Endohttps://en.backend.curie.fr/node/9853/edit?destination=https%3A//fr.backend.curie.fr/admin/content#edit-group-generalcytic Trafficking and Intracellular Delivery
Research in the Endocytic Trafficking and Intracellular Delivery team focuses on the mechanisms by which glycans regulate the cell surface dynamics of plasma membrane proteins and their polarized intracellular distribution, and mediate the cellular entry of pathogens. We further harness these principles to develop intracellular delivery strategies for immunotherapy.
Endocytosis allows for the internalization of extracellular materials and cell surface receptors, using membrane-bound carriers. Our team discovered a mechanism by which tubular endocytic pits are generated through the interaction of oligomeric lectins with glycans on a specific class of glycosylated lipids, the glycosphingolipids (GSLs)(1,2). Pathogenic lectins exploit this mechanism to drive the GSL-dependent uptake of bacterial toxins (e.g., Shiga toxin and cholera toxin) and animal viruses (e.g., polyomaviruses)(3). Endogenous lectins, known as galectins, in turn mediate the GSL-dependent internalization of glycoprotein cargos, including cell adhesion molecules (e.g., integrins, CD44) and scavenger receptors (e.g., LRP1)(4). In both cases, cargo uptake proceeds independently of the canonical clathrin-mediated endocytic machinery. We coined the term GlycoLipid–Lectin (GL-Lect) driven endocytosis to describe this distinct uptake process (5, Figure1).
Research themes
A first major focus of our current research is on the elucidation of structural, molecular, and pathophysiological principles governing GL-Lect driven endocytosis, with a particular emphasis on polarized trafficking across intestinal enterocytes, epithelial–mesenchymal plasticity in breast cancer, and immune checkpoints in head and neck squamous cell carcinoma (HNSCC). To address these questions, we combine state-of-the-art imaging approaches, including lattice light-sheet microscopy (6, 7, 8), fluorescence orientation microscopy, and spectral imaging, with membrane protein reconstitution (9, 10), cryogenic electron microscopy (6), DNA origami (11), multi-omics analyses, organoid systems, and genetically engineered mouse models.
Figure 1: The GL-Lect hypothesis for the formation of endocytic pits driven by galectin-3 (Gal3) in the biogenesis of clathrin-independent carriers (CLICs). Monomeric Gal3 is recruited to membranes by binding to glycosylated cargo proteins, such as CD44 and α5β1-integrin. Membrane-bound Gal3 oligomerizes and acquires the ability to bind functional glycosphingolipids (GSLs), endowing the Gal3/GSL complexes with active curvature properties, i.e. the ability to induce and/or detect membrane curvature. The cargo and glycosylated lipids are then clustered in tubular endocytic pits, from which clathrin-independent carriers (CLICs) are formed for endocytic uptake into the cellsLakshminarayan et al, 2014, Nature Cell Biology 16 : 595-606.
A second major focus of our research program is chemical biology. We identify small-molecule modulators of GSL metabolism as candidate therapeutics for lysosomal storage disorders. We define their cellular targets, elucidate their molecular and cellular mechanisms of action, and evaluate their therapeutic efficacy in mouse models of disease. In parallel, we develop synthetic chemical tools to reconstitute and functionally interrogate GSLs in physiological and pathological contexts, with particular emphasis on cancer and immunity. We have successfully used a similar approach in the past to find small-molecule modulators of bacterial toxin trafficking and intoxication (see Figure 2).
Figure 2. Identification of two toxin inhibitors by cell-based high-throughput screening. Cells are protected against the plant toxin ricin and bacterial Shiga-like toxins. Inhibitors selectively block toxin trafficking at endosome-TGN interface. One compound protects mice from lethal nasal challenge with ricin. Stechmann et al. Cell. 2010 Apr 16;141(2):231-42.
A third major research direction exploits GSL ligands to promote the targeted intracellular delivery of antigenic peptides and proteins to professional antigen-presenting cells, namely dendritic cells. Our goal is to induce immune modulation at mucosal surfaces, enabling preventive and durable vaccination against infectious diseases, such as COVID-19, as well as therapeutic vaccination against mucosal malignancies, including lung cancer and gastric cancer. To this end, we develop both mRNA- and protein subunit-based vaccine platforms, including the chemical synthesis of our dendritic cell-targeting vector STxB, a 69-amino-acid protein that we assemble in vitro into its homopentameric structure.
Figure 3: Shiga toxin trafficking into cells. Left: Shiga toxin (STx) is composed of a homo-pentamer STxB (green) and a catalytic A-subunit (red). STxB binds to the glycosphingolipid Gb3 (not shown) at the plasma membrane of target cells, clusters, induces membrane bending, formation of endocytic pits, and subsequent clathrin-independent generated vesicles for toxin trafficking to early endosomes. From there, the holotoxins are transported via retrograde trafficking route to the endoplasmic reticulum (ER), via the Golgi apparatus. The catalytic A-subunit is then translocated to the cytosol where it inhibits protein biosynthesis by modifying ribosomal RNAs (not shown).Right: In STxB (green)-based vaccines, engineered STxB is linked via covalent bonds to the antigene (blue). The endocytic process operates as for Shiga holotoxin. While STxB-antigen conjugates also undergo retrograde trafficking (not shown), a small fraction of them escapes from the lumen of endosomes to reach the cytosol (endosomal escape). Here, proteasomes process the antigens to generate antigenic peptides, that are then imported into the lumen of the ER (or of endo/phagosomal processing compartments; not shown) for loading onto MHC class I molecules and subsequent presentation at the plasma membrane to CD8+ T cells Toxins (Basel). 2022 Mar 10;14(3):202.
