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Estudos estruturais e funcionais da proteina stanniocalcina-1 humana, um novo marcador de microambiente de leucemia / Structural and functional studies of human protein stanniocalcina-1, a new microenvironmental marker of leukemiaTrindade, Daniel Maragno 13 August 2018 (has links)
Orientadores: Jorg Kobarg, Jose Andres Yunes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T22:20:50Z (GMT). No. of bitstreams: 1
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Previous issue date: 2009 / Resumo: Staniocalcinas (STCs) representam uma pequena família de hormônios glicoprotéicos, encontrados em todos os vertebrados e composta por STC1 e STC2, que foram inicialmente implicados na homeostase de cálcio e recentemente também foram implicados em vários outros processos. Stanniocalcina-1 (STC1), o primeiro membro encontrado, foi originalmente descoberto em peixes ósseos e posteriormente identificado em humanos onde parece ter um papel, embora ainda obscuro, na carcinogênese e angiogênese. Nos seres humanos STC1 pode ser encontrado em duas formas: um dímero ou um grupo de variantes de alto peso molecular coletivamente chamados bigSTC. Uma vez que ambas as células leucêmicas e os tumores sólidos dependem vascularização, a angiogênese é um passo fundamental na interação tumorhospedeiro e essencial para a progressão do câncer. Análises prévias de microarray resultaram na identificação de vários genes ativados em células endoteliais da medula óssea (BMEC) modulados pela presença de células leucêmicas. Apresentamos aqui STC1 como um marcador microambiente de medula óssea (BMM) durante leucemia linfoblástica aguda (LLA) pela validação dos dados prévios de microarray através de PCR em tempo real quantitativos. Em vista da falta de informações funcionais e estruturais sobre STC1 realizamos: (1) um screen no sistema de duplo híbrido em levedura, a fim de encontrar algumas das proteínas que interagem com a STC1 humana, e (2) uma caracterização estrutural inicial à baixa resolução desta proteína. Fomos capazes de construir um mapa interação proteína-proteína, obtido a partir dos 22 interactantes encontrados em screen de duplo híbrido em levedura. As proteínas encontradas apresentam-se em vários compartimentos celulares nos quais STC1 já foi demonstrada para estar presente, e essas novas informações podem ajudar a esclarecer como e qual o papel STC1 desempenha nesses locais. A fim de fornecer informação estrutural sobre STC1, realizamos análises bioquímicas e estruturais da proteína recombinante STC1 com 6xHis tag produzida em células de inseto utilizando o sistema baculovírus. A análise de dicroísmo circular confirmou a predição in silico do elevado conteúdo de alfa-helices. A análise por espectrometria de massas forneceu os dados experimentais que confirmaram o padrão conservado de pontes dissulfeto anteriormente descrito para STC1 de peixes. Finalmente, os dados de espalhamento de raios-X a baixo ângulo demonstraram que, STC1 adota uma estrutura dimérica, ligeiramente alongada em solução fornecendo deste modo os primeiros dados estruturais à baixa resolução desta família de proteínas. Além disso, foi possível obter proteína suficiente e de elevado grau de
pureza para realizar ensaios cristalização que resultaram em cristais os quais difrataram a boa resolução. / Abstract: Staniocalcins (STCs) represent a small family of glycoprotein hormones found in all vertebrates composed by STC1 and STC2, which have been initially implicated in calcium homeostasis and also recently implicated in several other processes. Stanniocalcin-1 (STC1), the first member found, was originally discovered in bony fishes and later identified in humans were it seems to have a role, although still unclear, in carcinogenesis and angiogenesis. In humans STC1 can be found in two forms: a dimer or a group of higher molecular weigh variants collectively called bigSTC. Since both leukemic cells and solid tumors depend on vascularization, angiogenesis is a fundamental step in tumor-host interaction and essential to the cancer progression. Previous microarray analysis resulted in the identification of several activated genes in bone marrow (BM) endothelial cells (BMEC) modulated by presence of leukemic cells. Here we present STC1 as a BM microenvironment marker during acute lymphoblastic leukemia (ALL) by validating previous microarray data by quantitative RealTime-PCR. In view of the lack of functional and structural information on STC1 we performed (1) a yeast two hybrid screen in order to find some of the human STC1 interacting proteins and (2) a initial structural lowresolution
characterization of this protein. We were able to construct a protein-protein interaction map, derived from the 22 interactants found in our yeast two-hybrid screen. The proteins found are located in several cellular compartments in which STC1 has already been shown to be present, and the new information might help to clarify how and which role it performs at these sites. As a means to provide structural information about STC1, we performed biochemical and structural analyses of recombinant STC1 6xHis tagged protein produced in
insect cells by using the baculovirus system. Circular dichroism analysis confirmed the in silico predicted high alpha-helical content. By mass spectroscopy analysis we provided experimental data that confirmed the conserved disulfide pattern previously described for fish STC1. Finally Small Angle X-ray Scattering data demonstrated that STC1 adopts a dimeric, slightly elongated structure in solution, providing there by the first low resolution structural data of this family of proteins. Additionally, we could obtain enough protein of high purity to perform crystallization trials that resulted in crystals diffracting at good resolution. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular
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A análise do interactoma de SCI1 (Stigma/Style Cell Cycle Inhibitor 1) revela possíveis mecanismos de controle da proliferação celular / The analysis of the interactome of SCI1 (Stigma/Style Cell Cycle Inhibitor 1) reveals possible mechanisms controlling cell proliferationEdward José Strini 05 May 2014 (has links)
A biologia da reprodução de plantas é um campo de grande interesse, já que a maioria dos alimentos consumidos pelo homem é composta de partes reprodutivas das plantas (frutos e sementes). O pistilo é o órgão reprodutivo feminino, composto de estigma, estilete e ovário. Devido à importância central do pistilo no sucesso da reprodução de plantas, faz-se necessário um melhor conhecimento dos genes e processos que regulam seu desenvolvimento e funcionamento. Estudos comparativos da expressão gênica nos órgãos vegetativos e reprodutivos de Nicotiana tabacum revelaram genes de expressão preferencial nos órgãos reprodutivos, entre eles alguns codificando proteínas de função ainda desconhecida. Um destes genes foi caracterizado e denominado SCI1 (Stigma/style Cell-cycle Inhibitor 1), por apresentar um papel importante no desenvolvimento do estigma/estilete, atuando como um inibidor de ciclo celular tecido-específico (DePaoli et al., 2011). O presente trabalho teve como objetivo estudar os mecanismos moleculares pelos quais NtSCI1 regula o ciclo celular, investigando seus parceiros de interação. Em um ensaio de pull-down, utilizando-se extrato proteico nuclear de estigmas/estiletes de N. tabacum, vários putativos reguladores de ciclo celular foram identificados, sendo a interação entre NtSCI1 e NtCDKG;2 confirmada por BiFC e localizada no nucléolo. Uma biblioteca de cDNAs de estigmas/estiletes de N. tabacum, no sistema de duplo-híbrido de levedura, foi construída com sucesso. O screening desta biblioteca, utilizando BD-NtSCI1 como \"isca\", permitiu a identificação de vários parceiros de interação com NtSCI1, entre eles: uma helicase de RNA DEAD-BOX, a proteína 14-3-3D2, dois fatores de transcrição (HOMEOBOX-22 e STOREKEEPER), um fator de splicing portador do domínio SWAP, uma quinase de adenosina e uma transposase. As interações entre NtSCI1 e os três primeiros parceiros citados já foram confirmadas por BiFC (observadas no núcleo e nucléolo) e a interação entre NtSCI1 e Nt14-3-3D2 foi confirmada também por co-imunoprecipitação. O envolvimento de NtSCI1 com a regulação do ciclo celular foi corroborado pela interação entre NtSCI1 e a proteína NtCICLINA-L1 (subunidade regulatória de CDKG;2), confirmada por duplo-híbrido e por BiFC, no nucléolo. A interação entre NtSCI1 e NtCICLINA-RELATED também foi confirmada por BiFC. Para entender a dinâmica de NtSCI1 no nucléolo, foi estudada a localização subcelular da proteína de fusão NtSCI1-GFP durante as fases do ciclo celular. NtSCI1-GFP foi observada no nucléolo de células BY-2 em interfase e prófase, desaparecendo na metáfase e anáfase e reaparecendo no nucléolo no final da telófase, mostrando que a presença de NtSCI1 na célula é controlada pelo ciclo celular. A construção de uma primeira versão do interactoma de NtSCI1 mostrou seu envolvimento direto e indireto com proteínas relacionadas ao metabolismo de RNAs, controle da transcrição e regulação do ciclo celular. Estes resultados sugerem que NtSCI1 possa atuar no controle do ciclo celular de forma não canônica, por meio de múltiplos processos paralelos que interconectam aspectos da regulação da transcrição e o processamento de RNAs com o controle do ciclo celular. / The biology of plant reproduction is a field of great interest, since most of the food consumed by humans is composed of reproductive parts of plants (fruits and seeds). The pistil is the female reproductive organ, composed of stigma, style and ovary. Due to the central importance of the pistil in the success of plant reproduction, a better knowledge of the genes and processes that regulate pistil development and function is necessary. Comparative studies of gene expression in vegetative and reproductive organs of Nicotiana tabacum have revealed genes preferentially expressed in the reproductive organs, among them some encoding proteins of unknown function. One of these genes was characterized and denominated SCI1 (Stigma/style Cell-cycle Inhibitor 1), since it has an important role in stigma/style development, acting as a tissue-specific cell-cycle inhibitor (DePaoli et al., 2011). The objective of the present work was to study the molecular mechanisms through which NtSCI1 regulates the cell cycle investigating its interaction partners. In a pull-down assay, using nuclear protein extracts from N. tabacum stigmas/styles, several putative cell cycle regulators were identified. Among them, the interaction between NtSCI1 and NtCDKG;2 was confirmed by BiFC and localized in the nucleolus. A N. tabacum stigma/style cDNA library in the yeast two-hybrid system was successfully constructed. The screening of this library, using BD-NtSCI1 as bait, allowed the identification of several NtSCI1 interaction partners, among them: a DEAD-BOX RNA helicase; the 14-3-3D2 protein; two transcription factors (HOMEOBOX-22 and STOREKEEPER); a splicing factor containing a SWAP domain; an adenosine kinase; and a transposase. The interactions between NtSCI1 and the first three mentioned partners have already been confirmed by BiFC (observed in the nucleus and nucleolus) and the interaction between NtSCI1 and Nt14-3-3D2 was also wconfirmed by co-immunoprecipitation. The NtSCI1 involvement in cell cycle regulation was corroborated by the interaction between NtSCI1 and the NtCYCLIN-L1 (a regulatory subunit of CDKG;2), which was confirmed by two-hybrid and BiFC in the nucleolus. The interaction between NtSCI1 and NtCYCLIN-RELATED was also confirmed by BiFC. To understand the dynamics of NtSCI1 in the nucleolus, the subcellular localization of the fusion protein NtSCI1-GFP was studied during the different cell cycle phases. NtSCI1-GFP was observed in the nucleolus of BY-2 cells at interphase and prophase, disappearing at metaphase and anaphase and reappearing in the nucleolus at the end of telophase, showing that NtSCI1 presence in the cell is controlled by the cell cycle. The construction of the first version of NtSCI1 interactome showed its direct and indirect involvement with proteins related to RNA metabolism, transcription control and cell cycle regulation. These results suggest that NtSCI1 may act in cell cycle control in a non-canonical way, through multiple parallel processes interconnecting aspects of transcription regulation, RNA processing and cell cycle control.
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Interaction entre yOgg1, une ADN glycosylase de la voie BER, et l’ADN polymérase réplicative Polε chez Saccharomyces cerevisiae / yOgg1, a Saccharomyces cerevisiae bifunctional DNA glycosylase involved in base excision repair of oxidative DNA damage, interacts with the replicative DNA polymerase, PolεEssalhi, Kadija 12 December 2013 (has links)
Les dommages oxydatifs de l’ADN sont impliqués dans les processus pathologiques que sont le cancer, les maladies neurodégénératives ou le vieillissement. Ces dommages résultent en partie de l’action des espèces réactives de l’oxygène (ERO), qui proviennent du métabolisme cellulaire ou d’agents exogènes (physiques ou chimiques), et qui conduisent à différents types de lésions parmi lesquelles l’oxydation des bases de l’ADN (8-oxoguanine, 8-oxoG) ou la formation de sites abasiques AP (apurique/apyrimidique). Ces lésions, qui si elles ne sont pas éliminées conduisent à des processus de mutagenèse ou de mort cellulaire, sont prises en charge spécifiquement par le système de réparation de l’ADN par excision de base ou BER. Le BER est initié par l’action d’une ADN glycosylase, telles que la 8-oxoG-ADN glycosylase (Ogg1) chargée d’éliminer la 8-oxoG, une lésion très abondante. Une étude par « double-hybride » initiatrice de ce projet a révélé l’existence d’une interaction in vivo chez S. cerevisiae entre la protéine yOgg1 et la sous-unité catalytique de l’ADN polymérase réplicative Polε (yPol2), également impliquée dans la voie BER chez la levure. Nos travaux démontrent que yOgg1 et yPol2 interagissent bien physiquement entre elles et de façon spécifique. Une étude par troncations et mutagenèse dirigée nous a permis d’identifier le domaine 3’→5’ exonucléase de yPol2 comme faisant partie de la forme tronquée minimale de yPol2 capable d’interagir avec yOgg1. La poche du site actif de yOgg1 et/ou son voisinage immédiat pourrait contenir pour partie le site d’interaction pour yPol2. Nous observons d’ailleurs une corrélation nette entre l’activité de yOgg1 et sa capacité à interagir avec yPol2 dans la levure. De même, l’activité 3’→5’ exonucléase de yPol2 pourrait être liée à son interaction avec yOgg1. D’un point de vue fonctionnel, yPol2 stimulerait l’activité AP lyase de yOgg1 et le couplage entre l’activité ADN glycosylase et AP lyase de l’enzyme, permettant ainsi une meilleure coordination de l’étape d’excision du nucléoside endommagé et l’étape de resynthèse de l’ADN dans la voie BER. / Oxidative DNA damages are involved in pathological processes such as cancer, neurodegenerative diseases and aging. Part of these damages results from the action of reactive oxygen species (ROS), which are produced by cellular metabolism or (physical or chemical) exogenous agents. They lead to different types of DNA lesions including DNA base oxidation (8-oxoguanine, 8-oxoG) and abasic site formation (AP, apuric/apyrimidic). If not removed, these lesions lead to mutagenesis or cell death. Most of base lesions are dealt specifically by the base excision repair (BER) pathway. BER is initiated by a DNA glycosylase, such as 8-oxoG-DNA glycosylase (Ogg1) which is responsible for the removal of 8-oxoG. In previous unpublished work, a yeast two-hybrid study revealed the existence in S. cerevisiae of an interaction between yOgg1 and the catalytic subunit of the replicative DNA polymerase Polε (yPol2), also involved in the BER pathway in eukaryotes. Our work shows that yOgg1 and yPol2 physically and specifically interact with each other. Truncation and site-directed mutagenesis studies allowed us to identify the 3 ' → 5' exonuclease activity domain of yPol2 as part of the minimal form of yPol2 still able to interact with yOgg1. The active site of yOgg1 and/or its immediate vicinity may contain part of its interaction domain with yPol2. Besides, we observe a clear correlation between yOgg1 catalytic activity and its ability to interact with yPol2 in vivo. Similarly, the 3'→5' exonuclease activity of yPol2 could be useful to its interaction with yOgg1. From a functional point of view, yPol2 stimulates in vitro the AP lyase activity of yOgg1 and the coupling of both DNA glycosylase and AP lyase enzyme activity. The interaction yOgg1/yPol2 could allow a better coordination of damaged nucleoside excision and DNA re-synthesis steps in BER.
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Étude de l’expression et de la fonction du gène Ankyrin-repeat and SOCS-Box protein 9 (ASB9) dans le follicule ovulatoire bovinBenoit, Gabriel 04 1900 (has links)
No description available.
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Tribbles pseudokinase 2 (TRIB2) dans le contrôle moléculaire de la fonction ovarienne bovineWarma, Aly 11 1900 (has links)
Au cours des processus de croissance folliculaire et d'ovulation, les cellules stéroïdogéniques, y compris les cellules de granulosa (CG), jouent un rôle crucial dans la maturation et la libération de l'ovocyte. Notre laboratoire a identifié et caractérisé pour la première fois tribbles pseudokinase 2 (TRIB2) dans les CG de follicules ovariens. Des études ont démontré que TRIB2 joue un rôle dans la coordination de la mitose et la morphogenèse chez la drosophile alors que chez la souris, son expression dans les cellules du cumulus serait liée à la maturation ovocytaire. Cependant, le rôle exact de TRIB2 dans la fonction des CG et ses effets sur les voies de signalisation impliquées dans la croissance folliculaire restait à être défini. La présente étude de doctorat avait donc pour but d’étudier la fonction et les partenaires de liaison de TRIB2 dans les CG de follicules ovariens bovins. À l’aide d’un modèle d’étude in vivo consistant en des CG obtenues à partir de follicules à différents stades de développement, nous avons démontré une régulation à la baisse de TRIB2 par l’hormone lutéinisante (LH) et la gonadotrophine chorionique humaine (hCG) aussi bien au niveau du messager que de la protéine. De plus, les analyses utilisant un modèle in vitro de CG en culture ont montré que la FSH stimule l'expression de TRIB2 tandis que l'inhibition de TRIB2 via CRISPR/Cas9 entraîne une réduction significative de la prolifération des CG (P<0,05). Les analyses Western blot ont montré une augmentation des niveaux de phosphorylation d’ERK1/2 (MAPK3/1) et p38MAPK (MAPK14) suite à la surexpression de TRIB2. Ces résultats suggèrent un rôle de TRIB2 dans la croissance folliculaire et la modulation des voies MAPK. Avec l’approche double hybride chez la levure, nous avons identifié CALM1, INHBA, INPPL1, NT5E, SCD, SDHB et RAB14 comme partenaires de liaison de TRIB2. Les analyses RT-PCR ont montré que ces partenaires sont régulés différemment au cours du développement folliculaire et les manipulations de l’expression de TRIB2 (inhibition ou surexpression) résulte en une régulation différente (augmentation ou diminution) de l’expression des partenaires dans les CG. Ces résultats suggèrent un rôle de TRIB2 dans la régulation de cibles effectrices en lien avec la fonction des CG et le développement folliculaire. Enfin, un modèle de CG provenant de vaches en période post-partum a été utilisé pour mieux comprendre le contrôle de l’activité des CG. Des analyses complémentaires avec ce modèle ont révélé une réduction significative de TRIB2 chez les vaches ayant un taux élevé de BHB (>1.4mmol/L) comparé à celles ayant un faible taux de BHB (<1.2mmol/L). Cette réduction était concomitante à une augmentation d’interleukines pro-inflammatoires et une réduction d’interleukines anti-inflammatoires dans les CG. L’ensemble de ces résultats supporte un rôle de TRIB2 dans la modulation de la signalisation MAPK dans les CG, apporte une preuve solide que TRIB2 pourrait agir comme un régulateur de la prolifération et de la fonction des CG et de l’expression de gènes cibles, et suggère que TRIB2 pourrait affecter la stéroïdogenèse au cours du développement folliculaire et lors de la période post-partum. / During the processes of follicular growth and ovulation, steroidogenic cells, including granulosa
cells, play a crucial role in the maturation and release of the oocyte. Our lab identified and
characterized for the first time tribble pseudokinase 2 (TRIB2) in GC of ovarian follicles. Previous
studies have shown that TRIB2 plays a role in the coordination of mitosis and morphogenesis in
Drosophila while in mice its expression in cumulus cells is linked to oocyte maturation. However,
the exact mechanism of action of TRIB2 as well as its function and effects on signaling pathways
in GC during follicular growth remained to be defined. This study aimed therefore to further
investigate TRIB2 function and identify its binding partners in GC. TRIB2 inhibition and
overexpression experiments were conducted using, respectively, CRISPR/Cas9 technology and the
pQE1 system. Using an in vivo model consisting of GC obtained from follicles at different stages
of development, we demonstrated a downregulation of TRIB2 by the endogenous luteinizing
hormone (LH) and human Chorionic Gonadotropin (hCG) at both the messenger and protein levels.
In addition, analyzes using an in vitro model of cultured GC, we showed that FSH stimulates the
expression of TRIB2 while inhibition of TRIB2 via CRISPR-Cas9 resulted in a significant
reduction in GC proliferation (P<0.05). Western blot analyzes showed an increase in the
phosphorylation levels of ERK1/2 (MAPK3/1) and p38MAPK (MAPK14) following TRIB2
overexpression. These results suggested a role of TRIB2 in follicular growth and modulation of
MAPK pathways. In the second part of the thesis, we identified CALM1, INHBA, INPPL1, NT5E,
SCD, SDHB and RAB14 as binding partners of TRIB2 in GC using the yeast two-hybrid approach.
RT-qPCR analyzes showed that all of these partners are present in the dominant follicles but are
differently regulated during follicular development. Moreover, TRIB2 manipulation (inhibition or
overexpression) results in a different regulation (up- or down-regulation) of these partners
expression in GC. These results suggest a role of TRIB2 in the regulation of effector targets genes
related to follicular development and GC activity. In the third part of the thesis, a GC model from
postpartum cows was also used to better understand the control of GC activity. Further analyses
using this model revealed a significant decrease of TRIB2 in cows with high level of BHB (>1.4
mmol/L) as compared to those with a low BHB levels (<1.2 mmol/L). This reduction of TRIB2
was concomitant with an increase in pro-inflammatory interleukins and a reduction in anti-inflammatory interleukins in GC. Overall, these results support a role of TRIB2 in the modulation
of MAPK signaling in GC, provide strong evidence that TRIB2 could act as a regulator of GC
proliferation and function as well as expression of target genes in GC, and suggests that TRIB2
might affect steroidogenesis during follicular development and during the post-partum period.
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A proteome-wide screen utilizing second generation sequencing for the identification of lysine and arginine methyltransferase protein interactionsWeimann, Mareike 13 September 2012 (has links)
Proteinmethylierung spielt eine immer größere Rolle in der Regulierung zellulärer Prozesse. Die Entwicklung effizienter proteomweiter Methoden zur Detektion von Methylierung auf Proteinen ist limitiert und technisch schwierig. In dieser Arbeit haben wir einen neuen Hefe-Zwei-Hybrid-Ansatz (Y2H) entwickelt, der Proteine, die miteinander wechselwirken, mit Hilfe von Sequenzierungen der zweiten Generation identifiziert (Y2H-Seq). Der neue Y2H-Seq-Ansatz wurde systematisch mit dem Y2H-Seq-Ansatz verglichen. Dafür wurde ein Bait-Set von 8 Protein-Arginin-Methyltransferasen, 17 Protein-Lysin-Methyltransferasen und 10 Demethylasen gegen 14,268 Prey-Proteine getestet. Der Y2H-Seq-Ansatz ist weniger arbeitsintensiv, hat eine höhere Sensitivität als der Standard Y2H-Matrix-Ansatz und ist deshalb besonders geeignet, um schwache Interaktionen zwischen Substraten und Protein-Methyltransferasen zu detektieren. Insgesamt wurden 523 Wechselwirkungen zwischen 22 Bait-Proteinen und 324 Prey-Pr oteinen etabliert, darunter 11 bekannte Methyltransferasen-Substrate. Netzwerkanalysen zeigen, dass Methyltransferasen bevorzugt mit Transkriptionsregulatoren, DNA- und RNA-Bindeproteinen wechselwirken. Diese Daten repräsentieren das erste proteomweite Wechselwirkungsnetzwerk über Protein-Methyltransferasen und dienen als Ressource für neue potentielle Methylierungssubstrate. In einem in vitro Methylierungsassay wurden exemplarisch mit Hilfe massenspektrometrischer Analysen die methylierten Aminosäurereste einiger Kandidatenproteine bestimmt. Von neun getesteten Proteinen waren sieben methyliert, zu denen gehören SPIN2B, DNAJA3, QKI, SAMD3, OFCC1, SYNCRIP und WDR42A. Wahrscheinlich sind viele Methylierungssubstrate im Netzwerk vorhanden. Das vorgestellte Protein-Protein-Wechselwirkungsnetzwerk zeigt, dass Proteinmethylierung sehr unterschiedliche zelluläre Prozesse beeinflusst und ermöglicht die Aufstellung neuer Hypothesen über die Regulierung Molekularer Mechanismen durch Methylierung. / Protein methylation on arginine and lysine residues is a largely unexplored posttranslational modification which regulates diverse cellular processes. The development of efficient proteome-wide approaches for detecting protein methylation is limited and technically challenging. We developed a novel workload reduced yeast-two hybrid (Y2H) approach to detect protein-protein interactions utilizing second generation sequencing. The novel Y2H-seq approach was systematically evaluated against our state of the art Y2H-matrix screening approach and used to screen 8 protein arginine methyltransferases, 17 protein lysine methyltransferases and 10 demethylases against a set of 14,268 proteins. Comparison of the two approaches revealed a higher sensitivity of the new Y2H-seq approach. The increased sampling rate of the Y2H-seq approach is advantageous when assaying transient interactions between substrates and methyltransferases. Overall 523 interactions between 22 bait proteins and 324 prey proteins were identified including 11 proteins known to be methylated. Network analysis revealed enrichment of transcription regulator activity, DNA- and RNA-binding function of proteins interacting with protein methyltransferases. The dataset represents the first proteome-wide interaction network of enzymes involved in methylation and provides a comprehensively annotated resource of potential new methylation substrates. An in vitro methylation assay coupled to mass spectrometry revealed amino acid methylation of candidate proteins. Seven of nine proteins tested were methylated including SPIN2B, DNAJA3, QKI, SAMD3, OFCC1, SYNCRIP and WDR42A indicating that the interaction network is likely to contain many putative methyltransferase substrate pairs. The presented protein-protein interaction network demonstrates that protein methylation is involved in diverse cellular processes and can inform hypothesis driven investigation into molecular mechanisms regulated through methylation.
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Functional analysis of Zfp819 in pluripotency and embryonic developmentTan, Xiaoying 02 November 2012 (has links)
Pluripotenz wird durch viele Stammzell-spezifische Transkriptionsfaktoren wie Oct3/4, Nanog und Sox2 sowie deren Funktion in ihrem regulatorischen Netzwerk etabliert und aufrechterhalten. Viele Studien haben gezeigt, wie diese Pluripotenz-assoziierten Faktoren ihre Zielgene regulieren. Dies geschieht durch die Interaktion mit bekannten und unbekannten Interaktionspartnern. In der vorliegenden Arbeit haben wir Zfp819 als einen neuen Pluripotenz-assoziierten Faktor beschrieben und dessen Funktion in pluripotenten Stammzellen untersucht.
Im ersten Teil der vorliegenden Arbeit haben wir zwei cDNA-Banken für Yeast two Hybdrid (Y2H)-Assays aus unterschiedlichen pluripotenten Stammzelltypen generiert. Dies hatte zum Ziel, potentielle Interaktionspartner eines Kandidatenproteins zu identifizieren um dadurch Eindrücke über die Funktion des Proteins zu gewinnen. Für die Identifizierung von potentiellen Interaktionspartnern von Zfp819 haben wir die cDNA-Bank aus embryonalen Stammzellen benutzt. Wir konnten 17 putative Interaktionspartner identifizieren und daraus ein hypothetisches „Interaktom“ von Zfp819 generieren. Die Einordnung der putativen Interaktionspartner nach ihrer Gen-Ontologie (GO) ließ vermuten, dass Zfp819 eine Rolle in der Regulation der Transkription, der Aufrechterhaltung der genetischen Integrität und im Zellzyklus bzw. bei der Apoptose spielt.
Im zweiten Teil der vorliegenden Arbeit wurde die sehr intensive Expression von Zfp819 in undifferenzierten pluripotenten Zelllinien gezeigt. Desweiteren konnte die Promotorregion von Zfp819 identifiziert werden, und es wurde gezeigt, dass diese mit epigenetischen Mustern ausgestattet ist. Zusätzlich konnten wir Regionen im Zfp819-Gen identifizieren, die für die nukleäre Lokalisation von Zfp819 verantwortlich sind. Desweiteren konnten wir zeigen, dass Zfp819 in der transkriptionellen Repression von spezifischen endogenen, retroviralen Elementen (ERVs) in pluripotenten Zellen eine Rolle spielt. Durch zelluläre und biochemische Studien konnten wir zeigen, dass Zfp819 mit vielen Proteinen interagiert (z.B. Kap1 und Chd4), welche für die Aufrechterhaltung der genomischen Integrität von Bedeutung sind. Tatsächlich resultierte der Verlust von Zfp819 in embryonalen Stammzellen in einer erhöhten Anfälligkeit für DNA-Schäden und in einer verminderten DNA-Reparatur.
Zusammenfassend lassen die Identifizierung der Interaktionspartner sowie die Ergebnisse der molekularen und der funktionellen Studien vermuten, dass Zfp819 durch die Unterdrückung von ausgewählten ERVs eine Rolle in der Regulation der genomischen Stabilität von pluripotenten Zellen spielt.
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Characterizing Protein-Protein Interactions of B0238.11, a Previously Uncharacterized Caenorhabditis elegans Intergenic Spacer Binding ProteinOmar, Syed A. A. 11 May 2012 (has links)
A protein, B0238.11, was identified in a yeast one-hybrid screen to bind to the ribosomal intergenic spacer region (IGS) of Caenorhabditis elegans. Proteins interacting with this region of the DNA have been implicated in ribosome biogenesis in other model organisms, so it is also possible that B0238.11 plays a role in RNA transcription by interacting with RNA polymerase I or other transcription machinery. Thus, the goal of this study was to further characterize the structure and function of B0238.11. I used yeast two-hybrid experiments to identify proteins that interact with B0238.11 within the nucleus. RPS-0, K04G2.2, DPY-4, EFT-3, PAL-1, and B0238.11, itself, were found to bind to B0238.11. Additionally, I analysed the amino acid sequence of B0238.11 using in silico bioinformatics methods to determine its structure and putative function and also to identify and characterize the other interacting proteins. I found that B0238.11 contains a high-mobility group box domain, which is also found in HMO1P in yeast and UBF in vertebrates. These other proteins also bind to the IGS, are known to form homodimers and have been implicated in the initiation of ribosomal RNA transcription. Here I scrutinize the validity of the interaction between each protein and B0238.11. I conclude that B0238.11 is likely to be a C. elegans homolog of UBF and present an updated interactome map for B0238.11. / Synopsis: I carried out yeast two-hybrid assay to find proteins interacting with B0238.11 (O16487_CAEEL). I found that this protein's DNA-binding profile and protein interaction profile mimic other HMG-box containing proteins UBF and HMO1P which are involved in ribosomal RNA transcription initiation. Acknowledgements: I would like to thank my supervisor, Dr. Teresa J. Crease, for not only giving me the opportunity to investigate an interesting topic in Molecular Biology, but also for her patient guidance, encouragement and sound advice. I feel extremely lucky to have a supervisor who cared so much about my work, who responded to my questions and queries so promptly, and was always available to discuss project and career related matters. I would also like to thank Dr. Todd Gillis and Dr. Terry Van Raay for their careful consideration of this project and timely constructive criticisms that helped shape my project. I would like to thank all the members of my committee for helping me see things from different perspectives and helping me develop and critical and mature understanding of the scientific process.
I must also express my gratitude to Dr. Robin Floyd for allowing me to build upon his work and Dr. Marian Walhout, at the University of Massachusetts, for providing the Caenorhabditis elegans complimentary DNA library. A large part of this project would not have been possible without the people at the genomics facility in the Department of Integrative Biology, I commend their professionalism and punctuality in delivering results. Completing this work would have been all the more difficult were it not for the support and friendship provided by my peers Shannon Eagle, Tyler Elliott, Nick Jeffery, Joao Lima, Sabina Stanescu, Fatima Mitterboeck and Paola Pierossi. And finally, I would like to thank my parents and siblings Sara Omar and Ali Omar for their continued support through good times and bad, and letting me use their laptops when mine broke down.
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Virus host interactome du polyomavirus à cellules de Merkel / Merkel cell polyomavirus virus host interactomeFerté-Chaudoy, Marion 15 September 2017 (has links)
Le polyomavirus à cellules de Merkel est aujourd’hui reconnu comme l’agent étiologique du carcinome à cellules de Merkel (CCM). Le cycle viral et les mécanismes de l’oncogenèse viro-induite sont peu connus et les connaissances se basent essentiellement sur les études menées notamment sur le polyomavirus SV40. L’objectif des travaux de thèse était d’identifier les interactions entre les protéines virales et les protéines cellulaires lors de l’infection ou dans le contexte du carcinome à cellules de Merkel (CCM). Pour identifier ces interactions, nous avons réalisé des cribles double hybride en levures sur les oncogènes du MCPyV et du BKPyV. Afin valider les interactions obtenues en levures, nous avons utilisé une méthode orthogonale de validation par complémentation en cellules de mammifères reposant sur la restauration de la luciférase de Gaussia princeps. La combinaison de ses deux techniques nous a permis de valider des interactions avec des partenaires cellulaires impliqués dans la régulation du cycle cellulaire ou encore de la voie Akt-mTOR. Les précédents travaux du laboratoire, qui portaient sur l’interactome des protéines mineures de capsides VP2/VP3, avaient également permis d’identifier des interactions avec des protéines de la voie NF-kB. Nous avons alors testé les interactions entre les oncogènes et la protéine mineure de capside VP2 avec des protéines cellulaires impliquées dans cette voie. Ces travaux nous ont conduits à tester l’activation de la voie, l’expression des gènes sous le contrôle de NF-kB et la régulation de l’apoptose. Les résultats obtenus montrent une action de la protéine VP2 sur l’activation de la voie NF-kB et une induction de l’apoptose. / The Merkel cell polyomavirus is now recognized as the etiologic agent of Merkel cell carcinoma (MCC). The viral cycle and viro-induced oncogenesis mechanisms are not fully understood and the knowledge is mainly based on the studies carried out particularly on the SV40 polyomavirus. The aim of our work is to identify interactions between viral proteins and cellular proteins during productive infection or in MCC context. To identify these interactions, we performed yeast two hybrid screens on MCPyV and BKPyV oncogenes, as control. To validate the interactions obtained in yeasts, we used an orthogonal method of validation by complementation in mammalian cells based on the restoration of Gaussia princeps luciferase. The combination of these two orthogonal techniques allowed us to validate interactions with cellular partners involved in cell cycle regulation or Akt-mTOR pathway. Previous lab work on VP2/VP3 minor capsid proteins allowed the identification of interactions with NF-kB pathway involved proteins. We examined the interactions between oncogenes, VP2, with the cellular proteins involved in this pathway. This work led us to evaluate pathway activation, genes expression under the control of NF-kB and apoptosis regulation. These results evidenced an action of the VP2 protein on the activation of NF-kB pathway and an induction of apoptosis.
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IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs)Rodríguez Solovey, Leisa Natacha 16 December 2015 (has links)
[EN] ABSTRACT
Abscisic acid (ABA) signaling plays a critical role in regulating root growth and root system architecture. ABA-mediated growth promotion and root tropic response under water stress are key responses for plant survival under limiting water conditions. In this work, we have explored the role of Arabidopsis (Arabidopsis thaliana) PYR/PYL/RCAR receptors (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS) for root ABA signaling. As a result, we discovered that PYL8 plays a nonredundant role for the regulation of root ABA sensitivity. Unexpectedly, given the multigenic nature and partial functional redundancy observed in the PYR/PYL family, the single pyl8 mutant showed reduced sensitivity to ABA-mediated root growth inhibition. This effect was due to the lack of PYL8-mediated inhibition of several clade A phosphatases type 2C (PP2Cs), since PYL8 interacted in vivo with at least five PP2Cs, namely HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 as revealed by tandem affinity purification and mass spectrometry proteomic approaches.
Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calciumdependent interactions of PYR/PYL/RCAR ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL/RCAR receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL/RCAR function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL/RCAR-interacting partners that mediate a transient Ca2+-dependent interaction with phospholipid vesicles, which affects PYR/PYL/RCAR subcellular localization and positively regulates ABA signaling. / [ES] RESUMEN
La señalización por la hormona vegetal ácido abscísico (ABA) desempeña un papel crítico en la regulación del crecimiento de la raíz y en la arquitectura del sistema radical. La promoción de crecimiento de la raíz en condiciones de estrés hídrico mediada por ABA es clave para la supervivencia de las plantas bajo condiciones limitantes de agua. En este trabajo, hemos explorado el papel de los receptores PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) de Arabidopsis (Arabidopsis thaliana) en la ruta de señalización de ABA en raíz. Así, hemos descubierto que el receptor de ABA PYL8 juega un papel no redundante en la regulación de la percepción de ABA en raíz. Inesperadamente, dada la naturaleza multigénica y la redundancia funcional parcial observada en la familia PYR/PYL/RCAR, el mutante pyl8 fue el único mutante sencillo de pérdida de función de los receptores PYR/PYL/RCAR que mostraba una sensibilidad reducida a la inhibición del crecimiento mediada por ABA en raíz. Este efecto se debe a la falta de inhibición mediada por PYL8 de varias fosfatasas del grupo A tipo 2C (PP2Cs), ya que PYL8 es capaz de interactuar in vivo con al menos cinco PP2Cs, denominadas HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 según lo han revelado la purificación por afinidad en tándem (TAP por sus siglas en inglés) y estudios proteómicos de espectrometría de masas.
La transducción de la señal del ABA localizada en la membrana plasmática celular
juega un papel crucial en los pasos iniciales de la señalización de la fitohormona, pero los mecanismos moleculares que unen los componentes básicos de la señalización y la membrana plasmática no están claros. Estudiando las interacciones de los receptores del ABA PYR/PYL/RCAR con la membrana plasmática hemos encontrado que éstos pueden interaccionar transitoriamente con ella de forma dependiente de calcio gracias a una familia de proteínas con dominios C2 relacionadas con la ruta de señalización de ABA (denominadas C2-domain ABA-related (CAR) proteins). Específicamente, se encontró que PYL4 interacciona de manera independiente de ABA con CAR1 tanto en la membrana plasmática como en el núcleo de las células vegetales. La proteína CAR1 pertenece a una familia multigénica constituida por 10 miembros en Arabidopsis thaliana, desde CAR1 hasta CAR10, y que solo se encuentra en plantas. Los ensayos de complementación bi-molecular de fluorescencia y de co-immunoprecipitación confirmaron la interacción en células vegetales tanto de PYL4-CAR1 como de otras parejas de PYR/PYL-CAR. La cristalización de la proteína CAR4 reveló que, además de un dominio C2 clásico de unión a lípidos dependiente de calcio, las proteínas de la familia CAR presentan un dominio específico que probablemente es responsable de la interacción con los receptores PYR/PYL/RCAR y de su posterior reclutamiento a las vesículas de fosfolípidos. Esta interacción es relevante para la función de los receptores PYR/PYL/RCAR en la señalización del ABA, ya que diferentes mutantes triples car de pérdida de función, que tienen afectados los genes CAR1, CAR4, CAR5, y CAR9, demostraron una reducción de la sensibilidad al ABA en ensayos de establecimiento de plántula y crecimiento de la raíz. En resumen, hemos identificado nueva familia de proteínas que son capaces mediar las interacciones transitorias dependientes de Ca2+ con vesículas de fosfolípidos, lo que a su vez afecta localización de PYR/PYL/RCAR y regula positivamente la señalización de ABA. / [CAT] RESUM
La senyalització per l'hormona vegetal àcid abcíssic (ABA) exerceix un paper crític en la regulació del creixement de l'arrel i també en l'arquitectura del sistema radical. La promoció del creixement de l'arrel en condicions d'estrés hídric, regulada per ABA és clau per la supervivència de les plantes sota condicions limitants d'aigua. Amb aquest treball, hem investigat el paper dels receptors PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) d'Arabidopsis (Arabidopsis thaliana) en el camí de senyalització d'ABA en arrel. Així, hem descobert que el receptor d'ABA PYL8 exerceix un paper no redundant en la regulació de la percepció d'ABA en arrel. Inesperadament, donada la naturalesa multigènica i la redundància funcional parcial que s'observa en la família PYR/PYL/RCAR, el mutant pyl8 va ser l'únic mutant senzill de pèrdua de funció dels receptors PYR/PYL/RCAR que mostrava una sensibilitat reduïda a la inhibició del creixement mitjançada per l'ABA en l'arrel. Doncs aquest efecte es deu a la falta d'inhibició regulada per PYL8 de diverses fosfatases del grup A tipus 2C (PP2Cs), ja que PYL8 té la capacitat d'interactuar in vivo almenys amb cinc PP2Cs, anomenades HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABAHYPERSENSITIVE GERMINATION3 segons ho han revelat per una banda la purificació per afinitat en tàndem (TAP són les seues sigles en anglés) i per altra banda, estudis proteòmics d'espectrometria de masses.
Pel que fa a la transducció del senyal del l'ABA, la qual es localitza en la membrana plasmàtica cel¿lular, juga un paper molt important en els primers instants de la senyalització de la fitohormona, no obstant això els mecanismes moleculars que uneixen els components bàsics d'aquesta senyalització amb la membrana plasmàtica, no es troben del tot clars. Per tant, s'han estudiat les interaccions que tenen els receptors del ABA PYR/PYL/RCAR amb la membrana plasmàtica, i hem trobat que aquests tenen la capacitat d'interaccionar transitòriament amb la membrana de forma dependent al calci, gràcies a una família de proteïnes amb domini C2, les quals es troben relacionades amb la ruta de senyalització d'ABA(anomenades C2domain ABArelated (CAR) proteins).Específicament, es va trobar que PYL4 interacciona d'una manera independent al ABA amb CAR1, tant en la membrana plasmàtica, com en el nucli de les cèl¿lules vegetals. La proteïna CAR1 pertany a la família multigènica constituïda per 10 components en Arabidopsis thaliana, des de CAR1 fins CAR10, que tan sols es troba en plantes. Els assajos de complementació bimolecular de fluorescència i de co-immunoprecipitació, van confirmar la interacció en cèl¿lules vegetals, tant de PYL4CAR1 com d'altres parelles de PYR/PYL-CAR. La cristal¿lització de la proteïna CAR4 va revelar que, a més d'un domini C2 clàssic de unió a lípids dependent del calci, les proteïnes de la família CAR presenten un domini PYR/PYL/RCAR, i del seu posterior reclutament a les vesícules fosfolipídiques. Doncs, aquesta interacció és rellevant en la funció dels receptors PYR/PYL/RCAR, ja que participa en la senyalització del l'ABA. Aquesta interacció es clau per a la funció dels receptors, ja que diferents mutants triples car de pèrdua de funció, els quals posseïxen afectats els gens CAR1, CAR4, CAR5 i CAR9, van mostrar una reducció de la sensibilitat a l'ABA en assajos d'establiment de plàntula i creixement de l'arrel. En conclusió, hem identificat una nova família de proteïnes amb la capacitat d'organitzar les interaccions transitòries dependents del calci amb vesícules de fosfolípids, fet que al seu torn afecta la localització de PYR/PYL/RCAR i regula positivament la senyalització d'ABA. / Rodríguez Solovey, LN. (2015). IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58862 / TESIS
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