Please use this identifier to cite or link to this item: http://hdl.handle.net/2445/57264
Title: Functional studies of 4F2hc-CD147 interaction = Estudios funcionales de la interacción 4F2hc‐CD147
Author: Delgado Martin, Gonzalo
Director: Palacín Prieto, Manuel
Keywords: Ectodomini
Ectodomain
Proteïnes de membrana
Interacció cel·lular
Aminoàcids
Membrane proteins
Cell interaction
Amino acids
Issue Date: 14-Jul-2014
Publisher: Universitat de Barcelona
Abstract: [spa]En la presente tesis identificamos CD147 (proteína inductora de metaloproteinasas) por su interacción con el ectodominio de 4F2hc (proteína de membrana plasmática con función dual como subunidad de transportadores de aminoácidos y como modulador positivo de integrinas beta 1 y 3). La interacción entre 4F2hc-ED y el ectodominio de CD147 (CD147-ED) se confirmó mediante estudio del complejo 4F2hc-ED / CD147-ED por espectrometría de masas y surface plasmon resonance, obteniendo una afinidad de interacción baja (KD en el rango de µM). La falta de producción del complejo determinada por cromatografía de exclusión, tras expresar por separada cada una de las dos proteínas en E. coli, no ha permitido cristalizar el complejo. Para superar este inconveniente se utilizó resonancia magnética nuclear, pero no se obtuvieron resultados significativos. Finalmente cálculos de docking entre ambos ectodominios ofrecieron dos posibles soluciones que son compatibles con el modelo de heterodímero 4F2hc/LAT2 (Rosell et al. 2014) y, una de ellas, con el modelo de embigin/MCT (Hallestrap, 2013). Por otro lado, concluimos que LAT1 es la subunidad ligera de 4F2hc que muestra mayor interacción con CD147, ya sea porque interacciona a su vez con CD147/MCT o bien porque dirige el heterodímero 4F2hc/LAT1 hacia zonas de la membrana plasmática ricas en CD147. El estudio funcional de la interacción mostró que la expresión de MMP2 depende de 4F2hc. Así, células knockout (KO) para 4F2hc, fibroblastos derivados de células madre embrionarias KO para 4F2hc y fibroblastos derivados de epidermis de ratón condicional KO para 4F2hc no expresan MMP2. Concomitantemente, ambos tipos celulares presentan migración celular defectiva. Este resultado es relevante a la luz de que la expresión de 4F2hc está aumentada en muchos tipos de tumores. Sorprendentemente, ninguno de los dos tipos celulares KO para 4F2hc recupera la expresión y función de MMP2 al ser transducidas con cualquiera de las dos isoformas de 4F2hc expresadas en los fibroblastos salvajes. De la misma forma, cambios en el estado de fosforilación de p38 y ERK1/2 y en la expresión de IKβ-α que ocurren en los fibroblastos KO no se restauran al recuperar la expresión de 4F2hc. Por el contrario, la recuperación de la expresión de 4F2hc restaura las actividades de transporte. Estos resultados indican que la ablación total de 4F2hc en fibroblastos reprograma a estas células para poder ser viables en ausencia de esta proteína.
[eng]We identified proteins that interacted with the ectodomain of 4F2hc, a plasma membrane protein with dual function as heavy subunit of the amino acid transporters and positive regulator of β1 and β3 integrins. Initially, affinity chromatography experiments identified CD147, a plasma membrane protein inducer of matrix metalloproteinases (MMPs). Both proteins colocalize in cell plasma membranes, especially in the leading edge of the cell, suggesting a role in cell adhesion and migration. Moreover, both proteins co-immunoprecipitate in mild detergents. The interaction was confirmed by the mass spectrometry and by surface plasmon resonance, indicating that it was specific but weak/transient, with a KD in the µM range, and it was mainly electrostatic. The study by NMR was unsuccessful, as no significative peak shifts were observed preventing the characterization of the interface. Co-elution in size exclusion chromatography was negative, precluding co-crystallization of the protein complex. Finally, a protein docking between both ectodomains was performed, resulting in two solutions compatible with the current model of heterodimer 4F2hc/LAT2 (Rosell, et al. 2014). One solution showed the Ig1 domain interacting with the ectodomain of 4F2hc, what is also compatible with the current model of embigin/MCT (Hallestrap, 2013). CD147 is immunoprecipitated preferentially with 4F2hc/LAT1 heterodimers in comparison the other light subunits, suggesting that LAT1 either localizes the heterodimer in CD147-enriched membrane domains or that it directly interacts with CD147/MCT. The studies of their respective functions showed that CD147 does not modulate the transport activity of 4F2hc-associated light subunits. Conversely, the expression of MMP-2, is dependent on 4F2hc. Studies in 4F2hc-knockout (KO) cells, fibroblasts derived from embryonic stem cells KO for 4F2hc and fibroblasts obtained from epidermis of conditional 4F2hc-KO mice, show that these cells do not express MMP-2. Moreover, both cell types have impaired cell migration. This is relevant as 4F2hc is overexpressed in many types of tumors. Wild type mouse fibroblasts express two isoforms of 4F2hc, whose expression is ablated in the KO cells. Strikingly, none of the 4F2hc-KO cell types recovers the expression and function of MMP-2 when any of the 4F2hc isoforms is virally transduced. Similarly, the phosphorylation levels of p38 and ERK1/2, and the expression of IκB-α, are not restored after 4F2hc recovery. Conversely, ablation of 4F2hc eliminates the transport activities of LAT1, y+LAT2 and xCT, and they are completely recovered when any of the isoforms of 4F2hc is reexpressed in any of the two 4F2hc-KO cells. These results indicate that ablation of 4F2hc in fibroblasts reprograms the cell to be able to survive, and the expression of MMP-2 no longer depends on 4F2hc.
URI: http://hdl.handle.net/2445/57264
Appears in Collections:Tesis Doctorals - Departament - Bioquímica i Biologia Molecular (Biologia)

Files in This Item:
File Description SizeFormat 
GDM_THESIS.pdf12.24 MBAdobe PDFView/Open


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.