Characterization of single chain antibody targets through yeast two hybrid

<p>Abstract</p> <p>Background</p> <p>Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful <it>in vitro </it>selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain.</p> <p>Results</p> <p>We describe an approach that allows detailed characterization of a given antibody's target(s) using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID), efficiently narrowing the epitope-containing region.</p> <p>Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed.</p> <p>Conclusions</p> <p>Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise domain mapping for linear epitopes, confirmation of non-linear epitopes for conformational sensors, and detection of secondary binding partners. This approach may thus prove to be an elegant and rapid method for the target characterization of newly obtained scFv antibodies. It may be considered prior to any research application and particularly before any use of such recombinant antibodies in clinical medicine.</p

ARF6 controls post-endocytic recycling through its downstream exocyst complex effector

The small guanosine triphosphate (GTP)–binding protein ADP-ribosylation factor (ARF) 6 regulates membrane recycling to regions of plasma membrane remodeling via the endocytic pathway. Here, we show that GTP–bound ARF6 interacts with Sec10, a subunit of the exocyst complex involved in docking of vesicles with the plasma membrane. We found that Sec10 localization in the perinuclear region is not restricted to the trans-Golgi network, but extends to recycling endosomes. In addition, we report that depletion of Sec5 exocyst subunit or dominant inhibition of Sec10 affects the function and the morphology of the recycling pathway. Sec10 is found to redistribute to ruffling areas of the plasma membrane in cells expressing GTP-ARF6, whereas dominant inhibition of Sec10 interferes with ARF6-induced cell spreading. Our paper suggests that ARF6 specifies delivery and insertion of recycling membranes to regions of dynamic reorganization of the plasma membrane through interaction with the vesicle-tethering exocyst complex

The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA

Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases

An aPKC-Exocyst Complex Controls Paxillin Phosphorylation and Migration through Localised JNK1 Activation

The exocyst/aPKC complex controls the spatiotemporal activation of the kinases JNK and ERK at the leading edge of migrating cells and thereby controls the dynamic behaviour of the adhesion protein paxillin during cell migration

CDC25, modulateur de l'activite des proteines RAS et regulateur du cycle de division cellulaire de la levure Saccharomyces cerevisiae

SIGLEINIST TD 19864 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Une approche génétique et cellulaire des Drosophila melanogaster révèle de nouvelles fonctions de la GTPase Ral, un acteur essentiel en oncogenèse

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocSudocFranceF

En aval des proto-oncogènes Ras, la voie de signalisation Ral-Rlip (identification de nouveaux partenaires moléculaires de Rlip)

PARIS-BIUP (751062107) / SudocSudocFranceF

L'ubiquitination des GTPases Ral (Un nouveau mécanisme de régulation diu trafic intracellulaire de Ral et des micro-domaines menmbranaires lipidiques)

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocSudocFranceF

« Entre systèmes modèles et conservation phylogénique, le projet DrosoMan de protéomique comparative entre l’Homme et la Drosophile ou l’association de moyens industriels et de la science artisanale pour établir les maillons faibles des ensembles protéiques »

L’annotation fonctionnelle est un défi pour les biologistes confrontés aux quantités d’informations fournies par les projets de séquences des génomes des Vertébrés, des organismes modèles, des micro-organismes. Mais le savoir-faire de la recherche fondamentale « classique », qui se prête mal à des approches massives, est à la fois indispensable pour une compréhension approfondie, et insuffisant pour affronter l’énormité de la tâche. Inversement, quelques techniques sont industrialisables et permettent une description en termes de « protéomique fonctionnelle », en aval de la génomique. L’Institut Curie et Hybrigenics ont pris conscience de leur complémentarité et ont établi une alliance dans le domaine du cancer et de la signalisation. Cette alliance s’est concrétisée autour d’un projet de protéomique fonctionnelle comparative entre l’Homme et la Drosophile. De nouveaux partenaires de protéines pourtant très étudiées ont été ainsi définis, de nouveaux réseaux sont apparus, et des « domaines biologiques » qui paraissaient indépendants sont connectés grâce à la profondeur des cribles et des analyses subséquentes