Molecular Basis of the activation of pre-B Cell Rerceptor (pre-BCR)

Le stade pre-B représente un point de contrôle crucial du développement des lymphocytes B dans la moelle osseuse. A ce stade, il y aura formation d'un récepteur intermédiaire nommé pre-BCR. Le pre-BCR est constitué de deux chaines lourdes Igµ et de deux pseudo-chaines légères (SLCs). Chaque SLC est constituée des protéines λ5 et VpreB, qui possèdent des régions « uniques » à leur extrémité N et C-terminale, respectivement. Ces régions uniques sont cruciales pour les fonctions du récepteur. La première partie de mes travaux sur l'étude du domaine λ5-UR nous a permis de proposer un modèle original d'assemblage du pre-BCR et d'apporter les bases structurales du rôle de chaperonne intramoléculaire de λ5-UR. L'activation du récepteur est permis par la formation d'une synapse immunologique. Des interactions entre la galectine-1 et λ5-UR vont permettre la formation d'un treillis d'interactions. L'étude structurale du complexe GAL1/λ5-UR, réalisée dans la seconde partie de ma thèse, a permis de déterminer la structure du complexe. L'interaction GAL1/λ5-UR engendre une modification d'affinité de GAL1 pour le lactose. Ce résultat suggère que l'interaction entre le pre-BCR et la galectine-1 peut influencer l'équilibre des interactions au niveau de la lattice en modulant l'affinité de la galectine-1 pour certains glycans. Dans la troisième partie de mon travail de thèse, des approches de glycomique fonctionnelle et structurale nous a permis l'élaboration d'un mécanisme de formation-dissolution de la synapse pre-B basés sur une modification d'affinité de GAL1 pour certains carbohydrates en présence de λ5-UR.The pre-B stage represents a critical checkpoint in the development of B cells in the bone marrow. At this stage , there will be formation of a receptor intermediate called pre-BCR . The pre -BCR is composed of two heavy chains Igμ and two surrogate light chains ( SLCs ) . Each SLC consists of two proteins: λ5 and VpreB , which have "unique region" to their N-terminus and C -terminus, respectively. These unique regions are crucial for the functions of the receptor. The first part of my work on the domain λ5-UR has allowed us to propose an original model for assembling the pre -BCR and provide the structural basis of the role of intramolecular chaperone of λ5-UR. Receptor activation is allowed by the formation of an immunological synapse. Interactions between galectin-1 and λ5-UR will allow the formation of a lattice interactions. The structural study of complex GAL1/λ5-UR , conducted in the second part of my thesis, has allowed to determine the structure of the complex. These interactions GAL1/λ5-UR generate a modification of affinity of GAL1 for lactose. This result suggests that the interaction between the pre-BCR and galectin-1 may affect the balance of interactions at the lattice by modulating the affinity for galectin-1 for some glycans. In the third part of my thesis, approaches to structural and functional glycomics has allowed us to develop a mechanism of formation-dissolution of the synapse pre-B based on a modified affinity of GAL1 for certain carbohydrates in presence of λ5-UR

Structural basis for galectin-1-dependent pre-B cell receptor (pre-BCR) activation.

International audienceDuring B cell differentiation in the bone marrow, the expression and activation of the pre-B cell receptor (pre-BCR) constitute crucial checkpoints for B cell development. Both constitutive and ligand-dependent pre-BCR activation modes have been described. The pre-BCR constitutes an immunoglobulin heavy chain (Ig?) and a surrogate light chain composed of the invariant ?5 and VpreB proteins. We previously showed that galectin-1 (GAL1), produced by bone marrow stromal cells, is a pre-BCR ligand that induces receptor clustering, leading to efficient pre-BII cell proliferation and differentiation. GAL1 interacts with the pre-BCR via the unique region of ?5 (?5-UR). Here, we investigated the solution structure of a minimal ?5-UR motif that interacts with GAL1. This motif adopts a stable helical conformation that docks onto a GAL1 hydrophobic surface adjacent to its carbohydrate binding site. We identified key hydrophobic residues from the ?5-UR as crucial for the interaction with GAL1 and for pre-BCR clustering. These residues involved in GAL1-induced pre-BCR activation are different from those essential for autonomous receptor activation. Overall, our results indicate that constitutive and ligand-induced pre-BCR activation could occur in a complementary manner

Pre-B cell receptor binding to galectin-1 modifies galectin-1/carbohydrate affinity to modulate specific galectin-1/glycan lattice interactions.

International audienceGalectins are glycan-binding proteins involved in various biological processes including cell/cell interactions. During B-cell development, bone marrow stromal cells secreting galectin-1 (GAL1) constitute a specific niche for pre-BII cells. Besides binding glycans, GAL1 is also a pre-B cell receptor (pre-BCR) ligand that induces receptor clustering, the first checkpoint of B-cell differentiation. The GAL1/pre-BCR interaction is the first example of a GAL1/unglycosylated protein interaction in the extracellular compartment. Here we show that GAL1/pre-BCR interaction modifies GAL1/glycan affinity and particularly inhibits binding to LacNAc containing epitopes. GAL1/pre-BCR interaction induces local conformational changes in the GAL1 carbohydrate-binding site generating a reduction in GAL1/glycan affinity. This fine tuning of GAL1/glycan interactions may be a strategic mechanism for allowing pre-BCR clustering and pre-BII cells departure from their niche. Altogether, our data suggest a novel mechanism for a cell to modify the equilibrium of the GAL1/glycan lattice involving GAL1/unglycosylated protein interactions

Zinc Fingers in HIV-1 Gag precursor are not equivalent for gRNA Recruitment at the Plasma Membrane

International audienceThe human immunodeficiency virus type 1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs) or a nonmyristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the accumulation of the ribonucleoprotein complexes at the PM. Finally, the myristate group, which is mandatory for anchoring the complexes at the PM, was found to be dispensable for the association of Gag with the gRNA in the cytosol