11 research outputs found
Identificação e análise das interações entre proteínas estruturais do vírus da dengue e proteínas de células hospedeiras humanas
Orientadora: Profa. Almeriane Maria Weffort SantosCo-orientador: Prof. Luís Roberto Benghi SoaresDissertação (Mestrado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Ciências Farmacêuticas. Defesa: Curitiba, 31/03/2009Bibliografia : fls. 66-68Resumo: O vírus de Dengue necessita de diversas proteínas da célula hospedeira humana para que possa completar o ciclo de infecção viral. Nas diversas etapas do ciclo, ocorrem interações com diferentes tipos de proteínas da célula. O presente trabalho, utilizando-se de ferramentas de biologia molecular e de bioinformática, teve como objetivo identificar e analisar, pela construção de um interatoma, as interações entre as proteínas do vírus da Dengue e as da célula hospedeira humana, a fim de identificar aquelas essenciais ao ciclo viral e ao desenvolvimento do quadro fisiopatológico da doença. Para tanto, realizou-se o cruzamento entre as proteínas estruturais Capsídeo, pré-Membrana e Envelope do vírus da Dengue, clonadas no vetor pBT, com as bibliotecas de cDNA construídas de células de cérebro e fígado humanos. Os dados obtidos por este cruzamento, assim como as respectivas seqüências, foram analisados e utilizados para alimentar o programa Cytoscape, no qual uma rede foi gerada. Análise desta rede permitiu a identificação de proteínas com funções relacionadas à infecção viral da célula, como a Clusterina, envolvida na ativação do sistema complemento e na resposta apoptótica, e das moléculas COX-1, -2 e -3, envolvidas na resposta inflamatória. Subseqüentemente, os dados obtidos no duplo-híbrido foram cruzados com dados de expressão gênica obtidos de células infectadas com o vírus da Dengue no programa Matisse e uma segunda rede foi gerada, possibilitando a identificação de módulos funcionais, dentre os quais o da apoptose, de interesse do presente trabalho, visto não ser interessante para o vírus promover apoptose da célula que o abriga, ao menos nos momentos iniciais da infecção. A seqüência das proteínas estruturais do vírus foi analisada, o que possibilitou a identificação de um domínio anti-apoptótico Bcl-2 na seqüência da proteína de Membrana. Este domínio também foi encontrado na seqüência protéica de outros vírus que apresentam função anti-apoptótica conhecida. A abordagem diferenciada adotada neste trabalho possibilitou não só observar e elaborar hipóteses sobre o modo pelo qual o vírus atua na célula hospedeira após ser infectada, como também abordar, de forma global, aspectos envolvidos nos processos celulares da interação entre o vírus da Dengue e as células humanas, os quais deverão ser biologicamente validados, a fim de comprovar ou refutar sua essencialidade.Abstract: The Dengue virus uses several of the human host cell proteins to complete the cycle of viral infection. Therefore, during the progressive stages of this cycle, many interactions with different types of cellular proteins take place. The aim of this work, which involved bioinformatics, was to identify and analyse some essential interactions for the virus replication cycle and for the development of the host disease established between the proteins from the Dengue virus and from the human host cells, resulting in a protein interactome. To achieve this, a screening of the in vitro binding between the Dengue virus Capsid, Premembrane and Envelope structural proteins, which were cloned in the pBT vector, and human brain and liver cDNA libraries was performed. The binding data obtained were analysed and the respective protein sequences involved determined. Both results were then used to feed the Cytoscape software in which a network of interacting proteins was generated. Analyses of this network identified proteins with functions related to cellular viral infection, such as Clusterin, involved in the activation of the complement system and cellular apoptosis, and Cox-1, -2 and 3, all involved in the inflammatory response. Subsequently, using the Matisse software, data obtained with the double-hybrid system were matched up against gene expression results obtained from cells infected with the Dengue virus. The network generated allowed the identification of some cellular functional modules, such as the apoptosis one, which is of particular interest in this work as interference in this cellular process is expected given the fact that, at least initially, it is not advantageous for the virus to kill the host cell. Analyses of the structural proteins sequences revealed the presence of a Bcl-2 anti-apoptotic domain in the Membrane viral protein, which has been reported for some other viruses with already known anti-apoptotic activity. The innovative approach used in this study not only allowed the formulation of questions and hypothesis about the way the virus behaves inside cells after infection, but also to speculate, in a more global view, about some of the cellular aspects involved in the relationship between the proteins of the Dengue virus and host cell interaction, which must be biologically validated in the future in order to confirm or refute their essentiality
Fructose-1,6-bisphosphate and its role on the flux-dependent regulation of metabolism
We hebben de rol van fructose-1,6-bisfosfaat (FBP) onderzocht als een flux-signalerend metaboliet en zijn participatie in de regulatie van metabolisme. We hebben de vermeende interactie tussen Hxk2 en FBP biochemisch getest, die door een nieuwe massaspectrometrie methode werd gesuggereerd waar Hxk2 conformatie veranderingen liet zien in de aanwezigheid van FBP. We vonden echter geen indicatie van een directe interactie met FBP, wat ertoe heeft geleid om de mogelijkheden te onderzoeken van secundaire effectoren, zoals metaalionen. We vonden dat de stabiliteit en activiteit van Hxk2 verzwakt wordt door zink, en dat FBP dit herstelt, werkend als een chelator. Verder hebben we de rol van FBP onderzocht in de conformatie veranderingen van zeven andere proteïnen waarvan gesuggereerd werd dat deze een interactie zouden hebben met FBP. Onze resultaten bevestigen dat er geen directe interactie is tussen FBP en de bestudeerde proteïnen, echter, we veronderstellen dat FBP metaalionen bindt en de activiteit van enzymen moduleert in een flux-afhankelijke manier. Vervolgens onderzochten we de rol van FBP in de regulatie van twee transcriptiefactoren: Cra en CggR. In het geval van Cra, waar de interactie met FBP nog steeds een discussiepunt was in de literatuur, hebben we nu experimenteel bewijs verschaft dat FBP geen interactie heeft met Cra, noch de activiteit moduleert. Bij CggR stelden we vast dat millimolaire concentraties van FBP nodig zijn om de interactie met de DNA operator te reguleren. Daarom concludeerden we dat FBP, werkend als een flux-signalerend metaboliet, een essentiële link verschaft tussen flux-signalen en genexpressie door de activiteit van CggR te moduleren
Identificação e análise das interações entre proteínas estruturais do vírus da dengue e proteínas de células hospedeiras humanas
Orientadora: Profa. Almeriane Maria Weffort SantosCo-orientador: Prof. Luís Roberto Benghi SoaresDissertação (Mestrado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Ciências Farmacêuticas. Defesa: Curitiba, 31/03/2009Bibliografia : fls. 66-68Resumo: O vírus de Dengue necessita de diversas proteínas da célula hospedeira humana para que possa completar o ciclo de infecção viral. Nas diversas etapas do ciclo, ocorrem interações com diferentes tipos de proteínas da célula. O presente trabalho, utilizando-se de ferramentas de biologia molecular e de bioinformática, teve como objetivo identificar e analisar, pela construção de um interatoma, as interações entre as proteínas do vírus da Dengue e as da célula hospedeira humana, a fim de identificar aquelas essenciais ao ciclo viral e ao desenvolvimento do quadro fisiopatológico da doença. Para tanto, realizou-se o cruzamento entre as proteínas estruturais Capsídeo, pré-Membrana e Envelope do vírus da Dengue, clonadas no vetor pBT, com as bibliotecas de cDNA construídas de células de cérebro e fígado humanos. Os dados obtidos por este cruzamento, assim como as respectivas seqüências, foram analisados e utilizados para alimentar o programa Cytoscape, no qual uma rede foi gerada. Análise desta rede permitiu a identificação de proteínas com funções relacionadas à infecção viral da célula, como a Clusterina, envolvida na ativação do sistema complemento e na resposta apoptótica, e das moléculas COX-1, -2 e -3, envolvidas na resposta inflamatória. Subseqüentemente, os dados obtidos no duplo-híbrido foram cruzados com dados de expressão gênica obtidos de células infectadas com o vírus da Dengue no programa Matisse e uma segunda rede foi gerada, possibilitando a identificação de módulos funcionais, dentre os quais o da apoptose, de interesse do presente trabalho, visto não ser interessante para o vírus promover apoptose da célula que o abriga, ao menos nos momentos iniciais da infecção. A seqüência das proteínas estruturais do vírus foi analisada, o que possibilitou a identificação de um domínio anti-apoptótico Bcl-2 na seqüência da proteína de Membrana. Este domínio também foi encontrado na seqüência protéica de outros vírus que apresentam função anti-apoptótica conhecida. A abordagem diferenciada adotada neste trabalho possibilitou não só observar e elaborar hipóteses sobre o modo pelo qual o vírus atua na célula hospedeira após ser infectada, como também abordar, de forma global, aspectos envolvidos nos processos celulares da interação entre o vírus da Dengue e as células humanas, os quais deverão ser biologicamente validados, a fim de comprovar ou refutar sua essencialidade.Abstract: The Dengue virus uses several of the human host cell proteins to complete the cycle of viral infection. Therefore, during the progressive stages of this cycle, many interactions with different types of cellular proteins take place. The aim of this work, which involved bioinformatics, was to identify and analyse some essential interactions for the virus replication cycle and for the development of the host disease established between the proteins from the Dengue virus and from the human host cells, resulting in a protein interactome. To achieve this, a screening of the in vitro binding between the Dengue virus Capsid, Premembrane and Envelope structural proteins, which were cloned in the pBT vector, and human brain and liver cDNA libraries was performed. The binding data obtained were analysed and the respective protein sequences involved determined. Both results were then used to feed the Cytoscape software in which a network of interacting proteins was generated. Analyses of this network identified proteins with functions related to cellular viral infection, such as Clusterin, involved in the activation of the complement system and cellular apoptosis, and Cox-1, -2 and 3, all involved in the inflammatory response. Subsequently, using the Matisse software, data obtained with the double-hybrid system were matched up against gene expression results obtained from cells infected with the Dengue virus. The network generated allowed the identification of some cellular functional modules, such as the apoptosis one, which is of particular interest in this work as interference in this cellular process is expected given the fact that, at least initially, it is not advantageous for the virus to kill the host cell. Analyses of the structural proteins sequences revealed the presence of a Bcl-2 anti-apoptotic domain in the Membrane viral protein, which has been reported for some other viruses with already known anti-apoptotic activity. The innovative approach used in this study not only allowed the formulation of questions and hypothesis about the way the virus behaves inside cells after infection, but also to speculate, in a more global view, about some of the cellular aspects involved in the relationship between the proteins of the Dengue virus and host cell interaction, which must be biologically validated in the future in order to confirm or refute their essentiality
Assessment of the interaction between the flux-signaling metabolite fructose-1,6-bisphosphate and the bacterial transcription factors CggR and Cra
Bacteria regulate cell physiology in response to extra- and intracellular cues. Recent work showed that metabolic fluxes are reported by specific metabolites, whose concentrations correlate with flux through the respective metabolic pathway. An example of a flux-signaling metabolite is fructose-1,6-bisphosphate (FBP). In turn, FBP was proposed to allosterically regulate master regulators of carbon metabolism, Cra in Escherichia coli and CggR in Bacillus subtilis. However, a number of questions on the FBP-mediated regulation of these transcription factors is still open. Here, using thermal shift assays and microscale thermophoresis we demonstrate that FBP does not bind Cra, even at millimolar physiological concentration, and with electrophoretic mobility shift assays we also did not find FBP-mediated impairment of Cra's affinity for its operator site, while fructose-1-phosphate does. Furthermore, we show for the first time that FBP binds CggR within the millimolar physiological concentration range of the metabolite, and decreases DNA-binding activity of this transcription factor. Molecular docking experiments only identified a single FBP binding site CggR. Our results provide the long thought after clarity with regards to regulation of Cra activity in E. coli and reveals that E. coli and B. subtilis use distinct cellular mechanism to transduce glycolytic flux signals into transcriptional regulation. This article is protected by copyright. All rights reserved