Ehlers–Danlos syndrome (EDS) periodontal-type (EDS-VIII) is a connective-tissue disorder characterized by aggressive periodontitis and various joint and skin manifestations. It is inherited as an autosomal dominant trait. In 2003, a genetic study on three families established linkage to an interval on chromosome 12p13. Our new and unpublished investigations started with linkage and exome analysis in a large Tyrolean family with this disease and was subsequently extended to a large number of families with EDS periodontal-type. This led to the identification of heterozygous missense mutations in the complement C1R or C1S genes in affected individuals from an additional set of 14 families. Up to now, the proteases C1r and C1s are mainly known to play a role in the activation of the classical complement cascade, a major element of antimicrobial host defense. Individuals with homozygous null mutations in C1R or C1S are highly susceptible to microbial infections and to have greater risk in developing autoimmune diseases like systemic lupus erythematosus. Heterozygous null mutations in C1R / C1S appear to be asymptomatic and there has been no previous reports on a connection of aggressive periodontitis or Ehlers-Danlos syndrome with C1R and C1S mutations. The aim of this project is to clarify both the clinical symptoms as well as the underlying molecular mechanisms of EDS periodontal type in a multidisciplinary approach. Starting with the identification of C1R or C1S mutation in patients, the diagnosis of EDS periodontal type will be complemented by various clinical investigations on oral, joint, and skin features, autoimmunological symptoms as well as vascular disease. The structural etiology of oral soft tissues will be explored by histological and immunochistochemical analyses. Alterations of the protease function in terms of interaction with C1q and activation of the classical pathway of complement will be investigated using recombinant mutant proteins and patient serum. Since C1r and C1s interact with the collagen-like structure of C1q, we will explore the hypotheses of abnormal interaction of mutant C1r and C1s with soluble procollagens as well as possible interference with their processing. The structural impact of mutations will be investigated in relation to the identified functional impact to better characterize the molecular defects. Proving the C1r/C1s mutations as the hub of connective tissue disorders implicates yet unknown functional properties and may result in new ways of understanding crosslinks between inflammatory diseases and connective tissue homeostasis.

Understanding how asymmetry emerges from an initial symmetrical condition is a major scientific goal. Our project will focus on the emergence of LR asymmetry or Chirality in biological systems, using a complementary, multiscale approach. Our previous work identified Myosin 1D (Myo1D) as a unique chiral factor essential for LR asymmetry and capable of breaking symmetry at all biological scales in Drosophila. While the actin cytoskeleton emerges as a pivotal and conserved pathway for chirality formation, the exact cytoskeletal factors involved in this process, their interplay and the molecular mechanisms involved remain to be explored. Collaborative work between Partners will study molecular-to-organismal chirality through the following tasks: 1. New regulators of Myo1D chiral function and development of the tracheal model system 2. Role of the Myo1D-DAAM interaction to induce cytoskeleton chirality in vitro 3. Role of Myo1D and DAAM in actin organization in cells, minimal tissues and organs in vivo.

Metals are both essential for the development of organisms and toxic when found at elevated concentrations. Recent work has demonstrated that the broad-spectrum IRT1 metal transporter from the model plant Arabidopsis not only acts as a transporter, but also as a metal sensor. IRT1 is the founding member of the family of ZIP transporters, with homologs being found from bacteria to humans, and thus serves as a model for the study of this family for which little is known. We therefore propose in the course of NUTRISENSE to 1) determine the mode of transport of metals via the IRT1 transporter, 2) obtain the first 3D structure of a eukaryotic ZIP transporter, and 3) use this information to modify the capabilities of transport and perception of metals by living organisms. For this, advanced approaches in biochemistry, structural biology and molecular biology will be implemented. The knowledge gained will not only benefit plant biologists but also medical scientists.

--- BACKGROUND --- Light-activated proteins of the rhodopsin family, found in all three domains of life, have also been found to be encoded by viruses, specifically giant viruses. Little is known on these virus-encoded rhodopsins and on their possible role in viral infection. Their structure, their oligomeric organization, and their function in terms of transport and substrates are unknown. In spite of significant amino-acid sequence similarity, we have sufficient preliminary data to show that viral rhodopsins differ greatly from known rhodopsins, and can assemble in complexes resembling human pentameric ligand-gated ion channels, where the agonist is light. These proteins could therefore involve novel biological mechanisms and sustain possible applications in optogenetics. --- OBJECTIVES --- Our aims are to decipher the structure and the function of viral rhodopsins, and to examine the possibility of using these proteins as light-sensitive actuators in mammalian tissues. The results should shed light on the role of viral rhodopsins in host infection and could form the basis of new tools for optogenetic applications. --- CONSORTIUM --- The consortium assembles three partners with demonstrated, complementary expertise in the fields of structural biology (IBS-Membrane, Institut de Biologie Structurale, Grenoble), electrophysiology in model cells (IBS-Channels, Institut de Biologie Structurale, Grenoble), and electrophysiology and optogenetics in mammalian native cells (iBV, Institut de Biologie Valrose, Nice). --- WORKFLOW --- Viral rhodopsins are separated in two phylogenetic groups. We will work on representatives of each group, OLPVRI and OLPVRII. Partner IBS-Membrane will perform structural characterization of OLPVRI and OLPVRII in their different conformational states by X-ray crystallography, time-resolved serial crystallography, and single-particle cryo-electron microscopy using advanced European instruments (Grenoble synchrotron, Hamburg X-ray free electron laser, and Grenoble Titan Krios). The partner will also perform in vitro functional characterization of the proteins. In parallel, partner IBS-Channels will characterize the function the viral rhodopsins in model cells and optimize their properties by protein engineering informed by structural data. Partner iBV will test the optogenetic potential of the proteins in neuronal cells and brain slices. The proposed tasks are extremely challenging. However, the feasibility of the project is asserted by solid preliminary data and proven successful experience of the partners in the corresponding fields of science and methodologies. In particular, a high-resolution structure of OLPVRII in the ground state has already been obtained and functional characterization of OLPVRI has been started.

Glycosaminoglycans, such as heparan sulfate and chondroitin sulfate, are long, complex polysaccharide chains found on the surface of all animal cells. They are covalently attached to a core protein and mediate the interaction with diverse cellular factors, including chemokines, pathogens and signalling receptors. An immense diversity in functional and pathological roles is associated with the complex glycosaminoglycan composition. A key step of glycosaminoglycan biosynthesis, which takes place in the Golgi lumen, is the polysaccharide chain elongation. Its molecular mechanism, however, is still unknown. Moreover, the intrinsic feature of the core protein mediating the specific addition of either a heparan sulfate or chondroitin sulfate chain remains to be elucidated. The objectives of this proposal are (i) to reveal the molecular basis of heparan sulfate and chondroitin sulfate chain polymerization, (ii) to uncover the intrinsic factor in the core protein that decides the fate of the generated glycosaminoglycan chain and (iii) to study the architecture of the heparan sulfate and chondroitin sulfate polymerase complexes in their native cellular environment. To achieve these aims, in vitro glycosyltransferase assays will be combined with in cellulo functional analysis. A tetrasaccharide-peptide library will be synthesized using a chemo-enzymatic approach, and high-resolution structures of substrate-bound complexes will be determined by single-particle cryo-electron microscopy and X-ray crystallography. Golgi-localized glycosyltransferase complexes will be characterized in situ using multitask nanobodies and cryo-electron tomography. This project will provide a comprehensive understanding of glycosaminoglycan chain polymerization across the biological scales, from molecules to cells, laying the corner stone for future research on the glycosaminoglycan function in health and disease.