Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection
TL;DR: 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.
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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.
Abstract: Antibodies are proteins that form part of an animal’s immune system and can identify and help eradicate infections. These proteins are also needed at many stages in biological research and represent one of the most promising tools in medical applications, from diagnostics to treatments. Traditionally, antibodies have been collected from animals that had been previously injected with a target molecule that the antibodies must recognize. An alternative strategy that uses bacteria and bacteria-infecting viruses instead of animals was developed several decades ago and allows researchers to obtain antibodies more quickly. However, the majority of the scientific community view these “in vitro selected antibodies” as inferior to those produced via the more traditional approach. Moutel, Bery et al. set out to challenge this widespread opinion, using a smaller kind of antibody known as nanobodies. The proteins were originally found in animals like llamas and camels and are now widely used in biological research. One particularly stable nanobody was chosen to form the backbone of the in vitro antibodies, and the DNA that encodes this nanobody was altered to make the protein more similar to human antibodies. Moutel, Bery et al. then changed the DNA sequence further to make billions of different versions of the nanobody, each one slightly different from the next in the region that binds to the target molecules. Transferring this DNA into bacteria resulted in a library (called the NaLi-H1 library) of bacterial clones that produce the nanobodies displayed at the surface of bacteria-infecting viruses. Moutel, Bery et al. then screened this library against various target molecules, including some from tumor cells, and showed that the fully in vitro selected antibodies worked just as well as natural antibodies in a number of assays. The in vitro antibodies could even be used to track, or inactivate, proteins within living cells. The NaLi-H1 library will help other researchers obtain new antibodies that bind strongly to their targets. The approaches developed to create the library could also see more people decide to create their own synthetic libraries, which would accelerate the identification of new antibodies in a way that is cheaper and requires fewer experiments to be done using animals. These in vitro selected antibodies could help to advance both fundamental and medical research.
219 citations
Cites methods from "Identification of a GTP-bound Rho s..."
...…libraries, immune or naı̈ve llama VHH libraries (Monegal et al., 2012; Olichon and Surrey, 2007) or from scFv libraries (Dimitrov et al., 2008; Goffinet et al., 2008; Nizak et al., 2003a) we then rationally designed CDR diversity with fixed CDR1 and CDR2 size and four CDR3 sizes (9, 12, 15…...
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TL;DR: This work grades every paper published in 2008 on a scale from A to F and outlines what features make a biosensor article fabulous, middling or abysmal and focuses on a few experimental, analysis and presentation mistakes that are alarmingly common.
Abstract: Optical biosensor technology continues to be the method of choice for label-free, real-time interaction analysis. But when it comes to improving the quality of the biosensor literature, education should be fundamental. Of the 1413 articles published in 2008, less than 30% would pass the requirements for high-school chemistry. To teach by example, we spotlight 10 papers that illustrate how to implement the technology properly. Then we grade every paper published in 2008 on a scale from A to F and outline what features make a biosensor article fabulous, middling or abysmal. To help improve the quality of published data, we focus on a few experimental, analysis and presentation mistakes that are alarmingly common. With the literature as a guide, we want to ensure that no user is left behind.
192 citations
TL;DR: The approaches used for antibody purification are critically examined with the aim of providing the reader with the principles and practical insights required to understand the intricacies of the procedures.
161 citations
TL;DR: It is shown that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display, and the highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design.
Abstract: Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library. The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×1010 independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations. The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.
105 citations
Additional excerpts
...pCANTAB3his [50], pCES [51], pHEN2 [52], pHAL14 [31], pIT2 [53],...
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TL;DR: Therapeutic approaches targeting Rho GTPase signaling may provide a novel alternative for anti-HCC therapy.
97 citations
References
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TL;DR: To the authors' knowledge, this is the first evidence that increased Rac signaling may inhibit invasion of epithelial tumor cells by up-regulation of TIMP-1 and TIMp-2.
110 citations
"Identification of a GTP-bound Rho s..." refers background in this paper
...Rho proteins are also implicated in participating in several steps of tumor progression and development of metastasis [4,5]....
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TL;DR: The role of Rho small GTP‐binding protein (Rho) in the progression of testicular germ cell tumour (GCT) is clarified by examining the expression levels of mRNAs of R Ho genes in testicular GCT.
Abstract: Objective To clarify the role of Rho small GTP-binding protein (Rho) in the progression of testicular germ cell tumour (GCT), by examining the expression levels of mRNAs of Rho genes in testicular GCT
Patients and methods The mRNA levels of the RhoA, RhoB and RhoC genes were analysed in the surgical specimens of testicular GCT tissues from 45 consecutive Japanese patients, and in the corresponding unaffected tissue originating from the same patient, using reverse transcription-polymerase chain reaction The expression levels in tumour tissues were compared with those in unaffected tissues and the relationship between their expression levels in tumours and tumour stage evaluated The expression levels of mRNAs of the Rho genes were also evaluated between tumours with seminoma only, and mixed tumours with seminoma and nonseminoma
Results The mRNA levels of RhoA were greater in tumour tissues than in unaffected tissues of the resected testis (P < 001); the mRNAs of RhoB and RhoC were not detected in either tissue The increase in RhoA mRNA levels was related to tumour stage (P < 005) The mRNA levels of RhoA in seminomatous and nonseminomatous areas where both were present were higher than those in tumours with seminoma only (P < 005)
Conclusions These results suggest that RhoA is involved in testicular germinal epithelial carcinogenesis and progression in testicular GCT, indicating that RhoA may be a useful prognostic marker for progression in testicular GCT
100 citations
"Identification of a GTP-bound Rho s..." refers background in this paper
...The involvement of RhoA in testicular human tumors was demonstrated by increased RhoA mRNA levels in relation to tumour grade [17]....
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TL;DR: These results provide the first demonstration of a physical interaction between a GPCR and p85, revealing a novel mechanism for negative regulation by ligand‐activated G PCR of PI3K‐dependent survival pathways, which may be an important molecular target for antineoplastic therapy.
Abstract: Phosphatidylinositol 3-kinase (PI3K) regulates many cellular functions including growth and survival, and its excessive activation is a hallmark of cancer. Somatostatin, acting through its G protein-coupled receptor (GPCR) sst2, has potent proapoptotic and anti-invasive activities on normal and cancer cells. Here, we report a novel mechanism for inhibiting PI3K activity. Somatostatin, acting through sst2, inhibits PI3K activity by disrupting a pre-existing complex comprising the sst2 receptor and the p85 PI3K regulatory subunit. Surface plasmon resonance and molecular modeling identified the phosphorylated-Y71 residue of a p85-binding pYXXM motif in the first sst2 intracellular loop, and p85 COOH-terminal SH2 as direct interacting domains. Somatostatin-mediated dissociation of this complex as well as p85 tyrosine dephosphorylation correlates with sst2 tyrosine dephosphorylation on the Y71 residue. Mutating sst2-Y71 disabled sst2 to interact with p85 and somatostatin to inhibit PI3K, consequently abrogating sst2's ability to suppress cell survival and tumor growth. These results provide the first demonstration of a physical interaction between a GPCR and p85, revealing a novel mechanism for negative regulation by ligand-activated GPCR of PI3K-dependent survival pathways, which may be an important molecular target for antineoplastic therapy.
88 citations
"Identification of a GTP-bound Rho s..." refers methods in this paper
...To measure the affinity of this scFv, surface plasmon resonance (SPR) experiments were performed on a Biacore 3000 instrument [27]....
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TL;DR: The principles and strategies used to develop Raichu-type FRET probes for Rho-family GTPases are described and practical tips for their optimization are provided.
Abstract: GFP‐based FRET probes that can visualize local activity changes in Rho GTPases in living cells are now available for examining the spatiotemporal regulation of these proteins. We previously developed FRET probes for Rho (and Ras) GTPases and collectively designated them “Ras and interacting protein chimeric unit” (Raichu) probes. In this chapter, we describe the principles and strategies used to develop Raichu‐type FRET probes for Rho‐family GTPases. The procedures for characterizing candidate probes, setting up the imaging system, and image acquisition/processing are also explained. An optimal FRET probe should: (1) have a wide dynamic range (i.e., a high sensitivity); (2) demonstrate high fluorescence intensity (i.e., a high signal‐to‐noise ratio); (3) show target specificity; and (4) cause minimal perturbation of endogenous signaling cascades. Although improvements of FRET probes should be executed in a trial‐and‐error manner, we provide practical tips for their optimization. In addition, some experimental results are presented to illustrate the expanding number of fields for the application of Raichu‐RhoA/Rac1/Cdc42, and the advantages and disadvantages of Raichu probes are discussed.
79 citations
"Identification of a GTP-bound Rho s..." refers background in this paper
...The Rho GTPases can be divided into six groups: Rho (RhoA, RhoB, RhoC), Rac (Rac1, Rac2, Rac3, RhoG), Cdc42 (Cdc42, TC10, TCL, Chp/Wrch-2, Wrch-1), Rnd (Rnd1, Rnd2, Rnd3/RhoE), RhoBTB (RhoBTB1 and RhoBTB2) and Miro (Miro-1 and Miro-2)....
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...Conclusion Binding domains of effector proteins have proven to be useful as conformational sensors in analyzing the spatiotemporal activation of GTPases [31,32]....
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...To further develop understanding of Rho activation, we identified a conformation-specific scFv against the active form of RhoA, RhoB and RhoC GTPases....
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...Whatever the level of gene expressions of Rho GTPases and assuming that high level of protein could be associate with higher concentration of activated Rho GTPase, the knowledge of accurate variations of the Rho activation under treatment would be a significant progress in the understanding of the biological role of Rho in oncogenesis....
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...Rho GTPases control a wide variety of signal transduction pathways regulating many fundamental processes of cell biology, such as organization of the actin cytoskeleton [2], gene expression, cell proliferation and survival [3]....
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TL;DR: In addition to the activation of specific kinases cascades, phospholipid‐derived messengers are candidates to compose some of the most critical elements associated to regulation of signaling cascades capable of discerning among life and death.
64 citations
"Identification of a GTP-bound Rho s..." refers background in this paper
...Furthermore, Lacal et al have shown that Rho GTPases are directly involved in signalling pathways that trigger either proliferation or cell death [14]....
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