Understanding the function of flexible biopolymers such as disordered proteins, glycans or oligonucleotides requires insight in their conformational ensembles. Liquid-state NMR is the leading technique reporting on this. Unfortunately, fast structural averaging on the NMR time scale strongly reduces chemical shift dispersion, which, given the many 1H-1H couplings, results in spectral overlap. This obstructs resolving 1H NMR data, such as residual dipolar couplings (RDCs), that report on long-range structural order and allosteric changes. In addition, molecular flexibility has until now greatly complicated the interpretation of RDC data. URANUS proposes a general strategy to solve both these issues for any biopolymer. First, it capitalizes on recent ‘pure shift’ methods to boost spectral resolution and RDC extraction by an order of magnitude. Second, a recently introduced method is used to treat RDC data in presence of molecular flexibility: Molecular Dynamics simulations with Orientational Constraints (MDOC). Both these approaches have recently been introduced for small molecule NMR, and in this project their use will be explored for the first time for conformational ensemble analysis of biopolymers. This approach will be directly applied to two biomedically relevant case-studies where insight in the conformational ensemble is key to comprehend the relation between molecular structure and biological activity. (1) The proline-rich region of the non-structural protein 5A (NS5A), a disordered protein region where it has been shown that transient conformations that are yet unidentified play an essential role in Hepatitis C virus RNA replication. (2) Oligosaccharides fragments of the structurally heterogeneous and flexible polysaccharide heparin, which is a clinically very important anticoagulant whose activity is determined by specific sulfation patterns, but conformational ensemble as a function of these patterns is not yet fully understood. URANUS aims to take the determination of conformational ensembles to a next level by maximizing the amount of spectral information and facilitating their translation into the ensemble. The goal is to provide a better description of conformational ensembles than currently feasible, allowing to resolve the subtleties necessary to clearly link biopolymers molecular structure to biological activity. On the mid-term, we will create a widely accessible toolbox of techniques that is generally applicable to any biopolymer.

The objective of the COVID-HEART project is to understand the pathophysiological mechanisms of long-term cardiometabolic complications in SARS-CoV-2 infection, aiming to identify predictive markers of these complications in COVID-19 patients. This project is related to the general thematic “Physiopathogénie et épidémiologie” and more precisely to “New preclinical research models to validate innovative therapeutic approaches” (the project is based on the use of a new experimental model of SARS-CoV-2 infection in obese and old hamsters) and to “Mid- and long-term consequences of COVID-19 in infected patients” (the project will evaluate the consequences of SARS-CoV-2 infection on the heart at different times post-infection). We will use an experimental model of infection in the hamster that allows longitudinal monitoring of the infection; it allows the collection of cardiac biopsies, pericardial adipose tissue and blood samples at different times after infection. This preclinical model is well-established and was found to mimic most of the clinical and pathological features of COVID-19. Thus, this model will be instrumental to validate innovative and effective therapeutic approaches after having characterized the cardiac impact of SARS-CoV-2 infection. There will be 3 groups, lean young adult hamsters, obese young adult hamsters (high-fat/high-sugar diet-induced obesity model), and aged hamsters (>21 months). The last two groups represent the classes of patients most at risk of long-term sequelae from COVID-19, including cardiac ones. The COVID-HEART project is divided into 3 workpackages (WP). The WP1 is dedicated to the molecular phenotyping of the heart and pericardial adipose tissues by histology, immunohistochemistry, gene expression analyses by RT-qPCR, and analyses of proteins involved in inflammation, oxidative stress, mitochondrial metabolism and autophagy by western blot. The WP2 is dedicated to the analysis of Clusterin, Renin-Angiotensin- System components and new targets of SARS-CoV-2 infection-induced severe cardiac outcomes by microarray. The WP3 is dedicated to deciphering markers for the early prediction of severe cardiac outcomes by using system biology analysis of the targets identified to be modulated in the heart following SARS-CoV-2 infection, by quantifying in blood samples the targets selected from the hamster cardiac biopsies and by validating the data using cardiac biopsies and associated blood samples obtained from patients died from COVID-19, who belong to the "LICORNE" cohort. The aims of the COVID-HEART project are: 1) to demonstrate that the preclinical model of SARS-CoV-2 infection in hamsters is useful to validate innovative and effective therapeutic approaches after having characterized the cardiac impact of SARS-CoV-2 infection, and 2) to identify markers predictive of severe cardiac outcomes following SARS-CoV-2 infection, notably in the most vulnerable populations of obese and aged individuals.

The Tau protein is a common pathophysiological player in cancers and neurodegenerative diseases and, overall, an important therapeutic target. Tau is mainly known as a cytosolic microtubule-associated protein that aggregates into filaments leading to neurodegeneration in pathological conditions referred to as tauopathies. In addition to microtubule dynamics, Tau’s physiological roles also include the protection and reparation of DNA following damage in the nucleus. However, Tau re-expression causes resistance to chemo- or radiotherapy during cancer treatment. The primary goal of the proposal NanoTarget is to develop new antibody-based tools for the intracellular targeting of Tau to modulate the protein’s binding to its partners. Within NanoTarget, the French and German project partners will combine their expertise in biological chemistry, structural biology, biochemistry and molecular physiology to generate cell-permeable Tau-specific nanobodies (Nbs) for their biological evaluation in living cells, including neurons. We will enable applications related to cancer and neurodegeneration, thus requiring delivery of the Nbs at a specific location inside the cell, to reach the core of the pathology. We hypothesize that Tau-specific Nbs could prevent Tau self-association inside neurons in a series of related pathologies called tauopathies, which include Alzheimer’s disease. Furthermore, building upon our ability to engineer Nbs for the compartment-specific cellular delivery, we plan to modulate nuclear Tau function. These cell-permeable Tau-specific Nbs will be chemically modified to monitor their location in cells, thus providing new tools for biological and biomedical research. NanoTarget’s ultimate goal is to provide unprecedented Tau-specific nanobody-based modalities with unmet potential as biological research tools and next-generation biopharmaceuticals. NanoTarget will support our research in the highly competitive field of tauopathies but also extend our original advances in the field of cancer. To date, the potential to reach Tau inside the cells with immunologic approaches has hardly been investigated. The intracellular delivery of functional proteins in living cells, which is essential for NanoTarget, is indeed a challenging task in the molecular life sciences, but it could prove extremely powerful for the development of new drugs. Key factors to be addressed in this context include the stability, cell permeability, and pharmacologic potency of the delivered proteins while maintaining their functional properties. The project’s viability to reach this ambitious goal is supported by the applicants’ proven expertise in protein chemistry and in particular cell-permeable Nb engineering, structural biology, cell biology, their solid track record in Tau research, and their previous successful collaborations.

Type 2 Diabetes (T2D) is characterized by high blood glucose levels and develops due to inadequate pancreatic ß-cell mass and function (i.e. insulin secretion) in the face of peripheral insulin resistance. ß-cell dysfunction is thought to have a major role in the pathogenesis of T2D. The restoration of ß-cell mass, identity and function has therefore become a field of intensive research seeking for the next generation of anti-diabetic drugs. Tremendous efforts and illuminating investigations focus on deciphering epigenetic regulations –the subtle and reversible chemical modifications on DNA or proteins– that control ß-cell function. Yet, the analysis and understanding of modifications on RNA in T2D physiopathology at the ß-cell level are currently lagging behind. In the current proposal, we aim to establish the concept that posttranscriptional modifications of RNAs, named epitranscriptome, may contribute to pancreatic ß-cell dysfunctions and defective insulin secretion during metabolic stress, thus favoring the susceptibility to T2D. Our research program aims to go beyond the current state-of-the-art by decoding the epitranscriptome and its underlying mechanisms within the ß cells. We propose to develop an innovative framework to uncover the physiological and pathophysiological roles of these epitranscriptomic modifications of RNA in the regulation of cellular responses during T2D development. This hypothesis will be tested combining unbiased next-generation sequencing technology, mouse models, CRISPR/Cas9, mouse and human cohort studies to dissect the RNA-modification-mediated regulated processes involved in the control of metabolic homeostasis. Thereby, we hope to identify novel targets, open new research fields and emerging concepts for the prevention, diagnosis and treatment of T2D. We believe that deciphering the epitranscriptomic code of ß cells will identify a new layer of information that controls gene expression, protein synthesis and tissue function.

Alzheimer’s disease (AD) is the most frequent neurodegenerative disease. Its burden on health and society is increasing and no treatment is validated. The study of genetic risk factors allows to identify pathways that may contribute to the pathophysiological process. This project focuses on the role of Pyk2, a non-receptor tyrosine kinase coded by a gene identified by Partner 2 as an AD risk factor. Partner 1 previously showed the importance of Pyk2 in synapse structure and plasticity. We shall join forces with physiologists (Partner 3) and protein chemists (Partner 4) to systematically explore the synaptic function of Pyk2, its role in the alteration and effects of two major proteins implicated in AD (Tau and beta-amyloid) and the consequences of manipulating Pyk2 on mouse models of AD. As a result of these studies combining multiple approaches we expect to precisely identify the role of Pyk2 in AD and determine whether it can be an interesting target for treatment.