Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection
Marine Goffinet,Patrick Chinestra,Isabelle Lajoie-Mazenc,Claire Medale-Giamarchi,Gilles Favre,Jean-Charles Faye +5 more
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TLDR
The success of C1-N1N2 in discriminating activated Rho in immunofluorescence studies implies that this new tool, in collaboration with currently used RhoA and B antibodies, has the potential to analyze Rho activation in cell function and tumor development.Abstract:
The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv) that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings. After five rounds of phage selection using a constitutively activated mutant of RhoB (RhoBQ63L), three scFvs (A8, C1 and D11) were selected for subsequent analysis. Further biochemical characterization was pursued for the single clone, C1, exhibiting an scFv structure. C1 was selective for the GTP-bound form of RhoA, RhoB, as well as RhoC, and failed to recognize GTP-loaded Rac1 or Cdc42, two other members of the Rho family. To enhance its production, soluble C1 was expressed in fusion with the N-terminal domain of phage protein pIII (scFv C1-N1N2), it appeared specifically associated with GTP-loaded recombinant RhoA and RhoB via immunoprecipitation, and endogenous activated Rho in HeLa cells as determined by immunofluorescence. We identified an antibody, C1-N1N2, specific for the GTP-bound form of RhoB from a phage library, and confirmed its specificity towards GTP-bound RhoA and RhoC, as well as RhoB. The success of C1-N1N2 in discriminating activated Rho in immunofluorescence studies implies that this new tool, in collaboration with currently used RhoA and B antibodies, has the potential to analyze Rho activation in cell function and tumor development.read more
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NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies
Sandrine Moutel,Sandrine Moutel,Sandrine Moutel,Nicolas Bery,Nicolas Bery,Virginie Bernard,Laura Keller,Laura Keller,Emilie Lemesre,Emilie Lemesre,Ario de Marco,Laetitia Ligat,Jean-Christophe Rain,Gilles Favre,Gilles Favre,Aurélien Olichon,Aurélien Olichon,Franck Perez,Franck Perez +18 more
TL;DR: This work describes the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1), based on a humanized synthetic singledomain antibody (hs2dAb) scaffold optimized for intracellular stability, which provides high affinity binders without animal immunization.
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Affinity chromatography as a tool for antibody purification.
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[8] Measurement of intrinsic nucleotide exchange and GTP hydrolysis rates
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TL;DR: This chapter describes methods to measure nucleotide exchange rates and GTP hydrolysis rates of Rho, Rac, and G25K and describes the Rho–GDP-dissociation inhibitor (GDI), Rho-GDI.
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Recombinant antibodies to the small GTPase Rab6 as conformation sensors.
TL;DR: An approach, based on antibody phage display, to generate molecular conformation sensors that could be applied to other molecules that can be locked in a particular conformation in vitro is reported.
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Designing biosensors for Rho family proteins — deciphering the dynamics of Rho family GTPase activation in living cells
Olivier Pertz,Klaus M. Hahn +1 more
TL;DR: Fluorescent probes that non-invasively report the changing subcellular location of Rho GTPase activity in single living cells are now allowing us to examine spatio-temporal regulation of the activity of these proteins, and are providing new biological insights.
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