Evaluación in vitro de la interacción del receptor de Vitamina D3 con Proteínas Sumo
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2016
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es
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Universidad Andrés Bello
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Resumen
La acción de Vitamina D3 está relacionada principalmente con la modulación transcripcional mediante la interacción con el receptor de la Vitamina D3 (VDR). VDR se ha asociado a diferentes vías de regulación de los procesos de desarrollo, reproductivos, metabólicos e inflamatorios. Existen evidencias de que VDR recluta diferentes complejos correguladores, generando diferentes respuestas en la regulación transcripcional de un mismo gen. En la actualidad, el mecanismo de cómo ocurre el reclutamiento de estos correguladores aún es desconocido. Se consideró la posibilidad de que VDR interactúe con correguladores que poseen modificaciones postraduccionales como proteínas SUMO (small ubiquitin-like modifier). Estudios preliminares realizados en el Centro de Investigaciones Biomédicas de la Universidad Andrés Bello permitieron definir que VDR poseería motivos de interacción a SUMO (SIM, por Sumo interaction motif) en el dominio de unión a ligando (LBD) y se demostró la interacción entre VDR y SUMO-1 in vitro. Por lo tanto, la SUMOilación de correguladores podría estar posibilitando su interacción con VDR. Por lo anterior, en esta Tesis se determinó si las proteínas SUMO se unen a VDR mediante los motivos de interacción SUMO (SIM) descritos bioinformáticamente.
Como principal objetivo, se evaluó la interacción del receptor de Vitamina D3 (VDR) y las proteínas SUMO-2 y SUMO-3 durante la respuesta a Vitamina D3. Se demostró esta interacción, mediante la generación de proteínas recombinantes de SUMO-2 y SUMO-3 con GST, el cual se obtuvo a través del subclonamiento de la secuencia de estos genes en el vector pGEX5X3, y posteriormente se realizó la sobreexpresión y purificación de GST-SUM02 y GST-SUM03 por incubación con la resina glutatión-sefarosa 4B. Finalmente. Se efectuaron ensayos de interacción proteína-proteína de VDR con GST-SUM02 y GST-SUM03 mediante GST-pulldown. Como segundo objetivo, se definieron cuáles son los motivos SIM involucrados en la interacción entre VDR y las proteínas SUMO-1, SUMO-2 y SUMO-3 durante la respuesta a Vitamina D3 in vitro. Se realizaron mutaciones sitio-dirigidas en tres motivos SIM, luego se hicieron transfecciones de las versiones mutantes de VDR en células HEK293FT. Posteriormente, se efectuaron ensayos de interacción proteína-proteína de las versiones mutantes de VDR con GST-SUMOl, GST-SUM02 y GST-SUM03 mediante GST -pulldown. En su conjunto estos análisis, hacen posible proponer un mecanismo por el cual VDR recluta diferentes complejos correguladores durante la regulación transcripcional de un gen en respuesta a Vitamina D3.
The action of vitamin D3 is mainly related with transcriptional modulation through interacting with the vitamin D3 receptor (VDR). VDR has been associated with different pathways that regúlate developmental, reproductive, metabolic and inflammatory processes. There is evidence that VDR recruits different coregulators complexes, generating different responses in the transcriptional regulation of the same gene. At present, the mechanism of how these coregulator complex recruitment occurs is still unknown. The possibility that VDR interacts with coregulators which have some posttranslational modifícations such as SUMO proteins (small ubiquitin-like modifier) was considered. Preliminary studies at the Center for Biomedical Research at the University Andrés Bello showed that VDR possess SUMO-interacting motifs (SIM) in the ligand binding domain (LBD) and an interaction was found between VDR and SUMO-1 in vitro. Therefore, SUMOylation of coregulators might make the interaction with VDR possible. Therefore, in this thesis it was determined whether the SUMO proteins bind to the VDR by SUMO interaction motifs (SIM) described for bioinformatics analysis. The first objective was to evalúate the interaction of Vitamin D3 receptor (VDR) and SUMO-2 and SUMO-3 during the response to Vitamin D3. This interaction was demonstrated by generating recombinant protein of SUMO-2 and SUMO-3 with GST, which was obtained through the subcloning of the sequence of these genes in the pGEX5X3 vector, and subsequently overexpression and purifícation of GST and GST-SUM02-SUM03 was performed by incubation with sepharose 4B glutathione resin. Finally, protein-protein interaction assays of VDR with GST- SUM02 and GST- SUM03 were made by GST-pulldown. The second objective was to defíne what motif SIM are involved in the interaction between VDR and the proteins SUMO-1, SUMO- 2 and SUMO-3 during the response to vitamin D3 in vitro. Site-directed mutations were made in three SIMs, then transfections of the mutant versions of VDR in HEK293FT cells were done. Subsequently, protein-protein interaction assays of mutant versions of VDR with GST-SUMOl, GST-SUM02 and GST-SUM03 using GST -pulldown were done. These experiments make it possible to propose a mechanism by which VDR recruits different coregulatory complexes during the transcriptional regulation of a gene in response to vitamin D3.
The action of vitamin D3 is mainly related with transcriptional modulation through interacting with the vitamin D3 receptor (VDR). VDR has been associated with different pathways that regúlate developmental, reproductive, metabolic and inflammatory processes. There is evidence that VDR recruits different coregulators complexes, generating different responses in the transcriptional regulation of the same gene. At present, the mechanism of how these coregulator complex recruitment occurs is still unknown. The possibility that VDR interacts with coregulators which have some posttranslational modifícations such as SUMO proteins (small ubiquitin-like modifier) was considered. Preliminary studies at the Center for Biomedical Research at the University Andrés Bello showed that VDR possess SUMO-interacting motifs (SIM) in the ligand binding domain (LBD) and an interaction was found between VDR and SUMO-1 in vitro. Therefore, SUMOylation of coregulators might make the interaction with VDR possible. Therefore, in this thesis it was determined whether the SUMO proteins bind to the VDR by SUMO interaction motifs (SIM) described for bioinformatics analysis. The first objective was to evalúate the interaction of Vitamin D3 receptor (VDR) and SUMO-2 and SUMO-3 during the response to Vitamin D3. This interaction was demonstrated by generating recombinant protein of SUMO-2 and SUMO-3 with GST, which was obtained through the subcloning of the sequence of these genes in the pGEX5X3 vector, and subsequently overexpression and purifícation of GST and GST-SUM02-SUM03 was performed by incubation with sepharose 4B glutathione resin. Finally, protein-protein interaction assays of VDR with GST- SUM02 and GST- SUM03 were made by GST-pulldown. The second objective was to defíne what motif SIM are involved in the interaction between VDR and the proteins SUMO-1, SUMO- 2 and SUMO-3 during the response to vitamin D3 in vitro. Site-directed mutations were made in three SIMs, then transfections of the mutant versions of VDR in HEK293FT cells were done. Subsequently, protein-protein interaction assays of mutant versions of VDR with GST-SUMOl, GST-SUM02 and GST-SUM03 using GST -pulldown were done. These experiments make it possible to propose a mechanism by which VDR recruits different coregulatory complexes during the transcriptional regulation of a gene in response to vitamin D3.
Notas
(Tesis) Magíster en Biotecnología
Palabras clave
Vitaminas, Uso Terapéutico, Chile, Viña del Mar, Región de Valpararaíso, Proteínas, Biotecnología