Base de datos de flora y fauna en Galicia

Sección: NoticiasLa Biblioteca del Instituto de Investigaciones Marinas de Vigo recopila, desde 1984 aproximadamente, una base de datos sobre Galicia que nació a partir de unas citas bibliográficas seleccionadas por un investigador de este centro y se fue ampliando en vista de la enorme solicitud de esta información que nos iban haciendo nuestros usuarios, sobre todo los externos al centroPeer reviewe

Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function

Metzincin metalloproteases have major roles in intercellular communication by modulating the function of membrane proteins. One of the proteases is the a-disintegrin-and-metalloprotease 10 (ADAM10) which acts as alpha-secretase of the Alzheimer\u27s disease amyloid precursor protein. ADAM10 is also required for neuronal network functions in murine brain, but neuronal ADAM10 substrates are only partly known. With a proteomic analysis of Adam10-deficient neurons we identified 91, mostly novel ADAM10 substrate candidates, making ADAM10 a major protease for membrane proteins in the nervous system. Several novel substrates, including the neuronal cell adhesion protein NrCAM, are involved in brain development. Indeed, we detected mistargeted axons in the olfactory bulb of conditional ADAM10-/- mice, which correlate with reduced cleavage of NrCAM, NCAM and other ADAM10 substrates. In summary, the novel ADAM10 substrates provide a molecular basis for neuronal network dysfunctions in conditional ADAM10-/- mice and demonstrate a fundamental function of ADAM10 in the brain

Stimulated release and functional activity of surface expressed metalloproteinase ADAM17 in exosomes.

By mediating proteolytic shedding on the cell surface the disintegrin and metalloproteinases ADAM10 and ADAM17 function as critical regulators of growth factors, cytokines and adhesion molecules. We here report that stimulation of lung epithelial A549 tumor cells with phorbol-12-myristate-13-acetate (PMA) leads to the downregulation of the surface expressed mature form of ADAM17 without affecting ADAM10 expression. This reduction could not be sufficiently explained by metalloproteinase-mediated degradation, dynamin-mediated internalization or microdomain redistribution of ADAM17. Instead, surface downregulation of ADAM17 was correlated with the presence of its mature form in exosomes. Exosomal ADAM17 release was also observed in monocytic and primary endothelial cells where it could be induced by stimulation with lipopolysaccharide. Antibody-mediated surface labelling of ADAM17 revealed that at least part of exosomal ADAM17 was oriented with the metalloproteinase domain outside and had been expressed on the cell surface. Suppression of iRHOM2-mediated ADAM17 maturation prevented surface expression and exosomal release of ADAM17. Further, deletion of the protease's C-terminus or cell treatment with a calcium chelator diminished exosomal release as well as surface downregulation of ADAM17, underlining that both processes are closely associated. Co-incubation of ADAM17 containing exosomes with cells expressing the ADAM17 substrates TGFalpha or amphiregulin lead to increased shedding of both substrates. This was prevented when exosomes were prepared from cells with shRNA-mediated ADAM17 knockdown. These data indicate that cell stimulation can downregulate expression of mature ADAM17 from the cell surface and induce release of exosomal ADAM17, which can then distribute and contribute to substrate shedding on more distant cells

The DRY motif is optimized for calcium signaling and chemotaxis.

<p>THP-1 cells were transduced with lentivirus encoding human CXCR6 variants or EV control. A/B: Cells were loaded with Fluo4-AM and stimulated with 10 nM soluble CXCL16 (A, n = 4), increasing concentrations of soluble CXCL16 (n = 4), or 3 nM CCL2 (control of general responsiveness, n = 3) (B). The increase of the Ca²⁺ signal was measured as increase in Fluo4-AM-fluorescence. Data were normalized to signals at 9 sec in A and quantified as AUC. In B, maximal fluorescence intensity was determined, and minimal fluorescence intensity was subtracted for each calcium response and expressed in relation to the response of EV cells for each concentration. C: Chemotaxis against increasing concentrations of soluble CXCL16 or 3 nM CCL2 (control of general migratory potential, n ≥ 4) was analyzed in a Boyden chamber assay (n ≥ 4). Statistical differences were analyzed by Student’s t-test. Asterisks indicate differences to control (EV in A, each buffer control in B, random migration in C), hashes indicate differences between receptor variants (*/#p<0.05, **p<0.01).</p

Rescue of the DRY motif in CXCR6 does not affect ligand binding or receptor recycling in HEK293 cells.

<p>HEK293 cells were transduced with lentivirus encoding the human CXCR6 variants or the EV control. A/C-E: Surface expression of CXCR6 in untreated cells (A, n = 4), after receptor internalization induced by 15 min treatment with soluble CXCL16 (C, n = 3), and receptor recycling (D, dyna = dynasore, n = 4) was determined by FACS analysis using an antibody against human CXCR6. Signals were expressed in relation to control (EV in A, untreated cells in C/D). Receptor recycling was further quantified as area under the curve of data shown in D (AUC, E). B: Ligand binding was analyzed by incubation with CXCL16-Fc fusion protein and FACS analysis (n ≥ 3, a.u. = arbitrary units). Statistical differences were analyzed by one-sample t-test (A and C, hypothetical value 1) or Student’s t-test (B, D and E). Asterisks indicate differences to control (*p<0.05, **p<0.01).</p

Rescue of the DRY motif in CXCR6 has no major impact on AKT signaling and adhesion.

<p>THP-1 cells were transduced with lentivirus encoding human CXCR6 variants or EV control. A/B: AKT activation upon stimulation with 10 nM soluble CXCL16 (A, n = 7) or with 3 nM CCL2 (B, 10 min, control of general responsiveness, n = 7) was measured as ratio of phosphorylated AKT (pAKT) to total AKT investigated by Western blot analysis. Data were expressed in relation to buffer control stimulated cells. C: Adhesion to immobilized CXCL16-Fc for 30 min was normalized to cells adhering to anti‑human-Fc (set = 1 for each cell-type, n = 4). Cells were pretreated with 100 nM soluble CXCL16 for 15 min to indicate specificity of CXCL16-CXCR6-mediated adhesion. D: Adhesion to immobilized CXCL16-Fc for 5, 15, and 30 min was normalized to cells adhering to anti‑human-Fc (set = 1 for each cell-type, n = 3). Statistical differences were analyzed by one-sample t-test (C for differences to adhesion to anti-human-Fc, hypothetical value 1) or Student’s t-test (A-D, with Welch’s correction in A). Asterisks indicate differences to control (untreated cells in B/C, EV in A/D), hashes indicate differences between receptor variants in A and D and CXCL16 preincubation in C (*/#p<0.05, **p<0.01, ***p<0.001).</p