The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF

Ariadne-1 (Ari-1) is an E3 ubiquitin-ligase essential for neuronal development, but whose neuronal substrates are yet to be identified. To search for putative Ari-1 substrates, we used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics of Drosophila heads. We identified 16 candidates that met the established criteria: a significant change of at least twofold increase on ubiquitination, with at least two unique peptides identified. Among those candidates, we identified Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release. Using a pull-down approach that relies on the overexpression and stringent isolation of a GFP-fused construct, we validate Comt/NSF to be an ubiquitination substrate of Ari-1 in fly neurons, resulting in the preferential monoubiquitination of Comt/NSF. We tested the possible functional relevance of this modification using Ari-1 loss-of-function mutants, which displayed a lower rate of spontaneous neurotransmitter release due to failures at the presynaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared with controls in a Cadependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. This phenotype distinction between spontaneous and evoked release suggests that NSF activity may discriminate between these two types of vesicle fusion. Our results thus provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination.This research was funded by grants BFU2015-65685 and PGC2018-094630-B-100 from the Spanish Ministry of Economy to A. F. and grant SAF2016-76898-P from the Spanish Ministry of Economy cofinanced with FEDER funds to U. M. J. R. was supported with a postdoctoral research fellowship from the University of the Basque Country (UPV/EHU)

Phosphoproteomic and functional analyses reveal sperm-specific protein changes downstream of kappa opioid receptor in human spermatozoa

G-protein coupled receptors (GPCRs) belong to the seven transmembrane receptor superfamily that transduce signals via G proteins in response to external stimuli to initiate different intracellular signaling pathways which culminate in specific cellular responses. The expression of diverse GPCRs at the plasma membrane of human spermatozoa suggests their involvement in the regulation of sperm fertility. However, the signaling events downstream of many GPCRs in spermatozoa remain uncharacterized. Here, we selected the kappa-opioid receptor (KOR) as a study model and applied phosphoproteomic approach based on TMT labeling and LC-MS/MS analyses. Quantitative coverage of more than 5000 proteins with over 3500 phosphorylation sites revealed changes in the phosphorylation levels of sperm-specific proteins involved in the regulation of the sperm fertility in response to a specific agonist of KOR, U50488H. Further functional studies indicate that KOR could be involved in the regulation of sperm fertile capacity by modulation of calcium channels. Our findings suggest that human spermatozoa possess unique features in the molecular mechanisms downstream of GPCRs which could be key regulators of sperm fertility and improved knowledge of these specific processes may contribute to the development of useful biochemical tools for diagnosis and treatment of male infertility

Intercellular communication via exosomes

Exosomes are small membrane bound vesicles between 30-100 nm in diameter of endocytic origin that are secreted into the extracellular environment by many different cell types. They play a role in intercellular communication by transferring proteins, lipids and RNA to recipient cells. The overall aim of this work has been to further investigate the mechanisms by which cells communicate with each other via exosomes. In Paper I we hypothesized that exosomes from human cells could be used as vectors to provide cells with therapeutic RNA. Herein, exogenous short interfering RNAs were successfully introduced into various kinds of human exosomes using electroporation. Flow cytometry, confocal microscopy and northern blot confirmed the presence of siRNA inside the exosomes. The results showed that exosomes from blood plasma could deliver the siRNA to human monocytes and lymphocytes. The siRNA delivered to the target cells was shown to be functional causing selective gene silencing of mitogen activated protein kinase 1. Our results imply that exosomes from human cells could be used as vectors for delivery of therapeutic exogenous nucleic acids to cells. In paper II we investigated if exosomes from activated CD3+ T cells could play a role in an immunological response by conveying signals from their secreting cells to recipient resting T cells in an in vitro autologous setting. The role of these exosomes was explored in IL-2 mediated T cell proliferation. The results showed that neither exosomes nor IL-2 alone could stimulate proliferation in resting T cells. However, exosomes from stimulated T cells together with IL-2 were able to induce proliferation. T cell cultures stimulated with exosomes and IL-2 showed a higher proportion of CD8+ T cells than cultures without exosomes. Moreover, a cytokine array showed significant changes in the levels of cytokines and chemokines when exosomes were present. The results indicate that activated CD3+ cells communicate with resting autologous T cells via exosomes. The main focus in paper III was to study the cellular mechanism by which esRNA is selectively packaged into exosome vesicles during their biosynthesis. Using RNA gel mobility shift assay, we showed the presence of RNA-binding proteins (RBPs) in exosomes. Moreover, we developed a method for the identification of exosomal RBPs able to bind to the esRNA and cellular microRNA. Using this method, we could identify 31 different RBPs in exosomes and 78 in cells. To evaluate the possible role of the identified RBPs in the transfer mechanism of RNA into intraluminal vesicles, five gene transcripts from the identified RBPs were silenced. The results revealed that a selective gene silencing of hnRNPA2B1 caused a reduction of RNA present in the extracellular vesicles. Thus, a novel transport mechanism was suggested for the packaging of esRNA into the exosomes. In conclusion, the studies presented in this thesis have implications for better understanding the RNA and protein transfer mechanism that occurs between cells via exosomes. The described ability of exosomes to deliver exogenous nucleic acids to cells may be of interest in clinical applications e.g. in gene therapy

Mutant JAK3 phosphoproteomic profiling predicts synergism between JAK3 inhibitors and MEK/BCL2 inhibitors for the treatment of T-cell acute lymphoblastic leukemia

Following the publication of this article the authors noted that data describing precisely where phosphorylation sites in proteins modulated following JAK1 or JAK3 inhibition in mutant T-ALL samples was not clearly annotated. Therefore an additional sheet has been added to Supplementary Table 2