Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Les cellules souches cancéreuses

Un remarquable changement de paradigme s'est produit ces dernières années concernant les origines biologiques du cancer. L'hypothèse des cellules souches cancéreuses est entrée en compétition avec les notions fondamentales du cancer, entrainant derrière elle son lot d'implications thérapeutiques profondes. Différentes preuves concordantes indiquent que des erreurs dans le développement d'une petite partie des cellules souches adultes peuvent conduire à l'apparition d'un cancer. Seule cette petite sous-population a le pouvoir inhérent de former une tumeur et de métastaser. Notre travail consiste en un état des lieux des connaissances actuelles sur le sujet émergeant des cellules souches cancéreuses en nous appuyant sur deux types de tumeurs, les tumeurs prostatiques et le mélanome. Il traite également des implications sur les plans thérapeutique, biologique et préventif induits par la validation de ce concept dans les différents types de tumeurs liquides ou solides.TOULOUSE3-BU Santé-Centrale (315552105) / SudocSudocFranceF

MAP1A light chain-2 interacts with GTP-RhoB to control epidermal growth factor (EGF)-dependent EGF receptor signaling.

International audienceRho GTPases have been implicated in the control of several cellular functions, including regulation of the actin cytoskeleton, cell proliferation, and oncogenesis. Unlike RhoA and RhoC, RhoB localizes in part to endosomes and controls endocytic trafficking. Using a yeast two-hybrid screen and a glutathione S-transferase pulldown assay, we identified LC2, the light chain of the microtubule-associated protein MAP1A, as a novel binding partner for RhoB. GTP binding and the 18-amino acid C-terminal hypervariable domain of RhoB are critical for its binding to MAP1A/LC2. Coimmunoprecipitation and immunofluorescence experiments showed that this interaction occurs in U87 cells. Down-regulation of MAP1A/LC2 expression decreased epidermal growth factor (EGF) receptor expression and modified the signaling response to EGF treatment. We concluded that MAP1A/LC2 is critical for RhoB function in EGF-induced EGF receptor regulation. Because MAP1A/LC2 is thought to function as an adaptor between microtubules and other molecules, we postulate that the RhoB and MAP1A/LC2 interactions facilitate endocytic vesicle trafficking and regulate the trafficking of signaling molecules

Generation of a single chain antibody variable fragment (scFv) to sense selectively RhoB activation.

Determining the cellular level of activated form of RhoGTPases is of key importance to understand their regulatory functions in cell physiopathology. We previously reported scFvC1, that selectively bind to the GTP-bound form of RhoA, RhoB and RhoC. In this present study we generate, by molecular evolution, a new phage library to isolate scFvs displaying high affinity and selectivity to RhoA and RhoB. Using phage display affinity maturation against the GTP-locked mutant RhoAL63, we isolated scFvs against RhoA active conformation that display Kd values at the nanomolar range, which corresponded to an increase of affinity of three orders of magnitude compared to scFvC1. Although a majority of these evolved scFvs remained selective towards the active conformation of RhoA, RhoB and RhoC, we identified some scFvs that bind to RhoA and RhoC but not to RhoB activated form. Alternatively, we performed a substractive panning towards RhoB, and isolated the scFvE3 exhibiting a 10 times higher affinity for RhoB than RhoA activated forms. We showed the peculiar ability of scFvE3 to detect RhoB but not RhoA GTP-bound form in cell extracts overexpressing Guanine nucleotide Exchange Factor XPLN as well as in EGF stimulated HeLa cells. Our results demonstrated the ability of scFvs to distinguish RhoB from RhoA GTP-bound form and provide new selective tools to analyze the cell biology of RhoB GTPase regulation

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-2

to the human immunoglobulins sequences (imgt). Sequences highlighted in red vary from the amino-acid sequence of scFv C1. Underlined sequences correspond to CDR. Asterisks indicate the missing amino-acid that would generate the usual CDR.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-7

sequence of bacterial pectate lyase that mediates secretion into the periplasmic space; : variable fragment of the heavy chain; : light chain; : 6 histidine-tag; : myc-tag; amber stop codon; : portions of the N- and C-term of phage capside protein pIII; and : primers used for sequencing the Vand Vdomain. Schematic construction of vectors encoding soluble scFv C1 and scFv C1-N1N2. The amber stop codon between the scFv and gene III in pHEN 2 was removed by mutagenesis (middle construct). The C-terminal portion of pIII was removed in the final pHEN C1-N1N2 vector (bottom construct), first by PCR amplification of pHEN C1-pIII, introducing an EcoRI site after N2, and subsequently by cloning the NcoI and EcoRI digested PCR product into the linearized pHEN C1-pIII plasmid at the NotI and EcoRI sites. : 6 histidine-tag; M: myc-tag.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p