Fish Glucose Transporter (GLUT)-4 Differs from Rat GLUT4 in Its Traffic Characteristics but Can Translocate to the Cell Surface in Response to Insulin in Skeletal Muscle Cells

In mammals, glucose transporter (GLUT)-4 plays an important role in glucose homeostasis mediating insulin action to increase glucose uptake in insulin-responsive tissues. In the basal state, GLUT4 is located in intracellular compartments and upon insulin stimulation is recruited to the plasma membrane, allowing glucose entry into the cell. Compared with mammals, fish are less efficient restoring plasma glucose after dietary or exogenous glucose administration. Recently our group cloned a GLUT4-homolog in skeletal muscle from brown trout (btGLUT4) that differs in protein motifs believed to be important for endocytosis and sorting of mammalian GLUT4. To study the traffic of btGLUT4, we generated a stable L6 muscle cell line overexpressing myc-tagged btGLUT4 (btGLUT4myc). Insulin stimulated btGLUT4myc recruitment to the cell surface, although to a lesser extent than rat-GLUT4myc, and enhanced glucose uptake. Interestingly, btGLUT4myc showed a higher steady-state level at the cell surface under basal conditions than rat-GLUT4myc due to a higher rate of recycling of btGLUT4myc and not to a slower endocytic rate, compared with rat-GLUT4myc. Furthermore, unlike rat-GLUT4myc, btGLUT4myc had a diffuse distribution throughout the cytoplasm of L6 myoblasts. In primary brown trout skeletal muscle cells, insulin also promoted the translocation of endogenous btGLUT4 to the plasma membrane and enhanced glucose transport. Moreover, btGLUT4 exhibited a diffuse intracellular localization in unstimulated trout myocytes. Our data suggest that btGLUT4 is subjected to a different intracellular traffic from rat-GLUT4 and may explain the relative glucose intolerance observed in fish.

Relevance of death receptors in nervous system: role in the pathogenesis of neurodegenerative diseases and targets for therapy

L’apoptosi és un procés fisiològic que controla el nombre de cèl·lules en organismes superiors. L’apoptosi està estrictament regulada i s’ha vist que està implicada en la patogènesi d’algunes malalties del sistema nerviós. En aquest sentit, un excés de mort cel·lular contribueix a les malalties neurodegenerati- ves, mentre que, el seu dèficit és una de les raons del desenvolupament de tumors. El punt principal de regulació del procés apoptòtic és l’activació de les caspases, cisteïna-proteases que tenen especificitat pels residus aspàrtic. Les caspases es poden activar per dos mecanismes principals: (1) alliberament de citocrom C dels mitocondris alterats al citoplasma i (2) l’activació dels receptors de la membrana anomenats receptors de mort (DR, de l’anglès death receptor). Aquests receptors s’han caracteritzat extensament en el sistema immunitari, mentre que en el sistema nerviós les seves funcions són encara desconegudes. El present article se centra en el paper dels DR en la patogènesi de malalties neurodegeneratives i suggereix el seu potencial des del punt de vista terapèutic. També es descriuen diverses molècules intracel·lulars caracteritzades per la seva habilitat en la modulació dels DR. Entre elles, presentem dues noves proteïnes – lifeguard i FAIM – que s’expressen específicament al sistema nerviós.

Lipid rafts act as specialised domains for tetanus toxin binding and internalisation in neurons

Tetanus (TeNT) is a zinc protease that blocks neurotransmission by cleaving the synaptic protein vesicle-associated membrane protein/synaptobrevin. Although its intracellular catalytic activity is well established, the mechanism by which this neurotoxin interacts with the neuronal surface is not known. In this study, we characterize p15s, the first plasma membrane TeNT binding proteins and we show that they are glycosylphosphatidylinositol-anchored glycoproteins in nerve growth factor (NGF)-differentiated PC12 cells, spinal cord cells, and purified motor neurons. We identify p15 as neuronal Thy-1 in NGF-differentiated PC12 cells. Fluorescence lifetime imaging microscopy measurements confirm the close association of the binding domain of TeNT and Thy-1 at the plasma membrane. We find that TeNT is recruited to detergent-insoluble lipid microdomains on the surface of neuronal cells. Finally, we show that cholesterol depletion affects a raft subpool and blocks the internalization and intracellular activity of the toxin. Our results indicate that TeNT interacts with target cells by binding to lipid rafts and that cholesterol is required for TeNT internalization and/or trafficking in neurons.

On the role of G protein-coupled receptors oligomerization

The existence of a supramolecular organization of the G protein-coupled receptor (GPCR) is now being widely accepted by the scientific community. Indeed, GPCR oligomers may enhance the diversity and performance by which extracellular signals are transferred to the G proteins in the process of receptor transduction, although the mechanism that underlies this phenomenon still remains unsolved. Recently, it has been proposed that a trans-conformational switching model could be the mechanism allowing direct inhibition/activation of receptor activation/inhibition, respectively. Thus, heterotropic receptor-receptor allosteric regulations are behind the GPCR oligomeric function. In this paper we want to revise how GPCR oligomerization impinges on several important receptor functions like biosynthesis, plasma membrane diffusion or velocity, pharmacology and signaling. In particular, the rationale of receptor oligomerization might lie in the need of sensing complex whole cell extracellular signals and translating them into a simple computational model.

Uncovering the functional constraints underlying the genomic organisation of the Odorant-Binding Protein genes

Animal olfactory systems have a critical role for the survival and reproduction of individuals. In insects, the odorant-binding proteins (OBPs) are encoded by a moderately sized gene family, and mediate the first steps of the olfactory processing. Most OBPs are organized in clusters of a few paralogs, which are conserved over time. Currently, the biological mechanism explaining the close physical proximity among OBPs is not yet established. Here, we conducted a comprehensive study aiming to gain insights into the mechanisms underlying the OBP genomic organization. We found that the OBP clusters are embedded within large conserved arrangements. These organizations also include other non-OBP genes, which often encode proteins integral to plasma membrane. Moreover, the conservation degree of such large clusters is related to the following: 1) the promoter architecture of the confined genes, 2) a characteristic transcriptional environment, and 3) the chromatin conformation of the chromosomal region. Our results suggest that chromatin domains may restrict the location of OBP genes to regions having the appropriate transcriptional environment, leading to the OBP cluster structure. However, the appropriate transcriptional environment for OBP and the other neighbor genes is not dominated by reduced levels of expression noise. Indeed, the stochastic fluctuations in the OBP transcript abundance may have a critical role in the combinatorial nature of the olfactory coding process.

Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP

Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP

Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP