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Rare individuals can naturally clear chronic hepatitis B virus (HBV) infection and acquire protection from reinfection as conferred by vaccination. To examine the protective humoral response against HBV, we cloned and characterized human antibodies specific to the viral surface glycoproteins (HBsAg) from memory B cells of HBV vaccinees and controllers. We found that human HBV antibodies are encoded by a diverse set of immunoglobulin genes and recognize various conformational HBsAg epitopes. Strikingly, HBsAg-specific memory B cells from natural controllers mainly produced neutralizing antibodies able to cross-react with several viral genotypes. Furthermore, monotherapy with the potent broadly neutralizing antibody Bc1.187 suppressed viremia in vivo in HBV mouse models and led to post-therapy control of the infection in a fraction of animals. Thus, human neutralizing HBsAg antibodies appear to play a key role in the spontaneous control of HBV and represent promising immunotherapeutic tools for achieving HBV functional cure in chronically infected humans., Competing Interests: Disclosures: V. Hehle reported a patent to anti-HBV antibodies and methods of use, pending. M. Beretta reported a patent to anti-HBV antibodies and methods of use, pending. M. Bourgine reported a patent to anti-HBV antibodies and methods of use, pending. M. Ait-Goughoulte reported a patent planned on the antibodies pending, "Roche." S. Pol reported personal fees from Gilead, Abbvie, BMS, Janssen, and Roche outside the submitted work. H. Strick-Marchand reported a patent to human neutralizing HBV antibodies and their use thereof, pending. N. Pelletier reported personal fees from Hoffmann-La Roche outside the submitted work; in addition, N. Pelletier had a patent planned to be submitted, pending "Roche Innovation Center Basel." H. Mouquet reported grants from Institut Roche during the conduct of the study; in addition, H. Mouquet had a patent to anti-HBV antibodies and methods of use, pending. No other disclosures were reported., (© 2020 Hehle et al.)

Toxoplasmosis is a zoonotic disease caused by the parasite Toxoplasma gondii. Up to a third of the global human population is estimated to carry a T. gondii infection, which can result in severe complications in immunocompromised individuals and pregnant women. Humans and animals can become infected by ingesting either tissue cysts containing T. gondii bradyzoites, from raw or undercooked meat, or sporulated oocysts from environmental sources. T. gondii oocysts are released in the faeces of cats and other felids, which are the parasite's definitive hosts, leading to environmental contamination. Therefore, vaccination of the feline host against T. gondii is an interesting strategy to interrupt the parasitic life cycle and subsequently limit contamination of intermediate hosts. With this goal in mind, we tested in cats, an attenuated live strain of T. gondii deleted for the Mic1 and Mic3 genes (Mic1-3KO) that was previously shown to be an efficient vaccine candidate in mouse and sheep models. Subcutaneous or oral vaccination routes induced a high specific antibody titer in the cat sera, indicating that the Mic1-3KO strain is immunogenic for cats. To assess protection induced by the vaccine candidate strain, we followed oocysts shedding by vaccinated cats, after oral challenge with a T. gondii wild-type strain. Surprisingly, a high antibody titer did not prevent cats from shedding oocysts from the challenge strain, regardless of the vaccination route. Our results show that the Mic1-3KO vaccine candidate is immunogenic in the feline host, is well tolerated and safe, but does not confer protection against oocysts shedding after natural infection with wild type T. gondii. This result highlights the particular relationship between T. gondii and its unique definitive host, which indicates the need for further investigations to improve vaccination strategies to limit environmental and livestock contaminations., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Animals included in the subcutaneous vaccination trial were bought by Vitamfero, of which S.M. and E.S are former employees. E.S. is now an independent consultant and S.M. is currently unemployed. The authors confirm that there are no financial or personal interest, or belief that could affect their objectivity in reporting on the results obtained in this study.]., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Photosynthetic eukaryotes are challenged by a fluctuating light supply, demanding for a modulated expression of nucleus-encoded light-harvesting proteins associated with photosystem II (LHCII) to adjust light-harvesting capacity to the prevailing light conditions. Here, we provide clear evidence for a regulatory circuit that controls cytosolic LHCII translation in response to light quantity changes. In the green unicellular alga Chlamydomonas reinhardtii, the cytosolic RNA-binding protein NAB1 represses translation of certain LHCII isoform mRNAs. Specific nitrosylation of Cys-226 decreases NAB1 activity and could be demonstrated in vitro and in vivo. The less active, nitrosylated form of NAB1 is found in cells acclimated to limiting light supply, which permits accumulation of light-harvesting proteins and efficient light capture. In contrast, elevated light supply causes its denitrosylation, thereby activating the repression of light-harvesting protein synthesis, which is needed to control excitation pressure at photosystem II. Denitrosylation of recombinant NAB1 is efficiently performed by the cytosolic thioredoxin system in vitro. To our knowledge, NAB1 is the first example of stimulus-induced denitrosylation in the context of photosynthetic acclimation. By identifying this novel redox cross-talk pathway between chloroplast and cytosol, we add a new key element required for drawing a precise blue print of the regulatory network of light harvesting., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

In photosynthetic organisms, thioredoxin-dependent redox regulation is a well established mechanism involved in the control of a large number of cellular processes, including the Calvin-Benson cycle. Indeed, 4 of 11 enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomics-based approaches suggested that not only four but all enzymes of the Calvin-Benson cycle may withstand redox regulation. Here, we characterized the redox features of the Calvin-Benson enzyme phosphoglycerate kinase (PGK1) from the eukaryotic green alga Chlamydomonas reinhardtii, and we show that C. reinhardtii PGK1 (CrPGK1) activity is inhibited by the formation of a single regulatory disulfide bond with a low midpoint redox potential (-335 mV at pH 7.9). CrPGK1 oxidation was found to affect the turnover number without altering the affinity for substrates, whereas the enzyme activation appeared to be specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses, and three-dimensional modeling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys(227) and Cys(361). Based on molecular mechanics calculation, the formation of the disulfide is proposed to impose structural constraints in the C-terminal domain of the enzyme that may lower its catalytic efficiency. It is therefore concluded that CrPGK1 might constitute an additional light-modulated Calvin-Benson cycle enzyme with a low activity in the dark and a TRX-dependent activation in the light. These results are also discussed from an evolutionary point of view., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Aims: Protein S-nitrosylation, a post-translational modification (PTM) consisting of the covalent binding of nitric oxide (NO) to a cysteine thiol moiety, plays a major role in cell signaling and is recognized to be involved in numerous physiological processes and diseases in mammals. The importance of nitrosylation in photosynthetic eukaryotes has been less studied. The aim of this study was to expand our knowledge on protein nitrosylation by performing a large-scale proteomic analysis of proteins undergoing nitrosylation in vivo in Chlamydomonas reinhardtii cells under nitrosative stress., Results: Using two complementary proteomic approaches, 492 nitrosylated proteins were identified. They participate in a wide range of biological processes and pathways, including photosynthesis, carbohydrate metabolism, amino acid metabolism, translation, protein folding or degradation, cell motility, and stress. Several proteins were confirmed in vitro by western blot, site-directed mutagenesis and activity measurements. Moreover, 392 sites of nitrosylation were also identified. These results strongly suggest that S-nitrosylation could constitute a major mechanism of regulation in C. reinhardtii under nitrosative stress conditions., Innovation: This study constitutes the largest proteomic analysis of protein nitrosylation reported to date., Conclusion: The identification of 381 previously unrecognized targets of nitrosylation further extends our knowledge on the importance of this PTM in photosynthetic eukaryotes. The data have been deposited to the ProteomeXchange repository with identifier PXD000569.

Triosephosphate isomerase (TPI) catalyzes the interconversion of glyceraldehyde-3-phosphate to dihydroxyacetone phosphate. Photosynthetic organisms generally contain two isoforms of TPI located in both cytoplasm and chloroplasts. While the cytoplasmic TPI is involved in the glycolysis, the chloroplastic isoform participates in the Calvin-Benson cycle, a key photosynthetic process responsible for carbon fixation. Compared with its cytoplasmic counterpart, the functional features of chloroplastic TPI have been poorly investigated and its three-dimensional structure has not been solved. Recently, several studies proposed TPI as a potential target of different redox modifications including dithiol/disulfide interchanges, glutathionylation, and nitrosylation. However, neither the effects on protein activity nor the molecular mechanisms underlying these redox modifications have been investigated. Here, we have produced recombinantly and purified TPI from the unicellular green alga Chlamydomonas reinhardtii (Cr). The biochemical properties of the enzyme were delineated and its crystallographic structure was determined at a resolution of 1.1 Å. CrTPI is a homodimer with subunits containing the typical (β/α)8-barrel fold. Although no evidence for TRX regulation was obtained, CrTPI was found to undergo glutathionylation by oxidized glutathione and trans-nitrosylation by nitrosoglutathione, confirming its sensitivity to multiple redox modifications.

Reversible redox post-translational modifications such as oxido-reduction of disulfide bonds, S-nitrosylation, and S-glutathionylation, play a prominent role in the regulation of cell metabolism and signaling in all organisms. These modifications are mainly controlled by members of the thioredoxin and glutaredoxin families. Early studies in photosynthetic organisms have identified the Calvin-Benson cycle, the photosynthetic pathway responsible for carbon assimilation, as a redox regulated process. Indeed, 4 out of 11 enzymes of the cycle were shown to have a low activity in the dark and to be activated in the light through thioredoxin-dependent reduction of regulatory disulfide bonds. The underlying molecular mechanisms were extensively studied at the biochemical and structural level. Unexpectedly, recent biochemical and proteomic studies have suggested that all enzymes of the cycle and several associated regulatory proteins may undergo redox regulation through multiple redox post-translational modifications including glutathionylation and nitrosylation. The aim of this review is to detail the well-established mechanisms of redox regulation of Calvin-Benson cycle enzymes as well as the most recent reports indicating that this pathway is tightly controlled by multiple interconnected redox post-translational modifications. This redox control is likely allowing fine tuning of the Calvin-Benson cycle required for adaptation to varying environmental conditions, especially during responses to biotic and abiotic stresses.

Nitrosylation is a reversible post-translational modification of protein cysteines playing a major role in cellular regulation and signaling in many organisms, including plants where it has been implicated in the regulation of immunity and cell death. The extent of nitrosylation of a given cysteine residue is governed by the equilibrium between nitrosylation and denitrosylation reactions. The mechanisms of these reactions remain poorly studied in plants. In this study, we have employed glycolytic GAPDH from Arabidopsis thaliana as a tool to investigate the molecular mechanisms of nitrosylation and denitrosylation using a combination of approaches, including activity assays, the biotin switch technique, site-directed mutagenesis, and mass spectrometry. Arabidopsis GAPDH activity was reversibly inhibited by nitrosylation of catalytic Cys-149 mediated either chemically with a strong NO donor or by trans-nitrosylation with GSNO. GSNO was found to trigger both GAPDH nitrosylation and glutathionylation, although nitrosylation was widely prominent. Arabidopsis GAPDH was found to be denitrosylated by GSH but not by plant cytoplasmic thioredoxins. GSH fully converted nitrosylated GAPDH to the reduced, active enzyme, without forming any glutathionylated GAPDH. Thus, we found that nitrosylation of GAPDH is not a step toward formation of the more stable glutathionylated enzyme. GSH-dependent denitrosylation of GAPC1 was found to be linked to the [GSH]/[GSNO] ratio and to be independent of the [GSH]/[GSSG] ratio. The possible importance of these biochemical properties for the regulation of Arabidopsis GAPDH functions in vivo is discussed.

Significance: In photosynthetic organisms, besides the well-established disulfide/dithiol exchange reactions specifically controlled by thioredoxins (TRXs), protein S-glutathionylation is emerging as an alternative redox modification occurring under stress conditions. This modification, consisting of the formation of a mixed disulfide between glutathione and a protein cysteine residue, can not only protect specific cysteines from irreversible oxidation but also modulate protein activities and appears to be specifically controlled by small disulfide oxidoreductases of the TRX superfamily named glutaredoxins (GRXs)., Recent Studies: In recent times, several studies allowed significant progress in this area, mostly due to the identification of several plant proteins undergoing S-glutathionylation and to the characterization of the molecular mechanisms and the proteins involved in the control of this modification., Critical Issues: This article provides a global overview of protein glutathionylation in photosynthetic organisms with particular emphasis on the mechanisms of protein glutathionylation and deglutathionylation and a focus on the role of GRXs. Then, we describe the methods employed for identification of glutathionylated proteins in photosynthetic organisms and review the targets and the possible physiological functions of protein glutathionylation., Future Directions: In order to establish the importance of protein S-glutathionylation in photosynthetic organisms, future studies should be aimed at delineating more accurately the molecular mechanisms of glutathionylation and deglutathionylation reactions, at identifying proteins undergoing S-glutathionylation in vivo under diverse conditions, and at investigating the importance of redoxins, GRX, and TRX, in the control of this redox modification in vivo.

Nanofilm biomaterials, formed by the layer-by-layer assembly of charged macromolecules, are important systems for a variety of cell-contacting biomedical and biotechnological applications. Mechanical rigidity and bioactivity are two key film properties influencing the behavior of contacting cells. Increased rigidity tends to improve cells attachment, and films may be rendered bioactive through the incorporation of proteins, peptides, or drugs. A key challenge is to realize films that are simultaneously rigid and bioactive. Chemical cross-linking of the polymer framework--the standard means of increasing a film's rigidity--can diminish bioactivity through deactivation or isolation of embedded biomolecules or inhibition of film biodegradation. We present here a strategy to decouple mechanical rigidity and bioactivity, potentially enabling nanofilm biomaterials that are both mechanically rigid and bioactive. Our idea is to selectively cross-link the outer region of the film, resulting in a rigid outer skin to promote cell attachment, while leaving the film interior (with any embedded bioactive species) unaffected. We propose an approach whereby an N-hydroxysulfosuccinimide (sulfo-NHS) activated poly(L-glutamic acid) is added as the terminal layer of a multilayer film and forms (covalent) amide bonds with amino groups of poly(L-lysine) placed previously within the film. We characterize film assembly and cross-linking extent via quartz crystal microbalance with dissipation monitoring (QCMD), Fourier transform infrared spectroscopy in attenuated total reflection mode (FTIR-ATR), and laser scanning confocal microscopy (LSCM) and measure the attachment and metabolic activity of preosteoblastic MC3T3-E1 cells. We show cross-linking to occur primarily at the film surface and the subsequent cell attachment and metabolic activity to be enhanced compared to native films. Our method appears promising as a means to realize films that are simultaneously mechanically rigid and bioactive.