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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.

Der Transkriptionsfaktor CCAAT/enhancer-binding Protein β (C/EBPβ) reguliert die Expression zahlreicher Gene, welche die Proliferation, Differenzierung und Seneszenz in hämatopoietischen Zellen, Adipozyten und Leukämiezellen kontrollieren. Um diese mannigfaltigen Aufgaben zu erfüllen interagiert C/EBPβ mit zahlreichen Kofaktoren und Proteinen der Transkriptionsregulations-Maschinerie. Da das funktionale Netzwerk von C/EBPβ und seinen zahlreichen Kooperationspartnern bis heute nicht vollständig entziffert ist, ist es das Ziel dieser Arbeit das Netzwerk aus Interaktionspartnern und C/EBPβ regulierten Proteinen in Leukämiezelllinien und darüber hinaus zu erforschen und aufzudecken. Das Interaktom von C/EBPβ wurde mittels einer Kombination aus einem membranbasierten Peptid-Interaktions Testverfahrens (APS) und endogener Immunprezipitationen mit gekoppelter MS-Analyse untersucht. Außerdem wurde die Proteinmenge von C/EBPβ und von potentiell von C/EBPβ regulierten Proteinen mittels proteomischer MS-Analyse in C/EBPβ Knock-out- und Leukämiezelllinien untersucht. Die Protein-Interaktionsversuche ergaben epigenetische und allgemeine transkriptionsregulierende Proteine, sowie Chromatinstruktur modellierende Faktoren, die mit C/EBPβ interagieren. Zusätzlich konnten neue Interaktionen von C/EBPβ mit Kondensin- und Kinetochorproteinen beobachtet werden. Die Versuchsergebnisse eröffnen überdies neue Interaktionen von C/EBPβ mit DNA Reparatur und Apoptose assoziierten Proteinen. Interessanterweise konnten auch Komponenten des Spliceosomes und RNA-prozessierende Proteine als Interaktoren von C/EBPβ identifiziert werden. Zusammenfassend ermöglicht diese Studie nicht nur die Verifikation von bereits bekannten Proteininteraktionen von C/EBPβ, sondern eröffnet zahlreiche weitere zukünftige Forschungsfelder bezüglich des Interaktionsnetzwerkes von C/EBPβ in Leukämien, sowie anderen Zellarten und Geweben. / The basic leucine zipper transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) regulates the expression of various genes that control the proliferation, differentiation and senescence of haematopoietic cells, adipocytes and leukemia cells. To facilitate its multifaceted functions C/EBPβ interacts with a collection of cofactors and proteins of the transcription regulation machinery. As the functional network of C/EBPβ and its numerous cooperation partners is still incomplete this study attempted to analyze interaction partners and downstream proteins of C/EBPβ in leukemia cells and beyond. A combinatory approach of an array based peptide-interaction screening (APS) and endogenous shotgun IP-MS from leukemia cell lines was applied to elucidate the interactome of C/EBPβ. Moreover, C/EBPβ abundance and potential C/EBPβ regulated proteins were determined by MS proteomics in C/EBPβ knockout and leukemia cell lines. The interaction screenings revealed proteins associated with the general and epigenetic regulation of transcription, with chromatin remodeling and mitotic chromatin organization as well as cell cycle regulation. Additionally, new interactions of C/EBPβ with condensin and kinetochore proteins could be elucidated. The data reports of novel C/EBPβ interactors involved in DNA repair and apoptosis. In addition, components of the spliceosome and RNA-processing were detected. Altogether this study verifies known and reveals various novel interactions of the transcription factor C/EBPβ and augments the network of previous reported interactions and potential cooperation partners. The here collected data discloses new subjects for further research concerning the interaction network of C/EBPβ during cell differentiation and in leukemia.

In C. elegans bietet die frühe Embryogenese ein attraktives Modellsystem, um Wechselwirkungen von Proteinen in vivo zu entschlüsseln. Zur präzisen Identifizierung von spezifischen Interaktionen im C. elegans Embryo wurde ein neuer quantitativer Ansatz entwickelt, welcher die Expression von Fusionsproteinen an grün fluoreszierendes Protein in vivo mit markierungsfreier Interaktionsproteomik kombiniert. Diese Strategie wurde angewandt, um die Interaktionspartner von acht Proteinen zu untersuchen, die in essentiellen biologischen Prozessen während der frühen Embryogenese involviert sind. Diese Studie liefert als Ergebnis ein erstes embryonales in vivo Interaktionsnetzwerk bestehend aus 559 Interaktionen zwischen 472 Proteinen. Dieses Netzwerk erfasst nicht nur bekannte Bindungen, sondern auch neue Interaktionen von hoher funktioneller Relevanz. Die Netzwerkinformationen wurden mit Experimenten auf Basis der Ribonukleinsäuren-Interferenz kombiniert um neue Regulatoren der sogenannten „P granules” ausfindig zu machen. Infolgedessen wurde das fadenwurmspezifische Protein GEI-12 als neuer Interaktionspartner der DYRK-Kinase MBK-2 und als wichtiger Regler für die Dynamik der „P granules“ und für die Aufrechterhaltung der Keimbahn identifiziert. Dies führt zu einem hypothetischen Modell in welchem der Phosphorylierungszustand von GEI-12 den Auf- und Abbau der „P granules“ während der frühen Embryogenese vermittelt. Darüber hinaus veranlasst GEI-12 auch die Entstehung von „P granules“ in Säugetierzellen und bindet an PP2A-Phosphatasen, was darauf hindeutet, dass die grundlegenden biophysikalischen Eigenschaften die zur Entstehung der Ribonukleoprotein-Körperchen notwendig sind, im Laufe der Evolution zwischen Spezies konserviert geblieben sind. Zusammenfassend stellt die in vivo Interaktionskartierung ein vielseitiges Werkzeug dar, welches nicht nur die funktionelle Organisation des Proteoms aufdeckt, sondern auch Einsichten in die tierische Entwicklungsbiologie liefert. / In C. elegans, early embryogenesis provides an attractive model system for mapping in vivo protein interactions. In order to accurately identify specific interactions in C. elegans embryos, a new quantitative approach was developed combining in vivo expressed GFP fusion proteins with label-free interaction proteomics. This strategy was applied to studying the interaction partners of eight bait proteins involved in essential biological processes during early embryogenesis. As a result, this study generated a pilot embryo in vivo interaction network composed of 559 interactions among 472 proteins. Importantly, this network captures not only well-characterized bindings but also new interactions of high functional relevance. Further utility of the network is demonstrated by combining it with RNAi perturbation to search for new regulators of P granule formation in early embryos. Consequently, a worm-specific protein GEI-12 was discovered as a novel interaction partner of the DYRK kinase MBK-2 and as an important regulator of P granule dynamics and germline maintenance. This leads to a hypothetical model in which the phosphorylation state of GEI-12 mediates P granule assembly and disassembly during early embryogenesis. In addition, GEI-12 also induces granule formation in mammalian cells and interacts with PP2A phosphatases, indicating that the fundamental biophysical properties required for ribonucleoprotein granule formation are conserved across species during evolution. In summary, in vivo interactome mapping is a versatile approach that not only unravels the functional organization of the proteome but also can reveal insights into animal development.

Protein-Protein Interaktions (PPI) Netzwerke liefern ein Grundgerüst für systematische Untersuchungen der komplexen molekularen Maschinerie in der Zelle. Die Komplexität von Protein-Wechselwirkungen stellt jedoch in Bezug auf ihre Identifizierung, Validierung und Annotation eine große experimentelle und rechnerische Herausforderung dar. In dieser Arbeit analysierte ich diese Probleme und lieferte Lösungen, um die Limitierungen aktueller humanen PPI Netzwerke zu überwinden. Meine Arbeit kann in zwei Teile aufgeteilt werden: Im ersten Teil führte ich eine kritischen Vergleich von acht unabhängig konstruierten humanen PPI Netzwerke durch, um mögliche experimentellen Verzerrungen zu erkennen. Die Ergebnisse zeigten starke Tendenzen bezüglich der Selektion und Detektion von Interaktionen, die in zukünftigen Anwendungen dieser Netzwerke berücksichtigt werden sollten. Einer der wichtigsten Schlussfolgerungen dieser Studie war, dass die derzeitigen humanen Interaktions Netzwerke komplementär sind und deshalb wurde eine Datenbank mit der Bezeichnung Unified Human Interaktome (UniHI) entwickelt, die menschliche PPI Daten aus zwölf wichtigsten Quellen integriert. Im zweiten Teil dieser Forschungsarbeit benutzte ich die Daten aus der UniHI Datenbank, die genetischen Modifikatoren in einer bestimmten Krankheit, Chorea Huntington (HD) eine autosomal dominante neurodegenerative Erkrankung, zu charakterisieren. Um die Proteine zu identifizieren, die den Krankheitsverlauf modifizieren können, wurden Protein Interaktion Daten mit Genexpressionsdaten von HD-Patienten in Kombination mit einem Mehrschritt-Filterungsverfahren integriert. Mit dem neuartigen Ansatz wurde ein Nucleus caudatus-spezifische Protein-Interaktion HD (PPI)-Netzwerk vorhergesagt, das 14 potentiell dysregulierten Proteine direkt oder indirekt mit dem Huntingtin-Protein verlinkt, mit mögliche Verbindung zu Molekularen Prozessen wie z.B. Apoptose, Metabolismus, neuronale Entwicklung. / Protein interaction networks aim to provide the scaffold maps for systematic studies of the complex molecular machinery in the cell. The complexity of protein interactions poses, however, large experimental and computational challenges regarding their identification, validation and annotation. Additionally, storage and linking is demanding since new data are rapidly accumulating. In this research work, I addressed these issues and provided solutions to overcome the limitations of current human protein-protein interaction (PPI) maps. In particular, my thesis can be partitioned into two parts: In the first part, I conducted a comparative assessment of eight recently constructed human protein-protein interaction networks to identify experimental biases. Results showed strong selection and detection biases which are necessary to take into consideration in future applications of these maps. One of the important conclusions of this study was that the current human interaction networks contain complementary information; hence, a database was developed, termed as Unified Human Interactome (UniHI), integrating human PPI data from twelve major sources. Several new tools were included for querying, analyzing and visualizing human PPI networks. In the second part of this research work, UniHI dataset was applied to characterize the genetic modifiers involved in a specific disease: Chorea Huntington (HD), an autosomal dominant neurodegenerative disease. To find the modifiers, a network-based modeling approach was implemented by integrating huntingtin-specific protein interaction network with gene expression data from HD patients in multiple steps. Using this approach, a Caudate Nucleus-specific HD protein interaction (PPI) network was predicted, connecting 14 potentially dysregulated proteins directly or indirectly to the disease protein, showing a possible link to molecular processes such as pro-apoptotic pathways, cell survival, anti-apoptotic, growth, and neuronal diseases.

Protein-Protein-Wechselwirkungen steuern zelluläre Funktionen auf molekularer Ebene. Posttranslationale Proteinmodifikationen beeinflussen diese Wechselwirkungen und erlauben dynamische Regulierung. Tyrosinphosphorylierung ist eine besonders relevante Modifikation, weil sie eng mit interzellulärer Regulation von Wachstum und Enticklung in Vielzellern verbunden ist. Da falsche Regulierung dieser Prozesse zu Krebs oder Autoimmunerkrankungen führen kann, ist sie auch von großem medizinischen Interesse. In Hefe-Zwei-Hybrid- Untersuchungen mit Volllängen-Proteinen im Genommaßstab wurde ein umfassender Satz von 292 größtenteils neuen phosphotyrosinabhängigen Proteinwechselwirkungen erster Güte ermittelt. Damit wurde eine Wissenslücke im Bereich der phosphotyrosinabhängigen Signalübertragung, der bisher hauptsächlich auf Peptidbindungs- und Affinitätsaufreinigungs-gekoppelten Massenspektronomieexperimenten fußte. Die Güte der Interaktionen wurde experimentell und informatisch, in Coimmunpräzipitations- und Proteinkomplementierungs-, sowie in Überrepräsentationsanalysen und Literaturvergleichen, gezeigt. Bekannte lineare Bindesequenzmotive kommen zwar gehäuft vor, können die Mehrzahl der Interaktionen aber offensichtlich nicht erklären. Die Wechselwirkungen bilden ein dichtes, einheitliches Netzwerk und widerspiegeln phosphotyrosinabhängige KEGG-Signalwege. Es hat ein Herzstück aus acht Genen, von denen sieben fest etablierte Signalverarbeitungshauptknotenpunkte darstellen. Dem achten, SH2D2A, scheint eine deutlich wichtigere Rolle zuzukommen als bisher wahrgenommen. Schliesslich wurde für eine Auswahl von GRB2-Interaktionen unterschiedliche subzelluläre Verortung vorgenommen. Zusammengenommen legen diese Ergebnisse nah, dass die hier veröffentlichten Wechselwirkungen einen wesentlichen Schritt für das Verstehen von Wachstum und Entwicklung markieren und zur Verbesserung der Behandlungsmöglichkeiten in wichtigen Medizinbereichen beitragen werden. / Protein-protein interactions govern cellular functions on the molecular level. Post-translational modifications alter these interactions allowing highly dynamic regulation. Protein tyrosine phosphorylation is an especially relevant post-translational modification, because it is tightly linked to intercellular regulation of growth and development in metazoans. Diseases like cancer or autoimmune disorders arise from misregulation of these processes generating great medical interest in protein tyrosine phosphorylation and processes relating to it. This study provides a comprehensive set of 292 mostly novel, high-quality phosphotyrosine- dependent protein-protein interactions detected in genome-scale yeast two-hybrid screens using full-length proteins filling a gap in phosphotyrosine signaling knowledge, which has so far been based largely on peptide binding and affinity purification-coupled mass spectrometry experiments. The high quality was demonstrated experimentally and computationally, in co-immunoprecipitation and protein complementation assays, as well as over-representation analyses and comparison to prior knowledge. Previously reported linear peptide motifs are reflected in the binding partners, but clearly do not account for most of the interactions, emphasizing the relevance of full-length protein context. The interactions were further shown to form an unusually dense, monolithic network with a central core and reflect and expand phosphotyrosine-related KEGG pathways. Seven of the eight core proteins are well-established signaling hubs. The eighth core gene, SH2D2A, seems to play a more central role than currently appreciated. Finally, selected interactions involving GRB2 were shown to occur in different specific subcellular localizations. Together, these results strongly suggest that the interactions presented here represent an important step toward understanding growth and development and will benefit treatment of pressing medical issues substantially.

Um mit ihrer Umgebung zu kommunizieren verfügen lebende Zellen über Rezeptoren, welche die umschließende Membran überbrücken. Die vorherrschende G-Protein-gekoppelte Rezeptoren (GPCR) erhalten Informationen von Außerhalb durch Bindung eines Liganden, wodurch der Rezeptor aktiviert wird. Während der Aktivierung bildet sich innerzellulär ein offener Spalt, in den ein G-Protein (Gαβγ, G) mit seinem C-terminalen Ende koppeln kann. Die Bindung an einen GPCR führt in der Gα-Untereinheit vom Gαβγ zu einen GDP/GTP-Austausch, welcher für die weitere Signalübertragung ins Zellinnere notwendig ist. Die Kopplung von Rezeptor und Gαβγ umfasst eine Reihe von dynamischen strukturellen Änderungen, die Geschwindigkeit und Spezifität der Interaktion regeln. Hier haben wir MD-Simulationen (Molekulardynamik) verwendet, um die molekularen Details der GPCR Gαβγ Kopplung vor und während der GPCR-Gαβγ-Komplexbildung bis hin zum GDP/GTP-Austausch zu untersuchen. / To communicate with their environment, living cells feature receptors that provide a bridge across the enclosing membrane. The prevalent G protein-coupled receptors (GPCR) receive outside information through the binding of a ligand, which activates the receptor. During activation, an open intracellular crevice forms, to which a G protein (Gαβγ, G) can couple with its Gα C-terminus. Binding to GPCRs triggers GDP/GTP exchange in the Gα subunit of Gαβγ, necessary for further signal transfer within the cell. The coupling between receptor and Gαβγ involves a series of dynamic structural changes that govern speed and specificity of the interaction. Here we used molecular dynamics (MD) simulations to elucidate molecular details of the GPCR Gαβγ coupling process before and during GPCR Gαβγ complex formation up to the GDP/GTP exchange.

Made available in DSpace on 2016-01-26T12:51:20Z (GMT). No. of bitstreams: 1 mariacarolinaferrerisarkismadrid_dissert.pdf: 2834086 bytes, checksum: 6c83e7649397cb555812544b997d81d3 (MD5) Previous issue date: 2007-12-04 / Yellow fever is an infectious disease caused by the yellow fever virus (YFV), a Flavivirus transmitted to humans by Aedes aegypti mosquitoes. Despite the existence of the yellow fever vaccine, the disease is endemic in South America and Africa, causing public health problems such as dispersed outbreaks, epidemics with variable impact and the risk of re-emergency of the urban cycle due to the occurrence of sylvatic disease. Aim. The knowledge of the components of YFV replication complex is still incipient but it is known that there are interactions among viral RNA, viral proteins and host proteins and, due to evidences of the existence of protein-protein interactions related to the NS5 protein of other Flavivirus, the target of our study was YFV NS5 protein. Once protein-protein interactions present basic importance for the activation, the regulation and the control of diverse biologic functions related to these interactions, the identification and the characterization of them are essential for a better comprehension of the pathogenesis and for the rational design of drugs for YFV. Material and Method. The YFV NS5 gene was divided in its two domains, which were independently cloned in a GAL4 DNA-BD plasmid, generating the methyltransferase (MT) and RNA polymerase (RNApol) baits. A two-hybrid system screening in Saccharomyces cerevisiae AH109 strain was performed utilizing RNApol bait and cDNA library of Hela cells, which was cloned in a GAL4 AD plasmid. MT bait showed to be toxic for the yeast. Results. All 204 obtained transformants were tested for activation of reporter genes HIS3, ADE2 and lacZ from AH109 and only 35 samples indicated positivity to, at least, two of the reporter genes assessed. Thirty three distinct cellular protein partners of the RNApol NS5 were identified after the sequencing of the clones and the comparison of its sequences with GenBank. Proteins Snf5, p54NRB, HMG20B, U1A, eIF3S6IP, GIPC PDZ and MIF were chosen for next experiments. A plasmid linkage with these proteins was performed to exclude the possibility of false-positive clones and to confirm the protein-protein interactions identified in the initial screening. RNApol regions responsible for the Snf5 and eIF3S6IP interactions were mapped and a region of approximately 80 aminoacids was identified as the minimum domain requested for the interactions, called fragment A. Conclusion. The prominence of this YFV fragment as a determinant of protein interactions became more evident when its sequence was compared to the sequences of other Flavivirus, signalizing a homology from aminoacid 20 to 80, demonstrating that this fragment is a conserved region. Moreover, the production of a similarity model of RNA polymerase domain of YFV NS5 protein, using the known DENV NS5 protein structure, showed that the region of interaction is exposed and potentially capable of forming interactions. / A febre amarela é uma doença infecciosa causada pelo vírus da febre amarela (yellow fever virus YFV), um Flavivirus transmitido ao homem pela picada do mosquito Aedes aegypti. Mesmo com a existência de uma vacina anti-amarílica, a enfermidade conserva-se endêmica na América do Sul e na África, gerando problemas de saúde pública que incluem surtos isolados, epidemias de impactos variáveis e, principalmente, o risco da possível re-emergência da sua forma urbana a partir da ocorrência de surtos silvestres. Objetivo. Embora sejam mínimas as informações sobre os componentes do complexo de replicação do YFV, sabe-se que nele estão envolvidas interações entre o RNA viral, proteínas virais e proteínas do hospedeiro e, devido às evidências de interações proteína-proteína relacionadas à proteína NS5 de outros Flavivirus, o alvo principal do nosso trabalho foi NS5 do YFV. Como interações protéicas são de fundamental importância para ativação, regulação e controle de diversas funções biológicas a elas relacionadas fica evidente a relevância da identificação e caracterização das interações participantes desse processo para uma melhor compreensão da patogênese e para o desenho racional de drogas contra a febre amarela. Material e Método. O gene NS5 de YFV foi dividido em seus dois domínios, os quais foram clonados independentemente no plasmídeo com DNA-BD de GAL4, gerando as iscas metiltransferase e RNA polimerase. Em seguida, foi realizado um screening em sistema duplo-híbrido com a isca RNApol contra biblioteca de cDNA de células Hela clonada em vetor com AD de GAL4, uma vez que MT mostrou-se tóxica para a levedura hospedeira do experimento Saccharomyces cerevisiae, linhagem AH109. Resultados. Os 204 transformantes obtidos foram testados quanto à capacidade de ativação dos genes repórteres HIS3, ADE2 e lacZ de AH109 quando, então, apenas 35 amostras mostraram-se positivas para pelo menos dois dos repórteres testados. Após o seqüenciamento nucleotídico desses clones e comparação das seqüências com o GenBank, os resultados indicaram seqüências nucleotídicas codificadoras para 33 proteínas celulares diferentes como parceiras interativas de RNApol NS5, dentre as quais foram eleitas as proteínas Snf5, p54NRB, HMG20B, U1A, eIF3S6IP, GIPC PDZ e MIF para o prosseguimento dos experimentos. Para excluir a possibilidade de pertencerem a uma classe de clones falso-positivos e confirmar as interações proteína-proteína identificadas na triagem inicial, foi efetuado o plasmid linkage. Após tal confirmação, foram mapeadas as regiões em RNApol responsáveis pelas interações com Snf5 e eIF3S6IP, tendo sido descoberta uma mesma região de aproximadamente 80 resíduos aminoácidos como o domínio mínimo requerido para tais interações, a qual foi denominada fragmento A. Conclusões. A relevância do fragmento A de YFV como determinante das interações protéicas tornou-se mais evidente quando sua seqüência foi comparada à de outros Flavivirus, mostrando a presença de uma homologia principalmente entre os aminoácidos 20 a 80, demonstrando que esse fragmento se comporta como uma região conservada entre os Flavivirus considerados. Além disso, a geração de um modelo de similaridade do domínio RNA polimerase da proteína NS5 de YFV, a partir de NS5 de DENV, demonstrou que a região de interação está exposta ao solvente, sendo, portanto, potencialmente capaz de formar interações.

A fosforilação reversível de proteínas em resíduos de serina e treonina, catalisada por quinases e fosfatases desempenha papel chave na regulação do crescimento e na diferenciação celular em eucariotos. As serina/treonina proteínas fosfatases (PSTPs) são atualmente divididas em três famílias denominadas PPP (PhosphoProtein Phosphatase), PPM (Phosphoprotein Phosphatase Magnesium-dependent) e FCP/SCP (RNA polymerase II CTD phosphatase), sendo que os membros da família PPP são, frequentemente, holoenzimas compostas de uma subunidade catalítica associada a uma ou mais subunidades reguladoras, as quais definem a função, localização e especificidade ao substrato da fosfatase. Neste trabalho, analisamos, através de RT-qPCR, o perfil de expressão dos genes codificadores de subunidades catalíticas de PPPs de Dictyostelium discoideum (PP1c, PP2Ac, PP4c, PP4c-like, PP6c e PP5c) e de 16 potenciais parceiros moleculares de algumas destas subunidades catalíticas, tais como DdI-2 e DdI-3, sabidamente inibidores da PP1c. Em resposta ao estresse térmico de células da fase de crescimento, detectamos o aumento dos níveis de transcritos de PP4c e PP6c e também de DdI-2, DdI-3 e DDB_G0292194, esta última, uma proteína de função desconhecida que interage com a PP1c em ensaios de duplo-híbrido em leveduras. Por outro lado, durante o estresse hiper-osmótico observamos a diminuição dos níveis de transcritos de quase todos os genes analisados com exceção de DdI-2 e DDB_G0292194. O nível de expressão de DdPP1c, DdI-2, DdI-3 e DDB_G0292194 também foi analisado em resposta ao estresse oxidativo e apenas o DDB_G0292194 foi induzido nesta condição. Os genes de PP1c, PP4, PP5c e PP6c são expressos durante todo o ciclo de vida de D. discoideum, mas a expressão de alguns dos genes analisados aumenta em uma fase definida do ciclo de desenvolvimento como é o caso de DDB_G0292194 que tem níveis de transcritos aumentados na fase de agregação. Este gene codifica uma proteína hipotética de 559 aminoácidos, que apresenta um domínio FHA (ForkHead-Associated) em sua região aminoterminal, além de uma sequência similar ao motivo consenso de ligação à PP1c. Ensaios no sistema de duplo-híbrido em leveduras confirmaram que a interação entre DDB_G0292194 e DdPP1c independe do domínio FHA. Verificamos, também, que o mutante nocaute de DDB_G0292194 apresenta uma morfologia alterada em condições padrões de cultivo, tanto na fase de crescimento como durante o desenvolvimento, além de uma maior sensibilidade ao estresse oxidativo causado pelo peróxido de hidrogênio quando comparado à linhagem selvagem. Em conjunto, nossos resultados evidenciam a importância das PPPs na resposta a diferentes tipos de estresse e para o crescimento e desenvolvimento de D. discoideum. / Reversible phosphorylation of proteins on serine and threonine residues, catalyzed by kinases and phosphatases plays a key role in growth and cell differentiation regulation in eukaryotes. Protein serine/threonine phosphatases (PSTPs) are currently divided into three families named PPP (Phosphoprotein Phosphatase), PPM (Phosphoprotein Phosphatase Magnesium-dependent) and FCP/SCP (RNA polymerase II CTD phosphatase). The PPP family members are often holoenzymes composed of a catalytic subunit associated with one or more regulatory subunits, which define function, localization and substrate specificity of the phosphatase. In this work, we have examined, by RT-qPCR, the expression profile of genes encoding PPP catalytic subunits of Dictyostelium discoideum (PP1c, PP2Ac, PP4c, PP4c-like, PP6c and PP5c) and 16 potential molecular partners for some of these catalytic subunits, such as DdI-2 and DdI-3, both known as PP1c inhibitors. In response to heat stress of growth phase cells, we detected increased levels of transcripts of PP4c and PP6c as well as of DdI-2, DdI-3, and DDB_G0292194, the latter a protein of unknown function that interacts with PP1c in yeast two-hybrid assays. Moreover, during the hyperosmotic stress we observed decreased transcript levels of nearly all genes examined except DdI-2 and DDB_G0292194. The expression level of DdPP1c, DdI-2, DdI-3 and DDB_G0292194 was also analyzed in response to oxidative stress and only DDB_G0292194 was induced in this condition. PP1c, PP4c, PP5c and PP6c genes are expressed throughout growth and development of D. discoideum while transcript levels of some the analysed genes were increased at a defined stage of the developmental cycle as in the case of DDB_G0292194, which increased during aggregation. This gene encodes a hypothetical protein of 559 amino acids bearing a FHA (ForkHead-Associated) domain in its aminoterminal region and a sequence matching the PP1c binding consensus motif. Yeast two-hybrid assays confirmed that DDB_G0292194 and DdPP1c interaction does not depend on FHA domain. We also found that DDB_G0292194 knockout mutant exibits an altered morphology on standard growth and developmental conditions and shows an increased sensitivity to oxidative stress induced by hydrogen peroxide in comparison to the wild type strain. Taken together, our results highlight the importance of PPPs in the response to different types of stress and for growth and development of D. discoideum.

Le palmier à huile est la première culture oléagineuse, avec environ 40% de la production mondiale, et son fruit accumule deux huiles de composition très différente dans le mésocarpe et l’amande. Chez les plantes, les acides gras sont assemblés en huile dans le réticulum endoplasmique, ceci par la voie dite de Kennedy à laquelle s’ajoutent des mécanismes d’édition impliquant le métabolisme de la phosphatidylcholine. Nous avons utilisé les outils de la lipidomique pour analyser la variabilité au sein de différentes populations de palmier ainsi que pour caractériser l’accumulation d’huile durant le développement du mésocarpe et de l’amande. Puis, nous avons entrepris de tester, dans le système du double hybride de levure, les interactions entre toutes les enzymes de la voie de Kennedy et celles responsables des mécanismes d’édition, et mis en évidence 241 interactions, dont 132 sont fortes, 73 moyennes et 36 faibles. Ces résultats suggèrent que ces enzymes pourraient s’assembler en complexes supra-moléculaires susceptibles de former des métabolons. Certaines isoformes d’une même enzyme ont des profils d’interaction distincts, ce qui ouvre des perspectives pour de futures recherches. De plus, nous avons caractérisé, par expression fonctionnelle dans un mutant de levure dépourvu de TAG, une acyltransférase présumée (EgWSD1-like) ainsi que les trois formes majeures de diacylglycérol acyltransférases du mésocarpe. EgWSD1-like ne restaure que l’activité de synthèse d’esters de cire dans le mutant, tandis que les trois DGAT complémentent toutes la déficience en TAG du mutant, avec d’apparentes spécificités distinctes vis-à-vis des acides gras. / Oil palm is the highest oil-yielding crop-plant, accounting for approximately 40% of the total world vegetable oil production. The fruit accumulates oil, made of triacylglycerol (TAG) molecules, in both mesocarp and kernel with totally different fatty acid profiles. Fatty acids are assembled into oil through Kennedy pathway in the endoplasmic reticulum, which is complicated by editing processes involving phosphatidylcholine metabolism. To investigate oil assembly in oil palm, we use lipidomics as a tool to analyze different populations of palm to search for TAG structural diversity, and to further characterize changes in lipid content and composition in mesocarp and kernel during fruit ripening. We used yeast two-hybrid system (split ubiquitin) to test protein-protein interactions for almost all the enzymes (32) involved in oil assembly pathway, and we demonstrated 241 interactions, including 132 strong interactions, 73 medium interactions and 36 weak interactions. Our results suggest that all enzymes might assemble into one or several complexes that may form metabolons. In addition, different isoforms of enzymes showed distinct interaction profiles, providing hints for future studies. Moreover, we also characterized the in vivo function of a putative acyltransferase (designated EgWSD1-like) possibly involved in oil assembly and the three major diacylglycerol acyltransferase (DGAT) isoforms of palm mesocarp in the mutant yeast H1246, which is devoid of neutral lipid synthesis. EgWSD1-like only shows wax ester synthase activity in yeast, while three EgDGATs all can restore TAG biosynthesis in yeast with different substrate specificities.