Piona Dariavach

Variable region genes in the human T-cell rearranging gamma (TRG) locus: V-J junction and homology with the mouse genes.

The locus of the human T cell rearranging gamma (TRG) or T cell receptor gamma chain genes comprises at least 14 variable genes (TRGV) belonging to four subgroups, five joining segments (TRGJ) and two constant region genes (TRGC). Nine V gamma genes belong to subgroup I, whereas subgroups II, III and IV each consists of a single gene respectively designated V9, V10 and V11. T cells expressing the gamma chain (TRG+) and recognized by the anti‐Ti gamma A monoclonal antibody have been shown to rearrange the V9 gene. In order to assess the N diversity at the V‐J junction in the TRG+ cells, the germline sequences of the segments involved in the V‐J rearrangements must be known. In this paper, we report the sequences of the germline V9 and V10 genes. Comparison of the V‐J junction and N region from transcripts or rearranged TRG genes belonging to the different subgroups shows no evidence of D segments in the human TRG locus. Sequences of the rearranged V11 gene from the JM cell line and those of the VA and VB pseudogenes, located upstream of V9 and V11 respectively, are given. Our results bring the number of human V gamma genes whose sequence is known to 13 and reveal unexpected homology with the mouse V gamma genes.

A novel druglike spleen tyrosine kinase binder prevents anaphylactic shock when administered orally

BACKGROUND The spleen tyrosine kinase (Syk) is recognized as a potential pharmaceutical target for the treatment of type I hypersensitivity reactions including allergic rhinitis, urticaria, asthma, and anaphylaxis because of its critical position upstream of immunoreceptor signaling complexes that regulate inflammatory responses in leukocytes. OBJECTIVE Our aim was to improve the selectivity of anti-Syk therapies by impeding the interaction of Syk with its cellular partners, instead of targeting its catalytic site. METHODS We have previously studied the inhibitory effects of the anti-Syk intracellular antibody G4G11 on Fc epsilonRI-induced release of allergic mediators. A compound collection was screened by using an antibody displacement assay to identify functional mimics of G4G11 that act as potential inhibitors of the allergic response. The effects of the selected druglike compounds on mast cell activation were evaluated in vitro and in vivo. RESULTS We discovered compound 13, a small molecule that inhibits Fc epsilonRI-induced mast cell degranulation in vitro and anaphylactic shock in vivo. Importantly, compound 13 was efficient when administered orally to mice. Structural analysis, docking, and site-directed mutagenesis allowed us to identify the binding cavity of this compound, located at the interface between the 2 Src homology 2 domains and the interdomain A of Syk. CONCLUSION We have isolated a new class of druglike compounds that modulate the interaction of Syk with some of its macromolecular substrates implicated in the degranulation pathway in mast cells.

Intracellular Single-Chain Variable Fragments Directed to the Src Homology 2 Domains of Syk Partially Inhibit FcεRI Signaling in the RBL-2H3 Cell Line

Intracellular expression of Ab fragments has been efficiently used to inactivate therapeutic targets, oncogene products, and to induce viral resistance in plants. Ab fragments expressed in the appropriate cell compartment may also help to elucidate the functions of a protein of interest. We report in this study the successful targeting of the protein tyrosine kinase Syk in the RBL-2H3 rat basophilic leukemia cell line. We isolated from a phage display library human single-chain variable fragments (scFv) directed against the portion of Syk containing the Src homology 2 domains and the linker region that separates them. Among them, two scFv named G4G11 and G4E4 exhibited the best binding to Syk in vivo in a yeast two-hybrid selection system. Stable transfectants of RBL-2H3 cells expressing cytosolic G4G11 and G4E4 were established. Immunoprecipitation experiments showed that intracellular G4G11 and G4E4 bind to Syk, but do not inhibit the activation of Syk following FcεRI aggregation, suggesting that the scFv do not affect the recruitment of Syk to the receptor. Nevertheless, FcεRI-mediated calcium mobilization and the release of inflammatory mediators are inhibited, and are consistent with a defect in Bruton’s tyrosine kinase and phospholipase C-γ2 tyrosine phosphorylation and activation. Interestingly, FcεRI-induced mitogen-activated protein kinase phosphorylation is not altered, suggesting that intracellular G4G11 and G4E4 do not prevent the coupling of Syk to the Ras pathway, but they selectively inhibit the pathway involving phospholipase C-γ2 activation.

Tyrosine Kinase Syk Non-Enzymatic Inhibitors and Potential Anti-Allergic Drug-Like Compounds Discovered by Virtual and In Vitro Screening

In the past decade, the spleen tyrosine kinase (Syk) has shown a high potential for the discovery of new treatments for inflammatory and autoimmune disorders. Pharmacological inhibitors of Syk catalytic site bearing therapeutic potential have been developed, with however limited specificity towards Syk. To address this topic, we opted for the design of drug-like compounds that could impede the interaction of Syk with its cellular partners while maintaining an active kinase protein. To achieve this challenging task, we used the powerful potential of intracellular antibodies for the modulation of cellular functions in vivo, combined to structure-based in silico screening. In our previous studies, we reported the anti-allergic properties of the intracellular antibody G4G11. With the aim of finding functional mimics of G4G11, we developed an Antibody Displacement Assay and we isolated the drug-like compound C-13, with promising in vivo anti-allergic activity. The likely binding cavity of this compound is located at the close vicinity of G4G11 epitope, far away from the catalytic site of Syk. Here we report the virtual screen of a collection of 500,000 molecules against this new cavity, which led to the isolation of 1000 compounds subsequently evaluated for their in vitro inhibitory effects using the Antibody Displacement Assay. Eighty five compounds were selected and evaluated for their ability to inhibit the liberation of allergic mediators from mast cells. Among them, 10 compounds inhibited degranulation with IC50 values ≤10 µM. The most bioactive compounds combine biological activity, significant inhibition of antibody binding and strong affinity for Syk. Moreover, these molecules show a good potential for oral bioavailability and are not kinase catalytic site inhibitors. These bioactive compounds could be used as starting points for the development of new classes of non-enzymatic inhibitors of Syk and for drug discovery endeavour in the field of inflammation related disorders.

Membrane immunoglobulin without sheath or anchor.

The canonical form of the B cell antigen receptor is composed of membrane immunoglobulin sheathed by the alpha/beta heterodimer. Whereas membrane IgM cannot be transported to the cell surface in the absence of alpha/beta, both IgD and IgG2b can be expressed naked (i.e. without alpha/beta) on the surface of myeloma transfectants. In the case of one cell-line, such naked IgD has been shown to be inserted into the membrane by a glycosyl-phosphatidylinositol anchor. Here, however, we show that both IgD and IgG2b (but not IgM) can be expressed on the surface of myeloma transfectants without either sheath or anchor. This distinction between the isotypes is attributable to differences in the region of the transmembrane segment.

Characterisation and specificity of two single-chain Fv antibodies directed to the protein tyrosine kinase Syk

In order to obtain single chain Fv fragments (scFv) specific for the protein tyrosine kinase Syk, we screened a human synthetic phage-display library. Two glutathione S-transferase (GST):Syk fusion proteins containing both SH2 domains of Syk were used to perform three rounds of selection of the library. Among the scFv fragments resulting from the third round of selection, the ones specific for the GST portion of the fusion proteins were eliminated by performing enzyme-linked immunosorbent assay tests on GST:Syk versus GST coated plates, and the monoclonal scFv fragments binding only to the GST:Syk coated plates with high affinities were further analysed. We report here the in vitro characterisation of G4G11 and G6G2 anti-Syk scFvs. G4G11 shows the best performance in immunoprecipitation and immunofluorescence experiments, and G6G2 is able to detect Syk in immunoprecipitation, immunofluorescence and on Western blots. Both scFvs are also able to detect the phosphorylated form of Syk, and neither of them binds to Zap-70, the other member of the Syk family of protein tyrosine kinases.

The promoter regions of the T‐cell receptor V9 γ (TRGV9) and V2 δ (TRDV2) genes display short direct repeats but no TATA box

Promoter region; Receptor, γ T‐cell; Receptor, δ T‐cell; Gene, variable; Transcription; (Human)

In-Cell Intrabody Selection from a Diverse Human Library Identifies C12orf4 Protein as a New Player in Rodent Mast Cell Degranulation

The high specificity of antibodies for their antigen allows a fine discrimination of target conformations and post-translational modifications, making antibodies the first choice tool to interrogate the proteome. We describe here an approach based on a large-scale intracellular expression and selection of antibody fragments in eukaryotic cells, so-called intrabodies, and the subsequent identification of their natural target within living cell. Starting from a phenotypic trait, this integrated system allows the identification of new therapeutic targets together with their companion inhibitory intrabody. We applied this system in a model of allergy and inflammation. We first cloned a large and highly diverse intrabody library both in a plasmid and a retroviral eukaryotic expression vector. After transfection in the RBL-2H3 rat basophilic leukemia cell line, we performed seven rounds of selection to isolate cells displaying a defect in FcεRI-induced degranulation. We used high throughput sequencing to identify intrabody sequences enriched during the course of selection. Only one intrabody was common to both plasmid and retroviral selections, and was used to capture and identify its target from cell extracts. Mass spectrometry analysis identified protein RGD1311164 (C12orf4), with no previously described function. Our data demonstrate that RGD1311164 is a cytoplasmic protein implicated in the early signaling events following FcεRI-induced cell activation. This work illustrates the strength of the intrabody-based in-cell selection, which allowed the identification of a new player in mast cell activation together with its specific inhibitor intrabody.