BattAllox aims at targeting multiple electron transfer and tuneable redox properties for enhanced energy storage systems. Using bioinspired design principles and molecular engineering, this project focuses on interfacing redox-active isoalloxazine and alloxazine units with coordination chemistry to deliver highly tuneable and versatile redox systems. Redox behaviour at the molecular and higher levels will be studied on small-molecule organic units, organometallic complexes and Coordination Polymers. These redox species will be the basis of a new class of robust multi-electron transfer materials, for electrode materials, for example, and will be incorporated in redox-flow batteries. This multi-scale and multidisciplinary approach bring together molecular design, electrochemical studies, coordination networks and redox-flow batteries.

IgA nephropathy (IgAN), one of the most common kidney diseases worldwide, is associated with microbial infections at mucosal sites and represents a major cause of renal failure. Its pathogenesis involves IgA1 complexes containing degalactosylated IgA1 (Gd-IgA1), autoantibodies against IgA1, and soluble IgA Fc receptor (sCD89), which are deposited in the renal mesangium. However, the origin of this abnormal IgA and the trigger for IgA immune complex formation is not clear. The IgA system is very different between humans and mice. In humans, two types of IgA are present. They differ in the hinge region: IgA1 has a longer and more heavily glycosylated hinge region compared to IgA2. As there is no homologue for human CD89 and only one type of IgA in the mouse, we previously developed humanized mice expressing both human IgA1 and CD89 (alpha1KI-CD89 transgenic (Tg) mice) as a model of IgAN. These mice spontaneously develop IgA1 mesangial deposits associated with the production of IgA1-sCD89 complexes. Moreover, IgA is differentially distributed between the systemic and mucosal immune system, and plays a key role in mucosal protection, notably secretory IgA (SIgA). In germ-free mice, the production of IgA is very low and the presence of a microbiota induces the production of mucosal SIgA. Reciprocally, IgA secretion limits the expansion of particular bacteria and regulates the microbial composition while, in turn, bacteria are capable of degrading SIgA, including removal of the secretory-component. Moreover, an intestinal microbiota dysbiosis was found in Italian IgAN patients. Recent GWAS data showed that genes involved in intestinal immunity, notably CARD9 and defensins, are linked to IgAN. Our preliminary data show that IgAN patient microbiota is associated with increased proportions of bacterial genera such as Alistipes and Ruminococcus (Firmicutes), Victivallis (Lentisphaerae), Akkermansia (Verrucomicrobia) and Bilophila (Deltaproteobacteria) as compared to control patients with other chronic kidney diseases (CKD). Moreover, faecal levels of Akkermansia muciniphilia correlated significantly with circulating degalactosylated IgA1 levels in IgAN patients. These results suggest a link between microbiota dysbiosis and IgAN. To further understand the role of the microbiota in IgAN we took advantage of our humanized mouse model, the alpha1KICD89Tg mice. Treatment of 12 w-old alpha1KICD89Tg mice with an antibiotic cocktail prevented the disease by abolishing proteinuria and decreasing mesangial IgA1 deposits, but not affecting serum IgA1 levels. In vitro results show that A. muciniphila promotes IgA1 degalactosylation inducing Gd-IgA1 retrotranscytosis by epithelial cells. In addition, alpha-defensin-6 levels are decreased in the stools of patients. In vitro results reveal that A. muciniphila is sensitive to this defensin opening a new therapeutic aproach. In addition, additional experiments have shown that CARD9 is involved in the activation of CD89. These results highlight the important role of the microbiota, defensins and CARD9 in disease development as well as pointing towards a possible origin of nephrotoxic IgA1 from mucosal sites. Our aim is to explore the relationship between the intestinal microbiota, IgA, CD89 and CARD9 using our humanized mouse model and samples from patients. Our tasks are: 1/ To characterize the intestinal microbiota in IgAN patients and in mice 2/ To define the role of mucin-degrading bacteria in IgAN pathogenesis 3/ To characterize the role of CARD9 and CD89 in microbiota dysbiosis during IgAN development 4/ To develop new therapeutic tools in alpha1KICD89Tg mice.

Bacterial communities colonize and attach to solid surfaces thanks to adhesive molecules exposed on the bacterial outer envelope. While a substantial number of molecular actors involved in bacterial adhesion have been characterized, their dynamics and their coordination on the bacterial envelope remain out of sight because the secretion machineries interfere with the fluorescence of standard probes. Recently, we showed thanks to mechanical assays that adhesive molecules were enriched at the old pole of bacteria. From this polar localization at single cell level, it results that microcolonies composed of rod-shaped bacteria develop into dense aggregates rather than into chains where bacteria would be highly exposed to their environment. This organization at the level of the community has a large impact in terms of biofilm tolerance to antibiotics and causes major health concerns by generating nosocomial diseases. In this project, we propose to use a new generation of fluorescent reporters, in order to measure the spatial dynamics of adhesive proteins exposed on the cell envelope of E. coli. By comparing physical modeling and experiments, we will aim at understanding the microscopic mechanisms that are responsible for adhesion polarity at the single cell level and how antibiotics could perturb this polarity and thus the structure of bacterial communities.

Superoxide dismutases (SOD) are very efficient redox metalloproteins, which protect the cell from oxidative stress. Their catalytic activity of superoxide dismutation can be reproduced by low-molecular weight Mn-complexes, called SOD-mimics (SODm) and the overall characteristics of SODs (tuned redox potential, electrostatic guidance of superoxide, compartmentation in organelles) can serve as a guideline in the design of efficient SODm. Oxidative stress, mainly produced in the mitochondria, is involved in inflammation, including Inflammatory Bowel Diseases (IBD), chosen as the biological target. We wish to develop manganese SODm directly inspired from the mitochondrial Mn-SOD that could exert an anti-inflammatory effect through an intracellular antioxidant activity. These will be studied in cellular models of oxidative stress relevant to IBD. This project will involve several steps. First, using a modular approach, we will conjugate a SODm, already developed by the consortium and known to be active in cells, to various vectors and probes to obtain a series of SOD-mimics with tuned cell-penetration properties or organelle targeting, which could be detected inside cells. We will also develop a new series of peptide-based Mn SODm. We will then determine their intrinsic anti-superoxide activity —kinetics of the reaction with superoxide. Their anti-inflammatory effects on several cell models relevant for IBD, intestinal epithelial cells and monocytes/macrophages, will be evaluated by measuring markers of oxidative stress and inflammation and reactive oxygen species (ROS). We will determine the intracellular content in complexes and their sub-cellular location by innovative imaging techniques. What are the main challenges in this project? This project aims at performing inorganic chemistry inside cells and is thus in line with emergent studies dealing with the control and characterization of small metal complexes in cells. This is a very active new field in inorganic chemical biology for which we need to translate the chemical knowledge we have acquired in the chemist’s round-bottom flasks into cells. Enhancing cell penetration and controlling the targeting of SODm to specific organelles is a real challenge, as is the determination of their speciation (or nature) in cells. Physico-chemical techniques to quantify and map metal cations at the sub-cellular level are now emerging: we will apply conventional fluorescence with tagged complexes but also the most recent techniques, such as X-fluorescence for direct sub-cellular mapping of Mn. Success here will certainly lead to a breakthrough in bio-inorganic chemistry as, at present, little information is available on the subcellular distribution of Mn-complexes SODm. This approach will provide guidelines for the rational improvement of antioxidant SODm with an intracellular activity. The project in inorganic biological chemistry dealing with bio-inspired catalytic SOD-mimics design, evaluation and characterization in cells, and sub-cellular imaging will be developed by a consortium with multidisciplinary expertise.

Protein-protein interactions during the secretion of neurotransmitters and local hormones or events downstream of receptor binding are difficult to study in a physiological context in situ. Although many of the players and their interactions are known, the dynamics of interactions within a cell physiology context are not because they are difficult to measure. One approach is to apply an inhibitory peptide in a spatially localised and time-resolved experiment, measuring the resulting changes in amplitude and time-course of physiological responses. This proposal takes developments in the photochemistry of one or two-photon uncaging and of peptide chemistry, particularly cell penetrating peptides (CPPs), to develop tools for the time resolved dissection of protein-protein interactions during cellular secretion and synaptic transmission. The approach is interdisciplinary, bringing together expertise in one and two-photon excitation, in development and application of CPPs, and cellullar secretion and synaptic transmission to probe protein-protein interactions during intracellular processes. By ‘caging’ critical residues to block binding, inhibitory peptides will be developed that become active only upon photolysis. Because the processes to be targeted are mainly intracellular, CPPs will permit access to the intracellular compartments; the spatial and time-resolved precision will be provided by one or two-photon photolysis in the experimental microscope. To develop specific probes there are 3 technical aspects that need to be optimised and integrated. First, intracellular access of caged peptides is usually by microinjection or perfusion from patch pipettes. CPPs will be developed to permit access of the inactive caged peptide to many cells within a tissue before release of the active peptide by photolysis localised to the structure of interest. Second, caging groups with enhanced two-photon cross-sections will be developed. Third, the caged CPP-peptide inhibitor conjugates will be tested for reduced binding affinity and biological/pharmacological inertness. Cell penetrating peptides. The proposal will develop efficient intracellular delivery for bioactive cargoes, quantifying uptake and mechanisms with MALDI-TOF mass spectrometry. New approaches are CPPs with basic domains functionalised with fatty acids, entering cells mainly by direct translocation. Rigid cyclic peptides will be developed and structures favoring direct translocation selected. Methods of ligation are also being developed to facilitate synthesis. Identifying structural elements that promote cytosolic translocation in combination with functional assays of CPP-caged inhibitors should lead to the development of new CPPs with improved properties. Photochemistry of one and two-photon cages. Caging groups with high one and two photon cross-sections, high water solubility and suitable for protecting amino N-terminal or lysine groups, sulfhydryls, alcohols or carboxylates are being developed around dimethylamino quinoline photochemistry. Endothelial cell secretion. Vascular endothelial cells regulate hemostasis, inflammation, vessel growth and repair and also provide a good model test system in culture for protein interactions during secretion. The molecules involved have been identified and molecular interventions and optical miroscopy are readily applied. Experiments will target syntaxin 2 related peptides, calmodulin inhibitory peptides and PKC inhibitory peptide. Synaptic transmission. Extracellular caged peptide inhibitors of AMPA subtype of glutamate receptor will be used to distinguish fast single site transmission from slow multi synapse spill-over and parasynaptic transmission at specific synapses in the cerebellar cortex. Intracellularly, caged peptides that interfere with receptor localisation at identifiable synapses after photolysis will be used to probe the time-course of changes in synaptic strength during burst stimulation.