Búsquedas y caracterización de nuevas asociaciones moleculares de las colas citosólicas de las MT-MMPs

Matrix metalloproteinases (MMPs) are involved in a variety of biological processes by their ability of remodelling the extracellular microenvironment, modulating the activity of transmembrane receptors and regulating signaling cascades. Membrane type-MMPs (MT1-, MT2-, MT3- and MT5-MMP) mainly act in pericellular proteolysis but they could also connect to intracellular events through their interaction with cytosolic proteins. We hypothesize that binding of MT-MMP cysolic tails to distinct partners can contribute to their specific functions. In this work, we have identified novel binding proteins of the MT-MMP cytosolic tails, characterized the residues involved in these interactions and explored their possible biological functions. The tyrosine present in the middle region of MT1-MMP cytosolic tail has been shown to be necessary for p130Cas binding and this binding participates in the promotion of myeloid progenitor migration and fusion. MT1-, MT2- and MT3-MMP have been shown to bind the ERM protein moesin; juxtamembrane and middle polybasic regions of MT1-MMP cytosolic tail act as key binding domains for this interaction that may be regulating MT1-MMP localization at the plasma membrane. Finally, ZO-1 has been identified as a differentially associated protein of MT-MMPs; MT2-, MT3- and MT5-MMP are able to bind ZO-1 in contrast to MT1-MMP. Essential residues in the PDZ binding motif responsible for this different association pattern have also been identified. We have also observed that MT2-MMP colocalizes with ZO-1 in MT2-MMP stably transfected MDCK cells and that MT2-MMP expression affects F-actin polarization in these epithelial cells. In summary, this work identifies three actin-linker proteins as new molecular partners differentially associated with MT-MMPs; this differential association may contribute to MTMMP specific functions in diverse pathophysiological scenarios. [RESUMEN] Las metaloproteinasas de matriz (MMPs) están implicadas en varios procesos biológicos por su capacidad de remodelar el microambiente extracelular, modular la actividad de los receptores transmembrana y regular las cascadas de señalización celular. Las MMPs de membrana (MT1-, MT2-, MT3- y MT5-MMP) actúan principalmente en la proteolisis pericelular pero pueden además participar en los eventos intracelulares a través de su interacción con proteínas citosólicas. Nuestra hipótesis es que la interacción de las colas citosólicas de las MT-MMP a distintas proteínas puede contribuir a sus funciones específicas. En este trabajo hemos identificado nuevas proteínas que interaccionan con las colas citosólicas de las MT-MMPs, caracterizándose los residuos implicados en estas interacciones y explorándose sus posibles funciones biológicas. Se ha demostrado que la tirosina presente en la región central de la cola citosólica de MT1-MMP es necesaria para la unión de p130Cas y esta unión participa en la migración y fusión de los progenitores mieloides. Se ha detectado la unión de MT1-, MT2- y MT3-MMP a la proteína ERM moesina; las regiones polibásicas yuxtamembranal y central de la cola citosólica de MT1-MMP actúan como dominios clave y pueden regular la localización de MT1-MMP en la membrana plasmática. Finalmente, ZO-1 ha sido identificada como una proteína diferencialmente asociada a las MT-MMPs; MT2-, MT3- y MT5-MMP son capaces de interaccionar con ZO-1 al contrario que MT1-MMP. Los residuos en el motivo de unión a dominios PDZ responsables de este diferente patrón de asociación han sido también identificados. Además, hemos observado que MT2-MMP colocaliza con ZO-1 en células MDCK transfectadas establemente con MT2-MMP y que la expresión de MT2-MMP afecta la polarización de F-actina en estas células epiteliales. En resumen, en este trabajo se han identificado tres proteínas de unión a actina como nuevas proteínas asociadas diferencialmente a las MT-MMPs; esta asociación diferencial puede contribuir a las funciones específicas de las MT-MMP en distintos escenarios fisiopatológicos

MT1-MMP is required for myeloid cell fusion via regulation of Rac1 signaling

13 páginas, 7 figuras -- PAGS nros. 77-89Cell fusion is essential for fertilization, myotube formation, and inflammation. Macrophages fuse under various circumstances, but the molecular signals involved in the distinct steps of their fusion are not fully characterized. Using null mice and derived cells, we show that the protease MT1-MMP is necessary for macrophage fusion during osteoclast and giant-cell formation in vitro and in vivo. Specifically, MT1-MMP is required for lamellipodia formation and for proper cell morphology and motility of bone marrow myeloid progenitors prior to membrane fusion. These functions of MT1-MMP do not depend on MT1-MMP catalytic activity or downstream pro-MMP-2 activation. Instead, MT1-MMP null cells show a decreased Rac1 activity and reduced membrane targeting of Rac1 and the adaptor protein p130Cas. Retroviral rescue experiments and protein binding assays delineate a signaling pathway in which MT1-MMP, via its cytosolic tail, contributes to macrophage migration and fusion by regulating Rac1 activity through an association with p130CasThis work was supported by National Institutes of Health grant AR47074 (to S.S.A.), the Fundación Ramón Areces and Spanish Fondo de Investigación Sanitaria grant RD06/0014/1016 (to A.G.A.), the Spanish Ministry of Science and Innovation through grants SAF2008-02100 to M.A.d.P. and SAF2008-02104 to A.G.A., and EUROHORCS (European Heads of Research Councils) and the European Science Foundation (ESF) through a EURYI (European Young Investigator) award to M.A.d.P. P.G. was funded by the Juan de la Cierva program and the Fondo de Investigación Sanitaria. M.C.G. and M.V.H.-R. are supported by fellowships BES-2006-13204 and 12790 from the Spanish Ministry of Science and Innovation, respectively. The CNIC is supported by the Spanish Ministry of Science and Innovation and the Pro-CNIC Foundation. J.R. is one of the inventors of the ICTP assay, but the royalty period has expiredPeer reviewe