Antibody-mediated neutralization of galectin-3 as a strategy for the treatment of systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune, inflammatory and fibrotic disease with limited treatment options. Developing new therapies is therefore crucial to address patient needs. To this end, we focused on galectin-3 (Gal-3), a lectin known to be associated with several pathological processes seen in SSc. Using RNA sequencing of whole-blood samples in a cross-sectional cohort of 249 patients with SSc, Gal-3 and its interactants defined a strong transcriptomic fingerprint associated with disease severity, pulmonary and cardiac malfunctions, neutrophilia and lymphopenia. We developed new Gal-3 neutralizing monoclonal antibodies (mAb), which were then evaluated in a mouse model of hypochlorous acid (HOCl)-induced SSc. We show that two of these antibodies, D11 and E07, reduced pathological skin thickening, lung and skin collagen deposition, pulmonary macrophage content, and plasma interleukin-5 and -6 levels. Moreover, E07 changed the transcriptional profiles of HOCl-treated mice, resulting in a gene expression pattern that resembled that of control mice. Similarly, pathological pathways engaged in patients with SSc were counteracted by E07 in mice. Collectively, these findings demonstrate the translational potential of Gal-3 blockade as a therapeutic option for SSc.

Further assessments of ligase LplA-mediated modifications of proteins in vitro and in cellulo.

BACKGROUND: Posttranslational modifications of proteins are catalyzed by a large family of enzymes catalyzing many chemical modifications. One can hijack the natural use of those enzymes to modify targeted proteins with synthetic chemical moieties. The lipoic acid ligase LplA mutants can be used to introduce onto the lysine sidechain lipoic acid moiety synthetic analogues. Substrate protein candidates of the ligase must obey a few a priori rules. METHODS AND RESULTS: In the present report, we technically detailed the use of a cell line stably expressing both the ligase and a model protein (thioredoxin). Although the goal can be reach, and the protein visualized in situ, many experimental difficulties must be fixed. The sequence of events comprises (i) in cellulo labeling of the target protein with a N3-lipoic acid derivative catalyzed by the mutant ligase, (ii) the further introduction by click chemistry onto this lysine sidechain of a fluorophore and (iii) the following of the labeled protein in living cells. One of the main difficulties was to assess the click chemistry step onto the living cells, because images from both control and experimental cells were similar. Alternatively, we describe at that stage, the preferred use of another technique: the Halo-Tag one that led to the obtention of clear images of the targeted protein in its cellular context. Although the ligase-mediated labeling of protein in situ is a rich domain for which many cellular tools must be developed, many difficulties must be considered before entering a systematic use of this approach. CONCLUSIONS: In the present contribution, we added several steps of analytical characterization, both in vitro and in cellulo that were previously lacking. Furthermore, we show that the use of the click chemistry should be manipulated with care, as the claimed specificity might be not complete whenever living cells are used. Finally, we added another approach-the Halo Tag-to complete the previously suggested approaches for labelling proteins in cells, as we found difficult to strictly apply the previously reported methodology.

Biochemistry, structure, and cellular internalization of a four nanobody-bearing Fc dimer.

VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a set of interesting proteins derived from antibodies that maintain their capacity to recognize the antigen, despite their relatively small molecular weight (in the 12,000 Da range). Continuing our exploration of the possibilities of those molecules, we chose to design alternative molecules with maintained antigen recognition, but enhanced capacity, by fusing four VHH with one Fc, the fragment crystallizable region of antibodies. In doing so, we aimed at having a molecule with superior quantitative antigen recognition (×4) while maintaining its size below the 110 kDa. In the present paper, we described the building of those molecules that we coined VHH2 -Fc-VHH2 . The structure of VHH2 -Fc-VHH2 in complex with HER2 antigen was determined using electronic microscopy and modeling. The molecule is shown to bind four HER2 proteins at the end of its flexible arms. VHH2 -Fc-VHH2 also shows an internalization capacity via HER2 receptor superior to the reference anti-HER2 monoclonal antibody, Herceptin®, and to a simple fusion of two VHH with one Fc (VHH2 -Fc). This new type of molecules, VHH2 -Fc-VHH2 , could be an interesting addition to the therapeutic arsenal with multiple applications, from diagnostic to therapy.

S95021, a novel selective and pan-neutralizing anti interferon alpha (IFN-α) monoclonal antibody as a candidate treatment for selected autoimmune rheumatic diseases.

Increased interferon-α (IFN-α) production is a critical component in the pathophysiology of systemic lupus erythematosus (SLE) and other rheumatic autoimmune diseases. Herein, we report the characterization of S95021, a fully human IgG1 anti-IFN-α monoclonal antibody (mAb) as a novel therapeutic candidate for targeted patient populations. S95021 was expressed in CHOZN GS-/- cells, purified by chromatography and characterized by using electrophoresis, size exclusion chromatography and liquid chromatography-mass spectrometry. High purity S95021 was obtained as a monomeric entity comprising different charge variants mainly due to N-glycosylation. Surface plasmon resonance kinetics experiments showed strong association rates with all IFN-α subtypes and estimated KDs below picomolar values. Pan-IFN-α-binding properties were confirmed by immunoprecipitation assays and neutralization capacity with reporter HEK-Blue IFN-α/ß cells. S95021 was IFN-α-selective and exhibited superior potency and broader neutralization profile when compared with the benchmark anti-IFN-α mAbs rontalizumab and sifalimumab. STAT-1 phosphorylation and the type I IFN gene signature induced in human peripheral blood mononuclear cells by recombinant IFN-α subtypes or plasmas from selected autoimmune patients were efficiently reduced by S95021 in a dose-dependent manner. Together, our results show that S95021 is a new potent, selective and pan IFN-α-neutralizing mAb. It is currently further evaluated as a valid therapeutic candidate in selected autoimmune diseases in which the IFN-α pro-inflammatory pathway is dysregulated.

VHH characterization.Recombinant VHHs: Production, characterization and affinity.

Among the biological approaches to therapeutics, are the cells, such as CAR-T cells engineered or not, the antibodies armed or not, and the smaller protein scaffolds that can be modified to render them specific of other proteins, à la façon of antibodies. For several years, we explored ways to substitute antibodies by nanobodies (also known as VHHs), the smallest recognizing part of camelids' heavy-chain antibodies: production of those small proteins in host microorganisms, minute analyses, characterization, and qualification of their affinity towards designed targets. Here, we present three standard VHHs described in the literature: anti-albumin, anti-EGF receptor and anti-HER2, a typical cancer cell surface -associated protein. Because they differ slightly in global structure, they are good models to assess our body of analytical methodologies. The VHHs were expressed in several bacteria strains in order to identify and overcome the bottlenecks to obtain homogeneous preparations of this protein. A large panel of biophysical tools, ranging from spectroscopy to mass spectrometry, was here combined to assess VHH structural features and the impact of the disulfide bond. The routes are now ready to move to more complex VHHs raised against specific targets in numerous areas including oncology.

Screening ubiquitin specific protease activities using chemically synthesized ubiquitin and ubiquitinated peptides.

Ubiquitin, a 76 amino acid protein, is a key component that contributes to cellular protein homeostasis. The specificity of this modification is due to a series of enzymes: ligases, attaching the ubiquitin to a lysine, and deubiquitinases, which remove it. More than a hundred of such proteins are implicated in the regulation of protein turnover. Their specificities are only partially understood. We chemically synthesized ubiquitin, attached it to lysines belonging to the protein sequences known to be ubiquitinated. We chose the model protein "murine double minute 2" (mdm2), a ubiquitin ligase, itself ubiquitinated and deubiquitinated. We folded the ubiquitinated peptides and checked their tridimensional conformation. We assessed the use of these substrates with a series of fifteen deubiquitinases to show the potentiality of such an enzymological technique. By manipulating the sequence of the peptide on which ubiquitin is attached, we were able to detect differences in the enzyme/substrate recognition, and to determine that these differences are deubiquitinase-dependent. This approach could be used to understand the substrate/protein relationship between the protagonists of this reaction. The methodology could be customized for a given substrate and used to advance our understanding of the key amino acids responsible for the deubiquitinase specificities.

Total chemical synthesis, refolding, and crystallographic structure of fully active immunophilin calstabin 2 (FKBP12.6).

Synthetic biology (or chemical biology) is a growing field to which the chemical synthesis of proteins, particularly enzymes, makes a fundamental contribution. However, the chemical synthesis of catalytically active proteins (enzymes) remains poorly documented because it is difficult to obtain enough material for biochemical experiments. We chose calstabin, a 107-amino-acid proline isomerase, as a model. We synthesized the enzyme using the native chemical ligation approach and obtained several tens of milligrams. The polypeptide was refolded properly, and we characterized its biophysical properties, measured its catalytic activity, and then crystallized it in order to obtain its tridimensional structure after X-ray diffraction. The refolded enzyme was compared to the recombinant, wild-type enzyme. In addition, as a first step of validating the whole process, we incorporated exotic amino acids into the N-terminus. Surprisingly, none of the changes altered the catalytic activities of the corresponding mutants. Using this body of techniques, avenues are now open to further obtain enzymes modified with exotic amino acids in a way that is only barely accessible by molecular biology, obtaining detailed information on the structure-function relationship of enzymes reachable by complete chemical synthesis.

Detergent-free Isolation of Functional G Protein-Coupled Receptors into Nanometric Lipid Particles.

G protein-coupled receptors (GPCRs) are integral membrane proteins that play a pivotal role in signal transduction. Understanding their dynamics is absolutely required to get a clear picture of how signaling proceeds. Molecular characterization of GPCRs isolated in detergents nevertheless stumbles over the deleterious effect of these compounds on receptor function and stability. We explored here the potential of a styrene-maleic acid polymer to solubilize receptors directly from their lipid environment. To this end, we used two GPCRs, the melatonin and ghrelin receptors, embedded in two membrane systems of increasing complexity, liposomes and membranes from Pichia pastoris. The styrene-maleic acid polymer was able, in both cases, to extract membrane patches of a well-defined size. GPCRs in SMA-stabilized lipid discs not only recognized their ligand but also transmitted a signal, as evidenced by their ability to activate their cognate G proteins and recruit arrestins in an agonist-dependent manner. Besides, the purified receptor in lipid discs undergoes all specific changes in conformation associated with ligand-mediated activation, as demonstrated in the case of the ghrelin receptor with fluorescent conformational reporters and compounds from distinct pharmacological classes. Altogether, these data highlight the potential of styrene-maleic stabilized lipid discs for analyzing the molecular bases of GPCR-mediated signaling in a well-controlled membrane-like environment.

Mutagenic analysis in a pure molecular system shows that thioredoxin-interacting protein residue Cys247 is necessary and sufficient for a mixed disulfide formation with thioredoxin.

The human thioredoxin (TRX)-interacting protein is found in multiple subcellular compartments and plays a major role in redox homeostasis, particularly in the context of metabolism (e.g., lipidemia and glycemia) and apoptosis. A molecular approach to the protein's modus operandi is still needed because some aspects of the TRX-interacting protein-mediated regulation of TRX are not clearly understood. To this end, His-tagged TRX-interacting proteins were over-expressed in Escherichia coli. Because the protein is expressed mainly in inclusion bodies, it was denatured in high concentrations of guanidium hydrochloride, centrifuged, and purified by Ni-NTA affinity chromatography. His-TRX-interacting protein was then refolded by dialysis and its restructuring monitored by circular dichroism spectrometry. This preparation resulted in the formation of a covalent complex with recombinant human TRX, demonstrating that association occurs without the intervention of other partner proteins. Multiple cysteine-to-serine mutants of TRX-interacting protein were produced and purified. These mutations were efficient in limiting the formation of disulfide-linked homo-oligomers in an oxidizing environment. The mutants were also used to gain functional insight into the formation of the TRX-interacting protein-TRX complexes. These complexes were able to form in the absence of internal disulfide bridges. A mutant with all but one cysteine changed to serine (Cys ²47) also showed an enhanced capacity to form complexes with TRX demonstrating, in a pure molecular system, that this particular cysteine is likely responsible for the disulfide bridge between TRX-interacting protein and TRX.

Structural and functional characterization of the recombinant human mitochondrial trifunctional protein.

The α and ß subunits of the human mitochondrial trifunctional protein (TFP), the multienzyme complex involved in fatty acid ß-oxidation, were coexpressed in Escherichia coli and purified to homogeneity by nickel affinity chromatography. The resulting α/His-ß construct was analyzed by gel filtration, sedimentation velocity, and electron microscopy, indicating a predominance of α(2)ß(2) and α(4)ß(4) complexes, with higher order oligomers. Electron microscopy indicated that the elementary species α(2)ß(2) had overall structural similarity with its bacterial homologue. As shown by cosedimentation and surface plasmon resonance analyses, recombinant TFP interacted strongly with cardiolipin and phosphatidylcholine, suggesting that the natural complex associates with the inner mitochondrial membrane through direct interactions with phospholipids. Recombinant TFP displayed 2-enoyl-CoA hydratase (ECH), l-3-hydroxyacyl-CoA dehydrogenase (HACD), and 3-ketoacyl-CoA thiolase (KACT) activities, and ECH and HACD each reached equilibrium when the downstream enzymes (HACD and KACT, respectively) were made inactive, indicating feed-back inhibition. The KACT activity was optimal at pH 9.5, sensitive to ionic strength, and inhibited at concentrations of its substrate 3-ketohexadecanoyl-CoA >5 µM. Its kinetic constants (k(cat) = 169 s(-1), K(m) = 4 µM) were consistent with those determined previously on a purified porcine TFP preparation. Using different assays, trimetazidine, an efficient antiaginal agent, had no significant inhibitory effect on any of the three enzymatic activities of the recombinant TFP preparation, in contrast with other reports. This study provides the first detailed structural and functional characterization of a recombinant human TFP preparation and opens the way to in-depth analyses through site-directed mutagenesis.

Molecular pharmacology of adipocyte-secreted autotaxin.

Autotaxin is a type II ecto-nucleotide pyrophosphate phosphodiesterase enzyme. It has been recently discovered that autotaxin also catalyses a lyso-phospholipase D activity. This enzyme probably provides most of the extracellular lyso-phosphatidic acid from lyso-phosphatidylcholine. There is almost no pharmacological tools available to study autotaxin. Indeed, all the reported inhibitors, thus far, are uneasy-to-use, lyso-phosphatidic acid derivatives. Initially, autotaxin was recognized as a phosphodiesterase (NPP2) [Bollen et al., Curr. Rev. Biochem. Biol. 35 (2000) 393-432], based on sequence similarity and enzymatic capability of autotaxin to catalyse ecto-nucleotidase activity. Phosphodiesterase forms a large family of enzymes characterized by a large number of chemically diverse inhibitors. None of them have been tested on autotaxin activity. For this reason, we screened those reported inhibitors, as well as a series of compounds, mostly kinase inhibitor-oriented, on autotaxin activity. Only two compounds of the various phosphodiesterase inhibitors (calmidazolium and vinpocetine) were potent enough to inhibit autotaxin catalytic activity. From the kinase inhibitor library, we found damnacanthal and hypericin, inhibiting phosphodiesterase activity in the 100-microM range, comparable to most of other available phospholipid-like inhibitors.

Murine and human autotaxin alpha, beta, and gamma isoforms: gene organization, tissue distribution, and biochemical characterization.

Autotaxin is a type II ectonucleotide pyrophosphate phosphodiesterase enzyme. It has been recently discovered that it also has a lysophospholipase D activity. This enzyme probably provides most of the extracellular lysophosphatidic acid from lysophosphatidylcholine. The cloning and tissue distribution of the three isoforms (imaginatively called alpha, beta, and gamma) from human and mouse are reported in this study, as well as their tissue distribution by PCR in the human and mouse. The fate of the alpha isoform from human was also studied after purification and using mass spectrometry. Indeed, this particular isoform expresses the intron 12 in which a cleavage site is present, leading to a rapid catabolism of the isoform. For the human isoform gamma and the total autotaxin mRNA expression, quantitative PCR is presented in 21 tissues. The isoforms were expressed in two different hosts, insect cells and Chinese hamster ovary cells, and were highly purified. The characteristics of the six purified isoforms (pH and temperature dependence, K(m) and V(max) values, and their dependence on metal ions) are presented in this study. Their sensitivity to a small molecule inhibitor, hypericin, is also shown. Finally, the specificity of the isoforms toward a large family of lysophosphatidylcholines is reported. This study is the first complete description of the reported autotaxin isoforms.