RESUMEN
The processes by which cells sense and respond to ambient oxygen concentration are fundamental to cell survival and function, and they commonly target gene regulatory events. To date, however, little is known about the link between the microRNA pathway and hypoxia signaling. Here, we show in vitro and in vivo that chronic hypoxia impairs Dicer (DICER1) expression and activity, resulting in global consequences on microRNA biogenesis. We show that von Hippel-Lindau-dependent down-regulation of Dicer is key to the expression and function of hypoxia-inducible factor α (HIF-α) subunits. Specifically, we show that EPAS1/HIF-2α is regulated by the Dicer-dependent microRNA miR-185, which is down-regulated by hypoxia. Full expression of hypoxia-responsive/HIF target genes in chronic hypoxia (e.g. VEGFA, FLT1/VEGFR1, KDR/VEGFR2, BNIP3L, and SLC2A1/GLUT1), the function of which is to regulate various adaptive responses to compromised oxygen availability, is also dependent on hypoxia-mediated down-regulation of Dicer function and changes in post-transcriptional gene regulation. Therefore, functional deficiency of Dicer in chronic hypoxia is relevant to both HIF-α isoforms and hypoxia-responsive/HIF target genes, especially in the vascular endothelium. These findings have relevance to emerging therapies given that we show that the efficacy of RNA interference under chronic hypoxia, but not normal oxygen availability, is Dicer-dependent. Collectively, these findings show that the down-regulation of Dicer under chronic hypoxia is an adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, thereby providing an essential mechanistic insight into the oxygen-dependent microRNA regulatory pathway.
Asunto(s)
Adaptación Fisiológica/fisiología , ARN Helicasas DEAD-box/biosíntesis , Endotelio Vascular/enzimología , Regulación Enzimológica de la Expresión Génica/fisiología , Oxígeno/metabolismo , Ribonucleasa III/biosíntesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Hipoxia de la Célula , ARN Helicasas DEAD-box/genética , Endotelio Vascular/citología , Transportador de Glucosa de Tipo 1/biosíntesis , Transportador de Glucosa de Tipo 1/genética , Células Hep G2 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , MicroARNs/biosíntesis , MicroARNs/genética , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Ribonucleasa III/genética , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genética , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
Nucleoside transporters (NTs) are integral membrane proteins necessary for the cellular entry of nucleoside analog drugs used in chemotherapeutic treatment of conditions such as cancer and viral or parasitic infections. NTs are also the targets of certain drugs used in the treatment of various cardiovascular conditions. Because of the importance of NTs in drug uptake, determination of the three-dimensional structure of these proteins, particularly hENT1, has the potential to improve these treatments through structure-based design of more specifically targeted and transported drugs. In this paper, we use NMR spectroscopy to investigate the structure of the large intracellular loop between transmembrane domains 6 and 7 and we also describe a method for the successful overexpression of full-length hENT1 in a bacterial system. Recombinant tandem histidine-affinity (HAT) and 3×FLAG tagged hENT1 was overexpressed in E. coli, affinity purified, and functionally characterized by nitrobenzylthioinosine (NBTI) binding. Anti-3×FLAG immunodetection confirmed the expression of N-HAT-3×FLAG-hENT1, while increased NBTI binding (3.2-fold compared with controls) confirmed the conformational integrity of the recombinant hENT1 within the bacterial inner membrane. Yields of recombinant hENT1 using this approach were ~15 µg/L of bacterial culture and this approach provides a basis for large-scale production of protein for a variety of purposes.
Asunto(s)
Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Escherichia coli/metabolismo , Secuencia de Aminoácidos , Animales , Tranportador Equilibrativo 1 de Nucleósido/química , Tranportador Equilibrativo 1 de Nucleósido/genética , Escherichia coli/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Equilibrative nucleoside transporters (ENTs) are integral membrane proteins that facilitate the movement of nucleosides and hydrophilic nucleoside analog (NA) drugs across cell membranes. ENTs are also targets for cardioprotectant drugs, which block re-uptake of the purine nucleoside adenosine, thereby enhancing purinergic receptor signaling pathways. ENTs are therefore important contributors to drug bioavailability and efficacy. Despite this important clinical role, very little is known about the structure and regulation of ENTs. Biochemical and structural studies on ENT proteins have been limited by their low endogenous expression levels, hydrophobicity and labile nature. To address these issues, we developed an approach whereby tagged mammalian ENT1 protein was over-expressed in mammalian cell lines, confirmed to be functional and isolated by affinity purification to sufficient levels to be analyzed using MALDI-TOF and tandem MS mass spectrometry. This proteomic approach will allow for a more detailed analysis of the structure, function and regulation of ENTs in the future.