; Yeo, Gene W

2016-10-20

RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of AOSC-binding proteins in neurons

NASA Astrophysics Data System (ADS)

Liu, Ming; Nie, Qin; Xin, Xianliang; Geng, Meiyu

2008-11-01

Acidic oligosaccharide sugar chain (AOSC), a D-mannuronic acid oligosaccharide, derived from brown algae polysaccharide, has been completed Phase I clinical trial in China as an anti-Alzheimer’s Disease (AD) drug candidate. The identification of AOSC-binding protein(s) in neurons is very important for understanding its action mechanism. To determine the binding protein(s) of AOSC in neurons mediating its anti-AD activities, confocal microscopy, affinity chromatography, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were used. Confocal microscopy analysis shows that AOSC binds to SH-SY5Y cells in concentration-, time-, and temperature-dependent fashions. The AOSC binding proteins were purified by affinity chromatography and identified by LC-MS/MS analysis. The results showed that there are 349 proteins binding AOSC, including clathrin, adaptor protein-2 (AP-2) and amyloid precursor protein (APP). These results suggest that the binding/entrance of AOSC to neurons is probably responsible for anti-AD activities.

Prediction of Carbohydrate Binding Sites on Protein Surfaces with 3-Dimensional Probability Density Distributions of Interacting Atoms

PubMed Central

Tsai, Keng-Chang; Jian, Jhih-Wei; Yang, Ei-Wen; Hsu, Po-Chiang; Peng, Hung-Pin; Chen, Ching-Tai; Chen, Jun-Bo; Chang, Jeng-Yih; Hsu, Wen-Lian; Yang, An-Suei

2012-01-01

Non-covalent protein-carbohydrate interactions mediate molecular targeting in many biological processes. Prediction of non-covalent carbohydrate binding sites on protein surfaces not only provides insights into the functions of the query proteins; information on key carbohydrate-binding residues could suggest site-directed mutagenesis experiments, design therapeutics targeting carbohydrate-binding proteins, and provide guidance in engineering protein-carbohydrate interactions. In this work, we show that non-covalent carbohydrate binding sites on protein surfaces can be predicted with relatively high accuracy when the query protein structures are known. The prediction capabilities were based on a novel encoding scheme of the three-dimensional probability density maps describing the distributions of 36 non-covalent interacting atom types around protein surfaces. One machine learning model was trained for each of the 30 protein atom types. The machine learning algorithms predicted tentative carbohydrate binding sites on query proteins by recognizing the characteristic interacting atom distribution patterns specific for carbohydrate binding sites from known protein structures. The prediction results for all protein atom types were integrated into surface patches as tentative carbohydrate binding sites based on normalized prediction confidence level. The prediction capabilities of the predictors were benchmarked by a 10-fold cross validation on 497 non-redundant proteins with known carbohydrate binding sites. The predictors were further tested on an independent test set with 108 proteins. The residue-based Matthews correlation coefficient (MCC) for the independent test was 0.45, with prediction precision and sensitivity (or recall) of 0.45 and 0.49 respectively. In addition, 111 unbound carbohydrate-binding protein structures for which the structures were determined in the absence of the carbohydrate ligands were predicted with the trained predictors. The overall prediction MCC was 0.49. Independent tests on anti-carbohydrate antibodies showed that the carbohydrate antigen binding sites were predicted with comparable accuracy. These results demonstrate that the predictors are among the best in carbohydrate binding site predictions to date. PMID:22848404

Structural and functional analyses of genes encoding VQ proteins in apple.

PubMed

Dong, Qinglong; Zhao, Shuang; Duan, Dingyue; Tian, Yi; Wang, Yanpeng; Mao, Ke; Zhou, Zongshan; Ma, Fengwang

2018-07-01

Recent studies with Arabidopsis and soybean have shown that a class of valine-glutamine (VQ) motif-containing proteins interacts with some WRKY transcription factors. However, little is known about the evolution, structures, and functions of those proteins in apple. Here, we examined their features and identified 49 apple VQ genes. Our evolutional analysis revealed that the proteins could be clustered into nine groups together with their homologues in 33 species. Historically, the main characteristics of proteins in Groups I, V, VI, VII, IX, and X were thought to have been generated before the monocot-dicot split, whereas those in Groups II, III + IV, and VIII were generated after that split. In the structural analysis, apple MdVQ proteins appeared to bind only with Group I and IIc MdWRKY proteins. Meanwhile, MdVQ1, MdVQ10, MdVQ15, and MdVQ36 interacted with multiple MdVQ proteins to form heterodimers but MdVQ15 formed a homodimer. The functional analysis indicated that overexpression of some apple MdVQs in Arabidopsis and tobacco plants effected their vegetative and reproductive growth. These results provide important information about the characteristics of apple MdVQ genes and can serve as a solid foundation for further studies about the role of WRKY-VQ interactions in regulating apple developmental and defense mechanisms. Copyright © 2018 Elsevier B.V. All rights reserved.

Drosophila Fragile X Mental Retardation Protein Developmentally Regulates Activity-Dependent Axon Pruning

PubMed Central

Tessier, Charles R.; Broadie, Kendal

2014-01-01

Summary Fragile X Syndrome (FraX) is a broad-spectrum neurological disorder with symptoms ranging from hyperexcitability to mental retardation and autism. Loss of the fragile X mental retardation 1 (fmr1) gene product, the mRNA-binding translational regulator FMRP, causes structural over-elaboration of dendritic and axonal processes as well as functional alterations in synaptic plasticity at maturity. It is unclear, however, whether FraX is primarily a disease of development, a disease of plasticity or both; a distinction vital for engineering intervention strategies. To address this critical issue, we have used the Drosophila FraX model to investigate the developmental roles of Drosophila FMRP (dFMRP). dFMRP expression and regulation of chickadee/profilin coincides with a transient window of late brain development. During this time, dFMRP is positively regulated by sensory input activity, and required to limit axon growth and for efficient activity-dependent pruning of axon branches in the Mushroom Body learning/memory center. These results demonstrate that dFMRP has a primary role in activity-dependent neural circuit refinement in late brain development. PMID:18321984

Alcohol exposure during development: Impact on the epigenome.

PubMed

Perkins, Amy; Lehmann, Claudia; Lawrence, R Charles; Kelly, Sandra J

2013-10-01

Fetal alcohol spectrum disorders represent a wide range of symptoms associated with in utero alcohol exposure. Animal models of FASD have been useful in determining the specific neurological consequences of developmental alcohol exposure, but the mechanisms of those consequences are unclear. Long-lasting changes to the epigenome are proposed as a mechanism of alcohol-induced teratogenesis in the hippocampus. The current study utilized a three-trimester rodent model of FASD to examine changes to some of the enzymatic regulators of the epigenome in adolescence. Combined pre- and post-natal alcohol exposureresulted in a significant increase in DNA methyltransferase activity (DNMT), without affecting histone deacetylase activity (HDAC). Developmental alcohol exposure also caused a change in gene expression of regulators of the epigenome, in particular, DNMT1, DNMT3a, and methyl CpG binding protein 2 (MeCP2). The modifications of the activity and expression of epigenetic regulators in the hippocampus of rodents perinatally exposed to alcohol suggest that alcohol's impact on the epigenome and its regulators may be one of the underlying mechanisms of alcohol teratogenesis. Copyright © 2013 ISDN. Published by Elsevier Ltd. All rights reserved.

Alcohol exposure during development: Impact on the epigenome

PubMed Central

Perkins, Amy; Lehmann, Claudia; Lawrence, R. Charles; Kelly, Sandra J.

2013-01-01

Fetal Alcohol Spectrum Disorders represent a wide range of symptoms associated with in utero alcohol exposure. Animal models of FASD have been useful in determining the specific neurological consequences of developmental alcohol exposure, but the mechanisms of those consequences are unclear. Long-lasting changes to the epigenome are proposed as a mechanism of alcohol-induced teratogenesis in the hippocampus. The current study utilized a three-trimester rodent model of FASD to examine changes to some of the enzymatic regulators of the epigenome in adolescence. Combined pre- and post-natal alcohol exposure resulted in a significant increase in DNA methyltransferase activity (DNMT), without affecting histone deacetylase activity (HDAC). Developmental alcohol exposure also caused a change in gene expression of regulators of the epigenome, in particular, DNMT1, DNMT3a, and methyl CpG binding protein 2 (MeCP2). The modifications of the activity and expression of epigenetic regulators in the hippocampus of rodents perinatally exposed to alcohol suggest that alcohol’s impact on the epigenome and its regulators may be one of the underlying mechanisms of alcohol teratogenesis. PMID:23542005

Human DAZL, DAZ and BOULE genes modulate primordial germ cell and haploid gamete formation

PubMed Central

Kee, Kehkooi; Angeles, Vanessa T; Flores, Martha; Nguyen, Ha Nam; Pera, Renee A Reijo

2009-01-01

The leading cause of infertility in men and women is quantitative and qualitative defects in human germ cell (oocyte and sperm) development. Yet, it has not been possible to examine the unique developmental genetics of human germ cell formation and differentiation due to inaccessibility of germ cells during fetal development. Although several studies have shown that germ cells can be differentiated from mouse and human embryonic stem cells, human germ cells differentiated in these studies generally did not develop beyond the earliest stages1-8. Here we used a germ cell reporter to quantitate and isolate primordial germ cells derived from both male and female hESCs. Then, by silencing and overexpressing genes that encode germ cell-specific cytoplasmic RNA-binding proteins (not transcription factors), we modulated human germ cell formation and developmental progression. We observed that human DAZL (Deleted in AZoospermia-Like) functions in primordial germ cell formation, whereas closely-related genes, DAZ and BOULE, promote later stages of meiosis and development of haploid gametes. These results are significant to the generation of gametes for future basic science and potential clinical applications. PMID:19865085

Novel noncoding RNA from human Y distal heterochromatic block (Yq12) generates testis-specific chimeric CDC2L2

PubMed Central

Jehan, Zeenath; Vallinayagam, Sambandam; Tiwari, Shrish; Pradhan, Suman; Singh, Lalji; Suresh, Amritha; Reddy, Hemakumar M.; Ahuja, Y.R.; Jesudasan, Rachel A.

2007-01-01

The human Y chromosome, because it is enriched in repetitive DNA, has been very intractable to genetic and molecular analyses. There is no previous evidence for developmental stage- and testis-specific transcription from the male-specific region of the Y (MSY). Here, we present evidence for the first time for a developmental stage- and testis-specific transcription from MSY distal heterochromatic block. We isolated two novel RNAs, which localize to Yq12 in multiple copies, show testis-specific expression, and lack active X-homologs. Experimental evidence shows that one of the above Yq12 noncoding RNAs (ncRNAs) trans-splices with CDC2L2 mRNA from chromosome 1p36.3 locus to generate a testis-specific chimeric β sv13 isoform. This 67-nt 5′UTR provided by the Yq12 transcript contains within it a Y box protein-binding CCAAT motif, indicating translational regulation of the β sv13 isoform in testis. This is also the first report of trans-splicing between a Y chromosomal and an autosomal transcript. PMID:17095710

Exploring DNA-binding Proteins with In Vivo Chemical Cross-linking and Mass Spectrometry

PubMed Central

Qiu, Haibo; Wang, Yinsheng

2009-01-01

DNA-binding proteins are very important constituents of proteomes of all species and play crucial roles in transcription, DNA replication, recombination, repair and other activities associated with DNA. Although a number of DNA-binding proteins have been identified, many proteins involved in gene regulation and DNA repair are likely still unknown because of their dynamic and/or weak interactions with DNA. In this report, we described an approach for the comprehensive identification of DNA-binding proteins with in vivo formaldehyde cross-linking and LC-MS/MS. DNA-binding proteins could be purified via the isolation of DNA-protein complexes and released from the complexes by reversing the cross-linking. By using this method, we were able to identify more than one hundred DNA-binding proteins, such as proteins involved in transcription, gene regulation, DNA replication and repair, and a large number of proteins which are potentially associated with DNA and DNA-binding proteins. This method should be generally applicable to the investigation of other nucleic acid-binding proteins, and hold great potential in the comprehensive study of gene regulation, DNA damage response and repair, as well as many other critical biological processes at proteomic level. PMID:19714816

Adrenocortical nuclear progesterone-binding protein: Identification by photoaffinity labeling and evidence for deoxyribonucleic acid binding and stimulation by adrenocorticotropin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Demura, T.; Driscoll, W.J.; Lee, Y.C.

1991-01-01

Nuclei of the guinea pig adrenal cortex contain a protein that specifically binds progesterone and that, biochemically, is clearly distinct from the classical progesterone receptor. The adrenocortical nuclear progesterone-binding protein has now been purified more than 2000-fold by steroid-affinity chromatography with a 75% yield. The purified protein preparation demonstrated three major bands on sodium dodecyl sulfate-polyacrylamide gel of 79K, 74K, and 50K. To determine which of the three might represent the progesterone-binding protein, steroid photoaffinity labeling was performed which resulted in the specific and exclusive labeling of a 50K band. Thus, the adrenocortical nuclear progesterone-binding protein appears to be distinctmore » from the classical progesterone receptor not only biochemically, but also on the basis of molecular size. To test whether the adrenocortical nuclear progesterone-binding protein can be hormonally stimulated, guinea pigs were treated with ACTH. The chronic administration of ACTH caused a 4- to 6-fold increase in the specific progesterone binding capacity without a change in the binding affinity. There appeared to be no significant difference in nuclear progesterone binding between the zona fasciculata and zona reticularis. This finding suggests a mediating role for the progesterone-binding protein in ACTH action. In addition, the nuclear progesterone-binding protein bound to nonspecific DNA sequences, further suggesting a possible transcriptional regulatory role.« less

Fibroblast growth factor regulates insulin-like growth factor-binding protein production by vascular smooth muscle cells.

PubMed

Ververis, J; Ku, L; Delafontaine, P

1994-02-01

Insulin-like growth factor I is an important mitogen for vascular smooth muscle cells, and its effects are regulated by several binding proteins. Western ligand blotting of conditioned medium from rat aortic smooth muscle cells detected a 24 kDa binding protein and a 28 kDa glycosylated variant of this protein, consistent with insulin-like growth factor binding protein-4 by size. Low amounts of a glycosylated 38 to 42 kDa doublet (consistent with binding protein-3) and a 31 kDa non-glycosylated protein also were present. Basic fibroblast growth factor markedly increased secretion of the 24 kDa binding protein and its 28 kDa glycosylated variant. This effect was dose- and time-dependent and was inhibited by co-incubation with cycloheximide. Crosslinking of [125I]-insulin-like growth factor I to cell monolayers revealed no surface-associated binding proteins, either basally or after agonist treatment. Induction of binding protein production by fibroblast growth factor at sites of vascular injury may be important in vascular proliferative responses in vivo.

Specificity in substrate binding by protein folding catalysts: tyrosine and tryptophan residues are the recognition motifs for the binding of peptides to the pancreas-specific protein disulfide isomerase PDIp.

PubMed Central

Ruddock, L. W.; Freedman, R. B.; Klappa, P.

2000-01-01

Using a cross-linking approach, we recently demonstrated that radiolabeled peptides or misfolded proteins specifically interact in vitro with two luminal proteins in crude extracts from pancreas microsomes. The proteins were the folding catalysts protein disulfide isomerase (PDI) and PDIp, a glycosylated, PDI-related protein, expressed exclusively in the pancreas. In this study, we explore the specificity of these proteins in binding peptides and related ligands and show that tyrosine and tryptophan residues in peptides are the recognition motifs for their binding by PDIp. This peptide-binding specificity may reflect the selectivity of PDIp in binding regions of unfolded polypeptide during catalysis of protein folding. PMID:10794419

Dominant Alcohol-Protein Interaction via Hydration-Enabled Enthalpy-Driven Binding Mechanism

PubMed Central

Chong, Yuan; Kleinhammes, Alfred; Tang, Pei; Xu, Yan; Wu, Yue

2015-01-01

Water plays an important role in weak associations of small drug molecules with proteins. Intense focus has been on binding-induced structural changes in the water network surrounding protein binding sites, especially their contributions to binding thermodynamics. However, water is also tightly coupled to protein conformations and dynamics, and so far little is known about the influence of water-protein interactions on ligand binding. Alcohols are a type of low-affinity drugs, and it remains unclear how water affects alcohol-protein interactions. Here, we present alcohol adsorption isotherms under controlled protein hydration using in-situ NMR detection. As functions of hydration level, Gibbs free energy, enthalpy, and entropy of binding were determined from the temperature dependence of isotherms. Two types of alcohol binding were found. The dominant type is low-affinity nonspecific binding, which is strongly dependent on temperature and the level of hydration. At low hydration levels, this nonspecific binding only occurs above a threshold of alcohol vapor pressure. An increased hydration level reduces this threshold, with it finally disappearing at a hydration level of h~0.2 (g water/g protein), gradually shifting alcohol binding from an entropy-driven to an enthalpy-driven process. Water at charged and polar groups on the protein surface was found to be particularly important in enabling this binding. Although further increase in hydration has smaller effects on the changes of binding enthalpy and entropy, it results in significant negative change in Gibbs free energy due to unmatched enthalpy-entropy compensation. These results show the crucial role of water-protein interplay in alcohol binding. PMID:25856773

Silencing of Aγ-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the −566 GATA Site▿ †

PubMed Central

Harju-Baker, Susanna; Costa, Flávia C.; Fedosyuk, Halyna; Neades, Renee; Peterson, Kenneth R.

2008-01-01

Autonomous silencing of γ-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the Aγ-globin gene was identified between −730 and −378 relative to the mRNA start site. A marked copy of the Aγ-globin gene inserted between locus control region 5′ DNase I-hypersensitive site 1 and the ɛ-globin gene was transcriptionally silenced in adult β-globin locus yeast artificial chromosome (β-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the −566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low γ-globin levels, whereas only a weak signal was detected when γ-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from β-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the Aγ-globin −566 or Gγ-globin −567 GATA site when γ-globin expression was low (day 18) but not when γ-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, γ-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the −566/−567 GATA sites of the proximal γ-globin promoters. PMID:18347053

Silencing of Agamma-globin gene expression during adult definitive erythropoiesis mediated by GATA-1-FOG-1-Mi2 complex binding at the -566 GATA site.

PubMed

Harju-Baker, Susanna; Costa, Flávia C; Fedosyuk, Halyna; Neades, Renee; Peterson, Kenneth R

2008-05-01

Autonomous silencing of gamma-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the (A)gamma-globin gene was identified between -730 and -378 relative to the mRNA start site. A marked copy of the (A)gamma-globin gene inserted between locus control region 5' DNase I-hypersensitive site 1 and the epsilon-globin gene was transcriptionally silenced in adult beta-globin locus yeast artificial chromosome (beta-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the -566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low gamma-globin levels, whereas only a weak signal was detected when gamma-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from beta-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the (A)gamma-globin -566 or (G)gamma-globin -567 GATA site when gamma-globin expression was low (day 18) but not when gamma-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, gamma-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the -566/-567 GATA sites of the proximal gamma-globin promoters.

Murine homeobox-containing gene, Msx-1: analysis of genomic organization, promoter structure, and potential autoregulatory cis-acting elements.

PubMed

Kuzuoka, M; Takahashi, T; Guron, C; Raghow, R

1994-05-01

Detailed molecular organization of the coding and upstream regulatory regions of the murine homeodomain-containing gene, Msx-1, is reported. The protein-encoding portion of the gene is contained in two exons, 590 and 1214 bp in length, separated by a 2107-bp intron; the homeodomain is located in the second exon. The two-exon organization of the murine Msx-1 gene resembles a number of other homeodomain-containing genes. The 5'-(GTAAGT) and 3'-(CCCTAG) splicing junctions and the mRNA polyadenylation signal (UAUAA) of the murine Msx-1 gene are also characteristic of other vertebrate genes. By nuclease protection and primer extension assays, the start of transcription of the Msx-1 gene was located 256 bp upstream of the first AUG. Computer analysis of the promoter proximal 1280-bp sequence revealed a number of potentially important cis-regulatory sequences; these include the recognition elements for Ap-1, Ap-2, Ap-3, Sp-1, a possible binding site for RAR:RXR, and a number of TCF-1 consensus motifs. Importantly, a perfect reverse complement of (C/G)TTAATTG, which was recently shown to be an optimal binding sequence for the homeodomain of Msx-1 protein (K.M. Catron, N. Iler, and C. Abate (1993) Mol. Cell. Biol. 13:2354-2365), was also located in the murine Msx-1 promoter. Binding of bacterially expressed Msx-1 homeodomain polypeptide to Msx-1-specific oligonucleotide was experimentally demonstrated, raising a distinct possibility of autoregulation of this developmentally regulated gene.

Translational co-regulation of a ligand and inhibitor by a conserved RNA element

PubMed Central

Zaucker, Andreas; Nagorska, Agnieszka; Kumari, Pooja; Hecker, Nikolai; Wang, Yin; Huang, Sizhou; Cooper, Ledean; Sivashanmugam, Lavanya; VijayKumar, Shruthi; Brosens, Jan; Gorodkin, Jan

2018-01-01

Abstract In many organisms, transcriptional and post-transcriptional regulation of components of pathways or processes has been reported. However, to date, there are few reports of translational co-regulation of multiple components of a developmental signaling pathway. Here, we show that an RNA element which we previously identified as a dorsal localization element (DLE) in the 3′UTR of zebrafish nodal-related1/squint (ndr1/sqt) ligand mRNA, is shared by the related ligand nodal-related2/cyclops (ndr2/cyc) and the nodal inhibitors, lefty1 (lft1) and lefty2 mRNAs. We investigated the activity of the DLEs through functional assays in live zebrafish embryos. The lft1 DLE localizes fluorescently labeled RNA similarly to the ndr1/sqt DLE. Similar to the ndr1/sqt 3′UTR, the lft1 and lft2 3′UTRs are bound by the RNA-binding protein (RBP) and translational repressor, Y-box binding protein 1 (Ybx1), whereas deletions in the DLE abolish binding to Ybx1. Analysis of zebrafish ybx1 mutants shows that Ybx1 represses lefty1 translation in embryos. CRISPR/Cas9-mediated inactivation of human YBX1 also results in human NODAL translational de-repression, suggesting broader conservation of the DLE RNA element/Ybx1 RBP module in regulation of Nodal signaling. Our findings demonstrate translational co-regulation of components of a signaling pathway by an RNA element conserved in both sequence and structure and an RBP, revealing a ‘translational regulon’. PMID:29059375

Factor H binds to the hypervariable region of many Streptococcus pyogenes M proteins but does not promote phagocytosis resistance or acute virulence.

PubMed

Gustafsson, Mattias C U; Lannergård, Jonas; Nilsson, O Rickard; Kristensen, Bodil M; Olsen, John E; Harris, Claire L; Ufret-Vincenty, Rafael L; Stålhammar-Carlemalm, Margaretha; Lindahl, Gunnar

2013-01-01

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems.

Factor H Binds to the Hypervariable Region of Many Streptococcus pyogenes M Proteins but Does Not Promote Phagocytosis Resistance or Acute Virulence

PubMed Central

Kristensen, Bodil M.; Olsen, John E.; Harris, Claire L.; Ufret-Vincenty, Rafael L.; Stålhammar-Carlemalm, Margaretha; Lindahl, Gunnar

2013-01-01

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems. PMID:23637608

Altered cell-matrix associated ADAM proteins in Alzheimer disease.

PubMed

Gerst, J L; Raina, A K; Pirim, I; McShea, A; Harris, P L; Siedlak, S L; Takeda, A; Petersen, R B; Smith, M A

2000-03-01

Alterations in cell-matrix 'contact' are often related to a disruption of cell cycle regulation and, as such, occur variously in neoplasia. Given the recent findings showing cell cycle alterations in Alzheimer disease, we undertook a study of ADAM-1 and 2 (A Disintegrin And Metalloprotease), developmentally-regulated, integrin-binding, membrane-bound metalloproteases. Our results show that whereas ADAM-1 and 2 are found in susceptible hippocampal neurons in Alzheimer disease, these proteins were not generally increased in similar neuronal populations in younger or age-matched controls except in association with age-related neurofibrillary alterations. This increase in both ADAM-1 and 2 in cases of Alzheimer disease was verified by immunoblot analysis (P < 0.05). An ADAM-induced loss of matrix integration would effectively "reset" the mitotic clock and thereby stimulate re-entry into the cell cycle in neurons in Alzheimer disease. Furthermore, given the importance of integrins in maintaining short-term memory, alterations in ADAM proteins or their proteolytic activity could also play a proximal role in the clinico-pathological manifestations of Alzheimer disease. Copyright 2000 Wiley-Liss, Inc.

[Glutamate-binding membrane proteins from human platelets].

PubMed

Gurevich, V S; Popov, Iu G; Gorodinskiĭ, A I; Dambinova, S A

1991-09-01

Solubilization of the total membrane fraction of human platelets in a 2% solution of sodium deoxycholate and subsequent affinity chromatography on glutamate agarose resulted in two protein fractions possessing a glutamate-binding activity. As can be evidenced from radioligand binding data, the first fraction contains two types of binding sites (Kd1 = 1 microM, Bmax 1 = 100 pmol/mg of protein; Kd2 = 9.3 microMm Bmax2 = 395 pmol/mg of protein). The second fraction has only one type of binding sites (Kd = 1 microM, Bmax = = 110 pmol/mg of protein). SDS-PAAG electrophoresis revealed the presence in the first fraction of proteins with Mr of 14, 24, 56 and 155 kDa, whereas the second fraction was found to contain 14, 46, 71 and 155 kDa proteins. Solid phase immunoenzymatic analysis using poly- and monoclonal specific antibodies against mammalian brain glutamate-binding proteins revealed a marked immunochemical similarity of the isolated protein fractions with human brain synaptic membrane glutamate-binding proteins.

A member of the polymerase beta nucleotidyltransferase superfamily is required for RNA interference in C. elegans.

PubMed

Chen, Chun-Chieh G; Simard, Martin J; Tabara, Hiroaki; Brownell, Daniel R; McCollough, Jennifer A; Mello, Craig C

2005-02-22

RNA interference (RNAi) is an ancient, highly conserved mechanism in which small RNA molecules (siRNAs) guide the sequence-specific silencing of gene expression . Several silencing machinery protein components have been identified, including helicases, RNase-related proteins, double- and single-stranded RNA binding proteins, and RNA-dependent RNA polymerase-related proteins . Work on these factors has led to the revelation that RNAi mechanisms intersect with cellular pathways required for development and fertility . Despite rapid progress in understanding key steps in the RNAi pathway, it is clear that many factors required for both RNAi and related developmental mechanisms have not yet been identified. Here, we report the characterization of the C. elegans gene rde-3. Genetic analysis of presumptive null alleles indicates that rde-3 is required for siRNA accumulation and for efficient RNAi in all tissues, and it is essential for fertility and viability at high temperatures. RDE-3 contains conserved domains found in the polymerase beta nucleotidyltransferase superfamily, which includes conventional poly(A) polymerases, 2'-5' oligoadenylate synthetase (OAS), and yeast Trf4p . These findings implicate a new enzymatic modality in RNAi and suggest possible models for the role of RDE-3 in the RNAi mechanism.

Developmental Effects of Acute, Chronic, and Withdrawal from Chronic Nicotine on Fear Conditioning

PubMed Central

Portugal, George S.; Wilkinson, Derek S.; Turner, Jill R.; Blendy, Julie A.; Gould, Thomas J.

2012-01-01

Pre-adolescence and adolescence are developmental periods associated with increased vulnerability for tobacco addiction, and exposure to tobacco during these periods may lead to long-lasting changes in behavioral and neuronal plasticity. The present study examined the short- and long-term effects of nicotine and nicotine withdrawal on fear conditioning in pre-adolescent, adolescent, and adult mice, and potential underlying substrates that may mediate the developmental effects of nicotine, such as changes in nicotinic acetylcholine receptor (nAChR) binding, CREB expression, and nicotine metabolism. Age-related differences existed in sensitivity to the effects of acute nicotine, chronic nicotine and nicotine withdrawal on contextual fear conditioning (no changes in cued fear conditioning were seen); younger mice were more sensitive to the acute effects and less sensitive to the effects of nicotine withdrawal 24 hours post treatment cessation. Developmental differences in nAChR binding were associated with the effects of nicotine withdrawal on contextual learning. Developmental differences in nicotine metabolism and CREB expression were also observed, but were not related to the effects of nicotine withdrawal on contextual learning 24 hours post treatment. Chronic nicotine exposure during pre-adolescence or adolescence, however, produced long-lasting impairments in contextual learning that were observed during adulthood, whereas adult chronic nicotine exposure did not. These developmental effects could be related to changes in CREB. Overall, there is a developmental shift in the effects of nicotine on hippocampus-dependent learning and developmental exposure to nicotine results in adult cognitive deficits; these changes in cognition may play an important role in the development and maintenance of nicotine addiction. PMID:22521799

Gene expression studies of developing bovine longissimus muscle from two different beef cattle breeds

PubMed Central

Lehnert, Sigrid A; Reverter, Antonio; Byrne, Keren A; Wang, Yonghong; Nattrass, Greg S; Hudson, Nicholas J; Greenwood, Paul L

2007-01-01

Background The muscle fiber number and fiber composition of muscle is largely determined during prenatal development. In order to discover genes that are involved in determining adult muscle phenotypes, we studied the gene expression profile of developing fetal bovine longissimus muscle from animals with two different genetic backgrounds using a bovine cDNA microarray. Fetal longissimus muscle was sampled at 4 stages of myogenesis and muscle maturation: primary myogenesis (d 60), secondary myogenesis (d 135), as well as beginning (d 195) and final stages (birth) of functional differentiation of muscle fibers. All fetuses and newborns (total n = 24) were from Hereford dams and crossed with either Wagyu (high intramuscular fat) or Piedmontese (GDF8 mutant) sires, genotypes that vary markedly in muscle and compositional characteristics later in postnatal life. Results We obtained expression profiles of three individuals for each time point and genotype to allow comparisons across time and between sire breeds. Quantitative reverse transcription-PCR analysis of RNA from developing longissimus muscle was able to validate the differential expression patterns observed for a selection of differentially expressed genes, with one exception. We detected large-scale changes in temporal gene expression between the four developmental stages in genes coding for extracellular matrix and for muscle fiber structural and metabolic proteins. FSTL1 and IGFBP5 were two genes implicated in growth and differentiation that showed developmentally regulated expression levels in fetal muscle. An abundantly expressed gene with no functional annotation was found to be developmentally regulated in the same manner as muscle structural proteins. We also observed differences in gene expression profiles between the two different sire breeds. Wagyu-sired calves showed higher expression of fatty acid binding protein 5 (FABP5) RNA at birth. The developing longissimus muscle of fetuses carrying the Piedmontese mutation shows an emphasis on glycolytic muscle biochemistry and a large-scale up-regulation of the translational machinery at birth. We also document evidence for timing differences in differentiation events between the two breeds. Conclusion Taken together, these findings provide a detailed description of molecular events accompanying skeletal muscle differentiation in the bovine, as well as gene expression differences that may underpin the phenotype differences between the two breeds. In addition, this study has highlighted a non-coding RNA, which is abundantly expressed and developmentally regulated in bovine fetal muscle. PMID:17697390

Hyperdiversity of Genes Encoding Integral Light-Harvesting Proteins in the Dinoflagellate Symbiodinium sp

PubMed Central

Boldt, Lynda; Yellowlees, David; Leggat, William

2012-01-01

The superfamily of light-harvesting complex (LHC) proteins is comprised of proteins with diverse functions in light-harvesting and photoprotection. LHC proteins bind chlorophyll (Chl) and carotenoids and include a family of LHCs that bind Chl a and c. Dinophytes (dinoflagellates) are predominantly Chl c binding algal taxa, bind peridinin or fucoxanthin as the primary carotenoid, and can possess a number of LHC subfamilies. Here we report 11 LHC sequences for the chlorophyll a-chlorophyll c 2-peridinin protein complex (acpPC) subfamily isolated from Symbiodinium sp. C3, an ecologically important peridinin binding dinoflagellate taxa. Phylogenetic analysis of these proteins suggests the acpPC subfamily forms at least three clades within the Chl a/c binding LHC family; Clade 1 clusters with rhodophyte, cryptophyte and peridinin binding dinoflagellate sequences, Clade 2 with peridinin binding dinoflagellate sequences only and Clades 3 with heterokontophytes, fucoxanthin and peridinin binding dinoflagellate sequences. PMID:23112815

Protein Binding: Do We Ever Learn?▿

PubMed Central

Zeitlinger, Markus A.; Derendorf, Hartmut; Mouton, Johan W.; Cars, Otto; Craig, William A.; Andes, David; Theuretzbacher, Ursula

2011-01-01

Although the influence of protein binding (PB) on antibacterial activity has been reported for many antibiotics and over many years, there is currently no standardization for pharmacodynamic models that account for the impact of protein binding of antimicrobial agents in vitro. This might explain the somewhat contradictory results obtained from different studies. Simple in vitro models which compare the MIC obtained in protein-free standard medium versus a protein-rich medium are prone to methodological pitfalls and may lead to flawed conclusions. Within in vitro test systems, a range of test conditions, including source of protein, concentration of the tested antibiotic, temperature, pH, electrolytes, and supplements may influence the impact of protein binding. As new antibiotics with a high degree of protein binding are in clinical development, attention and action directed toward the optimization and standardization of testing the impact of protein binding on the activity of antibiotics in vitro become even more urgent. In addition, the quantitative relationship between the effects of protein binding in vitro and in vivo needs to be established, since the physiological conditions differ. General recommendations for testing the impact of protein binding in vitro are suggested. PMID:21537013

Protein profile of mouse ovarian follicles grown in vitro.

PubMed

Anastácio, Amandine; Rodriguez-Wallberg, Kenny A; Chardonnet, Solenne; Pionneau, Cédric; Fédérici, Christian; Almeida Santos, Teresa; Poirot, Catherine

2017-12-01

Could the follicle proteome be mapped by identifying specific proteins that are common or differ between three developmental stages from the secondary follicle (SF) to the antrum-like stage? From a total of 1401 proteins identified in the follicles, 609 were common to the three developmental stages investigated and 444 were found uniquely at one of the stages. The importance of the follicle as a functional structure has been recognized; however, up-to-date the proteome of the whole follicle has not been described. A few studies using proteomics have previously reported on either isolated fully-grown oocytes before or after meiosis resumption or cumulus cells. The experimental design included a validated mice model for isolation and individual culture of SFs. The system was chosen as it allows continuous evaluation of follicle growth and selection of follicles for analysis at pre-determined developmental stages: SF, complete Slavjanski membrane rupture (SMR) and antrum-like cavity (AF). The experiments were repeated 13 times independently to acquire the material that was analyzed by proteomics. SFs (n = 2166) were isolated from B6CBA/F1 female mice (n = 42), 12 days old, from 15 l. About half of the follicles isolated as SF were analyzed as such (n = 1143) and pooled to obtain 139 μg of extracted protein. Both SMR (n = 359) and AF (n = 124) were obtained after individual culture of 1023 follicles in a microdrop system under oil, selected for analysis and pooled, to obtain 339 μg and 170 μg of protein, respectively. The follicle proteome was analyzed combining isoelectric focusing (IEF) fractionation with 1D and 2D LC-MS/MS analysis to enhance protein identification. The three protein lists were submitted to the 'Compare gene list' tool in the PANTHER website to gain insights on the Gene Ontology Biological processes present and to Ingenuity Pathway Analysis to highlight protein networks. A label-free quantification was performed with 1D LC-MS/MS analyses to emphasize proteins with different expression profiles between the three follicular stages. Supplementary western blot analysis (using new biological replicates) was performed to confirm the expression variations of three proteins during follicle development in vitro. It was found that 609 out of 1401 identified proteins were common to the three follicle developmental stages investigated. Some proteins were identified uniquely at one stage: 71 of the 775 identified proteins in SF, 181 of 1092 in SMR and 192 of 1100 in AF. Additional qualitative and quantitative analysis highlighted 44 biological processes over-represented in our samples compared to the Mus musculus gene database. In particular, it was possible to identify proteins implicated in the cell cycle, calcium ion binding and glycolysis, with specific expressions and abundance, throughout in vitro follicle development. Data are available via ProteomeXchange with identifier PXD006227. The proteome analyses described in this study were performed after in vitro development. Despite fractionation of the samples before LC-MS/MS, proteomic approaches are not exhaustive, thus proteins that are not identified in a group are not necessarily absent from that group, although they are likely to be less abundant. This study allowed a general view of proteins implicated in follicle development in vitro and it represents the most complete catalog of the whole follicle proteome available so far. Not only were well known proteins of the oocyte identified but also proteins that are probably expressed only in granulosa cells. This study was supported by the Portuguese Foundation for Science and Technology, FCT (PhD fellowship SFRH/BD/65299/2009 to A.A.), the Swedish Childhood Cancer Foundation (PR 2014-0144 to K.A.R-.W.) and Stockholm County Council to K.A.R-.W. The authors of the study have no conflict of interest to report. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d.

PubMed

Doxey, Andrew C; Cheng, Zhenyu; Moffatt, Barbara A; McConkey, Brendan J

2010-08-03

Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB) for coplanar aromatic motifs similar to those found in known glycan-binding proteins. The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO) enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192) in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry. Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.

Ion-binding properties of Calnuc, Ca2+ versus Mg2+--Calnuc adopts additional and unusual Ca2+-binding sites upon interaction with G-protein.

PubMed

Kanuru, Madhavi; Samuel, Jebakumar J; Balivada, Lavanya M; Aradhyam, Gopala K

2009-05-01

Calnuc is a novel, highly modular, EF-hand containing, Ca(2+)-binding, Golgi resident protein whose functions are not clear. Using amino acid sequences, we demonstrate that Calnuc is a highly conserved protein among various organisms, from Ciona intestinalis to humans. Maximum homology among all sequences is found in the region that binds to G-proteins. In humans, it is known to be expressed in a variety of tissues, and it interacts with several important protein partners. Among other proteins, Calnuc is known to interact with heterotrimeric G-proteins, specifically with the alpha-subunit. Herein, we report the structural implications of Ca(2+) and Mg(2+) binding, and illustrate that Calnuc functions as a downstream effector for G-protein alpha-subunit. Our results show that Ca(2+) binds with an affinity of 7 mum and causes structural changes. Although Mg(2+) binds to Calnuc with very weak affinity, the structural changes that it causes are further enhanced by Ca(2+) binding. Furthermore, isothermal titration calorimetry results show that Calnuc and the G-protein bind with an affinity of 13 nm. We also predict a probable function for Calnuc, that of maintaining Ca(2+) homeostasis in the cell. Using Stains-all and terbium as Ca(2+) mimic probes, we demonstrate that the Ca(2+)-binding ability of Calnuc is governed by the activity-based conformational state of the G-protein. We propose that Calnuc adopts structural sites similar to the ones seen in proteins such as annexins, c2 domains or chromogrannin A, and therefore binds more calcium ions upon binding to Gialpha. With the number of organelle-targeted G-protein-coupled receptors increasing, intracellular communication mediated by G-proteins could become a new paradigm. In this regard, we propose that Calnuc could be involved in the downstream signaling of G-proteins.

In Situ Protein Binding Assay Using Fc-Fusion Proteins.

PubMed

Padmanabhan, Nirmala; Siddiqui, Tabrez J

2017-01-01

This protocol describes an in situ protein-protein interaction assay between tagged recombinant proteins and cell-surface expressed synaptic proteins. The assay is arguably more sensitive than other traditional protein binding assays such as co-immunoprecipitation and pull-downs and provides a visual readout for binding. This assay has been widely used to determine the dissociation constant of binding of trans-synaptic adhesion proteins. The step-wise description in the protocol should facilitate the adoption of this method in other laboratories.

Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

PubMed Central

Choe, Weonu; Durgannavar, Trishaladevi A.; Chung, Sang J.

2016-01-01

The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed. PMID:28774114

21 CFR 866.5765 - Retinol-binding protein immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Retinol-binding protein immunological test system...

Developmental expression of the neuroligins and neurexins in fragile X mice.

PubMed

Lai, Jonathan K Y; Doering, Laurie C; Foster, Jane A

2016-03-01

Neuroligins and neurexins are transsynaptic proteins involved in the maturation of glutamatergic and GABAergic synapses. Research has identified synaptic proteins and function as primary contributors to the development of fragile X syndrome. Fragile X mental retardation protein (FMRP), the protein that is lacking in fragile X syndrome, binds neuroligin-1 and -3 mRNA. Using in situ hybridization, we examined temporal and spatial expression patterns of neuroligin (NLGN) and neurexin (NRXN) mRNAs in the somatosensory (S1) cortex and hippocampus in wild-type (WT) and fragile X knockout (FMR1-KO) mice during the first 5 weeks of postnatal life. Genotype-based differences in expression included increased NLGN1 mRNA in CA1 and S1 cortex, decreased NLGN2 mRNA in CA1 and dentate gyrus (DG) regions of the hippocampus, and increased NRXN3 mRNA in CA1, DG, and S1 cortex between female WT and FMR1-KO mice. In male mice, decreased expression of NRXN3 mRNA was observed in CA1 and DG regions of FMR1-KO mice. Sex differences in hippocampal expression of NLGN2, NRXN1, NRXN2, and NRXN3 mRNAs and in S1 cortex expression of NRXN3 mRNAs were observed WT mice, whereas sex differences in NLGN3, NRXN1, NRXN2, and NRXN3 mRNA expression in the hippocampus and in NLGN1, NRXN2 and NRXN3 mRNA expression in S1 cortex were detected in FMR1-KO mice. These results provide a neuroanatomical map of NLGN and NRXN expression patterns over postnatal development in WT and FMR1-KO mice. The differences in developmental trajectory of these synaptic proteins could contribute to long-term differences in CNS wiring and synaptic function. © 2015 Wiley Periodicals, Inc.

Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm.

PubMed

Helledie, T; Antonius, M; Sorensen, R V; Hertzel, A V; Bernlohr, D A; Kølvraa, S; Kristiansen, K; Mandrup, S

2000-11-01

Peroxisome proliferator-activated receptors (PPARs) are activated by a variety of fatty acids, eicosanoids, and hypolipidemic and insulin-sensitizing drugs. Many of these compounds bind avidly to members of a family of small lipid-binding proteins, the fatty acid-binding proteins (FABPs). Fatty acids are activated to CoA esters, which bind with high affinity to the acyl-CoA-binding protein (ACBP). Thus, the availability of known and potential PPAR ligands may be regulated by lipid-binding proteins. In this report we show by transient transfection of CV-1 cells that coexpression of ACBP and adipocyte lipid-binding protein (ALBP) exerts a ligand- and PPAR subtype-specific attenuation of PPAR-mediated trans-activation, suggesting that lipid-binding proteins, when expressed at high levels, may function as negative regulators of PPAR activation by certain ligands. Expression of ACBP, ALBP, and keratinocyte lipid-binding protein (KLBP) is induced during adipocyte differentiation, a process during which PPARgamma plays a prominent role. We present evidence that endogenous ACBP, ALBP, and KLBP not only localize to the cytoplasm but also exhibit a prominent nuclear localization in 3T3-L1 adipocytes. In addition, forced expression of ACBP, ALBP, and KLBP in CV-1 cells resulted in a substantial accumulation of all three proteins in the nucleus. These results suggest that lipid-binding proteins, contrary to the general assumption, may exert their action in the nucleus as well as in the cytoplasm.

Unconventional RNA-binding proteins: an uncharted zone in RNA biology.

PubMed

Albihlal, Waleed S; Gerber, André P

2018-06-16

RNA-binding proteins play essential roles in the post-transcriptional regulation of gene expression. While hundreds of RNA-binding proteins can be predicted computationally, the recent introduction of proteome-wide approaches has dramatically expanded the repertoire of proteins interacting with RNA. Besides canonical RNA-binding proteins that contain characteristic RNA-binding domains, many proteins that lack such domains but have other well-characterised cellular functions were identified; including metabolic enzymes, heat shock proteins, kinases, as well as transcription factors and chromatin-associated proteins. In the context of these recently published RNA-protein interactome datasets obtained from yeast, nematodes, flies, plants and mammalian cells, we discuss examples for seemingly evolutionary conserved "unconventional" RNA-binding proteins that act in central carbon metabolism, stress response or regulation of transcription. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

The hepta-beta-glucoside elicitor-binding proteins from legumes represent a putative receptor family.

PubMed

Mithöfer, A; Fliegmann, J; Neuhaus-Url, G; Schwarz, H; Ebel, J

2000-08-01

The ability of legumes to recognize and respond to beta-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound beta-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-beta-glucoside conjugate (Kd = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (Kd = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex.

Isolation from genomic DNA of sequences binding specific regulatory proteins by the acceleration of protein electrophoretic mobility upon DNA binding.

PubMed

Subrahmanyam, S; Cronan, J E

1999-01-21

We report an efficient and flexible in vitro method for the isolation of genomic DNA sequences that are the binding targets of a given DNA binding protein. This method takes advantage of the fact that binding of a protein to a DNA molecule generally increases the rate of migration of the protein in nondenaturing gel electrophoresis. By the use of a radioactively labeled DNA-binding protein and nonradioactive DNA coupled with PCR amplification from gel slices, we show that specific binding sites can be isolated from Escherichia coli genomic DNA. We have applied this method to isolate a binding site for FadR, a global regulator of fatty acid metabolism in E. coli. We have also isolated a second binding site for BirA, the biotin operon repressor/biotin ligase, from the E. coli genome that has a very low binding efficiency compared with the bio operator region.

Engineered proteins as specific binding reagents.

PubMed

Binz, H Kaspar; Plückthun, Andreas

2005-08-01

Over the past 30 years, monoclonal antibodies have become the standard binding proteins and currently find applications in research, diagnostics and therapy. Yet, monoclonal antibodies now face strong competition from synthetic antibody libraries in combination with powerful library selection technologies. More recently, an increased understanding of other natural binding proteins together with advances in protein engineering, selection and evolution technologies has also triggered the exploration of numerous other protein architectures for the generation of designed binding molecules. Valuable protein-binding scaffolds have been obtained and represent promising alternatives to antibodies for biotechnological and, potentially, clinical applications.

Odorant-binding proteins from a primitive termite.

PubMed

Ishida, Yuko; Chiang, Vicky P; Haverty, Michael I; Leal, Walter S

2002-09-01

Hitherto, odorant-binding proteins (OBPs) have been identified from insects belonging to more highly evolved insect orders (Lepidoptera, Coleoptera, Diptera, Hymenoptera, and Hemiptera), whereas only chemosensory proteins have been identified from more primitive species, such as orthopteran and phasmid species. Here, we report for the first time the isolation and cloning of odorant-binding proteins from a primitive termite species, the dampwood termite. Zootermopsis nevadensis nevadensis (Isoptera: Termopsidae). A major antennae-specific protein was detected by native PAGE along with four other minor proteins, which were also absent in the extract from control tissues (hindlegs). Multiple cDNA cloning led to the full characterization of the major antennae-specific protein (ZnevOBP1) and to the identification of two other antennae-specific cDNAs, encoding putative odorant-binding proteins (ZnevOBP2 and ZnevOBP3). N-terminal amino acid sequencing of the minor antennal bands and cDNA cloning showed that olfaction in Z. n. nevadensis may involve multiple odorant-binding proteins. Database searches suggest that the OBPs from this primitive termite are homologues of the pheromone-binding proteins from scarab beetles and antennal-binding proteins from moths.

Roles of Copper-Binding Proteins in Breast Cancer.

PubMed

Blockhuys, Stéphanie; Wittung-Stafshede, Pernilla

2017-04-20

Copper ions are needed in several steps of cancer progression. However, the underlying mechanisms, and involved copper-binding proteins, are mainly elusive. Since most copper ions in the body (in and outside cells) are protein-bound, it is important to investigate what copper-binding proteins participate and, for these, how they are loaded with copper by copper transport proteins. Mechanistic information for how some copper-binding proteins, such as extracellular lysyl oxidase (LOX), play roles in cancer have been elucidated but there is still much to learn from a biophysical molecular viewpoint. Here we provide a summary of copper-binding proteins and discuss ones reported to have roles in cancer. We specifically focus on how copper-binding proteins such as mediator of cell motility 1 (MEMO1), LOX, LOX-like proteins, and secreted protein acidic and rich in cysteine (SPARC) modulate breast cancer from molecular and clinical aspects. Because of the importance of copper for invasion/migration processes, which are key components of cancer metastasis, further insights into the actions of copper-binding proteins may provide new targets to combat cancer.

Special AT-rich sequence binding protein 1 promotes tumor growth and metastasis of esophageal squamous cell carcinoma.

PubMed

Ma, Jun; Wu, Kaiming; Zhao, Zhenxian; Miao, Rong; Xu, Zhe

2017-03-01

Esophageal squamous cell carcinoma is one of the most aggressive malignancies worldwide. Special AT-rich sequence binding protein 1 is a nuclear matrix attachment region binding protein which participates in higher order chromatin organization and tissue-specific gene expression. However, the role of special AT-rich sequence binding protein 1 in esophageal squamous cell carcinoma remains unknown. In this study, western blot and quantitative real-time polymerase chain reaction analysis were performed to identify differentially expressed special AT-rich sequence binding protein 1 in a series of esophageal squamous cell carcinoma tissue samples. The effects of special AT-rich sequence binding protein 1 silencing by two short-hairpin RNAs on cell proliferation, migration, and invasion were assessed by the CCK-8 assay and transwell assays in esophageal squamous cell carcinoma in vitro. Special AT-rich sequence binding protein 1 was significantly upregulated in esophageal squamous cell carcinoma tissue samples and cell lines. Silencing of special AT-rich sequence binding protein 1 inhibited the proliferation of KYSE450 and EC9706 cells which have a relatively high level of special AT-rich sequence binding protein 1, and the ability of migration and invasion of KYSE450 and EC9706 cells was distinctly suppressed. Special AT-rich sequence binding protein 1 could be a potential target for the treatment of esophageal squamous cell carcinoma and inhibition of special AT-rich sequence binding protein 1 may provide a new strategy for the prevention of esophageal squamous cell carcinoma invasion and metastasis.

Interaction between G Protein-Coupled Receptor 143 and Tyrosinase: Implications for Understanding Ocular Albinism Type 1.

PubMed

De Filippo, Elisabetta; Schiedel, Anke C; Manga, Prashiela

2017-02-01

Developmental eye defects in X-linked ocular albinism type 1 are caused by G-protein coupled receptor 143 (GPR143) mutations. Mutations result in dysfunctional melanosome biogenesis and macromelanosome formation in pigment cells, including melanocytes and retinal pigment epithelium. GPR143, primarily expressed in pigment cells, localizes exclusively to endolysosomal and melanosomal membranes unlike most G protein-coupled receptors, which localize to the plasma membrane. There is some debate regarding GPR143 function and elucidating the role of this receptor may be instrumental for understanding neurogenesis during eye development and for devising therapies for ocular albinism type I. Many G protein-coupled receptors require association with other proteins to function. These G protein-coupled receptor-interacting proteins also facilitate fine-tuning of receptor activity and tissue specificity. We therefore investigated potential GPR143 interaction partners, with a focus on the melanogenic enzyme tyrosinase. GPR143 coimmunoprecipitated with tyrosinase, while confocal microscopy demonstrated colocalization of the proteins. Furthermore, tyrosinase localized to the plasma membrane when coexpressed with a GPR143 trafficking mutant. The physical interaction between the proteins was confirmed using fluorescence resonance energy transfer. This interaction may be required in order for GPR143 to function as a monitor of melanosome maturation. Identifying tyrosinase as a potential GPR143 binding protein opens new avenues for investigating the mechanisms that regulate pigmentation and neurogenesis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Myelin management by the 18.5–kDa and 21.5–kDa classic myelin basic protein isoforms

PubMed Central

Harauz, George; Boggs, Joan M.

2013-01-01

The classic myelin basic protein (MBP) splice isoforms range in nominal molecular mass from 14 to 21.5 kDa, and arise from the gene in the oligodendrocyte lineage (Golli) in maturing oligodendrocytes. The 18.5-kDa isoform that predominates in adult myelin adheres the cytosolic surfaces of oligodendrocyte membranes together, and forms a two-dimensional molecular sieve restricting protein diffusion into compact myelin. However, this protein has additional roles including cytoskeletal assembly and membrane extension, binding to SH3-domains, participation in Fyn-mediated signaling pathways, sequestration of phosphoinositides, and maintenance of calcium homeostasis. Of the diverse post-translational modifications of this isoform, phosphorylation is the most dynamic, and modulates 18.5-kDa MBP’s protein-membrane and protein-protein interactions, indicative of a rich repertoire of functions. In developing and mature myelin, phosphorylation can result in microdomain or even nuclear targeting of the protein, supporting the conclusion that 18.5-kDa MBP has significant roles beyond membrane adhesion. The full-length, early-developmental 21.5-kDa splice isoform is predominantly karyophilic due to a non-traditional P-Y nuclear localization signal, with effects such as promotion of oligodendrocyte proliferation. We discuss in vitro and recent in vivo evidence for multifunctionality of these classic basic proteins of myelin, and argue for a systematic evaluation of the temporal and spatial distributions of these protein isoforms, and their modified variants, during oligodendrocyte differentiation. PMID:23398367

A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

NASA Technical Reports Server (NTRS)

Safadi, F.; Reddy, V. S.; Reddy, A. S.

2000-01-01

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

Effects of mutations in the Arabidopsis Cold Shock Domain Protein 3 (AtCSP3) gene on leaf cell expansion.

PubMed

Yang, Yongil; Karlson, Dale

2012-08-01

The cold shock domain is among the most evolutionarily conserved nucleic acid binding domains from prokaryotes to higher eukaryotes, including plants. Although eukaryotic cold shock domain proteins have been extensively studied as transcriptional and post-transcriptional regulators during various developmental processes, their functional roles in plants remains poorly understood. In this study, AtCSP3 (At2g17870), which is one of four Arabidopsis thaliana c old s hock domain proteins (AtCSPs), was functionally characterized. Quantitative RT-PCR analysis confirmed high expression of AtCSP3 in reproductive and meristematic tissues. A homozygous atcsp3 loss-of-function mutant exhibits an overall reduced seedling size, stunted and orbicular rosette leaves, reduced petiole length, and curled leaf blades. Palisade mesophyll cells are smaller and more circular in atcsp3 leaves. Cell size analysis indicated that the reduced size of the circular mesophyll cells appears to be generated by a reduction of cell length along the leaf-length axis, resulting in an orbicular leaf shape. It was also determined that leaf cell expansion is impaired for lateral leaf development in the atcsp3 loss-of-function mutant, but leaf cell proliferation is not affected. AtCSP3 loss-of-function resulted in a dramatic reduction of LNG1 transcript, a gene that is involved in two-dimensional leaf polarity regulation. Transient subcellular localization of AtCSP3 in onion epidermal cells confirmed a nucleocytoplasmic localization pattern. Collectively, these data suggest that AtCSP3 is functionally linked to the regulation of leaf length by affecting LNG1 transcript accumulation during leaf development. A putative function of AtCSP3 as an RNA binding protein is also discussed in relation to leaf development.

Androgen Modulation of Foxp1 and Foxp2 in the Developing Rat Brain: Impact on Sex Specific Vocalization

PubMed Central

Perez-Pouchoulen, Miguel; Roby, Clinton R.; Ryan, Timothy E.; McCarthy, Margaret M.

2014-01-01

Sex differences in vocal communication are prevalent in both the animals and humans. The mechanism(s) mediating gender differences in human language are unknown, although, sex hormones, principally androgens, play a central role in the development of vocalizations in a wide variety of animal species. The discovery of FOXP2 has added an additional avenue for exploring the origins of language and animal communication. The FOXP2 gene is a member of the forkhead box P (FOXP) family of transcription factors. Prior to the prenatal androgen surge in male fetuses, we observed no sex difference for Foxp2 protein levels in cultured cells. In contrast, 24 hours after the onset of the androgen surge, we found a sex difference for Foxp2 protein levels in cultured cortical cells with males having higher levels than females. Furthermore, we observed the potent nonaromatizable androgen dihydrotestosterone altered not only Foxp2 mRNA and protein levels but also Foxp1. Androgen effects on both Foxp2 and Foxp1 were found to occur in the striatum, cerebellar vermis, and cortex. Immunofluorescence microscopy and coimmunoprecipitation demonstrate Foxp2 and the androgen receptor protein interact. Databases for transcription factor binding sites predict a consensus binding motif for androgen receptor on the Foxp2 promoter regions. We also observed a sex difference in rat pup vocalization with males vocalizing more than females and treatment of females with dihydrotestosterone eliminated the sex difference. We propose that androgens might be an upstream regulator of both Foxp2 and Foxp1 expression and signaling. This has important implications for language and communication as well as neuropsychiatric developmental disorders involving impairments in communication. PMID:25247470

Androgen modulation of Foxp1 and Foxp2 in the developing rat brain: impact on sex specific vocalization.

PubMed

Bowers, J Michael; Perez-Pouchoulen, Miguel; Roby, Clinton R; Ryan, Timothy E; McCarthy, Margaret M

2014-12-01

Sex differences in vocal communication are prevalent in both the animals and humans. The mechanism(s) mediating gender differences in human language are unknown, although, sex hormones, principally androgens, play a central role in the development of vocalizations in a wide variety of animal species. The discovery of FOXP2 has added an additional avenue for exploring the origins of language and animal communication. The FOXP2 gene is a member of the forkhead box P (FOXP) family of transcription factors. Prior to the prenatal androgen surge in male fetuses, we observed no sex difference for Foxp2 protein levels in cultured cells. In contrast, 24 hours after the onset of the androgen surge, we found a sex difference for Foxp2 protein levels in cultured cortical cells with males having higher levels than females. Furthermore, we observed the potent nonaromatizable androgen dihydrotestosterone altered not only Foxp2 mRNA and protein levels but also Foxp1. Androgen effects on both Foxp2 and Foxp1 were found to occur in the striatum, cerebellar vermis, and cortex. Immunofluorescence microscopy and coimmunoprecipitation demonstrate Foxp2 and the androgen receptor protein interact. Databases for transcription factor binding sites predict a consensus binding motif for androgen receptor on the Foxp2 promoter regions. We also observed a sex difference in rat pup vocalization with males vocalizing more than females and treatment of females with dihydrotestosterone eliminated the sex difference. We propose that androgens might be an upstream regulator of both Foxp2 and Foxp1 expression and signaling. This has important implications for language and communication as well as neuropsychiatric developmental disorders involving impairments in communication.

Role of Chondroitin Sulfate (CS) Modification in the Regulation of Protein-tyrosine Phosphatase Receptor Type Z (PTPRZ) Activity: PLEIOTROPHIN-PTPRZ-A SIGNALING IS INVOLVED IN OLIGODENDROCYTE DIFFERENTIATION.

PubMed

Kuboyama, Kazuya; Fujikawa, Akihiro; Suzuki, Ryoko; Tanga, Naomi; Noda, Masaharu

2016-08-26

Protein-tyrosine phosphatase receptor type Z (PTPRZ) is predominantly expressed in the developing brain as a CS proteoglycan. PTPRZ has long (PTPRZ-A) and short type (PTPRZ-B) receptor forms by alternative splicing. The extracellular CS moiety of PTPRZ is required for high-affinity binding to inhibitory ligands, such as pleiotrophin (PTN), midkine, and interleukin-34; however, its functional significance in regulating PTPRZ activity remains obscure. We herein found that protein expression of CS-modified PTPRZ-A began earlier, peaking at approximately postnatal days 5-10 (P5-P10), and then that of PTN peaked at P10 at the developmental stage corresponding to myelination onset in the mouse brain. Ptn-deficient mice consistently showed a later onset of the expression of myelin basic protein, a major component of the myelin sheath, than wild-type mice. Upon ligand application, PTPRZ-A/B in cultured oligodendrocyte precursor cells exhibited punctate localization on the cell surface instead of diffuse distribution, causing the inactivation of PTPRZ and oligodendrocyte differentiation. The same effect was observed with the removal of CS chains with chondroitinase ABC but not polyclonal antibodies against the extracellular domain of PTPRZ. These results indicate that the negatively charged CS moiety prevents PTPRZ from spontaneously clustering and that the positively charged ligand PTN induces PTPRZ clustering, potentially by neutralizing electrostatic repulsion between CS chains. Taken altogether, these data indicate that PTN-PTPRZ-A signaling controls the timing of oligodendrocyte precursor cell differentiation in vivo, in which the CS moiety of PTPRZ receptors maintains them in a monomeric active state until its ligand binding. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl-like molecules binding.

PubMed

Isvoran, Adriana; Craciun, Dana; Martiny, Virginie; Sperandio, Olivier; Miteva, Maria A

2013-06-14

Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins.

Erythropoietin binding protein from mammalian serum

DOEpatents

Clemons, Gisela K.

1997-01-01

Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

Erythropoietin binding protein from mammalian serum

DOEpatents

Clemons, G.K.

1997-04-29

Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

Impact of germline and somatic missense variations on drug binding sites.

PubMed

Yan, C; Pattabiraman, N; Goecks, J; Lam, P; Nayak, A; Pan, Y; Torcivia-Rodriguez, J; Voskanian, A; Wan, Q; Mazumder, R

2017-03-01

Advancements in next-generation sequencing (NGS) technologies are generating a vast amount of data. This exacerbates the current challenge of translating NGS data into actionable clinical interpretations. We have comprehensively combined germline and somatic nonsynonymous single-nucleotide variations (nsSNVs) that affect drug binding sites in order to investigate their prevalence. The integrated data thus generated in conjunction with exome or whole-genome sequencing can be used to identify patients who may not respond to a specific drug because of alterations in drug binding efficacy due to nsSNVs in the target protein's gene. To identify the nsSNVs that may affect drug binding, protein-drug complex structures were retrieved from Protein Data Bank (PDB) followed by identification of amino acids in the protein-drug binding sites using an occluded surface method. Then, the germline and somatic mutations were mapped to these amino acids to identify which of these alter protein-drug binding sites. Using this method we identified 12 993 amino acid-drug binding sites across 253 unique proteins bound to 235 unique drugs. The integration of amino acid-drug binding sites data with both germline and somatic nsSNVs data sets revealed 3133 nsSNVs affecting amino acid-drug binding sites. In addition, a comprehensive drug target discovery was conducted based on protein structure similarity and conservation of amino acid-drug binding sites. Using this method, 81 paralogs were identified that could serve as alternative drug targets. In addition, non-human mammalian proteins bound to drugs were used to identify 142 homologs in humans that can potentially bind to drugs. In the current protein-drug pairs that contain somatic mutations within their binding site, we identified 85 proteins with significant differential gene expression changes associated with specific cancer types. Information on protein-drug binding predicted drug target proteins and prevalence of both somatic and germline nsSNVs that disrupt these binding sites can provide valuable knowledge for personalized medicine treatment. A web portal is available where nsSNVs from individual patient can be checked by scanning against DrugVar to determine whether any of the SNVs affect the binding of any drug in the database.

Patterns and plasticity in RNA-protein interactions enable recruitment of multiple proteins through a single site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valley, Cary T.; Porter, Douglas F.; Qiu, Chen

2012-06-28

mRNA control hinges on the specificity and affinity of proteins for their RNA binding sites. Regulatory proteins must bind their own sites and reject even closely related noncognate sites. In the PUF [Pumilio and fem-3 binding factor (FBF)] family of RNA binding proteins, individual proteins discriminate differences in the length and sequence of binding sites, allowing each PUF to bind a distinct battery of mRNAs. Here, we show that despite these differences, the pattern of RNA interactions is conserved among PUF proteins: the two ends of the PUF protein make critical contacts with the two ends of the RNA sites.more » Despite this conserved 'two-handed' pattern of recognition, the RNA sequence is flexible. Among the binding sites of yeast Puf4p, RNA sequence dictates the pattern in which RNA bases are flipped away from the binding surface of the protein. Small differences in RNA sequence allow new modes of control, recruiting Puf5p in addition to Puf4p to a single site. This embedded information adds a new layer of biological meaning to the connections between RNA targets and PUF proteins.« less

The Intrinsically Disordered Regions of the Drosophila melanogaster Hox Protein Ultrabithorax Select Interacting Proteins Based on Partner Topology

PubMed Central

Hsiao, Hao-Ching; Gonzalez, Kim L.; Catanese, Daniel J.; Jordy, Kristopher E.; Matthews, Kathleen S.; Bondos, Sarah E.

2014-01-01

Interactions between structured proteins require a complementary topology and surface chemistry to form sufficient contacts for stable binding. However, approximately one third of protein interactions are estimated to involve intrinsically disordered regions of proteins. The dynamic nature of disordered regions before and, in some cases, after binding calls into question the role of partner topology in forming protein interactions. To understand how intrinsically disordered proteins identify the correct interacting partner proteins, we evaluated interactions formed by the Drosophila melanogaster Hox transcription factor Ultrabithorax (Ubx), which contains both structured and disordered regions. Ubx binding proteins are enriched in specific folds: 23 of its 39 partners include one of 7 folds, out of the 1195 folds recognized by SCOP. For the proteins harboring the two most populated folds, DNA-RNA binding 3-helical bundles and α-α superhelices, the regions of the partner proteins that exhibit these preferred folds are sufficient for Ubx binding. Three disorder-containing regions in Ubx are required to bind these partners. These regions are either alternatively spliced or multiply phosphorylated, providing a mechanism for cellular processes to regulate Ubx-partner interactions. Indeed, partner topology correlates with the ability of individual partner proteins to bind Ubx spliceoforms. Partners bind different disordered regions within Ubx to varying extents, creating the potential for competition between partners and cooperative binding by partners. The ability of partners to bind regions of Ubx that activate transcription and regulate DNA binding provides a mechanism for partners to modulate transcription regulation by Ubx, and suggests that one role of disorder in Ubx is to coordinate multiple molecular functions in response to tissue-specific cues. PMID:25286318

Label-free proteome profiling reveals developmental-dependent patterns in young barley grains.

PubMed

Kaspar-Schoenefeld, Stephanie; Merx, Kathleen; Jozefowicz, Anna Maria; Hartmann, Anja; Seiffert, Udo; Weschke, Winfriede; Matros, Andrea; Mock, Hans-Peter

2016-06-30

Due to its importance as a cereal crop worldwide, high interest in the determination of factors influencing barley grain quality exists. This study focusses on the elucidation of protein networks affecting early grain developmental processes. NanoLC-based separation coupled to label-free MS detection was applied to gain insights into biochemical processes during five different grain developmental phases (pre-storage until storage phase, 3days to 16days after flowering). Multivariate statistics revealed two distinct developmental patterns during the analysed grain developmental phases: proteins showed either highest abundance in the middle phase of development - in the transition phase - or at later developmental stages - within the storage phase. Verification of developmental patterns observed by proteomic analysis was done by applying hypothesis-driven approaches, namely Western Blot analysis and enzyme assays. High general metabolic activity of the grain with regard to protein synthesis, cell cycle regulation, defence against oxidative stress, and energy production via photosynthesis was observed in the transition phase. Proteins upregulated in the storage phase are related towards storage protein accumulation, and interestingly to the defence of storage reserves against pathogens. A mixed regulatory pattern for most enzymes detected in our study points to regulatory mechanisms at the level of protein isoforms. In-depth understanding of early grain developmental processes of cereal caryopses is of high importance as they influence final grain weight and quality. Our knowledge about these processes is still limited, especially on proteome level. To identify key mechanisms in early barley grain development, a label-free data-independent proteomics acquisition approach has been applied. Our data clearly show, that proteins either exhibit highest expression during cellularization and the switch to the storage phase (transition phase, 5-7 DAF), or during storage product accumulation (10-16 DAF). The results highlight versatile cellular metabolic activity in the transition phase and strong convergence towards storage product accumulation in the storage phase. Notably, both phases are characterized by particular protective mechanism, such as scavenging of oxidative stress and defence against pathogens, during the transition and the storage phase, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Nuclear proteins that bind the human gamma-globin gene promoter: alterations in binding produced by point mutations associated with hereditary persistence of fetal hemoglobin.

PubMed Central

Gumucio, D L; Rood, K L; Gray, T A; Riordan, M F; Sartor, C I; Collins, F S

1988-01-01

The molecular mechanisms responsible for the human fetal-to-adult hemoglobin switch have not yet been elucidated. Point mutations identified in the promoter regions of gamma-globin genes from individuals with nondeletion hereditary persistence of fetal hemoglobin (HPFH) may mark cis-acting sequences important for this switch, and the trans-acting factors which interact with these sequences may be integral parts in the puzzle of gamma-globin gene regulation. We have used gel retardation and footprinting strategies to define nuclear proteins which bind to the normal gamma-globin promoter and to determine the effect of HPFH mutations on the binding of a subset of these proteins. We have identified five proteins in human erythroleukemia cells (K562 and HEL) which bind to the proximal promoter region of the normal gamma-globin gene. One factor, gamma CAAT, binds the duplicated CCAAT box sequences; the -117 HPFH mutation increases the affinity of interaction between gamma CAAT and its cognate site. Two proteins, gamma CAC1 and gamma CAC2, bind the CACCC sequence. These proteins require divalent cations for binding. The -175 HPFH mutation interferes with the binding of a fourth protein, gamma OBP, which binds an octamer sequence (ATGCAAAT) in the normal gamma-globin promoter. The HPFH phenotype of the -175 mutation indicates that the octamer-binding protein may play a negative regulatory role in this setting. A fifth protein, EF gamma a, binds to sequences which overlap the octamer-binding site. The erythroid-specific distribution of EF gamma a and its close approximation to an apparent repressor-binding site suggest that it may be important in gamma-globin regulation. Images PMID:2468996

CXCL4 is a novel nickel-binding protein and augments nickel allergy.

PubMed

Kuroishi, T; Bando, K; Tanaka, Y; Shishido, K; Kinbara, M; Ogawa, T; Muramoto, K; Endo, Y; Sugawara, S

2017-08-01

Nickel (Ni) is the most frequent metal allergen and induces a TH 1 -dependent type-IV allergy. Although Ni 2+ is considered to bind to endogenous proteins, it currently remains unclear whether these Ni-binding proteins are involved in Ni allergy in vivo. We previously reported the adjuvant effects of lipopolysaccharide (LPS) in a Ni allergy mouse model. As LPS induces a number of inflammatory mediators, we hypothesized that Ni-binding protein(s) are also induced by LPS. The objective of this study was to purify and identify Ni-binding protein(s) from serum taken from LPS-injected mice (referred as LPS serum) and examined the augmenting effects of these Ni-binding protein(s) on Ni allergy in an in vivo model. BALB/cA mice were sensitized with an i.p. injection of NiCl 2 and LPS. Ten days after sensitization, mice were challenged with NiCl 2 by an i.d. injection into ear pinnae. Ni-binding protein(s) were purified by Ni-affinity column chromatography and gel filtration. Lipopolysaccharide serum, but not serum taken from saline-injected mice, augmented ear swelling induced by Ni-allergic inflammation. Ni-binding, but not non-binding fraction, purified from LPS serum augmented Ni-allergic inflammation. Mass spectrometry and Western blotting detected CXCL4 in the active fraction. A batch analysis with Ni-sepharose and a surface plasmon resonance analysis revealed direct binding between CXCL4 and Ni 2+ . Recombinant CXCL4 augmented Ni-allergic inflammation and exerted adjuvant effects at the sensitization phase. These results indicate that CXCL4 is a novel Ni-binding protein that augments Ni allergy at the elicitation and sensitization phases. This is the first study to demonstrate that the Ni-binding protein augments Ni allergy in vivo. © 2017 John Wiley & Sons Ltd.

Binding Linkage in a Telomere DNA–Protein Complex at the Ends of Oxytricha nova Chromosomes

PubMed Central

Buczek, Pawel; Orr, Rochelle S.; Pyper, Sean R.; Shum, Mili; Ota, Emily Kimmel Irene; Gerum, Shawn E.; Horvath, Martin P.

2005-01-01

Alpha and beta protein subunits of the telomere end binding protein from Oxytricha nova (OnTEBP) combine with telomere single strand DNA to form a protective cap at the ends of chromosomes. We tested how protein–protein interactions seen in the co-crystal structure relate to DNA binding through use of fusion proteins engineered as different combinations of domains and subunits derived from OnTEBP. Joining alpha and beta resulted in a protein that bound single strand telomere DNA with high affinity (KD-DNA=1.4 nM). Another fusion protein, constructed without the C-terminal protein–protein interaction domain of alpha, bound DNA with 200-fold diminished affinity (KD-DNA=290 nM) even though the DNA-binding domains of alpha and beta were joined through a peptide linker. Adding back the alpha C-terminal domain as a separate protein restored high-affinity DNA binding. The binding behaviors of these fusion proteins and the native protein subunits are consistent with cooperative linkage between protein-association and DNA-binding equilibria. Linking DNA–protein stability to protein–protein contacts at a remote site may provide a trigger point for DNA–protein disassembly during telomere replication when the single strand telomere DNA must exchange between a very stable OnTEBP complex and telomerase. PMID:15967465