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Tesis Doctoral Bases estructurales del transporte de Mg2+ mediado por los factores homeostáticos CNNM2 y CNNM4, y de su interacción oncogénica con las fosfatasas PRL. Paula Giménez Mascarell 2019 Tesis dirigida por el Dr. L. Alfonso Martínez de la Cruz (c)2019 PAULA GIMENEZ MASCARELL Tabla de contenido Listado de abreviaturas....................................................................................................... 1 Resumen .............................................................................................................................. 7 1. Introducción ............................................................................................................... 11 1.1. HOMEOSTASIS ..................................................................................................... 13 1.2. TRANSPORTE DE IONES Y MOLÉCULAS A TRAVÉS DE LAS MEMBRANAS CELULARES: Canales y transportadores ................................................................................................ 13 1.3. EL MAGNESIO ...................................................................................................... 16 1.3.1. Homeostasis del Mg2+ en células procariotas. ............................................................................... 17 1.3.1.1. Moléculas que median el transporte de Mg2+ en células procariotas ................................. 17 1.3.2. Homeostasis del Mg2+ en organismos eucariotas. ........................................................................ 23 1.4. FACTORES HOMEOSTÁTICOS CNNM. .................................................................... 35 1.4.1. Estructura tridimensional de los factores homeostáticos CNNMs. ........................................... 40 1.4.2. Ligandos y moléculas que interaccionan con los CNNMs. ........................................................... 44 1.4.3. Macromoléculas que interactúan con los CNNMs: PRLs ............................................................. 47 2. Objetivos .................................................................................................................... 51 3. Materiales y métodos ............................................................................................... 55 3.1. PRODUCCIÓN DE PROTEÍNAS RECOMBINANTES.................................................... 57 3.1.1. Sobreexpresión y purificación del constructo CNNM2BAT............................................................ 57 3.1.2. Sobreexpresión y purificación del constructo CNNM2cNMP ......................................................... 59 3.1.3. Sobreexpresión y purificación de la fosfatasa PRL-1. ................................................................... 60 3.1.4. Purificación del complejo CNNM2BAT·PRL-1 ................................................................................... 61 3.1.5. Sobreexpresión y purificación de CNNM4BAT ................................................................................. 62 3.1.6. Sobreexpresión y purificación de CNNM4cNMP............................................................................... 63 3.1.7. Producción de CNNM4cNMP marcada con Selenio-Metionina ..................................................... 64 3.1.8. Sobreexpresión y purificación de la región intracelular de CNNM4 (CNNM4BAT-cNMP-Ctail) 65 3.1.9. Producción de mutantes CNNM4cNMP y CNNM4BAT-cNMP-Ctail F631A ............................................. 65 3.1.10. Producción del complejo CNNM4BAT-cNMP-Ctail·PRL-1................................................................. 66 3.2. TÉCNICAS COMPLEMENTARIAS EN LA PURIFICACIÓN DE PROTEÍNA....................... 67 3.2.1. Electroforesis en gel de acrilamida .................................................................................................. 67 3.2.2. Determinación de la concentración de proteína........................................................................... 67 3.2.3. Determinación de parámetros estándar de proteínas ................................................................. 68 3.3. CRISTALIZACIÓN DE PROTEÍNAS ........................................................................... 69 3.3.1. Cristalización del complejo CNNM2BAT·PRL-1................................................................................. 69 3.3.2. Cristalización de CNNM4BAT............................................................................................................... 71 3.3.3. Cristalización de CNNM4cNMP ............................................................................................................ 71 3.4. RESOLUCIÓN ESTRUCTURAL POR TÉCNICAS CRISTALOGRÁFICAS DE RAYOS X. ....... 72 3.4.1. Resolución estructural del complejo CNNM2BAT·PRL-1. ............................................................... 72 3.4.2. Resolución estructural de CNNM4BAT .............................................................................................. 74 3.4.3. Resolución estructural de CNNM4cNMP............................................................................................ 74 3.5. DISPERSIÓN DE RAYOS X A BAJO ÁNGULO (SAXS) ................................................. 75 3.6. DISPERSIÓN DE LUZ MULTIÁNGULO (MALS).......................................................... 76 4. Resultados ................................................................................................................. 79 4.1. DISEÑO DE CONSTRUCTOS DE LAS PROTEÍNAS CNNM ........................................... 81 Capítulo 1. Estructura de CNNM4 ..................................................................................... 85 4.2. MÓDULO BATEMAN DE CNNM4 (CNNM4BAT) ........................................................ 87 4.2.1. Estructura cristalina de CNNM4BAT................................................................................................... 88 4.3. ESTRUCTURA DEL DOMINIO CNMP. ...................................................................... 99 4.3.1. Sobreexpresión y purificación del dominio cNMP de CNNM2 y CNNM4. .............................. 100 4.3.2. Cristalización del dominio cNMP.................................................................................................... 101 4.3.3. Estudio cristalográfico preliminar de CNNM4cNMP....................................................................... 103 4.3.4. Estructura cristalina de CNNM4cNMP. ............................................................................................. 105 4.4. ESTRUCTURA DE LA REGIÓN INTRACELULAR DE LAS PROTEÍNAS CNNM. ...............116 4.4.1. Estructura en solución de la región intracelular de CNNM4 ..................................................... 117 4.4.2. Función estructural del dominio cNMP de CNNM4 .................................................................... 123 Capítulo 2. Bases estructurales de la interacción oncogénica de los CNNMs con las fosfatasas de la familia PRL. ........................................................................................... 125 4.5. CNNM2BAT INTERACCIONA CON LAS PROTEÍNAS PRLS...........................................127 4.6. COMPLEJO CNNM2BAT ·PRL-1. ..............................................................................128 4.6.1. Cristalización del complejo CNNM2BAT ·PRL-1. ............................................................................. 128 4.6.2. Estructura cristalina del complejo CNNM2BAT·PRL-1. ................................................................. 130 4.6.3. CNNM2cNMP no interacciona con CNNM2BAT y PRL-1. ................................................................. 134 4.6.4. La unión de PRL-1 aplana el módulo CBS de CNNM2. ................................................................ 135 4.6.5. La unión de ATP no varía conformacionalmente el complejo CNNM2 BAT·PRL-1. .................. 137 4.7. ESTRUCTURA DEL COMPLEJO CNNM4BAT-CNMP-CTAIL CON PRL-1. ...............................139 5. Discusión .................................................................................................................. 143 6. Conclusiones ............................................................................................................ 161 7. Bibliografía .............................................................................................................. 165 8. Publicaciones ........................................................................................................... 187 Listado de abreviaturas 1 2 Abreviaturas Listado de abreviaturas La siguiente lista describe el significado de diferentes abreviaturas y acrónimos utilizados a lo largo del texto de esta tesis. ACD: “Ancient Conserved Domain” (Dominio ancestral conservado) ACDP: “Ancient Conserved Domain Protein” (Proteína de dominio ancestral conservado) ADN: Ácido desoxirribonucleico ADP: Adenosín difosfato ADPnP: Adenosina 5’-(β, γ-imido) trifosfato AMP: Adenosín monofosfato AMPc: Adenosín monofosfato cíclico AP: “Adaptor protein” Proteína adaptadora de clatrina ARN: Ácido ribonucleico ATP: Adenosín trifosfato A.U: “Asymmetric Unit” (Unidad Asimétrica) BSA: “Bovine serum albumin” (Albúmina de suero bovina) CBS: Cistationina β-sintasa CDK: “Cyclin-dependent kinase” (Proteína kinasa dependiente de ciclina) CNBD: “Cyclic nucleotide binding domain” (Dominio de unión a nucleótidos cíclicos) CNGC: “Cyclic Nucleotide Gated Channel” (Canal regulado por nucleótidos cíclicos) cNMP: “Cyclic Nucleotide MonoPhosphate” (Nucleótido cíclico monofosfato) CNNM: “Cyclin and CBS domain divalent metal cation transport mediators”
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