We report the identification and molecular characterization of Dtrk, a Drosophila gene encoding a receptor tyrosine kinase highly related to the trk family of mammalian neurotrophin receptors. The product of the Dtrk gene, gp160Dtrk, is dynamically expressed during Drosophila embryogenesis in several areas of the developing nervous system, including neurons and fasciculating axons. gp160Dtrk has structural homology with neural cell adhesion molecules of the immunoglobulin superfamily and promotes cell adhesion in a homophilic, Ca2+ independent manner. More importantly, this adhesion process specifically activates its tyrosine protein kinase activity. These findings suggest that gp160Dtrk represents a new class of neural cell adhesion molecules that may regulate neuronal recognition and axonal guidance during the development of the Drosophila nervous system.

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The integration of therapeutic biomolecules, such as proteins and peptides, in nanovesicles is a widely used strategy to improve their stability and efficacy. However, the translation of these promising nanotherapeutics to clinical tests is still challenged by the complexity involved in the preparation of functional nanovesicles and their reproducibility, scalability, and cost production. Here we introduce a simple one-step methodology based on the use of CO2-expanded solvents to prepare multifunctional nanovesicle-bioactive conjugates. We demonstrate high vesicle-to-vesicle homogeneity in terms of size and lamellarity, batch-to-batch consistency, and reproducibility upon scaling-up. Importantly, the procedure is readily amenable to the integration/encapsulation of multiple components into the nanovesicles in a single step and yields sufficient quantities for clinical research. The simplicity, reproducibility, and scalability render this one-step fabrication process ideal for the rapid and low-cost translation of nanomedicine candidates from the bench to the clinic.

Phosphorylation of the ␣ subunit of the eukaryotic initiation factor 2 (eIF-2␣) is one of the best-characterized mechanisms for downregulating protein synthesis in mammalian cells in response to various stress conditions. In Drosophila, such a regulatory mechanism has not been elucidated. We report the molecular cloning and characterization of DGCN2, a Drosophila eIF-2␣ kinase related to yeast GCN2 protein kinase. DGCN2 contains all of the 12 catalytic subdomains characteristic of eukaryotic Ser/Thr protein kinases and the conserved sequence of eIF-2␣ kinases in subdomain V. A large insert of 94 amino acids, which is characteristic of eIF-2␣ kinases, is also present between subdomains IV and V. It is particularly notable that DGCN2 possesses an amino acid sequence related to class II aminoacyl-tRNA synthetases, a unique feature of yeast GCN2 protein kinase. DGCN2 expression is developmentally regulated. During embryogenesis, DGCN2 mRNA is dynamically expressed in several tissues. Interestingly, at later stages this expression becomes restricted to a few cells of the central nervous system. Affinity-purified antibodies, raised against a synthetic peptide based on the predicted DGCN2 sequence, specifically immunoprecipitated an eIF-2␣ kinase activity and recognized an ϳ175 kDa phosphoprotein in Western blots of Drosophila embryo extracts.Protein synthesis is mainly regulated at the initiation of mRNA translation. The best-characterized mechanism of translational regulation in eukaryotes involves the phosphorylation of the ␣-subunit of eukaryotic initiation factor 2 (eIF-2␣)

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Lysosomal storage disorders (LSD) are caused by lysosomal dysfunction usually as a consequence of deficiency of a single enzyme required for the metabolism of macromolecules, such as lipids, glycoproteins, and mucopolysaccharides. For instance, the lack of α-galactosidase A (GLA) activity in Fabry disease patients causes the accumulation of glycosphingolipids in the vasculature leading to multiple organ pathology. Enzyme replacement therapy, which is the most common treatment of LSD, exhibits several drawbacks mainly related to the instability and low efficacy of the exogenously administered therapeutic enzyme. In this work, the unprecedented increased enzymatic activity and intracellular penetration achieved by the association of a human recombinant GLA to nanoliposomes functionalized with Arginine-Glycine-Aspartic acid (RGD) peptides is reported. Moreover, these new GLA loaded nanoliposomes lead to a higher efficacy in the reduction of the GLA substrate named globotriasylceramide in a cellular model of Fabry disease, than that achieved by the same concentration of the free enzyme. The preparation of these new liposomal formulations by DELOS-SUSP, based on the depressurization of a CO2 -expanded liquid organic solution, shows the great potential of this CO2 -based methodology for the one-step production of protein-nanoliposome conjugates as bioactive nanomaterials with therapeutic interest.

Bivalent ligands have emerged as chemical tools to study G protein-coupled receptor dimers. Using a combination of computational, chemical, and biochemical tools, here we describe the design of bivalent ligand 13 with high affinity (K DB1 = 21 pM) for the dopamine D 2 receptor (D 2 R) homodimer. Bivalent ligand 13 enhances the binding affinity relative to monovalent compound 15 by 37-fold, indicating simultaneous binding at both protomers. Using synthetic peptides with amino acid sequences of transmembrane (TM) domains of D 2 R, we provide evidence that TM6 forms the interface of the homodimer. Notably, the disturber peptide TAT-TM6 decreased the binding of bivalent ligand 13 by 52-fold and had no effect on monovalent compound 15, confirming the D 2 R homodimer through TM6 ex vivo. In conclusion, by using a versatile multivalent chemical platform, we have developed a precise strategy to generate a true bivalent ligand that simultaneously targets both orthosteric sites of the D 2 R homodimer.

In this study we have used the yeast two-hybrid system to identify proteins that interact with the carboxylcytoplasmic domain (residues 464 -509) of the insulinsensitive glucose transporter GLUT4 (C-GLUT4). Using as bait C-GLUT4, we have isolated the carboxyl domain of Daxx (C-Daxx), the adaptor protein associated with the Fas and the type II TGF-␤ (T␤RII) receptors (1, 2). The two-hybrid interaction between C-GLUT4 and CDaxx is validated by the ability of in vitro translated C-GLUT4 to interact with in vitro translated full-length Daxx and C-Daxx. C-Daxx does not interact with the C-cytoplasmic domain of GLUT1, the ubiquitous glucose transporter homologous to GLUT4. Replacement of alanine and serine for the dileucine pair (Leu 489 -Leu 490 ) critical for targeting GLUT4 from the trans-Golgi network to the perinuclear intracellular store as well as for its surface internalization by endocytosis inhibits 2-fold the interaction of C-GLUT4 with Daxx. Daxx is pulled down with GLUT4 immunoprecipitated from lysates of 3T3-L1 fibroblasts stably transfected with GLUT4 and 3T3-L1 adipocytes expressing physiological levels of the two proteins. Similarly, GLUT4 is recovered with antiDaxx immunoprecipitates. Using an established cell fractionation procedure we present evidence for the existence of two distinct intracellular Daxx pools in the nucleus and low density microsomes. Confocal immunofluorescence microscopy studies localize Daxx to promyelocytic leukemia nuclear bodies and punctate cytoplasmic structures, often organized in strings and underneath the plasma membrane. Daxx and GLUT4 are SUMOlated as shown by their reaction with an anti-SUMO1 antibody and by the ability of this antibody to pull down Daxx and GLUT4.The cytoplasmic domain of membrane proteins plays important roles in their transport, signal transduction, organization of protein scaffolds, and regulation of their turnover. Trafficking of GLUT4 in adipose and skeletal muscle cells is regulated by insulin and muscle contraction and is critical for the control of glucose levels in blood. Upon increase in insulin levels and muscle contraction the GLUT4 retained in intracellular stores is translocated to the plasma membrane, where it facilitates glucose transport (3). Trafficking of GLUT4 is mediated by motifs localized to the amino and carboxyl-cytoplasmic domains of the protein, though their characterization and the identification of the factors involved in their reading is incomplete.SUMO (also called sentrin, PIC1, and GMP1), a 101-amino acid ubiquitin-like modifier protein that is highly conserved from yeast to human, appears to control protein turnover and compartmentalization (4). Three members of the SUMO family have been described in vertebrates. They show major structural differences in the sequences of their N-extensions, which are absent in ubiquitin. It has been shown recently that Ubc9, the only E2-type SUMO1-conjugating enzyme described in vertebrates, interacts with the carboxyl-cytoplasmic domain of GLUT4 as part of a mechanism that slows its...