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Caracterización Proteómica De La Interacción Y La Inserción De Proteínas De Membrana

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Caracterización Proteómica De La Interacción Y La Inserción De Proteínas De Membrana TESIS DOCTORAL Valencia, Octubre 2019 Doctorado en Biomedicina y Biotecnología Caracterización proteómica de la interacción y la inserción de proteínas de membrana Dirigida por: Dr. Manuel Mateo Sánchez del Pino Dr. Luis Martínez Gil Doctorado en Biomedicina y Biotecnología Departamento de Bioquímica y Biología Molecular Facultad de Biología, Campus Burjassot Valencia, Octubre 2019 Caracterización proteómica de la interacción y la inserción de proteínas de membrana Memoria realizada por Dª Natalia Mara Vera Velasco para optar al Grado de Doctor por la Universitat de València Dirigida por: Dr. Manuel Mateo Sánchez del Pino Dr. Luis Martínez Gil OBJETIVOS • Objetivo 1 Estudio del interactoma asociado a la inserción de proteínas de membrana. • Objetivo 2 Estudio de las proteínas humanas incluidas en el virión del virus Nipah. 1 2 Objetivo 1 Estudio del interactoma asociado a la inserción de proteínas de membrana. 3 4 CONTENIDOS OBJETIVO 1 OBJETIVOS ................................................................................................................................ 1 ABREVIATURAS ....................................................................................................................... 7 INTRODUCCIÓN ....................................................................................................................... 9 I1.1 Componentes lipídicos ..................................................................................................... 12 I1.2 Componentes proteicos .................................................................................................... 12 I2. Direccionamiento de las proteínas de membrana .................................................................. 14 I2.1 Mecanismo de inserción cotraduccional .......................................................................... 15 I2.2 Mecanismo de inserción postraduccional ........................................................................ 17 I3. Orientación de las proteínas en la membrana ........................................................................ 21 I4. Interacción proteína-proteína ................................................................................................. 23 I5. Espectrometría de masas ........................................................................................................ 25 I5. 1 Tipos de adquisición de los espectros de fragmentación ................................................ 28 I5.2 Cuantificación de proteínas .............................................................................................. 30 MATERIALES Y MÉTODOS ................................................................................................. 35 M1. Biología molecular y cultivos .............................................................................................. 37 M1.1 Descripción de las quimeras TM y ΔTM ....................................................................... 37 M1.2 Expresión de proteínas ................................................................................................... 37 M1.2.1 Quimeras TM y ΔTM en E. coli BL21 pLysS ........................................................ 37 M1.2.2 Proteínas HslU y MetH en la librería ASKA .......................................................... 38 M1.3 PCR en tiempo real (qPCR) .......................................................................................... 39 M2. Métodos bioquímicos........................................................................................................... 41 M2.1 Purificación de proteínas ............................................................................................... 41 M2.2 Electroforesis SDS-PAGE ............................................................................................. 42 M2.3 Estabilidad de las proteínas quiméricas TM y ΔTM ..................................................... 43 M2.4 Fraccionamiento subcelular ........................................................................................... 44 M2.5 Ensayos de purificación del interactoma ....................................................................... 45 M2.6 Electroforesis de dos dimensiones ................................................................................. 46 M2.7 Análisis de proteína total ............................................................................................... 48 M2.8 Medidas de la actividad ATPasa de HslU ..................................................................... 49 M2.9 Cromatografía de exclusión molecular .......................................................................... 50 M2.10 Interacción proteína-proteína por dispersión de luz..................................................... 50 M2.11 Análisis de entrecruzamiento con proteína purificada ................................................. 51 M2.12 Construcción de la biblioteca de espectros de masas .................................................. 53 5 M3. Proteómica ........................................................................................................................... 54 M3.1 Cromatografía líquida de péptidos (nanoHPLC) ........................................................... 54 M3.2 Espectrometría de masas DDA o shotgun ..................................................................... 54 M3.3 Espectrometría de masas DIA o SWATH ..................................................................... 55 M3.4 Identificación de proteínas ............................................................................................. 56 M3.5 Cuantificación de proteínas por espectrometría de masas: Top3 y SWATH. ............... 56 M3.5.1 Técnica del top3 ...................................................................................................... 56 M3.5.2 SWATH .................................................................................................................. 56 RESULTADOS .......................................................................................................................... 59 R1. Análisis de las quimeras ....................................................................................................... 61 R1.1 Expresión de las proteínas quiméricas ............................................................................ 61 R1.2 Estabilidad de las proteínas quiméricas .......................................................................... 62 R1.3 Análisis de la inserción de las quimeras TM y ΔTM en la membrana ........................... 64 R2. Identificación de las proteínas asociadas a las quimeras ...................................................... 65 R2.1 Ensayos de purificación del interactoma ........................................................................ 65 R2.2 Electroforesis de dos dimensiones .................................................................................. 68 R3. Análisis de proteína total ...................................................................................................... 71 R4. PCR en tiempo real (qPCR).................................................................................................. 73 R5. Análisis de la interacción entre TM con las proteínas HslU y MetH ................................... 74 R5.1 Ensayos funcionales ....................................................................................................... 74 R5.2.1 Análisis de la actividad ATPasa .............................................................................. 75 R5.2.2 Análisis de la interacción mediante luz dispersada Rayleigh .................................. 75 R5.2.3 Análisis de la interacción mediante cromatografía de filtración en gel ................... 77 R5.2 Análisis de entrecruzamiento con proteína purificada .................................................... 80 DISCUSIÓN ............................................................................................................................... 85 CONCLUSIONES ..................................................................................................................... 93 6 ABREVIATURAS AAA - ATPases associated to a variety of cellular activities AdoMet - Adenosilmetionina AF – Ácido fórmico APEX - Absolute Protein Expresion BiP - Immunoglobulin heavy chain Binding Protein CAML – Calcium Modulating cyclophilin Ligand CAN - Acetonitrilo DDA - Data-Dependent Acquisition DIA - Data-Independent Acquisition DTSSP - 3,3'-dithiobis sulfosuccinimidyl propionate DTT - Dithiothreitol emPAI - exponentially modified Protein Abundance Index ER - Endoplasmic Reticulum ESI - Electrospray Ionization FDR – False Discovery Rate GET3 – Guided Entry of Tail-anchored proteins-3 GFP – Green Fluorescent Protein GO - Gene Ontology GpA – Glicoforina A Grp78 - Endoplasmic reticulum chaperone BiP Hcy - Homocisteína Hsp - Heat Shock Protein IEC – Cromatografía de intercambio iónico LB - medio Luria-Bertani LC-MS/MS – Liquid Chromatography tandem Mass Spectrometry MALDI – Matrix Assisted Laser Desorption/Ionization NBF – dominios ATPasa de SecA NMR - Magnetic Resonance Spectroscopy NSAF - Normalized Spectral Abundance Index PAI - Protein Abundance Index PANTHER - Protein ANalysis THrough Evolutionary Relationships pI – Punto isoeléctrico PSA- Persulfato de amonio Q – Quadrupole mass analyzer RNC – Ribosome Nascent chain Complex RP – Cromatografía de fase reversa SDS-PAGE - Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEC – Cromatografía
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