Tetraspanin-6 negatively regulates exosome production.

Exosomes, extracellular vesicles (EVs) of endosomal origin, emerge as master regulators of cell-to-cell signaling in physiology and disease. Exosomes are highly enriched in tetraspanins (TSPNs) and syndecans (SDCs), the latter occurring mainly in proteolytically cleaved form, as membrane-spanning C-terminal fragments of the proteins. While both protein families are membrane scaffolds appreciated for their role in exosome formation, composition, and activity, we currently ignore whether these work together to control exosome biology. Here we show that TSPN6, a poorly characterized tetraspanin, acts as a negative regulator of exosome release, supporting the lysosomal degradation of SDC4 and syntenin. We demonstrate that TSPN6 tightly associates with SDC4, the SDC4-TSPN6 association dictating the association of TSPN6 with syntenin and the TSPN6-dependent lysosomal degradation of SDC4-syntenin. TSPN6 also inhibits the shedding of the SDC4 ectodomain, mimicking the effects of matrix metalloproteinase inhibitors. Taken together, our data identify TSPN6 as a regulator of the trafficking and processing of SDC4 and highlight an important physical and functional interconnection between these membrane scaffolds for the production of exosomes. These findings clarify our understanding of the molecular determinants governing EV formation and have potentially broad impact for EV-related biomedicine.

Lipids in Exosome Biology.

Extracellular vesicles (EVs), and exosomes in particular, were initially considered as "garbage bags" for secretion of undesired cellular components. This view has changed considerably over the last two decades, and exosomes have now emerged as important organelles controlling cell-to-cell signaling. They are present in biological fluids and have important roles in the communication between cells in physiological and pathological processes. They are envisioned for clinical use as carriers of biomarkers, therapeutic targets, and vehicles for drug delivery. Important efforts are being made to characterize the contents of these vesicles and to understand the mechanisms that govern their biogenesis and modes of action. This chapter aims to recapitulate the place given to lipids in our understanding of exosome biology. Besides their structural role and their function as carriers, certain lipids and lipid-modifying enzymes seem to exert privileged functions in this mode of cellular communication. By extension, the use of selective "lipid inhibitors" might turn out to be interesting modulators of exosomal-based cell signaling.

Syntenin mediates SRC function in exosomal cell-to-cell communication.

The cytoplasmic tyrosine kinase SRC controls cell growth, proliferation, adhesion, and motility. The current view is that SRC acts primarily downstream of cell-surface receptors to control intracellular signaling cascades. Here we reveal that SRC functions in cell-to-cell communication by controlling the biogenesis and the activity of exosomes. Exosomes are viral-like particles from endosomal origin that can reprogram recipient cells. By gain- and loss-of-function studies, we establish that SRC stimulates the secretion of exosomes having promigratory activity on endothelial cells and that syntenin is mandatory for SRC exosomal function. Mechanistically, SRC impacts on syndecan endocytosis and on syntenin-syndecan endosomal budding, upstream of ARF6 small GTPase and its effector phospholipase D2, directly phosphorylating the conserved juxtamembrane DEGSY motif of the syndecan cytosolic domain and syntenin tyrosine 46. Our study uncovers a function of SRC in cell-cell communication, supported by syntenin exosomes, which is likely to contribute to tumor-host interactions.

Phospholipase D and phosphatidic acid in the biogenesis and cargo loading of extracellular vesicles.

Extracellular vesicles released by viable cells (exosomes and microvesicles) have emerged as important organelles supporting cell-cell communication. Because of their potential therapeutic significance, important efforts are being made toward characterizing the contents of these vesicles and the mechanisms that govern their biogenesis. It has been recently demonstrated that the lipid modifying enzyme, phospholipase D (PLD)2, is involved in exosome production and acts downstream of the small GTPase, ARF6. This review aims to recapitulate our current knowledge of the role of PLD2 and its product, phosphatidic acid, in the biogenesis of exosomes and to propose hypotheses for further investigation of a possible central role of these molecules in the biology of these organelles.

Study of PDZ-Peptide and PDZ-Lipid Interactions by Surface Plasmon Resonance/BIAcore.

Surface plasmon resonance (SPR)/BIAcore technology enables the characterization of molecular interactions, including determination of affinities and kinetics. In BIAcore, one of the interaction partners (the ligand) is immobilized on a chip and the other (the analyte) is provided in solution. BIAcore allows to study association and dissociation rates in real time without the use of labeling. BIAcore can be applied to molecular interactions involving small compounds and biological macromolecules such as proteins, lipids, nucleic acids, or carbohydrates. Here we describe protocols for the measurements of PDZ domain-peptide (oriented biotinylated peptides), PDZ domain-liposomes (lipid membranes), and PDZ-lipid-peptide tripartite interactions.

PKCε controls the fusion of secretory vesicles in mast cells in a phosphatidic acid-dependent mode.

PKCε is highly expressed in mast cells and plays a fundamental role in the antigen-triggered activation of the allergic reaction. Although its regulation by diacylglycerols has been described, its regulation by acidic phospholipids and how this regulation leads to the control of downstream vesicle secretion is barely known. Here, we used structural and evolutionary studies to find the molecular mechanism that explains the selectivity of the C1B domain of PKCε by Phosphatidic Acid (PA). This resided in a collection of Arg residues that form a specific rim on the outer surface of the C1B domain, around the diacylglycerol binding cleft. In RBL-2H3 cells, this basic rim allowed the kinase to respond specifically to phosphatidic acid signals that induced its translocation to the plasma membrane and subsequent activation. Further experiments in cells that overexpress PKCε and a mutant of the PA binding site, showed that PA-dependent PKCε activation increased vesicle degranulation in RBL-2H3 cells, and this correlated with increased SNAP23 phosphorylation. Over-expression of PKCε in these cells also induced an increase in the number of docked vesicles containing SNAP23, when stimulated with PA. This accumulation could be attributed to the stabilizing effect of phosphorylation on the formation of the SNARE complex, which ultimately led to increased release of content in the presence of Ca2+ during the fusion process. Therefore, these findings reinforce the importance of PA signaling in the activation of PKCε, which could be an important target to inhibit the exacerbated responses of these cells in the allergic reaction.

Fragment-based drug design targeting syntenin PDZ2 domain involved in exosomal release and tumour spread.

Syntenin stimulates exosome production and its expression is upregulated in many cancers and implicated in the spread of metastatic tumor. These effects are supported by syntenin PDZ domains interacting with syndecans. We therefore aimed to develop, through a fragment-based drug design approach, novel inhibitors targeting syntenin-syndecan interactions. We describe here the optimization of a fragment, 'hit' C58, identified by in vitro screening of a PDZ-focused fragment library, which binds specifically to the syntenin-PDZ2 domain at the same binding site as the syndecan-2 peptide. X-ray crystallographic structures and computational docking were used to guide our optimization process and lead to compounds 45 and 57 (IC50 = 33 µM and 47 µM; respectively), two representatives of syntenin-syndecan interactions inhibitors, that selectively affect the syntenin-exosome release. These findings demonstrate that it is possible to identify small molecules inhibiting syntenin-syndecan interaction and exosome release that may be useful for cancer therapy.

Syntenin-knock out reduces exosome turnover and viral transduction.

Exosomal transfers represent an important mode of intercellular communication. Syntenin is a small scaffold protein that, when binding ALIX, can direct endocytosed syndecans and syndecan cargo to budding endosomal membranes, supporting the formation of intraluminal vesicles that compose the source of a major class of exosomes. Syntenin, however, can also support the recycling of these same components to the cell surface. Here, by studying mice and cells with syntenin-knock out, we identify syntenin as part of dedicated machinery that integrates both the production and the uptake of secreted vesicles, supporting viral/exosomal exchanges. This study significantly extends the emerging role of heparan sulfate proteoglycans and syntenin as key components for macromolecular cargo internalization into cells.

Frizzled 7 and PIP2 binding by syntenin PDZ2 domain supports Frizzled 7 trafficking and signalling.

PDZ domain-containing proteins work as intracellular scaffolds to control spatio-temporal aspects of cell signalling. This function is supported by the ability of their PDZ domains to bind other proteins such as receptors, but also phosphoinositide lipids important for membrane trafficking. Here we report a crystal structure of the syntenin PDZ tandem in complex with the carboxy-terminal fragment of Frizzled 7 and phosphatidylinositol 4,5-bisphosphate (PIP2). The crystal structure reveals a tripartite interaction formed via the second PDZ domain of syntenin. Biophysical and biochemical experiments establish co-operative binding of the tripartite complex and identify residues crucial for membrane PIP2-specific recognition. Experiments with cells support the importance of the syntenin-PIP2 interaction for plasma membrane targeting of Frizzled 7 and c-jun phosphorylation. This study contributes to our understanding of the biology of PDZ proteins as key players in membrane compartmentalization and dynamics.