RESUMEN
Ca2+ dynamics and oxidative signaling are fundamental mechanisms for mitochondrial bioenergetics and cell function. The MCU complex is the major pathway by which these signals are integrated in mitochondria. Whether and how these coactive elements interact with MCU have not been established. As an approach toward understanding the regulation of MCU channel by oxidative milieu, we adapted inflammatory and hypoxia models. We identified the conserved cysteine 97 (Cys-97) to be the only reactive thiol in human MCU that undergoes S-glutathionylation. Furthermore, biochemical, structural, and superresolution imaging analysis revealed that MCU oxidation promotes MCU higher order oligomer formation. Both oxidation and mutation of MCU Cys-97 exhibited persistent MCU channel activity with higher [Ca2+]m uptake rate, elevated mROS, and enhanced [Ca2+]m overload-induced cell death. In contrast, these effects were largely independent of MCU interaction with its regulators. These findings reveal a distinct functional role for Cys-97 in ROS sensing and regulation of MCU activity.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio , Calcio/metabolismo , Células Endoteliales/metabolismo , Activación del Canal Iónico , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Células COS , Canales de Calcio/química , Canales de Calcio/genética , Señalización del Calcio/efectos de los fármacos , Muerte Celular , Hipoxia de la Célula , Chlorocebus aethiops , Cisteína , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Metabolismo Energético , Glutatión/metabolismo , Células HEK293 , Células HeLa , Humanos , Activación del Canal Iónico/efectos de los fármacos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/patología , Mutación , Oxidación-Reducción , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Estructura Cuaternaria de Proteína , Relación Estructura-Actividad , Trombina/farmacología , Factores de Tiempo , TransfecciónRESUMEN
BACKGROUND: Prostate cancer (PCa) poses a major health concern in men worldwide. Retinoic Acid Receptor Responder (RARRES1)/ Tazarotene-induced gene-1 (TIG-1) is a putative tumor suppressor gene that exerts its tumor suppressor function via unknown mechanisms. Epigenetic silencing of RARRES1 leads to its loss in several types of cancer, including PCa. Determining the molecular mechanisms that mediate the tumor suppressor role of RARRES1 in PCa is the focus of our study. FINDINGS: Our data indicates that RARRES1 over expression in PCa cell lines represses mitogen-activated protein kinase (MAPK) activation. RARRES1 expression induces the levels of autophagy-related genes, beclin, ATG3 and increases LC3B-II conversion. A significant induction of SIRT1 along with mTOR inhibition is noted on RARRES1 expression. Furthermore, RARRES1 over expression elevates the levels of the antioxidant enzyme, catalase. Our results also indicate that RARRES1 expression inhibits angiogenesis in endothelial cells. CONCLUSIONS: In summary, the data presented here indicate that forced expression of RARRES1 in PCa cells (a) induces ER stress and autophagic response; (b) increases SIRT1 levels; and (c) higher levels of anti-oxidant enzymes. Our study also implicates the role of RARRES1 as a novel anti-angiogenic molecule. Overall this study reports the molecular players that RARRES1 modulates to serve as a tumor suppressor molecule. Future studies will help determine the in vivo mechanisms by which RARRES1 may serve as a target for therapeutic intervention both in cancer and in angiogenesis-related disorders.