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
Citations
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Book ChapterDOI
Use of phage display for the identification of molecular sensors specific for activated Rho.
TL;DR: The method described here has permitted to identify an scFv that discriminates between the activated and the inactivated form of the Rho subfamily.
Journal ArticleDOI
Generation of a single chain antibody variable fragment (scFv) to sense selectively RhoB activation
Patrick Chinestra,Aurélien Olichon,Claire Medale-Giamarchi,Isabelle Lajoie-Mazenc,Rémi Gence,Cyril Inard,Laetitia Ligat,Jean-Charles Faye,Gilles Favre +8 more
TL;DR: The ability of scFvs to distinguish RhoB from RhoA GTP-bound form is demonstrated and provide new selective tools to analyze the cell biology of Rho B GTPase regulation.
Dissertation
Wafer scale integration of coulomb blockade-based nanobiosensors with microfluidic channels for label-free detection of cancer biomarkers
TL;DR: In this paper, a novel type of nanobiosensor capable of high sensitivity detection of biomolecules was proposed and implemented on 4 inch wafer of the nano-ilots de nickel for label-free biodetection using quartz crystal microbalance.
Journal ArticleDOI
Screening for serum biomarkers in patients with chronic hepatitis B with hepatitis B surface antigen seroclearance, following pegylated interferon alpha therapy
Zheling Wang,Xiaofei Li,Changhe Shi,Min Zhang,Ru Chen,Wei Wu,Qingshun Hou,Wei Ke,Tianli Fan,Zirong Wen,Xinjie Hao,Naifang Qu +11 more
TL;DR: The newly identified mimic peptide IFNC1 showed a high predictive validity HBsAg seroclearance in patients with CHB, following peg-IFN-α therapy, and may be a potential serum biomarker, which could be used to predict the treatment outcomes of peg- IFN- α therapy.
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