Estudio de la vía de señalización mediada por IRE1 durante la respuesta a la acumulación de proteinas mal plagadas en arabidopsis thaliana
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2015
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es
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Universidad Andrés Bello
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Resumen
El plegamiento de proteínas al interior del retículo endoplasmatico (RE) es un proceso fundamental para la vida celular de los eucariontes, ya que se presumen que más de un tercio todas las proteínas sintetizadas en una célula eucariontes, son sintetizadas en este organelo. Al interior del retículo endoplasmatico, varias proteínas participan en el proceso de síntesis, plegamiento y degradación de proteínas. Específicamente, al interior del RE se llevan a cabo los procesos de: N-glicosilación, control de calidad del plegamiento de proteínas y degradación de proteínas asociada al RE. Todos estos procesos y mecanismos asociados buscan la correcta síntesis y plegamientos de proteínas al interior del RE o bien la destrucción de aquellas que no completaron correctamente algunos de los procesos anteriores, de manera de evitar una pérdida de la capacidad de producción de proteínas vitales para la célula. Sin embargo, existen situaciones en las cuales la demanda de síntesis de proteínas supera significativamente la capacidad de síntesis, plegamiento y degradación de proteínas del RE, generándose una situación de estrés en el RE, debido a la acumulación de proteínas mal plegadas. Es durante este tipo de eventos que las células eucariontes activan un mecanismo de respuesta conocido como respuesta a la acumulación de proteínas mal plegadas o por sus siglas en inglés, UPR.
La respuesta a la acumulación de proteínas mal plegadas es un fenómeno que ha sido estudiado ampliamente en levaduras y eucariontes superiores. Un gen fundamental para este proceso fue descrito en levaduras como “inositol requiring enzyme 1” o IRE1. La proteína codificada por este gen, es capaz de procesar el ARN mensajero de un factor de transcripción (conjuntamente con una tARN ligasa), generando un nuevo ARN mensajero que codifica para un factor de transcripción activo, el cual puede regular la expresión de genes asociados a la síntesis, plegamiento y degradación de proteínas en el retículo endoplasmatico. En levaduras el gen, a partir del cual se genera este ARN mensajero se conoce como HAC1, mientras que en mamíferos, insectos y peces se conoce como XBP1. Sin embargo, en plantas se desconocía cual era el gen, a partir del cual se genera este ARN mensajero procesado por IRE1, a pesar de haber documentado con anterioridad la existencia de IRE1 en organismos vegetales.
En la presente tesis, se describe el gen que da lugar al ARN mensajero que es procesado por IRE1 en la planta modelo Arabidopsis thaliana y que da lugar a un factor de transcripción activo, capaz de regular la expresión de genes involucrados en la síntesis, plegamiento y degradación de proteínas en el RE. Este gen corresponde un factor de transcripción del tipo cremallera de leucina conocido como AtbZIP60. Además, se describe en que procesos fisiopatológicos de la planta, el procesamiento de este ARN mensajero tiene lugar, al mismo tiempo que se entrega evidencia sobre los efectos que tiene la ausencia de ambos genes (IRE1, AtbZIP60) en la respuesta de la planta frente a la infección por patógenos bacterianos.
Por otra parte, se entregan antecedentes asociados a otra función de IRE1, la cual es degradar de forma selectiva ARN mensajeros que codifican para proteínas que debiesen ser sintetizadas en el RE. En la literatura, este evento se ha descrito en insectos, mamíferos y recientemente en levaduras mas no existían datos sobre este proceso en plantas antes del inicio de este trabajo. Al analizar los cambios en el transcriptoma de plantas mutantes de Arabidopsis thaliana que carecen de IRE1 y al comparar estos datos con los obtenidos desde plantas silvestres, se observa que la acumulación de un grupo de transcritos que codifican para proteínas cuya síntesis ocurre en el RE, disminuye significativamente durante la activación de la respuesta a la acumulación de proteínas mal plegadas y esta disminución es dependiente de la presencia de IRE1.
En conclusión, este trabajo entrega nueva información acerca de los componentes de la respuesta a la acumulación de proteínas mal plegadas en plantas y sugiere que este mecanismo es importante para la respuesta de las plantas a su entorno. Adicionalmente, se ha incluido un nuevo capitulo donde se muestra evidencia que sustenta la existencia de una nueva variante de AtbZIP60, cuya síntesis es independiente de IRE1 y tendría un rol de en la regulación transcripcional bajo condiciones de estrés osmotico. Esta observación sugiere fuertemente que el factor de transcripción AtbZIP60 participaría de dos vías de señalización distintas, donde IRE1 solo regularía una de ella. Este tipo de doble función de un factor de transcripción cuyo ARN mensajero es blanco de IRE1, no ha sido descrito en ningún otro organismo eucarionte hasta la fecha.
Folding of newly synthesized proteins inside endoplasmic reticulum (ER) is a fundamental process for eukaryotic cells. It is assume that more than one third of all cell proteins are synthesized on this organelle. Several proteins participate in the synthesis, folding and degradation of proteins inside of the ER. Process as N-glycosylation, protein quality control and ER associated degradation of proteins take place specifically at the ER. All these processes and it is associated mechanism assure the correct synthesis and folding of proteins or the destruction of any corrupted protein. This strict control avoids the loss of proteins synthesis capabilities, an important cue since some of proteins synthesized at the ER are associated to important cell functions. Nevertheless, some situations demand a high synthesis of proteins overwhelming the ER capabilities. In these cases, misfolded or unfolded proteins start to accumulate inside the ER. To overcome this situation, the eukaryotic cells activate the unfolded protein response (UPR). The UPR is a process widely studied on model organism as baker’s yeast and other higher eukaryotes. A key player on this process is known as IRE1 (inositol reuiring enzyme 1) and it was describe in baker’s yeast for first time. IRE1 protein has the capacity to modify the messenger RNA of a transcription factor (in conjunction with a tRNA ligase). The newly formed messenger encodes an active transcription factor that can regulate the expression of several genes involved in the synthesis, folding and degradation of proteins at the ER. In baker’s yeast, the messenger RNA that is substrate of IRE1 is transcribed from the gene HAC1. In mammals, insects and fishes this gene is known as XBP1. In plants, before this thesis was realized, it was unknown if a substrate of IRE1 exists although the existence of IRE1 has been described long time ago. On this thesis, the gene that gives place to the messenger RNA which is substrate of IRE1 and encodes an active transcription factor after IRE1 action is described for the model plant, Arabidopsis thaliana. This gene belongs to the basic leucine zipper family of transcription factor and it is known as AtbZIP60. The active form of this gene can regulated the expression of several genes involved in the synthesis, folding and degradation of proteins at the ER. In addition, the physiological processes that lead to the processing or modification of AtbZIP60 transcript are studied. At the same time, the role of AtbZIP60 or IRE1 during bacterial pathogens challenge is analyzed using with loss-of-function mutants for each gene. Furthermore, this work gives insights about RNAse role of IRE1. It has been described that IRE1 can degrade transcripts encoding proteins that must be synthesized on the ER. The literature describes this event on insects, mammals and recently on yeasts but in plants no reports were found at early stages of this thesis. In order to understand if this process take place on plants, the changes in the transcriptome of mutant plants that lack IRE1 were compared to wild type plants under treatments than induce the UPR. It was observed that several transcripts were down-regulated in wild type plants but no changes were observed for the same transcripts on mutants suggesting that IRE1 is responsible for the observed down-regulation. In conclusion, this work provides new information about the genes involved on the UPR in plants and suggests that this mechanism is important for the plant response to the environment. Finally, a new chapter has been added to this thesis, were the role of the unspliced version of AtbZIP60 is explored. The results obtained strongly suggest that AtbZIP60 can regulate gene expression under osmotic stress conditions in an IRE1 independent manner. As far as it is known, this is the first report of dual functionality of a transcription factor which messenger ARN is target or substrate of IRE1.
Folding of newly synthesized proteins inside endoplasmic reticulum (ER) is a fundamental process for eukaryotic cells. It is assume that more than one third of all cell proteins are synthesized on this organelle. Several proteins participate in the synthesis, folding and degradation of proteins inside of the ER. Process as N-glycosylation, protein quality control and ER associated degradation of proteins take place specifically at the ER. All these processes and it is associated mechanism assure the correct synthesis and folding of proteins or the destruction of any corrupted protein. This strict control avoids the loss of proteins synthesis capabilities, an important cue since some of proteins synthesized at the ER are associated to important cell functions. Nevertheless, some situations demand a high synthesis of proteins overwhelming the ER capabilities. In these cases, misfolded or unfolded proteins start to accumulate inside the ER. To overcome this situation, the eukaryotic cells activate the unfolded protein response (UPR). The UPR is a process widely studied on model organism as baker’s yeast and other higher eukaryotes. A key player on this process is known as IRE1 (inositol reuiring enzyme 1) and it was describe in baker’s yeast for first time. IRE1 protein has the capacity to modify the messenger RNA of a transcription factor (in conjunction with a tRNA ligase). The newly formed messenger encodes an active transcription factor that can regulate the expression of several genes involved in the synthesis, folding and degradation of proteins at the ER. In baker’s yeast, the messenger RNA that is substrate of IRE1 is transcribed from the gene HAC1. In mammals, insects and fishes this gene is known as XBP1. In plants, before this thesis was realized, it was unknown if a substrate of IRE1 exists although the existence of IRE1 has been described long time ago. On this thesis, the gene that gives place to the messenger RNA which is substrate of IRE1 and encodes an active transcription factor after IRE1 action is described for the model plant, Arabidopsis thaliana. This gene belongs to the basic leucine zipper family of transcription factor and it is known as AtbZIP60. The active form of this gene can regulated the expression of several genes involved in the synthesis, folding and degradation of proteins at the ER. In addition, the physiological processes that lead to the processing or modification of AtbZIP60 transcript are studied. At the same time, the role of AtbZIP60 or IRE1 during bacterial pathogens challenge is analyzed using with loss-of-function mutants for each gene. Furthermore, this work gives insights about RNAse role of IRE1. It has been described that IRE1 can degrade transcripts encoding proteins that must be synthesized on the ER. The literature describes this event on insects, mammals and recently on yeasts but in plants no reports were found at early stages of this thesis. In order to understand if this process take place on plants, the changes in the transcriptome of mutant plants that lack IRE1 were compared to wild type plants under treatments than induce the UPR. It was observed that several transcripts were down-regulated in wild type plants but no changes were observed for the same transcripts on mutants suggesting that IRE1 is responsible for the observed down-regulation. In conclusion, this work provides new information about the genes involved on the UPR in plants and suggests that this mechanism is important for the plant response to the environment. Finally, a new chapter has been added to this thesis, were the role of the unspliced version of AtbZIP60 is explored. The results obtained strongly suggest that AtbZIP60 can regulate gene expression under osmotic stress conditions in an IRE1 independent manner. As far as it is known, this is the first report of dual functionality of a transcription factor which messenger ARN is target or substrate of IRE1.
Notas
Tesis (Doctor en Biotecnología)
Palabras clave
Arabidopsis Thaliana, Proteínas, ARN Mensajero