Christophe Blanchetot

Regulation of receptor protein-tyrosine phosphatase α by oxidative stress

The presence of two protein‐tyrosine phosphatase (PTP) domains is a striking feature in most transmembrane receptor PTPs (RPTPs). The function of the generally inactive membrane‐distal PTP domain (RPTP‐D2) is unknown. Here we report that an intramolecular interaction between the spacer region (Sp) and the C‐terminus in RPTPα prohibited intermolecular interactions. Interestingly, stress factors such as H2O2, UV and heat shock induced reversible, free radical‐dependent, intermolecular interactions between RPTPα and RPTPα‐SpD2, suggesting an inducible switch in conformation and binding. The catalytic site cysteine of RPTPα‐SpD2, Cys723, was required for the H2O2 effect on RPTPα. H2O2 induced a rapid, reversible, Cys723‐dependent conformational change in vivo, as detected by fluorescence resonance energy transfer, with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) flanking RPTPα‐SpD2 in a single chimeric protein. Importantly, H2O2 treatment stabilized RPTPα dimers, resulting in inactivation. We propose a model in which oxidative stress induces a conformational change in RPTPα‐D2, leading to stabilization of RPTPα dimers, and thus to inhibition of RPTPα activity.

CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells

The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monoclonal antibodies. Here we report the isolation and characterization of functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method. The highly selective VHH-based immunoglobulin single variable domains competitively inhibited the CXCR4-mediated signaling and antagonized the chemoattractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to distinct but partially overlapping sites in the extracellular loops. Short peptide linkage of 238D2 with 238D4 resulted in significantly increased affinity for CXCR4 and picomolar activity in antichemotactic assays. Interestingly, the monovalent nanobodies behaved as neutral antagonists, whereas the biparatopic nanobodies acted as inverse agonists at the constitutively active CXCR4-N3.35A. The CXCR4 nanobodies displayed strong antiretroviral activity against T cell-tropic and dual-tropic HIV-1 strains. Moreover, the biparatopic nanobody effectively mobilized CD34-positive stem cells in cynomolgus monkeys. Thus, the nanobody platform may be highly effective at generating extremely potent and selective G protein-coupled receptor modulators.

Transforming growth factor-beta-stimulated clone-22 is a member of a family of leucine zipper proteins that can homo- and heterodimerize and has transcriptional repressor activity.

TGF-β-stimulatedclone-22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved during evolution. Two homologues are known that share a similar leucine zipper domain and another conserved domain (designated the TSC box). Only limited data are available on the function of TSC-22 and its homologues. TSC-22 is transcriptionally up-regulated by many different stimuli, including anti-cancer drugs and growth inhibitors, and recent data suggest that TSC-22 may play a suppressive role in tumorigenesis. In this paper we show that TSC-22 forms homodimers via its conserved leucine zipper domain. Using a yeast two-hybrid screen, we identified a TSC-22 homologue (THG-1) as heterodimeric partner. Furthermore, we report the presence of two more mammalian family members with highly conserved leucine zippers and TSC boxes. Interestingly, both TSC-22 and THG-1 have transcriptional repressor activity when fused to a heterologous DNA-binding domain. The repressor activity of TSC-22 appears sensitive for promoter architecture, but not for the histone deacetylase inhibitor trichostatin A. Mutational analysis showed that this repressor activity resides in the non-conserved regions of the protein and is enhanced by the conserved dimerization domain. Our results suggest that TSC-22 belongs to a family of leucine zipper-containing transcription factors that can homodimerize and heterodimerize with other family members and that at least two TSC-22 family members may be repressors of transcription.

Dimerization of Receptor Protein-Tyrosine Phosphatase alpha in living cells

BackgroundDimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs) involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs), are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined.ResultsIn order to assess RPTP dimerization, we have assayed Fluorescence Resonance Energy Transfer (FRET) between chimeric proteins of cyan- and yellow-emitting derivatives of green fluorescent protein, fused to RPTPα, using three different techniques: dual wavelength excitation, spectral imaging and fluorescence lifetime imaging. All three techniques suggested that FRET occurred between RPTPα -CFP and -YFP fusion proteins, and thus that RPTPα dimerized in living cells. RPTPα dimerization was constitutive, extensive and specific. RPTPα dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker. Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization.ConclusionsWe demonstrate here that RPTPα dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs.

Multimerization of the protein-tyrosine phosphatase (PTP)-like insulin-dependent diabetes mellitus autoantigens IA-2 and IA-2beta with receptor PTPs (RPTPs). Inhibition of RPTPalpha enzymatic activity.

Most receptor-type protein-tyrosine phosphatases (RPTPs) contain two tandem PTP domains. For some RPTPs the enzymatically inactive membrane-distal phosphatase domains (D2) were found to bind enzymatically active membrane proximal PTP (D1) domains, and oligomerization has been proposed as a general regulatory mechanism. The RPTP-like proteins IA-2 and IA-2β, major autoantigens in insulin-dependent diabetes mellitus, contain just a single enzymatically inactive PTP-like domain. Their physiological role is as yet enigmatic. To investigate whether the catalytically inactive cytoplasmic domains of IA-2 and IA-2β are involved in oligomerization, we exploited interaction trap assay in yeast and glutathione S-transferase pull-down and co-immunoprecipitation strategies on lysates of transfected COS-1 cells. The results show that IA-2 and IA-2β are capable of homo- and heterodimerization to which both the juxtamembrane region and the phosphatase-like segment can contribute. Furthermore, they can form heterodimers with some other RPTP members, most notably RPTPα and RPTPε, and down-regulate RPTPα enzymatic activity. Thus, in addition to homo-dimerization, the enzymatic activity of receptor-type PTPs can be regulated through heterodimerization with other RPTPs, including the catalytically inactive IA-2 and IA-2β.

Identification of p130cas as an in vivo substrate of receptor protein-tyrosine phosphatase alpha.

We have employed a substrate trapping strategy to identify physiological substrates of the receptor protein-tyrosine phosphatase α (RPTPα). Here we report that a substrate-trapping mutant of the RPTPα membrane proximal catalytic domain (D1), RPTPα-D1-C433S, specifically bound to tyrosine-phosphorylated proteins from pervanadate-treated cells. The membrane distal catalytic domain of RPTPα (D2) and mutants thereof did not bind to tyrosine-phosphorylated proteins. The pattern of tyrosine-phosphorylated proteins that bound to RPTPα-D1-C433S varied between cell lines, but a protein of approximately 130 kDa was pulled down from every cell line. This protein was identified as p130 cas . Tyrosine-phosphorylated p130 cas from fibronectin-stimulated NIH3T3 cells bound to RPTPα-D1-C433S as well, suggesting that p130 cas is a physiological substrate of RPTPα. RPTPα dephosphorylated p130 cas in vitro, and RPTPα co-localized with a subpopulation of p130 cas to the plasma membrane. Co-transfection experiments with activated SrcY529F, p130 cas , and RPTPα or inactive, mutant RPTPα indicated that RPTPα dephosphorylated p130 cas in vivo. Tyrosine-phosphorylated epidermal growth factor receptor was not dephosphorylated by RPTPα under these conditions, suggesting that p130 cas is a specific substrate of RPTPα in living cells. In conclusion, our results provide evidence that p130 cas is a physiological substrate of RPTPα in vivo.

Four individually druggable MET hotspots mediate HGF-driven tumor progression.

Activation of MET by HGF plays a key role in tumor progression. Using a recently developed llama platform that generates human-like immunoglobulins, we selected 68 different antibodies that compete with HGF for binding to MET. HGF-competing antibodies recognized 4 distinct hotspots localized in different MET domains. We identified 1 hotspot that coincides with the known HGF β chain binding site on blades 2-3 of the SEMA domain β-propeller. We determined that a second and a third hotspot lie within blade 5 of the SEMA domain and IPT domains 2-3, both of which are thought to bind to HGF α chain. Characterization of the fourth hotspot revealed a region across the PSI-IPT 1 domains not previously associated with HGF binding. Individual or combined targeting of these hotspots effectively interrupted HGF/MET signaling in multiple cell-based biochemical and biological assays. Selected antibodies directed against SEMA blades 2-3 and the PSI-IPT 1 region inhibited brain invasion and prolonged survival in a glioblastoma multiforme model, prevented metastatic disease following neoadjuvant therapy in a triple-negative mammary carcinoma model, and suppressed cancer cell dissemination to the liver in a KRAS-mutant metastatic colorectal cancer model. These results identify multiple regions of MET responsible for HGF-mediated tumor progression, unraveling the complexity of HGF-MET interaction, and provide selective molecular tools for targeting MET activity in cancer.

Neutralizing Nanobodies Targeting Diverse Chemokines Effectively Inhibit Chemokine Function

Background: Chemokines play a prominent role in inflammatory diseases. Results: Nanobodies targeting chemokines display high affinity and potently neutralize chemokine-induced receptor binding and signaling. Conclusion: Neutralizing Nanobodies targeting chemokines effectively inhibit chemokine function. Significance: Nanobodies directed against inflammatory and homeostatic chemokines form a promising new class of potent and specific inhibitors of chemokine function, to be used for research and therapeutic purposes. Chemokine receptors and their ligands play a prominent role in immune regulation but many have also been implicated in inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, allograft rejection after transplantation, and also in cancer metastasis. Most approaches to therapeutically target the chemokine system involve targeting of chemokine receptors with low molecular weight antagonists. Here we describe the selection and characterization of an unprecedented large and diverse panel of neutralizing Nanobodies (single domain camelid antibodies fragment) directed against several chemokines. We show that the Nanobodies directed against CCL2 (MCP-1), CCL5 (RANTES), CXCL11 (I-TAC), and CXCL12 (SDF-1α) bind the chemokines with high affinity (at nanomolar concentration), thereby blocking receptor binding, inhibiting chemokine-induced receptor activation as well as chemotaxis. Together, we show that neutralizing Nanobodies can be selected efficiently for effective and specific therapeutic treatment against a wide range of immune and inflammatory diseases.

Targeting chemokines and chemokine receptors with antibodies

Chemokines and their receptors are highly interesting therapeutic targets for pharmaceutical and biotechnology companies. In particular, industrial development pipelines are filled with new chemokine-targeting drugs to treat inflammatory diseases and malignancies. In this review, we specifically highlight antibody-based therapeutics and monoclonal antibodies (mAbs) targeting the chemokine system. Besides discussing pitfalls inherently linked to their discovery, we will elaborate on where progress can be made in the development of novel human therapeutic antibodies directed at the chemokine system.

Neutralisation of HIV-1 cell-cell spread by human and llama antibodies

BackgroundDirect cell-cell spread of HIV-1 is a very efficient mode of viral dissemination, with increasing evidence suggesting that it may pose a considerable challenge to controlling viral replication in vivo. Much current vaccine research involves the study of broadly neutralising antibodies (bNabs) that arise during natural infection with the aims of eliciting such antibodies by vaccination or incorporating them into novel therapeutics. However, whether cell-cell spread of HIV-1 can be effectively targeted by bNabs remains unclear, and there is much interest in identifying antibodies capable of efficiently neutralising virus transmitted by cell-cell contact.ResultsIn this study we have tested a panel of bNAbs for inhibition of cell-cell spread, including some not previously evaluated for inhibition of this mode of HIV-1 transmission. We found that three CD4 binding site antibodies, one from an immunised llama (J3) and two isolated from HIV-1-positive patients (VRC01 and HJ16) neutralised cell-cell spread between T cells, while antibodies specific for glycan moieties (2G12, PG9, PG16) and the MPER (2F5) displayed variable efficacy. Notably, while J3 displayed a high level of potency during cell-cell spread we found that the small size of the llama heavy chain-only variable region (VHH) J3 is not required for efficient neutralisation since recombinant J3 containing a full-length human heavy chain Fc domain was significantly more potent. J3 and J3-Fc also neutralised cell-cell spread of HIV-1 from primary macrophages to CD4+ T cells.ConclusionsIn conclusion, while bNabs display variable efficacy at preventing cell-cell spread of HIV-1, we find that some CD4 binding site antibodies can inhibit this mode of HIV-1 dissemination and identify the recently described llama antibody J3 as a particularly potent inhibitor. Effective neutralisation of cell-cell spread between physiologically relevant cell types by J3 and J3-Fc supports the development of VHH J3 nanobodies for therapeutic or prophylactic applications.