Unravelling the molecular mechanisms of tetraspanin CD9

This thesis focuses on a family of membrane proteins, called tetraspanins. As molecular facilitators, tetraspanins maintain the plasma membrane integrity by interacting with each other or with other macromolecules. If these master organizers are missing, if they are too abundant or defective, the membrane organization can be compromised and the cell functions affected, leading to diseases such as cancer and many others. Because of their crucial role on the cell surface, tetraspanins can also be regarded in some circumstances as targets of medicines. By targeting a tetraspanin we modulate their organization activities, aiming at restoring the normal cell function. The topic of this thesis is the biochemical, structural and functional characterization of human tetraspanin CD9. In Chapter 1 we introduce the main subjects of this thesis: the membrane microdomains, the tetraspanin family and post-translational modifications on tetraspanins. Finally, we present an overview of antibodies targeting CD9 and their properties. Membrane protein production for structural studies requires multiple steps, which are often hampered by many technical issues. In Chapter 2 we describe the efforts to produce a stable and homogeneous tetraspanin sample, its characterization and the isolation of CD9-specific nanobodies. Chapter 3 presents the structure and mode of action of a CD9-antibody fragment, derived from a melanoma patient, that binds CD9 extracellular loop (called EC2). This antibody binds tumor cells, but do not induce platelets aggregation, which is the major side-effect triggered by other reported CD9 antibodies. The structure of the Fab-CD9EC2 complex revealed that the antibody recognizes an extended and unfolded region of the extracellular loop. We observed that this unfolded region is also present in the CD9EC2 unbound structure and that, surprisingly, it corresponds to a CD9 homo-dimer interface. The antibody-binding mode sets the basis for the development of a CD9-specific drug for cancer therapy. Tetraspanins can be modified by the attachment of a fatty acid chain to cysteine residues, that are situated on protein regions close to the plasma membrane. Chapter 4 describes how this post-translational modification (called lipidation) affects tetraspanins function. We found that specific cysteines on CD9 are modified at different levels: three cysteines are lipidated at high extent, while the other three cysteines show variable lipidation. We observed that lipidation, occurring at specific sites, affects CD9 interaction with its major protein partners, while membrane localization and clustering of CD9 did not change. We analyzed the effect of lipidation on CD9 using a novel and simple tool. We mutated cysteine residues into tryptophans, to mimic the effect that lipidation might induce on the protein conformation. This strategy allowed us to make significant advances in understanding how tetraspanins associates by forming lateral interactions in the membrane microdomains. We finally discuss the main implications of this thesis in Chapter 5. In conclusion, we investigated the molecular mechanism of tetraspanin CD9 using different approaches. The dynamic association of tetraspanins in cell-surface clusters and their regulation is a crucial aspect in cell-cell signaling, adhesion, migration, fusion and many other cellular processes