Michel Vidal

Vesiclepedia: A Compendium for Extracellular Vesicles with Continuous Community Annotation

Vesiclepedia is a community-annotated compendium of molecular data on extracellular vesicles.

Exosome secretion, including the DNA damage-induced p53-dependent secretory pathway, is severely compromised in TSAP6/Steap3-null mice

TSAP6 (tumor suppressor-activated pathway 6), also known as Steap3, is a direct p53 transcriptional target gene. It regulates protein secretion, for example translationally controlled tumor protein (TCTP), which is implicated in tumor reversion. In keeping with the latter, we show herein that TSAP6 is a glycosylated protein present in the trans-Golgi network, endosomal–vesicular compartment and cytoplasmic membrane. To further investigate the physiological function of TSAP6, we have generated TSAP6-deficient mice. These mice exhibit microcytic anemia with abnormal reticulocyte maturation and deficient transferrin receptor downregulation, a process known to be dependent on exosomal secretion. Moreover, we provide direct evidence that exosome production is severely compromised in TSAP6-null cells. Finally, we show that the DNA damage-induced p53-dependent nonclassical exosomal secretory pathway is abrogated in TSAP6-null cells. Given the fact that exosomes are used as cell-free vaccines against cancer and that they could be involved in the biogenesis and spread of human immunodeficiency virus, it is important to understand their regulation. The results presented here provide the first genetic demonstration that exosome formation is a tightly controlled biological process dependent of TSAP6.

Galectin-5 is bound onto the surface of rat reticulocyte exosomes and modulates vesicle uptake by macrophages

Reticulocytes release small membrane vesicles termed exosomes during their maturation into erythrocytes. Exosomes are intraluminal vesicles of multivesicular endosomes released into the extracellular medium by fusion of these endosomal compartments with the plasma membrane. This secretion pathway contributes to reticulocyte plasma membrane remodeling by eliminating certain membrane glycoproteins. We show in this study that galectin-5, although mainly cytosolic, is also present on the cell surface of rat reticulocytes and erythrocytes. In addition, in reticulocytes, it resides in the endosomal compartment. We document galectin-5 translocation from the cytosol into the endosome lumen, leading to its secretion in association with exosomes. Galectin-5 bound onto the vesicle surface may function in sorting galactose-bearing glycoconjugates. Fittingly, we found that Lamp2, a major cellular glycoprotein presenting galectin-reactive poly-N-acetylactosamine chains, is lost during reticulocyte maturation. It is associated with released exosomes, suggestive of binding to galectin-5. Finally, we reveal that the uptake of rat reticulocyte exosomes by macrophages is dependent on temperature and the mechanoenzyme dynamin and that exosome uptake is decreased by adding galectin-5. These data imply galectin-5 functionality in the exosomal sorting pathway during rat reticulocyte maturation.

Exosome Secretion: The Art of Reutilizing Nonrecycled Proteins?

Multivesicular bodies contain membrane vesicles which either undergo lysosomal digestion or are released in the extracellular environment as exosomes. Evidence is accumulating that supports a physiological role for exosomes in, for example, antigen presentation or removal of transferrin receptor during reticulocyte development. Here, inspired by observations on exosomal release from reticulocytes, we discuss the potential involvement of the so‐called ESCRT mechanism in the entrapment of both lysosomal and exosomal cargo within the intralumenal vesicles of multivesicular bodies. We propose that this mechanism operates at different sites in the endocytic itinerary in different cells, thereby providing a tool for directional sorting. We also explore the possibility that the efficiency of sorting of molecules into exosomes increases when the recycling kinetics of molecules decreases, exosomal sorting being favored by intermolecular interactions occurring within lipid domains, or with protein webs, that slow lateral mobility. These considerations are mirrored in the context of current knowledge on the mechanism of protein sorting for degradation in lysosomes, and the hijacking of such mechanisms by some retroviruses for particle budding.

Degradation of AP2 During Reticulocyte Maturation Enhances Binding of Hsc70 and Alix to a Common Site on TfR for Sorting into Exosomes

Reticulocytes release small membrane vesicles termed exosomes during their maturation in erythrocytes. The transferrin receptor (TfR) is completely lost from the red cell surface by its segregation in the secreted vesicles where it interacts with the heat shock cognate 70 kDa protein (hsc70). We have now determined a region of the TfR that can potentially interact with hsc70. The peptide P1 (YTRFSLARQV) from the TfR cytosolic domain: (i) binds to hsc70 (ii) with an increased affinity in oxidative conditions, (iii) competes for binding of an unfolded protein to hsc70, and (iv) inhibits the interaction of hsc70 with a recombinant protein corresponding to the cytosolic domain of the receptor. This peptide encompasses the internalization motif (YTRF) of the receptor, and accordingly an affinity column made with the immobilized peptide retains hsc70 and also the AP2 adaptor complex. On the other hand, we show that AP2 is degraded by the proteasome system during reticulocyte maturation and that the presence of the proteasome inhibitor during in vitro red cell maturation inhibits AP2 degradation and specifically decreases TfR secretion via exosomes. Finally, coimmunoprecipitation of Alix with the exosomal TfR, and binding of P1 peptide to the Alix homolog PalA suggest that Alix also interacts with the YTRF motif and contributes to exosomal TfR sorting.

Reduced phosphatase activity of SHP-2 in LEOPARD syndrome: Consequences for PI3K binding on Gab1

LEOPARD (LS) and Noonan (NS) are overlapping syndromes associated with distinct mutations of SHP‐2. Whereas NS mutations enhance SHP‐2 catalytic activity, we show that the activity of three representative LS mutants is undetectable when assayed using a standard protein tyrosine phosphatase (PTP) substrate. A different assay using a specific SHP‐2 substrate confirms their decreased PTP activity, but also reveals a significant activity of the T468M mutant. In transfected cells stimulated with epidermal growth factor, the least active LS mutants promote Gab1/PI3K binding, validating our in vitro data. LS mutants thus display a reduced PTP activity both in vitro and in transfected cells.

Shifting the Paradigm: The Putative Mitochondrial Protein ABCB6 Resides in the Lysosomes of Cells and in the Plasma Membrane of Erythrocytes

ABCB6, a member of the adenosine triphosphate–binding cassette (ABC) transporter family, has been proposed to be responsible for the mitochondrial uptake of porphyrins. Here we show that ABCB6 is a glycoprotein present in the membrane of mature erythrocytes and in exosomes released from reticulocytes during the final steps of erythroid maturation. Consistent with its presence in exosomes, endogenous ABCB6 is localized to the endo/lysosomal compartment, and is absent from the mitochondria of cells. Knock-down studies demonstrate that ABCB6 function is not required for de novo heme biosynthesis in differentiating K562 cells, excluding this ABC transporter as a key regulator of porphyrin synthesis. We confirm the mitochondrial localization of ABCB7, ABCB8 and ABCB10, suggesting that only three ABC transporters should be classified as mitochondrial proteins. Taken together, our results challenge the current paradigm linking the expression and function of ABCB6 to mitochondria.

SH2 and SH3 domains as targets for anti-proliferative agents

The Src homology domains SH2 and SH3 are small modular protein motifs about 100 and 60 amino acids long, respectively. SH2 domains interact with phosphotyrosine residues, whereas SH3 domains recognize proline-rich motifs of their interacting partners. SH2 and SH3 domains are frequently found in signaling proteins such as small adaptors and in enzymes such as kinases, lipases and phosphatases, in which they differ from the catalytic motif and constitute recognition modules. SH2 and SH3 domains are also found in oncoproteins and in proteins overexpressed in deregulated signaling pathways in tumor cells. The highly folded structures of these domains have been characterized alone and complexed with the essential fragments of their targets. Therefore, based on molecular data, inhibitors of interactions with SH2 and SH3 domains are considered to be potential antitumor agents. Current results are very promising, as inhibitors with very efficient anti-proliferative activity in tumor cells have been reported. This paper describes SH2 and/or SH3 domain-containing proteins that may constitute targets for anticancer therapeutics. It also deals with the essential structural data concerning SH2 and SH3 domains, and the rational design of inhibitors. Some of the more recent pharmacological results obtained with these compounds are also discussed.

The Glut1 and Glut4 glucose transporters are differentially expressed during perinatal and postnatal erythropoiesis

Glucose is a major source of energy for living organisms, and its transport in vertebrates is a universally conserved property. Of all cell lineages, human erythrocytes express the highest level of the Glut1 glucose transporter with more than 200,000 molecules per cell. However, we recently reported that erythrocyte Glut1 expression is a specific trait of vitamin C-deficient mammalian species, comprising only higher primates, guinea pigs, and fruit bats. Here, we show that in all other tested mammalian species, Glut1 was transiently expressed in erythrocytes during the neonatal period. Glut1 was up-regulated during the erythroblast stage of erythroid differentiation and was present on the vast majority of murine red blood cells (RBCs) at birth. Notably though, Glut1 was not induced in adult mice undergoing anemia-induced erythropoiesis, and under these conditions, the up-regulation of a distinct transporter, Glut4, was responsible for an increased glucose transport. Sp3 and Sp1 transcriptions factors have been proposed to regulate Glut1 transcription, and we find that the concomitant repression of Glut1 and induction of Glut4 was associated with a significantly augmented Sp3/Sp1 ratio. Glucose transporter expression patterns in mice and human erythrocytes are therefore distinct. In mice, there is a postnatal switch from Glut1 to Glut4, with Glut4 further up-regulated under anemic conditions.

Mannose receptor contribution to Candida albicans phagocytosis by murine E‐clone J774 macrophages

Mannoproteins, as the main constituents of the outer layer of yeast cell walls, are able to interact with phagocytic cells in an opsonin‐independent manner through the mannose receptor (MR) and to induce yeast ingestion by the professional phagocytes. Moreover, the MR also mediates endocytosis of soluble ligands through clathrin‐coated pits. Here, we studied some aspects of the interaction between the MR and Candida albicans using murine E‐clone macrophages and the consequences on MR trafficking. Using a pull‐down assay involving mixture E‐clone macrophage detergent lysate with mannosylated Sepharose beads and glutaraldehyde‐fixed, heat‐killed (HK) C. albicans, we found that binding of solubilized MR to mannosylated particles occurred with characteristics similar to the receptor’s cell‐surface mannose‐binding activity. We then demonstrated that MR expressed on E‐clone macrophages contributed to phagocytosis of unopsonized, HK C. albicans and that yeast phagocytosis induced a decrease in MR endocytic activity without concomitant degradation of the receptor in the time lapse studied.