Crosstalk between ß2- and α2-Adrenergic Receptors in the Regulation of B16F10 Melanoma Cell Proliferation.

Adrenergic receptors (AR) belong to the G protein-coupled receptor superfamily and regulate migration and proliferation in various cell types. The objective of this study was to evaluate whether ß-AR stimulation affects the antiproliferative action of α2-AR agonists on B16F10 cells and, if so, to determine the relative contribution of ß-AR subtypes. Using pharmacological approaches, evaluation of Ki-67 expression by flow cytometry and luciferase-based cAMP assay, we found that treatment with isoproterenol, a ß-AR agonist, increased cAMP levels in B16F10 melanoma cells without affecting cell proliferation. Propranolol inhibited the cAMP response to isoproterenol. In addition, stimulation of α2-ARs with agonists such as clonidine, a well-known antihypertensive drug, decreased cancer cell proliferation. This effect on cell proliferation was suppressed by treatment with isoproterenol. In turn, the suppressive effects of isoproterenol were abolished by the treatment with either ICI 118,551, a ß2-AR antagonist, or propranolol, suggesting that isoproterenol effects are mainly mediated by the ß2-AR stimulation. We conclude that the crosstalk between the ß2-AR and α2-AR signaling pathways regulates the proliferative activity of B16F10 cells and may therefore represent a therapeutic target for melanoma therapy.

ß-blockers Reverse Agonist-Induced ß2-AR Downregulation Regardless of Their Signaling Profile.

Altered ß-adrenergic receptor (ß-AR) density has been reported in cells, animals, and humans receiving ß-blocker treatment. In some cases, ß-AR density is upregulated, but in others, it is unaffected or even reduced. Collectively, these results would imply that changes in ß-AR density and ß-blockade are not related. However, it has still not been clarified whether the effects of ß-blockers on receptor density are related to their ability to activate different ß-AR signaling pathways. To this aim, five clinically relevant ß-blockers endowed with inverse, partial or biased agonism at the ß2-AR were evaluated for their effects on ß2-AR density in both human embryonic kidney 293 (HEK293) cells expressing exogenous FLAG-tagged human ß2-ARs and human lymphocytes expressing endogenous ß2-ARs. Cell surface ß2-AR density was measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Treatment with propranolol, carvedilol, pindolol, sotalol, or timolol did not induce any significant change in surface ß2-AR density in both HEK293 cells and human lymphocytes. On the contrary, treatment with the ß-AR agonist isoproterenol reduced the number of cell surface ß2-ARs in the tested cell types without affecting ß2-AR-mRNA levels. Isoproterenol-induced effects on receptor density were completely antagonized by ß-blocker treatment. In conclusion, the agonistic activity of ß-blockers does not exert an important effect on short-term regulation of ß2-AR density.

Probiotics and antibiotic-associated diarrhea in children: A review and new evidence on Lactobacillus rhamnosus GG during and after antibiotic treatment.

Antibiotic associated diarrhea (AAD) is a common complication in childhood in the outpatient and inpatient settings. This review provides up to date information on the use of probiotics in the prevention and treatment of AAD, including that from Clostridium Difficile, in children. The most recently systematic reviews and subsequently published randomized controlleds trials are considered. Different single and multistrain probiotics are described; a specific recommendation for the use of Lactobacillus Rhamnosus GG (LGG) and Saccharomyces boulardii (Sb) emerges. New information on LGG survival under amoxicillin/clavulanate therapy in children is also provided. This information is relevant in view of the frequent use of this molecule in children, its association with AAD, and LGG's sensitivity to penicillin that might make this probiotic ineffective. In spite of a demonstrated positive effect of specific strains of probiotics on AAD, safety issues still remain among which the risk of associated severe infections and of antibiotic resistant gene exchange.

Genetically induced dysfunctions of Kir2.1 channels: implications for short QT3 syndrome and autism-epilepsy phenotype.

Short QT3 syndrome (SQT3S) is a cardiac disorder characterized by a high risk of mortality and associated with mutations in Kir2.1 (KCNJ2) channels. The molecular mechanisms leading to channel dysfunction, cardiac rhythm disturbances and neurodevelopmental disorders, potentially associated with SQT3S, remain incompletely understood. Here, we report on monozygotic twins displaying a short QT interval on electrocardiogram recordings and autism-epilepsy phenotype. Genetic screening identified a novel KCNJ2 variant in Kir2.1 that (i) enhanced the channel's surface expression and stability at the plasma membrane, (ii) reduced protein ubiquitylation and degradation, (iii) altered protein compartmentalization in lipid rafts by targeting more channels to cholesterol-poor domains and (iv) reduced interactions with caveolin 2. Importantly, our study reveals novel physiological mechanisms concerning wild-type Kir2.1 channel processing by the cell, such as binding to both caveolin 1 and 2, protein degradation through the ubiquitin-proteasome pathway; in addition, it uncovers a potential multifunctional site that controls Kir2.1 surface expression, protein half-life and partitioning to lipid rafts. The reported mechanisms emerge as crucial also for proper astrocyte function, suggesting the need for a neuropsychiatric evaluation in patients with SQT3S and offering new opportunities for disease management.

Ligands raise the constraint that limits constitutive activation in G protein-coupled opioid receptors.

Using a cell-free bioluminescence resonance energy transfer strategy we compared the levels of spontaneous and ligand-induced receptor-G protein coupling in δ (DOP) and µ (MOP) opioid receptors. In this assay GDP can suppress spontaneous coupling, thus allowing its quantification. The level of constitutive activity was 4-5 times greater at the DOP than at the MOP receptor. A series of opioid analogues with a common peptidomimetic scaffold displayed remarkable inversions of efficacy in the two receptors. Agonists that enhanced coupling above the low intrinsic level of the MOP receptor were inverse agonists in reducing the greater level of constitutive coupling of the DOP receptor. Yet the intrinsic activities of such ligands are identical when scaled over the GDP base line of both receptors. This pattern is in conflict with the predictions of the ternary complex model and the "two state" extensions. According to this theory, the order of spontaneous and ligand-induced coupling cannot be reversed if a shift of the equilibrium between active and inactive forms raises constitutive activation in one receptor type. We propose that constitutive activation results from a lessened intrinsic barrier that restrains spontaneous coupling. Any ligand, regardless of its efficacy, must enhance this constraint to stabilize the ligand-bound complexed form.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 modulates endosomal pH and protein trafficking in astrocytes: relevance to MLC disease pathogenesis.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in the gene encoding MLC1, a membrane protein mainly expressed in astrocytes in the central nervous system. Although MLC1 function is unknown, evidence is emerging that it may regulate ion fluxes. Using biochemical and proteomic approaches to identify MLC1 interactors and elucidate MLC1 function we found that MLC1 interacts with the vacuolar ATPase (V-ATPase), the proton pump that regulates endosomal acidity. Because we previously showed that in intracellular organelles MLC1 directly binds Na, K-ATPase, which controls endosomal pH, we studied MLC1 endosomal localization and trafficking and MLC1 effects on endosomal acidity and function using human astrocytoma cells overexpressing wild-type (WT) MLC1 or MLC1 carrying pathological mutations. We found that WT MLC1 is abundantly expressed in early (EEA1(+), Rab5(+)) and recycling (Rab11(+)) endosomes and uses the latter compartment to traffic to the plasma membrane during hyposmotic stress. We also showed that WT MLC1 limits early endosomal acidification and influences protein trafficking in astrocytoma cells by stimulating protein recycling, as revealed by FITC-dextran measurement of endosomal pH and transferrin protein recycling assay, respectively. WT MLC1 also favors recycling to the plasma-membrane of the TRPV4 cation channel which cooperates with MLC1 to activate calcium influx in astrocytes during hyposmotic stress. Although MLC disease-causing mutations differentially affect MLC1 localization and trafficking, all the mutated proteins fail to influence endosomal pH and protein recycling. This study demonstrates that MLC1 modulates endosomal pH and protein trafficking suggesting that alteration of these processes contributes to MLC pathogenesis.

Megalencephalic leukoencephalopathy with subcortical cysts protein 1 functionally cooperates with the TRPV4 cation channel to activate the response of astrocytes to osmotic stress: dysregulation by pathological mutations.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare leukodystrophy characterized by macrocephaly, subcortical fluid cysts and myelin vacuolation, has been linked to mutations in the MLC1 gene. This gene encodes a membrane protein that is highly expressed in astrocytes. Based on MLC pathological features, it was proposed that astrocyte-mediated defects in ion and fluid homeostasis could account for the alterations observed in MLC-affected brains. However, the role of MLC1 and the effects of pathological mutations on astrocyte osmoregulatory functions have still to be demonstrated. Using human astrocytoma cells stably overexpressing wild-type MLC1 or three known MLC-associated pathological mutations, we investigated MLC1 involvement in astrocyte reaction to osmotic changes using biochemical, dynamic video imaging and immunofluorescence techniques. We have found that MLC1 overexpressed in astrocytoma cells is mainly localized in the plasma membrane, is part of the Na,K-ATPase-associated molecular complex that includes the potassium channel Kir4.1, syntrophin and aquaporin-4 and functionally interacts with the calcium permeable channel TRPV4 (transient receptor potential vanilloid-4 cation channel) which mediates swelling-induced cytosolic calcium increase and volume recovery in response to hyposmosis. Pathological MLC mutations cause changes in MLC1 expression and intracellular localization as well as in the astrocyte response to osmotic changes by altering MLC1 molecular interactions with the Na,K-ATPase molecular complex and abolishing the increase in calcium influx induced by hyposmosis and treatment with the TRPV4 agonist 4αPDD. These data demonstrate, for the first time, that MLC1 plays a role in astrocyte osmo-homeostasis and that defects in intracellular calcium dynamics may contribute to MLC pathogenesis.

Benzodiazepine diazepam regulates cell surface ß1-adrenergic receptor density in human monocytes.

Downregulation of cell surface ß-adrenergic receptors (ß-AR) is an important adaptive response that prevents deleterious effects of receptor overstimulation. Various factors including reactive oxygen species cause ß-AR downregulation. In this study, we evaluated the effects of ligands of the peripheral benzodiazepine receptor (PBR), a key protein in regulating oxidative stress, on surface density of endogenous ß1-and ß2-ARs in highly differentiated cells such as human monocytes, which express both ß-AR subtypes. ß-AR expression in human monocytes was evaluated by flow cytometry, qPCR and western blotting. Monocyte treatment with ß-AR agonist isoproterenol did not change surface ß1-AR density while downregulating surface ß2-AR density. This effect was antagonized by the ß-blocker propranolol. An opposite response was observed with benzodiazepine diazepam that led to a time-dependent reduction in ß1-AR density. In particular, while no significant downregulation was observed after 3 h of treatment, only 63% of ß1-ARs were still present on the cell surface after 48 h of treatment with diazepam at 1 µM. Treatment with the PBR antagonist PK11195, but not with propranolol, antagonized the effects of diazepam. No change in ß1-AR-mRNA or protein levels was observed at any time after diazepam treatment. We also found that diazepam did not affect Gs-protein or ß-arrestin-2 recruitment for both ß-ARs in engineered fibroblasts, further suggesting that diazepam activity on ß1-AR density is mediated by PBR. Finally, no sex-related differences were found. Collectively, these results indicate that monocyte ß1-ARs are resistant to catecholamine-mediated downregulation and suggest that PBR plays an important role in regulating ß1-AR density.

Divergent agonist selectivity in activating ß1- and ß2-adrenoceptors for G-protein and arrestin coupling.

The functional selectivity of adrenergic ligands for activation of ß1- and ß2-AR (adrenoceptor) subtypes has been extensively studied in cAMP signalling. Much less is known about ligand selectivity for arrestin-mediated signalling pathways. In the present study we used resonance energy transfer methods to compare the ability of ß1- and ß2-ARs to form a complex with the G-protein ß-subunit or ß-arrestin-2 in response to a variety of agonists with various degrees of efficacy. The profiles of ß1-/ß2-AR selectivity of the ligands for the two receptor-transducer interactions were sharply different. For G-protein coupling, the majority of ligands were more effective in activating the ß2-AR, whereas for arrestin coupling the relationship was reversed. These data indicate that the ß1-AR interacts more efficiently than ß2-AR with arrestin, but less efficiently than ß2-AR with G-protein. A group of ligands exhibited ß1-AR-selective efficacy in driving the coupling to arrestin. Dobutamine, a member of this group, had 70% of the adrenaline (epinephrine) effect on arrestin via ß1-AR, but acted as a competitive antagonist of adrenaline via ß2-AR. Thus the structure of such ligands appears to induce an arrestin-interacting form of the receptor only when bound to the ß1-AR subtype.

Pharmacology of Kappa Opioid Receptors: Novel Assays and Ligands.

The present study investigated the in vitro pharmacology of the human kappa opioid receptor using multiple assays, including calcium mobilization in cells expressing chimeric G proteins, the dynamic mass redistribution (DMR) label-free assay, and a bioluminescence resonance energy transfer (BRET) assay that allows measurement of receptor interaction with G protein and ß-arrestin 2. In all assays, dynorphin A, U-69,593, and [D-Pro10]dyn(1-11)-NH2 behaved as full agonists with the following rank order of potency [D-Pro10]dyn(1-11)-NH2 > dynorphin A ≥ U-69,593. [Dmt1,Tic2]dyn(1-11)-NH2 behaved as a moderate potency pure antagonist in the kappa-ß-arrestin 2 interaction assay and as low efficacy partial agonist in the other assays. Norbinaltorphimine acted as a highly potent and pure antagonist in all assays except kappa-G protein interaction, where it displayed efficacy as an inverse agonist. The pharmacological actions of novel kappa ligands, namely the dynorphin A tetrameric derivative PWT2-Dyn A and the palmitoylated derivative Dyn A-palmitic, were also investigated. PWT2-Dyn A and Dyn A-palmitic mimicked dynorphin A effects in all assays showing similar maximal effects but 3-10 fold lower potency. In conclusion, in the present study, multiple in vitro assays for the kappa receptor have been set up and pharmacologically validated. In addition, PWT2-Dyn A and Dyn A-palmitic were characterized as potent full agonists; these compounds are worthy of further investigation in vivo for those conditions in which the activation of the kappa opioid receptor elicits beneficial effects e.g. pain and pruritus.

The CaMKII/MLC1 Axis Confers Ca2+-Dependence to Volume-Regulated Anion Channels (VRAC) in Astrocytes.

Astrocytes, the main glial cells of the central nervous system, play a key role in brain volume control due to their intimate contacts with cerebral blood vessels and the expression of a distinctive equipment of proteins involved in solute/water transport. Among these is MLC1, a protein highly expressed in perivascular astrocytes and whose mutations cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, chronic brain edema, cysts, myelin vacuolation, and astrocyte swelling. Although, in astrocytes, MLC1 mutations are known to affect the swelling-activated chloride currents (ICl,swell) mediated by the volume-regulated anion channel (VRAC), and the regulatory volume decrease, MLC1's proper function is still unknown. By combining molecular, biochemical, proteomic, electrophysiological, and imaging techniques, we here show that MLC1 is a Ca2+/Calmodulin-dependent protein kinase II (CaMKII) target protein, whose phosphorylation, occurring in response to intracellular Ca2+ release, potentiates VRAC-mediated ICl,swell. Overall, these findings reveal that MLC1 is a Ca2+-regulated protein, linking volume regulation to Ca2+ signaling in astrocytes. This knowledge provides new insight into the MLC1 protein function and into the mechanisms controlling ion/water exchanges in the brain, which may help identify possible molecular targets for the treatment of MLC and other pathological conditions caused by astrocyte swelling and brain edema.

Morphine-like opiates selectively antagonize receptor-arrestin interactions.

The addictive potential of opioids may be related to their differential ability to induce G protein signaling and endocytosis. We compared the ability of 20 ligands (sampled from the main chemical classes of opioids) to promote the association of mu and delta receptors with G protein or beta-arrestin 2. Receptor-arrestin binding was monitored by bioluminescence resonance energy transfer (BRET) in intact cells, where pertussis toxin experiments indicated that the interaction was minimally affected by receptor signaling. To assess receptor-G protein coupling without competition from arrestins, we employed a cell-free BRET assay using membranes isolated from cells expressing luminescent receptors and fluorescent Gbeta(1). In this system, the agonist-induced enhancement of BRET (indicating shortening of distance between the two proteins) was G alpha-mediated (as shown by sensitivity to pertussis toxin and guanine nucleotides) and yielded data consistent with the known pharmacology of the ligands. We found marked differences of efficacy for G protein and arrestin, with a pattern suggesting more restrictive structural requirements for arrestin efficacy. The analysis of such differences identified a subset of structures showing a marked discrepancy between efficacies for G protein and arrestin. Addictive opiates like morphine and oxymorphone exhibited large differences both at delta and mu receptors. Thus, they were effective agonists for G protein coupling but acted as competitive enkephalins antagonists (delta) or partial agonists (mu) for arrestin. This arrestin-selective antagonism resulted in inhibition of short and long term events mediated by arrestin, such as rapid receptor internalization and down-regulation.

MLC1 trafficking and membrane expression in astrocytes: role of caveolin-1 and phosphorylation.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy caused by mutations in the MLC1 gene that encodes a membrane protein of unknown function. In the brain MLC1 protein is mainly expressed in astrocyte end-feet, localizes in lipid rafts and associates with the dystrophin glycoprotein complex (DGC). Using pull-down and co-fractionation assays in cultured human and rat astrocytes, we show here that MLC1 intracellular domains pull-down the DGC proteins syntrophin, dystrobrevin, Kir4.1 and caveolin-1, the structural protein of caveolae, thereby supporting a role for DGC and caveolar structures in MLC1 function. By immunostaining and subcellular fractionation of cultured rat or human astrocytes treated with agents modulating caveolin-mediated trafficking, we demonstrate that MLC1 is also expressed in intracellular vesicles and endoplasmic reticulum and undergoes caveolae/raft-mediated endocytosis. Inhibition of endocytosis, cholesterol lowering and protein kinases A- and C-mediated MLC1 phosphorylation favour the expression of membrane-associated MLC1. Because pathological mutations prevent MLC1 membrane expression, the identification of substances regulating MLC1 intracellular trafficking is potentially relevant for the therapy of MLC.

Vasopressin receptor 2 mutations in the nephrogenic syndrome of inappropriate antidiuresis show different mechanisms of constitutive activation for G protein coupled receptors.

Vasopressin receptor 2 (V2R) mutations causing the nephrogenic syndrome of inappropriate antidiuresis (NSIAD) can generate two constitutively active receptor phenotypes. One type results from residue substitutions in several V2R domains and is sensitive to vaptan inverse agonists. The other is only caused by Arg 137 replacements and is vaptan resistant. We compared constitutive and agonist-driven interactions of the vaptan-sensitive F229V and vaptan-resistant R137C/L V2R mutations with ß-arrestin 1, ß-arrestin 2, and Gαs, using null fibroblasts reconstituted with individual versions of the ablated transduction protein genes. F229V displayed very high level of constitutive activation for Gs but not for ß-arrestins, and enhanced or normal responsiveness to agonists and inverse agonists. In contrast, R137C/L mutants exhibited maximal levels of constitutive activation for ßarrestin 2 and Gs, minimal levels for ß-arrestin 1, but a sharp decline of ligands sensitivity at all transducer interactions. The enhanced constitutive activity and reduced ligand sensitivity of R137 mutants on cAMP signaling persisted in cells lacking ß-arrestins, indicating that these are intrinsic molecular properties of the mutations, not the consequence of altered receptor trafficking. The results suggest that the two groups of NSIAD mutations represent two distinct molecular mechanisms of constitutive activation in GPCRs.

Megalencephalic Leukoencephalopathy with Subcortical Cysts Disease-Linked MLC1 Protein Favors Gap-Junction Intercellular Communication by Regulating Connexin 43 Trafficking in Astrocytes.

Astrocytes, the most numerous cells of the central nervous system, exert critical functions for brain homeostasis. To this purpose, astrocytes generate a highly interconnected intercellular network allowing rapid exchange of ions and metabolites through gap junctions, adjoined channels composed of hexamers of connexin (Cx) proteins, mainly Cx43. Functional alterations of Cxs and gap junctions have been observed in several neuroinflammatory/neurodegenerative diseases. In the rare leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), astrocytes show defective control of ion/fluid exchanges causing brain edema, fluid cysts, and astrocyte/myelin vacuolation. MLC is caused by mutations in MLC1, an astrocyte-specific protein of elusive function, and in GlialCAM, a MLC1 chaperon. Both proteins are highly expressed at perivascular astrocyte end-feet and astrocyte-astrocyte contacts where they interact with zonula occludens-1 (ZO-1) and Cx43 junctional proteins. To investigate the possible role of Cx43 in MLC pathogenesis, we studied Cx43 properties in astrocytoma cells overexpressing wild type (WT) MLC1 or MLC1 carrying pathological mutations. Using biochemical and electrophysiological techniques, we found that WT, but not mutated, MLC1 expression favors intercellular communication by inhibiting extracellular-signal-regulated kinase 1/2 (ERK1/2)-mediated Cx43 phosphorylation and increasing Cx43 gap-junction stability. These data indicate MLC1 regulation of Cx43 in astrocytes and Cx43 involvement in MLC pathogenesis, suggesting potential target pathways for therapeutic interventions.

Intermittent ß-adrenergic blockade downregulates the gene expression of ß-myosin heavy chain in the mouse heart.

Expression of the ß-myosin heavy chain (ß-MHC), a major component of the cardiac contractile apparatus, is tightly regulated as even modest increases can be detrimental to heart under stress. In healthy hearts, continuous inhibition of ß-adrenergic tone upregulates ß-MHC expression. However, it is unknown whether the duration of the ß-adrenergic inhibition and ß-MHC expression are related. Here, we evaluated the effects of intermittent ß-blockade on cardiac ß-MHC expression. To this end, the ß-blocker propranolol, at the dose of 15mg/kg, was administered once a day in mice for 14 days. This dosing schedule caused daily drug-free periods of at least 6 h as evidenced by propranolol plasma concentrations and cardiac ß-adrenergic responsiveness. Under these conditions, ß-MHC expression decreased by about 75% compared to controls. This effect was abolished in mice lacking ß1- but not ß2-adrenergic receptors (ß-AR) indicating that ß-MHC expression is regulated in a ß1-AR-dependent manner. In ß1-AR knockout mice, the baseline ß-MHC expression was fourfold higher than in wild-type mice. Also, we evaluated the impact of intermittent ß-blockade on ß-MHC expression in mice with systolic dysfunction, in which an increased ß-MHC expression occurs. At 3 weeks after myocardial infarction, mice showed systolic dysfunction and upregulation of ß-MHC expression. Intermittent ß-blockade decreased ß-MHC expression while attenuating cardiac dysfunction. In vitro studies showed that propranolol does not affect ß-MHC expression on its own but antagonizes catecholamine effects on ß-MHC expression. In conclusion, a direct relationship occurs between the duration of the ß-adrenergic inhibition and ß-MHC expression through the ß1-AR.

Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells.

Green bioluminescence in Renilla species is generated by a approximately 100% efficient RET (resonance energy transfer) process that is caused by the direct association of a blue-emitting luciferase [Rluc (Renilla luciferase)] and an RGFP (Renilla green fluorescent protein). Despite the high efficiency, such a system has never been evaluated as a potential reporter of protein-protein interactions. To address the question, we compared and analysed in mammalian cells the bioluminescence of Rluc and RGFP co-expressed as free native proteins, or as fused single-chain polypeptides and tethered partners of self-assembling coiled coils. Here, we show that: (i) no spontaneous interactions generating detectable BRET (bioluminescence RET) signals occur between the free native proteins; (ii) high-efficiency BRET similar to that observed in Renilla occurs in both fusion proteins and self-interacting chimaeras, but only if the N-terminal of RGFP is free; (iii) the high-efficiency BRET interaction is associated with a dramatic increase in light output when the luminescent reaction is triggered by low-quantum yield coelenterazine analogues. Here, we propose a new functional complementation assay based on the detection of the high-efficiency BRET signal that is generated when the reporters Rluc and RGFP are brought into close proximity by a pair of interacting proteins to which they are linked. To demonstrate its performance, we implemented the assay to measure the interaction between GPCRs (G-protein-coupled receptors) and beta-arrestins. We show that complementation-induced BRET allows detection of the GPCR-beta-arrestin interaction in a simple luminometric assay with high signal-to-noise ratio, good dynamic range and rapid response.

Genomic Comparison of Lactobacillus helveticus Strains Highlights Probiotic Potential.

Lactobacillus helveticus belongs to the large group of lactic acid bacteria (LAB), which are the major players in the fermentation of a wide range of foods. LAB are also present in the human gut, which has often been exploited as a reservoir of potential novel probiotic strains, but several parameters need to be assessed before establishing their safety and potential use for human consumption. In the present study, six L. helveticus strains isolated from natural whey cultures were analyzed for their phenotype and genotype in exopolysaccharide (EPS) production, low pH and bile salt tolerance, bile salt hydrolase (BSH) activity, and antibiotic resistance profile. In addition, a comparative genomic investigation was performed between the six newly sequenced strains and the 51 publicly available genomes of L. helveticus to define the pangenome structure. The results indicate that the newly sequenced strain UC1267 and the deposited strain DSM 20075 can be considered good candidates for gut-adapted strains due to their ability to survive in the presence of 0.2% glycocholic acid (GCA) and 1% taurocholic and taurodeoxycholic acid (TDCA). Moreover, these strains had the highest bile salt deconjugation activity among the tested L. helveticus strains. Considering the safety profile, none of these strains presented antibiotic resistance phenotypically and/or at the genome level. The pangenome analysis revealed genes specific to the new isolates, such as enzymes related to folate biosynthesis in strains UC1266 and UC1267 and an integrated phage in strain UC1035. Finally, the presence of maltose-degrading enzymes and multiple copies of 6-phospho-ß-glucosidase genes in our strains indicates the capability to metabolize sugars other than lactose, which is related solely to dairy niches.

Megalencephalic Leukoencephalopathy with Subcortical Cysts Protein-1 (MLC1) Counteracts Astrocyte Activation in Response to Inflammatory Signals.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 (MLC1) is a membrane protein expressed by perivascular astrocytes. MLC1 mutations cause MLC, an incurable leukodystrophy characterized by macrocephaly, brain edema, cysts, myelin vacuolation, and astrocytosis, leading to cognitive/motor impairment and epilepsy. Although its function is unknown, MLC1 favors regulatory volume decrease after astrocyte osmotic swelling and down-regulates intracellular signaling pathways controlling astrocyte activation and proliferation. By combining analysis of human brain tissues with in vitro experiments, here we investigated MLC1 role in astrocyte activation during neuroinflammation, a pathological condition exacerbating patient symptoms. MLC1 upregulation was observed in brain tissues from multiple sclerosis, Alzheimer's, and Creutzfeld-Jacob disease, all pathologies characterized by strong astrocytosis and release of inflammatory cytokines, particularly IL-1ß. Using astrocytoma lines overexpressing wild-type (WT) or mutated MLC1 and astrocytes from control and Mlc1 knock-out (KO) mice, we found that IL-1ß stimulated WT-MLC1 plasma membrane expression in astrocytoma cells and control primary astrocytes. In astrocytoma, WT-MLC1 inhibited the activation of IL-1ß-induced inflammatory signals (pERK, pNF-kB) that, conversely, were constitutively activated in mutant expressing cells or abnormally upregulated in KO astrocytes. WT-MLC1+ cells also expressed reduced levels of the astrogliosis marker pSTAT3. We then monitored MLC1 expression timing in a demyelinating/remyelinating murine cerebellar organotypic culture model where, after the demyelination and release of inflammatory cytokines, recovery processes occur, revealing MLC1 upregulation in these latter phases. Altogether, these findings suggest that by modulating specific pathways, MLC1 contributes to restore astrocyte homeostasis after inflammation, providing the opportunity to identify drug target molecules to slow down disease progression.

Delayed internalization and lack of recycling in a beta2-adrenergic receptor fused to the G protein alpha-subunit.

BACKGROUND: Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR) sequence to the N-terminus of the G protein alpha-subunit have been extensively used to investigate several aspects of GPCR signalling. Although both the receptor and the G protein generally maintain a fully functional state in such polypeptides, original observations made using a chimera between the beta2-adrenergic receptor (beta2AR) and Galphas indicated that the fusion to the alpha-subunit resulted in a marked reduction of receptor desensitization and down-regulation. To further investigate this phenomenon, we have compared the rates of internalization and recycling between wild-type and Galphas-fused beta2AR. RESULTS: The rate of agonist-induced internalization, measured as the disappearance of cell surface immunofluorescence in HEK293 cells permanently expressing N-terminus tagged receptors, was reduced three-fold by receptor-G protein fusion. However, both fused and non-fused receptors translocated to the same endocytic compartment, as determined by dual-label confocal analysis of cells co-expressing both proteins and transferrin co-localization. Receptor recycling, determined as the reversion of surface immunofluorescence following the addition of antagonist to cells that were previously exposed to agonist, markedly differed between wild-type and fused receptors. While most of the internalized beta2AR returned rapidly to the plasma membrane, beta2AR-Galphas did not recycle, and the observed slow recovery for the fusion protein immunofluorescence was entirely accounted for by protein synthesis. CONCLUSION: The covalent linkage between beta2AR and Galphas does not appear to alter the initial endocytic translocation of the two proteins, although there is reduced efficiency. It does, however, completely disrupt the process of receptor and G protein recycling. We conclude that the physical separation between receptor and Galpha is not necessary for the transit to early endosomes, but is an essential requirement for the correct post-endocytic sorting and recycling of the two proteins.