Conception de biosenseurs des protéines RhoA, RhoB, RhoC

Notre équipe s'intéresse à la compréhension des mécanismes de dérégulation des voies de signalisation cellulaire dans la survenue et la maintenance des processus tumoraux, ainsi que leurs conséquences dans la réponse aux thérapies antitumorales. Nous nous intéressons particulièrement aux protéines Rho et à leurs régulateurs. Ils interviennent dans les voies de signalisation des récepteurs cellulaires conduisant à des modifications de l'adhérence, de la prolifération, de la motilité et de la balance survie/mort cellulaire. Les protéines RhoA, RhoB et RhoC sont des petites GTPases passant d'un état actif (liées au GTP) à un état inactif (liées au GDP) et dont l'homologie est proche de 85%. La surexpression des protéines RhoA et RhoC a été décrite dans un grand nombre de tumeurs; à l'inverse, on observe une diminution de l'expression de RhoB dans le mélanome et dans le cancer du poumon. La conception de fragments d'anticorps sélectifs de la conformation active des Rho, appelés biosenseurs, permettant d'évaluer l'activation de ces protéines in situ dans des coupes de tissus sains et cancéreux, pourrait aboutir à un usage pronostique de ces outils voire à la définition de nouveaux marqueurs thérapeutiques et permettrait de répondre à des questions plus fondamentales comme leurs localisations cellulaire, leur rôle dans la migration cellulaire, leur activation spatio-temporelle. A partir du scFvC1 sélectif de la conformation active des trois protéines RhoA, RhoB et RhoC et isolé précédemment dans l'équipe, nous avons créé une banque secondaire par mutagénèse aléatoire et opéré une sélection par phage display avec un objectif double : 1°) augmenter l'affinité du scFvC1 pour améliorer ses capacités d'interaction avec les protéines Rho actives natives en vue d'améliorer ses performances en immunohistologie ainsi que pour une utilisation intracellulaire. 2°) sélectionner des variants spécifiques de chaque Rho malgré leur très forte homologie. Nous montrons que la stratégie mise en œuvre permet d'augmenter l'affinité des scFv et de modifier leur sélectivité puisqu'un variant se lie préférentiellement à RhoA et RhoC. De plus, contrairement au svFvC1, ces scFv sont immunoprécipitants pour les Rho actives produites dans des cellules eucaryotes. En parallèle, nous avons mis au point une méthodologie permettant le marquage d'un scFv anti-RhoB obtenu au laboratoire, par l'action d'un dérivé fluorescent de la biotine sur une intéine exprimée en fusion C-terminale du scFv. Ceci permettra d'améliorer les techniques immunohistologiques avec des biosenseurs fluorescents.Our team is interested in understanding the mechanisms of deregulation of cell signaling pathways in the development and maintenance of tumor processes and their consequences in response to anti-tumor therapies. We focuse on Rho proteins as well as on their regulators. They are involved in signaling pathways of cell receptors leading to changes in adhesion, proliferation, motility and balance survival / cellular death. RhoA, RhoB and RhoC are small GTPases switching from an active state (GTP-bound) to an inactive state (GDP-bound) and the homology of which is close to 85%. Overexpression of RhoA and RhoC protein has been described in many tumors; in contrast, there was a decreased expression of RhoB in melanoma and lung cancer. Engineering antibody fragment specific of Rho active conformations, namely biosensors, would allow in situ assessment of these proteins activation in healthy or tumour samples. These tools could be further developed towards diagnosis or prognosis usage, or could even define novel therapeutics markers and be used to answer more fundamental question as their cellular localization, their role in cell migration, or their spatio-temporal activation. Starting from the scFvC1 previously isolated in the group and which is selective for RhoA, RhoB and RhoC active conformations, we have created by random mutagenesis a second library and performed a phage display selection with two aims: 1°) increase the C1 scFv affinity to enhance its interaction potential with active native Rho proteins in order to improve its performance in immuno-histology or to use it as an intracellular antibody. 2°) select variants with a selectivity towards strictly only one of the three Rho excluding the others despite their strong homology. We show that our strategy allowed an affinity increase of scFv and also a selectivity modulation as one variant preferentially binds RhoA and C. Moreover, in contrast to scFvC1 these scFvs immuno-precipitate active endogenous Rho Proteins in eukaryotic cells. In parallel, we have established a method allowing the labelling of an anti-RhoB scFv from the lab, by fusing it to an intein that induce covalent binding of a biotin fluorescent analogue. This approach will improve immuno-histological techniques using fluorescent biosensors

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

Detection of label-free cancer biomarkers using nickel nanoislands and quartz crystal microbalance

We present a technique for the label-free detection and recognition of cancer biomarkers using metal nanoislands intended to be integrated in a novel type of nanobiosensor. His-tagged (scFv)-F7N1N2 is the antibody fragment which is directly immobilized, by coordinative bonds, onto ~5 nm nickel islands, then deposited on the surface of a quartz crystal of a quartz crystal microbalance (QCM) to validate the technique. Biomarker GTPase RhoA was investigated because it has been found to be overexpressed in various tumors and because we have recently isolated and characterized a new conformational scFv which selectively recognizes the active form of RhoA. We implemented a surface chemistry involving an antibiofouling coating of polyethylene glycol silane (PEG-silane) (<2 nm thick) and Ni nanoislands to reach a label-free detection of the active antigen conformation of RhoA, at various concentrations. The methodology proposed here proves the viability of the concept by using Ni nanoislands as an anchoring surface layer enabling the detection of a specific conformation of a protein, identified as a potential cancer biomarker. Hence, this novel methodology can be transferred to a nanobiosensor to detect, at lower time consumption and with high sensitivity, specific biomolecules

Detection of label-free cancer biomarkers using nickel nanoislands and quartz crystal microbalance

Adri&amp;aacute;n Mart&amp;iacute;nez-Rivas1,2, Patrick Chinestra3,4, Gilles Favre3,4, S&amp;eacute;bastien Pinaud1, Child&amp;eacute;rick S&amp;eacute;verac1,2, Jean-Charles Faye3,4, Christophe Vieu1,21LAAS-CNRS; Universit&amp;eacute; de Toulouse, Toulouse, France; 2Universit&amp;eacute; de Toulouse, UPS, INSA, INP, ISAE; LAAS; Toulouse, France; 3INSERM U563, Universit&amp;eacute; de Toulouse, CPTP, &amp;ldquo;Signalisation Cellulaire, GTPase Rho et cancers&amp;rdquo;, Toulouse, France; 4Institut Claudius Regaud, Biology Department, Toulouse, FranceAbstract: We present a technique for the label-free detection and recognition of cancer biomarkers using metal nanoislands intended to be integrated in a novel type of nanobiosensor. His-tagged (scFv)-F7N1N2 is the antibody fragment which is directly immobilized, by coordinative bonds, onto ~5 nm nickel islands, then deposited on the surface of a quartz crystal of a quartz crystal microbalance (QCM) to validate the technique. Biomarker GTPase RhoA was investigated because it has been found to be overexpressed in various tumors and because we have recently isolated and characterized a new conformational scFv which selectively recognizes the active form of RhoA. We implemented a surface chemistry involving an antibiofouling coating of polyethylene glycol silane (PEG-silane) (&amp;lt;2 nm thick) and Ni nanoislands to reach a label-free detection of the active antigen conformation of RhoA, at various concentrations. The methodology proposed here proves the viability of the concept by using Ni nanoislands as an anchoring surface layer enabling the detection of a specific conformation of a protein, identified as a potential cancer biomarker. Hence, this novel methodology can be transferred to a nanobiosensor to detect, at lower time consumption and with high sensitivity, specific biomolecules.Keywords: nickel nanoislands, cancer biomarkers, quartz crystal microbalance, PEG-silane, RhoA protein, nanobiosenso

Insulin and estrogen receptor ligand influence the FGF-2 activities in MCF-7 breast cancer cells.

From the MCF-7 cell line we have developed, a human mammary cancer cell subline with the same karyotype as the mother strain and named MCF-7(SF), able to grow in serum-free chemically defined medium. This cell subline was firstly used to analyze the effect of basic fibroblast growth factor (FGF-2) in estrogen-receptor-positive human breast cancer cells. FGF-2 like estradiol is able to increase cell proliferation and pS2 expression but was also found to inhibit progesterone receptor (PR) expression. The anti-estrogen tamoxifen partly counteracts the effects of FGF-2 and to discriminate between its two main mediators (estrogen receptor vs. anti-estrogen binding site, AEBS) we compare the efficacies of pure anti-estrogen (ICI 182,780) and AEBS ligand (PBPE). It appears that pure anti-estrogen counteracts cell growth and pS2 effects of FGF-2 since AEBS ligand inhibits the cell growth but has no activity on pS2 expression. Secondly, adding insulin (10(-6)M) in the culture medium induces a strong increase in cell proliferation, which then elicits an inhibitory effect of FGF-2 and addition of anti-estrogens, are less efficient to further decrease growth, since the effects of FGF-2 and anti-estrogens on pS2 expression are conserved

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-0

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

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-6

recombinant RhoA and RhoB loaded with either GTP or GDP were incubated with C1-N1N2 fixed on Ni-beads. An irrevelant scFv was used as control. Complexes on beads were resolved by SDS-PAGE and immunobloted with anti-RhoA and anti-RhoB. Western Blot is representative of 2 independent experiments. Immunofluorescence shows that scFv C1-N1N2 specifically binds to activated HeLa cells. Suspension containing scFv C1-N1N2 was incubated with GDP-loaded RhoA(and ) or GTPγS-preloaded RhoA beads (). Twenty-four hours after seeding, HeLa cells were serum-starved for 48 h and activated with 10% SVF and EGF (100 ng/ml) for 1 hour. Cells were fixed, permabilized and incubated with supernatants from scFv C1-N1N2-Rho incubation and anti-c-myc FITC conjugate secondary antibody. ) Non-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GDP-loaded RhoA beads, ) EGF-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GDP-loaded RhoA beads ) EGF-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GTPγS-loaded RhoA beads. () Non-activated HeLa cells incubated with the commercial Rhoa antibody, () EGF-activated HeLa cells incubated with the commercial RhoA antibody, () EGF-activated HeLa cells incubated with irrelevant scFv (anti-tyroglobulin). Pictures are representative of 2 independent experiments.<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