AFMB – Architecture et Fonction des Macromolécules Biologiques

Our team aims at establishing the relationships between the amino acid sequence of carbohydrate-active enzymes and their precise specificity. This work find developments in various areas, from the exploration of the gut microbiota to the search of novel enzymes for bio-fuel production or blood group conversion.

Carbohydrates are crucial for most organisms as carbon sources or as signaling molecules, but also for cell wall synthesis, host pathogen interactions, energy storage etc. We term carbohydrate-active enzymes (CAZymes) the enzymes that assemble and breakdown complex carbohydrates and carbohydrate polymers.

Unlike most other classes of enzymes whose sequences carry limited informative power, the peculiarities of CAZymes and of their substrates turn these enzymes into extremely powerful probes to examine and explain the lifestyle of living organisms. During the last 20 years we have developed a classification in sequence-based families that correlate with the structure and catalytic mechanism of CAZymes.

This classification currently includes 5 enzyme categories (glycoside hydrolases, glycosyltransferases, carbohydrate esterases, polysaccharide lyases and auxiliary activities) and their appended carbohydrate-binding modules. To make the classification available to the community, we have created the CAZy database (www.cazy.org), which has been meticulously curated and updated since its first version in 1998. Recently, we have coupled various bioinformatics tools to our database explore the CAZyme content of hundreds of eukaryotic and prokaryotic genomes, as well as many metagenomic datasets.

The main objective of the group is to identify, characterize and elucidate the functional role of intrinsically disordered proteins or protein regions in biological systems relevant to human health.

In particular, the team focuses on proteins of the replicative complex of major human pathogenic viruses, such as the measles, and the Nipah and Hendra viruses.

During the last twenty years, the “structure-function” paradigm has been called into question by the discovery of intrinsically disordered proteins (IDPs), namely proteins devoid of stable secondary and tertiary structure under physiological conditions of pH and salinity and in the absence of a partner or ligand. These proteins are however functional and very widespread in the living world. The group played a key role in discovering that the measles virus nucleoprotein (N) and phosphoprotein (P) possess very long disordered regions (up to 300 residues), and then extended these findings to the N and P proteins from the Nipah and Hendra viruses, two safety level 4 human pathogens.

More recently the group has started focusing on phase separation and transition phenomena mediated by intrinsically disordered protein regions. Liquid-liquid phase separation (LLPS) constitutes a new paradigm in biology and underlies the formation of membraneless organelles (MLO) and of viral factories in Mononegavirales members. These condensates allow fine spatio-temporal regulation of cellular activities. They can undergo “maturation” and nucleate amyloid-like fibers.

The group is particularly interested in phase separation and fibrillation of the V and W proteins from the Nipah and Hendra viruses. These proteins play a key role in evading the host innate immune response. The research is articulated around three axes:

• Elucidation of the molecular determinants and of the sequence properties that govern phase separation and fibrillation of the V and W proteins.
• Elucidation of the functional role of these phenomena in the cellular context.
• Assessment of the antiviral potential of fibrillation inhibitors.

Our research projects address the molecular architectures and functional mechanisms of carbohydrate-active enzymes (CAZymes), and of enzymes, receptors/channels, and cell adhesion molecules with a neurobiological interest.

The CAZymes govern the hydrolysis, conversion or recognition of sugar moieties from glycoconjuguates widely found in animals, fungi, plants and bacteria. These conjugates are involved in key biological processes such as development, quality control of protein folding, host-pathogen recognition, immune response, and they can be linked to diseases such as neurodegenerative metabolic diseases. In collaboration with the “Glycogenomics” team we pay particular attention to those glycoside hydrolases that are of interest for biomedical and biotechnological applications.

We also address the function, recognition properties and structure of receptors, enzymes and adhesion molecules involved in central and peripheral neurotransmission, with particular interest for their structure-function relationships, specificity of partner and ligand recognition, conformational dynamics and functional regulation. In particular we have accumulated expertise in the study of the enzyme acetylcholinesterase and of the extracellular ligand-binding domain of the nicotinic acetylcholine receptor, whose malfunctioning has been correlated with neurodegenerative diseases, and to cell-adhesion molecules structurally related to the cholinesterases or to other families of proteins and associated with developmental neuronal deficiencies.

Our research also focuses on the structural and functional study of membrane sensor proteins involved in biofilm formation in the pathogen Pseudomonas aeruginosa, or on enzymes involved in lipid metabolism in the pathogen Mycobacterium tuberculosis.

Our team focuses on understanding viral and pseudo-viral infection mechanisms, mainly ruled by the interplay of viral proteins, cellular factors and nucleic acids assembled into multifunctional complexes. In some cases, like many positive stranded RNA viruses, replication is associated to the generation of membrane organelles where replication and transcription occur. Our aim is to understand the mechanisms by which the multiple enzymatic activities are coordinated and regulated to efficiently carry out infection. For this purpose, understanding the interactions of viral proteins with cellular protein factors and membranes is crucial. To reach our goals we use a broad range of biochemical and biophysical technics combined with X-ray crystallography and electron microscopy.

Our team seeks to unravel the molecular mechanisms of emerging viruses by characterizing the structures and the enzymatic activities of proteins forming the viral replication and transcription complex. These studies are a prerequisite for the development of specific inhibitors of these enzymes and should allow the development of new antiviral strategies.

RNA genome viruses are regularly associated with the emergence of infectious diseases. Among the best known, epidemics related to Bunyavirales (LCMV, Lassa, Rift Valley…), Filovirales (Ebola), Nidovirales (Coronavirus), Flavivirales ( Chikungunya or Dengue virus) and chronic diseases (HIV, HCV, HBV, HEV) illustrate public health problems generated on a global scale.

Our studies focus not only on viral polymerases that are at the heart viral replication, but also on the enzymes involved in RNA modifications such as capping and proofreading and proteins involved in the regulation of replication. Beyond the study of the RTC, we are also interested in the interplay of these enzymes with the innate immunity defence mechanism of the cell. The viral replication proteins are privileged antiviral targets and understanding of their structure and function is essential for the design of antiviral molecules with a high potential. To support these research projects, the lab benefits from the support of an inhibitor-screening platform (PCML) and a platform dedicated to the expression of recombinant viral proteins.