Estudio de la respuesta a proteínas mal plegadas (UPR) en mitocondrias
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Fecha
2013
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es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Hoy en día existen muchas enfermedades que no tienen una cura defmitiva y que aún se
desconocen las causas o eventos que las provocan, como el cáncer. Algunas investigaciones
han determinado posibles causas relacionadas con fallas en el funcionamiento de las
mitocondrias, debido a la generación de especies reactivas de oxígeno (ROS) o por una
desregulación de factores de sobrevida y muerte celular. Recientemente se ha descrito en C.
elegans y cultivos celulares una posible vía de señalización en respuesta a proteínas mal
plegadas en la mitocondria (mtUPR), similar a la que existe en retículo endoplásmico
( erUPR). Esta respuesta podría estar involucrada en enfermedades como el cáncer, ya que se
ha descrito que drogas que afectan la actividad de chaperonas mitocondriales, así como la
inhibición de algunos componentes de mtUPR, disminuyen la viabilidad de células tumorales
in vivo. Sin embargo, sólo se han descrito algunos posibles participantes del mtUPR y se
desconoce la vía completa implicada en la respuesta. Más aún, no ha sido demostrado que una
efectiva acumulación de proteínas en la mitocondria promueva la muerte celular. El objetivo
de este trabajo fue estudiar de qué manera responden los componentes de mtUPR frente a
diversos tipos de estrés mitocondriales y si la acumulación de proteínas en la mitocondria es
capaz de afectar la viabilidad celular. Se realizaron ensayos de inducción de estrés
mitocondrial mediante la sobre expresión de una forma mutante de la enzima de matriz
mitocondrial Ornitina Transcarbamilasa (OTC-.-1), o la aplicación de las drogas A23187
(ionóforo de calcio que afecta el potencial de membrana mitocondrial) y rotenona (inhibidor
del complejo I que induce formación de ROS). Se observó que frente a la acumulación de
OTC-.-1 no habría una inducción de la expresión de HSP60, ClpP ni NDUFB2 (indicadores de
mtUPR). Sin embargo, se determinó que esta acumulación tendría un efecto negativo sobre la
viabilidad celular a las 48 h post transfección y también sería capaz de inducir la expresión de
la chaperona BiP (marcador de erUPR) en etapas tempranas (24 h). Por otro lado, se observó
que la expresión de HSP60 aumenta rápidamente tras la aplicación de las drogas A23187 o
rotenona, en forma dependiente de la concentración. Estos resultados indican que la
acumulación de proteínas mal plegadas en la mitocondria tendría un efecto negativo sobre la
viabilidad celular y sería capaz de inducir otras vías como el erUPR, lo cual no ha sido
previamente descrito. Aunque no se observó un aumento de HSP60 frente a este estímulo, sí
presentó una rápida respuesta frente al estrés mitocondrial causado por A23187 y rotenona,
sugiriendo que mtUPR podría responder ente a otros tipos de estrés mitocondrial.
Nota: resumen extraído de un programa de reconocimiento de texto, fórmulas pueden contener inexactitudes, consulte el texto original.
Evidence suggests a connection between the malfunction of mitochondria and the development of cancer and other pathologies possibly due to an overproduction of Reactive Oxygen Species (ROS) or misregulation of cell survival and death factors. Recently it was reported that similarly to the unfolded protein response in the endoplasmic reticulum ( erUPR), C. elegans and mammalian cells contain a pathway that responds to an accumulation of unfolded proteins in mitochondria (mtUPR). This pathway may provide new druggable targets since researchers have demonstrated that the inhibition of so me components of mtUPR and mitochondrial chaperones reduces the viability of cancerous cells both in vivo and in vitro. However, this pathway, particularly in mammalian cells, has not been fully described and few components are known. Additionally, it has not been demonstrated that the accumulation of unfolded proteins in mitochondria induces cell death. This work aims to study how particular components of mtUPR respond to different mitochondrial insults and to determine if mtUPR could affect cell viability. In order to do this we induced mitochondrial stress by the overexpression of a mutated form of the mitochondrial matrix enzyme Omithine Transcarbamylase (OTC-il) or by the application of the drugs A23187, a calcium ionophore that depolarizes the mitochondrial membrane, or rotenone, an inhibitor of Complex I that induces the production of ROS. We found that upon the over-expression ofOTC-il, the expression ofthe mtUPR markers HSP60, ClpP and NDUFB2 was not induced. The over-expression of OTC-L\ did have a negative effect over cell viability at 48h post-transfection. Surprisingly we observed a previously unreported induction of BiP (erUPR marker) at early stages of the response (24 h posttransfection). We found that treatrnent with either A23187 or Rotenone induced a concentration dependent, fast induction ofHPS60. These results suggest that the accumulation of unfolded proteins in the mitochondria have a negative effect on cell viability and suggest a possible connection with the erUPR. Although we did not see an increase in expression of HSP60 upon induction of OTC-L\, we observed a quick increase in levels of this chaperon upon treatment with A23187 and rotenone, suggesting that the mtUPR or its components could be participating in other stress response pathways.
Evidence suggests a connection between the malfunction of mitochondria and the development of cancer and other pathologies possibly due to an overproduction of Reactive Oxygen Species (ROS) or misregulation of cell survival and death factors. Recently it was reported that similarly to the unfolded protein response in the endoplasmic reticulum ( erUPR), C. elegans and mammalian cells contain a pathway that responds to an accumulation of unfolded proteins in mitochondria (mtUPR). This pathway may provide new druggable targets since researchers have demonstrated that the inhibition of so me components of mtUPR and mitochondrial chaperones reduces the viability of cancerous cells both in vivo and in vitro. However, this pathway, particularly in mammalian cells, has not been fully described and few components are known. Additionally, it has not been demonstrated that the accumulation of unfolded proteins in mitochondria induces cell death. This work aims to study how particular components of mtUPR respond to different mitochondrial insults and to determine if mtUPR could affect cell viability. In order to do this we induced mitochondrial stress by the overexpression of a mutated form of the mitochondrial matrix enzyme Omithine Transcarbamylase (OTC-il) or by the application of the drugs A23187, a calcium ionophore that depolarizes the mitochondrial membrane, or rotenone, an inhibitor of Complex I that induces the production of ROS. We found that upon the over-expression ofOTC-il, the expression ofthe mtUPR markers HSP60, ClpP and NDUFB2 was not induced. The over-expression of OTC-L\ did have a negative effect over cell viability at 48h post-transfection. Surprisingly we observed a previously unreported induction of BiP (erUPR marker) at early stages of the response (24 h posttransfection). We found that treatrnent with either A23187 or Rotenone induced a concentration dependent, fast induction ofHPS60. These results suggest that the accumulation of unfolded proteins in the mitochondria have a negative effect on cell viability and suggest a possible connection with the erUPR. Although we did not see an increase in expression of HSP60 upon induction of OTC-L\, we observed a quick increase in levels of this chaperon upon treatment with A23187 and rotenone, suggesting that the mtUPR or its components could be participating in other stress response pathways.
Notas
Tesis (Magister en Biotecnología)
Palabras clave
Proteínas, Muerte Celular