Caracterización de anticuerpos monoclonales funcionales específicos de ADAM-17

ADAM-17 es una proteína transmembrana que forma parte de la superfamilia de lasmetaloproteasas dependientes de zinc. Presenta una organización compleja en dominiosfuncionales y participa en gran variedad de procesos celulares. Por ello, es muy importanteprofundizar en el estudio de los mecanismos que regulan la actividad de ADAM-17. El trabajorealizado en esta tesis se ha centrado en obtener herramientas que permitan regular algunosde los procesos controlados por ADAM-17: i) adhesión celular, mediada por la interacción de laintegrina α5β1 con ADAM-17; ii) procesamiento proteolítico, determinado por el corte y laliberación de dos de los sustratos de ADAM-17 (ALCAM y TNF-α). Hemos obtenido ycaracterizado dos anticuerpos monoclonales (denominados 2A10 y YES-2) que reconocenespecíficamente el dominio desintegrina de ADAM-17 humano. La especificidad de estosanticuerpos monoclonales frente a ADAM17 se determinó mediante ensayos deinmunoabsorción ligada a enzimas (ELISA), citometría de flujo y western-blot. Estos anticuerposmonoclonales ejercen un efecto inhibitorio en la interacción celular de la integrina α5β1 conADAM-17 y con fibronectina en líneas celulares tumorales y leucocitarias y, en consecuencia,inhiben la adhesión mediada por la integrina α5β1. Además, los anticuerpos monoclonales 2A10y YES-2 aumentan la actividad sheddasa de ADAM-17 en linfocitos B y monocitos. Por lo tanto,estos anticuerpos podrían utilizarse como herramientas para modular la capacidad adhesiva yproteolítica de ADAM-17

Different states of integrin LFA-1 aggregation are controlled through its association with tetraspanin CD9

© 2015 Elsevier B.V. The tetraspanin CD9 has been shown to interact with different members of the β1 and β3 subfamilies of integrins, regulating through these interactions cell adhesion, migration and signaling. Based on confocal microscopy co-localization and on co-immunoprecipitation results, we report here that CD9 associates with the β2 integrin LFA-1 in different types of leukocytes including T, B and monocytic cells. This association is resistant to stringent solubilization conditions which, together with data from chemical crosslinking, in situ Proximity Ligation Assays and pull-down experiments, suggest a primary/direct type of interaction mediated by the Large Extracellular Loop of the tetraspanin. CD9 exerts inhibitory effects on the adhesive function of LFA-1 and on LFA-1-dependent leukocyte cytotoxic activity. The mechanism responsible for this negative regulation exerted by CD9 on LFA-1 adhesion does not involve changes in the affinity state of this integrin but seems to be related to alterations in its state of aggregation.SAF2012-34561 from the Spanish «Ministerio de Economía y Competitividad-MINECOPeer Reviewe

CD9 controls integrin α5β1-mediated cell adhesion by modulating its association with the metalloproteinase ADAM17

Integrin α5β1 is a crucial adhesion molecule that mediates the adherence of many cell types to the extracellular matrix through recognition of its classic ligand fibronectin as well as to other cells through binding to an alternative counter-receptor, the metalloproteinase ADAM17/TACE. Interactions between integrin α5β1 and ADAM17 may take place both in trans (between molecules expressed on different cells) or in cis (between molecules expressed on the same cell) configurations. It has been recently reported that the cis association between α5β1 and ADAM17 keeps both molecules inactive, whereas their dissociation results in activation of their adhesive and metalloproteinase activities. Here we show that the tetraspanin CD9 negatively regulates integrin α5β1-mediated cell adhesion by enhancing the cis interaction of this integrin with ADAM17 on the cell surface. Additionally we show that, similarly to CD9, the monoclonal antibody 2A10 directed to the disintegrin domain of ADAM17 specifically inhibits integrin α5β1-mediated cell adhesion to its ligands fibronectin and ADAM17.This work has been supported by the grant SAF2016-77096-R from Ministerio Español de Economía y Competitividad (MINECO) awarded to CC, by a grant from Fundación Ramón Areces Ayudas a la Investigación en Ciencias de la Vida y de la Materia, 2014 awarded to MY-M, and by the Deutsche Forschungsgemeinschaft (SFB 877, A6, Z3 and SPP1710) to JG and I

CD9 Controls Integrin α5β1-Mediated Cell Adhesion by Modulating Its Association With the Metalloproteinase ADAM17

Integrin α5β1 is a crucial adhesion molecule that mediates the adherence of many cell types to the extracellular matrix through recognition of its classic ligand fibronectin as well as to other cells through binding to an alternative counter-receptor, the metalloproteinase ADAM17/TACE. Interactions between integrin α5β1 and ADAM17 may take place both in trans (between molecules expressed on different cells) or in cis (between molecules expressed on the same cell) configurations. It has been recently reported that the cis association between α5β1 and ADAM17 keeps both molecules inactive, whereas their dissociation results in activation of their adhesive and metalloproteinase activities. Here we show that the tetraspanin CD9 negatively regulates integrin α5β1-mediated cell adhesion by enhancing the cis interaction of this integrin with ADAM17 on the cell surface. Additionally we show that, similarly to CD9, the monoclonal antibody 2A10 directed to the disintegrin domain of ADAM17 specifically inhibits integrin α5β1-mediated cell adhesion to its ligands fibronectin and ADAM17

Different states of integrin LFA-1 aggregation are controlled through its association with tetraspanin CD9

This is the author’s version of a work that was accepted for publication in Biochimica et Biophysica Acta - Mollecular Cell Research. A definitive version was subsequently published in Biochimica et Biophysica Acta - Mollecular Cell Research, 1853.10 (2015): 2464-2480 DOI: 10.1016/j.bbamcr.2015.05.018The tetraspanin CD9 has been shown to interact with different members of the β1 and β3 subfamilies of integrins, regulating through these interactions cell adhesion, migration and signaling. Based on confocal microscopy co-localization and on coimmunoprecipitation results, we report here that CD9 associates with the β2 integrin LFA-1 in different types of leukocytes including T, B and monocytic cells. This association is resistant to stringent solubilisation conditions which, together with data from chemical crosslinking, in situ Proximity Ligation Assays and pull-down experiments, suggests a primary/direct type of interaction mediated by the Large Extracellular Loop of the tetraspanin. CD9 exerts inhibitory effects on the adhesive function of LFA-1 and on LFA-1-dependent leukocyte cytotoxic activity. The mechanism responsible for this negative regulation exerted by CD9 on LFA-1 adhesion does not involve changes in the affinity state of this integrin but seems to be related to alterations in its state of aggregationThis work was supported by grant SAF2012-34561 from the Spanish «Ministerio de Economía y Competitividad-MINECO», (to C.C.). R.R.M. salary is supported by a «Profesor Ayudante» position from Departamento de Biología, Facutad de Ciencias, Universidad Autónoma de Madri

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements