Glycosylation-Dependent IFN-γR Partitioning in Lipid and Actin Nanodomains Is Critical for JAK Activation.

Understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge. We studied interferon-γ receptor (IFN-γR) signaling in fibroblasts from homozygous patients with a T168N mutation in IFNGR2. By adding a neo-N-glycan on IFN-γR2 subunit, this mutation blocks IFN-γ activity by unknown mechanisms. We show that the lateral diffusion of IFN-γR2 is confined by sphingolipid/cholesterol nanodomains. In contrast, the IFN-γR2 T168N mutant diffusion is confined by distinct actin nanodomains where conformational changes required for Janus-activated tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) activation by IFN-γ could not occur. Removing IFN-γR2 T168N-bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient cells, whereas adding galectins impaired these processes in control cells. These experiments prove the critical role of dynamic receptor interactions with actin and lipid nanodomains and reveal a new function for receptor glycosylation and galectins. Our study establishes the physiological relevance of membrane nanodomains in the control of transmembrane receptor signaling in vivo. VIDEO ABSTRACT.

Meningococcus Hijacks a ß2-adrenoceptor/ß-Arrestin pathway to cross brain microvasculature endothelium.

Following pilus-mediated adhesion to human brain endothelial cells, meningococcus (N. meningitidis), the bacterium causing cerebrospinal meningitis, initiates signaling cascades, which eventually result in the opening of intercellular junctions, allowing meningeal colonization. The signaling receptor activated by the pathogen remained unknown. We report that N. meningitidis specifically stimulates a biased ß2-adrenoceptor/ß-arrestin signaling pathway in endothelial cells, which ultimately traps ß-arrestin-interacting partners, such as the Src tyrosine kinase and junctional proteins, under bacterial colonies. Cytoskeletal reorganization mediated by ß-arrestin-activated Src stabilizes bacterial adhesion to endothelial cells, whereas ß-arrestin-dependent delocalization of junctional proteins results in anatomical gaps used by bacteria to penetrate into tissues. Activation of ß-adrenoceptor endocytosis with specific agonists prevents signaling events downstream of N. meningitidis adhesion and inhibits bacterial crossing of the endothelial barrier. The identification of the mechanism used for hijacking host cell signaling machineries opens perspectives for treatment and prevention of meningococcal infection.

STING agonists enable antiviral cross-talk between human cells and confer protection against genital herpes in mice.

In recent years, there has been an increasing interest in immunomodulatory therapy as a means to treat various conditions, including infectious diseases. For instance, Toll-like receptor (TLR) agonists have been evaluated for treatment of genital herpes. However, although the TLR7 agonist imiquimod was shown to have antiviral activity in individual patients, no significant effects were observed in clinical trials, and the compound also exhibited significant side effects, including local inflammation. Cytosolic DNA is detected by the enzyme cyclic GMP-AMP (2'3'-cGAMP) synthase (cGAS) to stimulate antiviral pathways, mainly through induction of type I interferon (IFN)s. cGAS is activated upon DNA binding to produce the cyclic dinucleotide (CDN) 2'3'-cGAMP, which in turn binds and activates the adaptor protein Stimulator of interferon genes (STING), thus triggering type I IFN expression. In contrast to TLRs, STING is expressed broadly, including in epithelial cells. Here we report that natural and non-natural STING agonists strongly induce type I IFNs in human cells and in mice in vivo, without stimulating significant inflammatory gene expression. Systemic treatment with 2'3'-cGAMP reduced genital herpes simplex virus (HSV) 2 replication and improved the clinical outcome of infection. More importantly, local application of CDNs at the genital epithelial surface gave rise to local IFN activity, but only limited systemic responses, and this treatment conferred total protection against disease in both immunocompetent and immunocompromised mice. In direct comparison between CDNs and TLR agonists, only CDNs acted directly on epithelial cells, hence allowing a more rapid and IFN-focused immune response in the vaginal epithelium. Thus, specific activation of the STING pathway in the vagina evokes induction of the IFN system but limited inflammatory responses to allow control of HSV2 infections in vivo.

Endoplasmic reticulum stress induces G2 cell-cycle arrest via mRNA translation of the p53 isoform p53/47.

p53 downstream pathways control G1 and G2 cell-cycle arrest, DNA repair, or apoptosis. However, it is still not clear how cells differentiate the cell-biological outcome of p53 activation in response to different types of stresses. The p53/47 isoform lacks the first 39 amino acids of full-length p53 including the Mdm2 binding site and the first trans-activation domain, and tetramers including p53/47 exhibit altered activity and biochemical properties. Here we show that endoplasmic reticulum stress promotes PERK-dependent induction of p53/47 mRNA translation and p53/47 homo-oligomerization. p53/47 induces 14-3-3sigma and G2 arrest but does not affect G1 progression. This is contrary to p53FL, which promotes G1 arrest but has no effect on the G2. These results show a unique role for p53/47 in the p53 pathway and illustrate how a cellular stress leads to a defined cell-biological outcome through expression of a p53 isoform.

Neutrophil recruitment to lymph nodes limits local humoral response to Staphylococcus aureus.

Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF-ß1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.

Dual agonist occupancy of AT1-R-α2C-AR heterodimers results in atypical Gs-PKA signaling.

Hypersecretion of norepinephrine (NE) and angiotensin II (AngII) is a hallmark of major prevalent cardiovascular diseases that contribute to cardiac pathophysiology and morbidity. Herein, we explore whether heterodimerization of presynaptic AngII AT1 receptor (AT1-R) and NE α2C-adrenergic receptor (α2C-AR) could underlie their functional cross-talk to control NE secretion. Multiple bioluminescence resonance energy transfer and protein complementation assays allowed us to accurately probe the structures and functions of the α2C-AR-AT1-R dimer promoted by ligand binding to individual protomers. We found that dual agonist occupancy resulted in a conformation of the heterodimer different from that induced by active individual protomers and triggered atypical Gs-cAMP-PKA signaling. This specific pharmacological signaling unit was identified in vivo to promote not only NE hypersecretion in sympathetic neurons but also sympathetic hyperactivity in mice. Thus, we uncovered a new process by which GPCR heterodimerization creates an original functional pharmacological entity and that could constitute a promising new target in cardiovascular therapeutics.

An essential role for RGS protein/Gαi2 interactions in B lymphocyte-directed cell migration and trafficking.

Chemokines engage B lymphocyte surface receptors, triggering heterotrimeric G protein Gαi subunit guanine nucleotide exchange. RGS proteins limit the duration that Gαi subunits remain GTP bound, and the loss of an individual RGS protein typically enhances chemokine receptor signaling. In this study, we show that B cells carrying a Gαi2 (G184S/G184S) mutation that disables all RGS protein/Gαi2 interactions exhibit an unexpectedly severe reduction in chemokine receptor signaling. The Gαi2 (G184S/G184S) B cells have markedly elevated basal calcium levels, but poor chemokine-induced increases, enhanced nonspecific migration, but extremely poor chemotaxis. In striking contrast, the Gαi2 (G184S/G184S) B cells exhibited enhanced sensitivity to sphingosine 1-phosphate (S1P). S1P elicited heightened intracellular calcium responses and enhanced S1P-triggered cell migration. Mice with the Gαi2 (G184S/G184S) mutation displayed excessive numbers of germinal center-like structures; abnormal serum Ig profiles; and aberrant B lymphocyte trafficking. These findings establish an essential role for RGS proteins in B cell chemoattractant signaling and for the proper position of B lymphocytes in lymphoid organs.

B Lymphocyte-Specific Loss of Ric-8A Results in a Gα Protein Deficit and Severe Humoral Immunodeficiency.

Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a highly evolutionarily conserved cytosolic protein initially identified in Caenorhabditis elegans, where it was assigned a regulatory role in asymmetric cell divisions. It functions as a guanine nucleotide exchange factor for Gαi, Gαq, and Gα12/13 and as a molecular chaperone required for the initial association of nascent Gα subunits with cellular membranes in embryonic stem cell lines. To test its role in hematopoiesis and B lymphocytes specifically, we generated ric8 (fl/fl) vav1-cre and ric8 (fl/fl) mb1-cre mice. The major hematopoietic cell lineages developed in the ric8 (fl/fl) vav1-cre mice, notwithstanding severe reduction in Gαi2/3, Gαq, and Gα13 proteins. B lymphocyte-specific loss of Ric-8A did not compromise bone marrow B lymphopoiesis, but splenic marginal zone B cell development failed, and B cells underpopulated lymphoid organs. The ric8 (fl/fl) mb1-cre B cells exhibited poor responses to chemokines, abnormal trafficking, improper in situ positioning, and loss of polarity components during B cell differentiation. The ric8 (fl/fl) mb1-cre mice had a severely disrupted lymphoid architecture and poor primary and secondary Ab responses. In B lymphocytes, Ric-8A is essential for normal Gα protein levels and is required for B cell differentiation, trafficking, and Ab responses.

SARS-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the MAVS/TRAF3/TRAF6 signalosome.

Coronaviruses (CoV) have recently emerged as potentially serious pathogens that can cause significant human morbidity and death. The severe acute respiratory syndrome (SARS)-CoV was identified as the etiologic agent of the 2002-2003 international SARS outbreak. Yet, how SARS evades innate immune responses to cause human disease remains poorly understood. In this study, we show that a protein encoded by SARS-CoV designated as open reading frame-9b (ORF-9b) localizes to mitochondria and causes mitochondrial elongation by triggering ubiquitination and proteasomal degradation of dynamin-like protein 1, a host protein involved in mitochondrial fission. Also, acting on mitochondria, ORF-9b targets the mitochondrial-associated adaptor molecule MAVS signalosome by usurping PCBP2 and the HECT domain E3 ligase AIP4 to trigger the degradation of MAVS, TRAF3, and TRAF 6. This severely limits host cell IFN responses. Reducing either PCBP2 or AIP4 expression substantially reversed the ORF-9b-mediated reduction of MAVS and the suppression of antiviral transcriptional responses. Finally, transient ORF-9b expression led to a strong induction of autophagy in cells. The induction of autophagy depended upon ATG5, a critical autophagy regulator, but the inhibition of MAVS signaling did not. These results indicate that SARS-CoV ORF-9b manipulates host cell mitochondria and mitochondrial function to help evade host innate immunity. This study has uncovered an important clue to the pathogenesis of SARS-CoV infection and illustrates the havoc that a small ORF can cause in cells.

Distinct functional outputs of PTEN signalling are controlled by dynamic association with ß-arrestins.

The tumour suppressor PTEN (phosphatase and tensin deleted on chromosome 10) regulates major cellular functions via lipid phosphatase-dependent and -independent mechanisms. Despite its fundamental pathophysiological importance, how PTEN's cellular activity is regulated has only been partially elucidated. We report that the scaffolding proteins ß-arrestins (ß-arrs) are important regulators of PTEN. Downstream of receptor-activated RhoA/ROCK signalling, ß-arrs activate the lipid phosphatase activity of PTEN to negatively regulate Akt and cell proliferation. In contrast, following wound-induced RhoA activation, ß-arrs inhibit the lipid phosphatase-independent anti-migratory effects of PTEN. ß-arrs can thus differentially control distinct functional outputs of PTEN important for cell proliferation and migration.

CXCR4-CCR5: a couple modulating T cell functions.

Chemokines and their receptors direct leukocyte migration among blood, lymph and tissues. Evidence has recently accumulated that, besides their chemotactic functions, chemokine receptors are highly versatile players that fine tune immune responses. During human T cell activation by antigen-presenting cells, the chemokine receptors CCR5 and CXCR4 are recruited into the immunological synapse, where they deliver costimulatory signals. However, the molecular mechanisms allowing signaling versatility of chemokine receptors are unknown. Here, we describe the functional interaction between CXCR4 and CCR5 to exert specific biological functions and modulate T lymphocyte responses. We demonstrate that simultaneous expression and cooperation between CCR5 and CXCR4 are required for chemokine-induced T cell costimulation at the immunological synapse. In addition, we provide evidence for a physical association of the two receptors in a signaling complex that activates distinct T cell functions. We suggest that cooperation between receptors represents one key strategy for the functional plasticity of chemokines.

beta-arrestin 2 oligomerization controls the Mdm2-dependent inhibition of p53.

beta-arrestins (beta-arrs), two ubiquitous proteins involved in serpentine heptahelical receptor regulation and signaling, form constitutive homo- and heterooligomers stabilized by inositol 1,2,3,4,5,6-hexakisphosphate (IP6). Monomeric beta-arrs are believed to interact with receptors after agonist activation, and therefore, beta-arr oligomers have been proposed to represent a resting biologically inactive state. In contrast to this, we report here that the interaction with and subsequent titration out of the nucleus of the protooncogene Mdm2 specifically require beta-arr2 oligomers together with the previously characterized nucleocytoplasmic shuttling of beta-arr2. Mutation of the IP6-binding sites impair oligomerization, reduce interaction with Mdm2, and inhibit p53-dependent antiproliferative effects of beta-arr2, whereas the competence for receptor regulation and signaling is maintained. These observations suggest that the intracellular concentration of beta-arr2 oligomers might control cell survival and proliferation.

The cGAS-STING pathway is a therapeutic target in a preclinical model of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer, and the incidence of HCC is increasing. Recently, cancer immunotherapy has emerged as an efficient treatment against some cancers. Here we have used a mouse model of mutagen-induced HCC to explore the therapeutic usefulness of targeting the DNA-activated STING pathway in HCC. STING-deficient mice exhibited unaltered initial development of HCC, but had higher number of large tumors at late stages of disease. In the liver of STING-deficient HCC mice, we observed reduced levels of phospho-STAT1, autophagy, and cleaved caspase3. These responses were activated in the liver by treatment with a cyclic dinucleotide (CDN) STING agonist. Importantly, CDN treatment of mice after HCC development efficiently reduced tumor size. Initiation of CDN treatment at an even later stage of disease to allow HCC detection by MR scanning revealed that the majority of tumors regressed in response to CDN, but new tumors were also detected, which were unresponsive to CDN treatment. Overall, the modulation of the STING pathway affects the development of HCC, and holds promise for a use as a treatment of this disease, most likely in combination with other immunomodulatory treatments such as PD1 inhibitors or with standard of care.

Gαi Signaling Promotes Marginal Zone B Cell Development by Enabling Transitional B Cell ADAM10 Expression.

The follicular (FO) versus marginal zone (MZ) B cell fate decision in the spleen depends upon BCR, BAFF, and Notch2 signaling. Whether or how Gi signaling affects this fate decision is unknown. Here, we show that direct contact with Notch ligand expressing stromal cells (OP9-Delta-like 1) cannot promote normal MZ B cell development when progenitor B cells lack Gαi proteins, or if Gi signaling is disabled. Consistent with faulty ADAM10-dependent Notch2 processing, Gαi-deficient transitional B cells had low ADAM10 membrane expression levels and reduced Notch2 target gene expression. Immunoblotting Gαi-deficient B cell lysates revealed a reduction in mature, processed ADAM10. Suggesting that Gαi signaling promotes ADAM10 membrane expression, stimulating normal transitional B cells with CXCL12 raised it, while inhibiting Gαi nucleotide exchange blocked its upregulation. Surprisingly, inhibiting Gαi nucleotide exchange in transitional B cells also impaired the upregulation of ADAM10 that occurs following antigen receptor crosslinking. These results indicate that Gαi signaling supports ADAM10 maturation and activity in transitional B cells, and ultimately Notch2 signaling to promote MZ B cell development.

Identification of differentially expressed genes in human varicose veins: involvement of matrix gla protein in extracellular matrix remodeling.

This study was designed to identify the global pattern of differentially expressed genes in human varicose veins. Using suppressive subtractive hybridization, we identified overexpression of genes known to be associated with extracellular matrix remodeling, including collagen III, tissue inhibitor of metalloproteinases I, dermatopontin, matrix Gla protein (MGP) and tenascin C. Real-time polymerase chain reaction analysis confirmed the differential expression of these genes. The overexpression of MGP transcript was associated with increased MGP level in varicose veins, in particular the undercarboxylated form of the protein. Smooth muscle cells from varicose veins showed increased proliferation rate and enhanced matrix mineralization. This observation correlated with the presence of ectopic mineralization areas in the varicose vein walls. The use of warfarin, to inhibit MGP activity, or siRNA targeting MGP transcript induced a reduction in the exacerbated proliferation of varicose vein smooth muscle cells. Our results suggest that high expression of MGP in varicose veins may contribute to venous wall remodeling by affecting proliferation and mineralization processes probably through impaired carboxylation of MGP. In addition, suppressive subtractive hybridization results also produce a profile of differentially expressed genes in varicose veins, in particular extracellular matrix components. Further study of these genes will provide insights into their specific roles in the etiology of venous disease.

Structure-activity relationships of a novel series of urotensin II analogues: identification of a urotensin II antagonist.

Urotensin II (U-II) is a potent vasoconstrictor peptide which has been identified as the endogenous ligand for the orphan G protein-coupled receptor GPR14 now renamed UT receptor. As the C-terminal cyclic hexapeptide of U-II (U-II(4-11), H-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH) possesses full biological activity, we have synthesized a series of U-II(4-11) analogues and measured their binding affinity on hGPR14-transfected CHO cells and their contractile activity on de-endothelialized rat aortic rings. The data indicate that a free amino group and a functionalized side-chain at the N-terminal extremity of the peptide are not required for biological activity. In addition, the minimal chemical requirement at position 9 of U-II(4-11) is the presence of an aromatic moiety. Most importantly, replacement of the Phe6 residue by cyclohexyl-Ala (Cha) led to an analogue, [Cha6]U-II(4-11), that was devoid of agonistic activity but was able to dose-dependently suppress the vasoconstrictor effect of U-II on rat aortic rings. These new pharmacological data, by providing further information regarding the structure-activity relationships of U-II analogues, should prove useful for the rational design of potent and nonpeptidic UT receptor agonists and antagonists.

Design, Synthesis, and Biological Evaluation of Novel Cyclic Adenosine-Inosine Monophosphate (cAIMP) Analogs That Activate Stimulator of Interferon Genes (STING).

We describe novel STING-activating cyclic dinucleotides whose constituent nucleosides are adenosine and inosine and that vary by ribose substitution, internucleotide linkage position, and phosphate modification. In mammalian cells in vitro, some of these cAIMP analogs induce greater STING-dependent IRF and NF-κB pathway signaling than do the reference agonists for murine (DMXAA) or human (2',3'-cGAMP) STING. In human blood ex vivo, they induce type I interferons (IFNs) and proinflammatory cytokines: for the former, 3',3'-cAIMP (9; EC50 of 6.4 µM) and analogs 52-56 (EC50 of 0.4-4.7 µM), which contain one or two 2'-fluoro-2'-deoxyriboses and/or bis-phosphorothioate linkages, are more potent than 2',3'-cGAMP (EC50 of 19.6 µM). Interestingly, 9 induces type I IFNs more strongly than do its linkage isomers 2',3'-cAIMP (10), 3',2'-cAIMP (23), and 2',2'-cAIMP (27). Lastly, some of the cAIMP analogs are more resistant than 2',3'-cGAMP to enzymatic cleavage in vitro. We hope to exploit our findings to develop STING-targeted immunotherapies.

Cardioprotective Angiotensin-(1-7) Peptide Acts as a Natural-Biased Ligand at the Angiotensin II Type 1 Receptor.

Hyperactivity of the renin-angiotensin-aldosterone system through the angiotensin II (Ang II)/Ang II type 1 receptor (AT1-R) axis constitutes a hallmark of hypertension. Recent findings indicate that only a subset of AT1-R signaling pathways is cardiodeleterious, and their selective inhibition by biased ligands promotes therapeutic benefit. To date, only synthetic biased ligands have been described, and whether natural renin-angiotensin-aldosterone system peptides exhibit functional selectivity at AT1-R remains unknown. In this study, we systematically determined efficacy and potency of Ang II, Ang III, Ang IV, and Ang-(1-7) in AT1-R-expressing HEK293T cells on the activation of cardiodeleterious G-proteins and cardioprotective ß-arrestin2. Ang III and Ang IV fully activate similar G-proteins than Ang II, the prototypical AT1-R agonist, despite weaker potency of Ang IV. Interestingly, Ang-(1-7) that binds AT1-R fails to promote G-protein activation but behaves as a competitive antagonist for Ang II/Gi and Ang II/Gq pathways. Conversely, all renin-angiotensin-aldosterone system peptides act as agonists on the AT1-R/ß-arrestin2 axis but display biased activities relative to Ang II as indicated by their differences in potency and AT1-R/ß-arrestin2 intracellular routing. Importantly, we reveal Ang-(1-7) a known Mas receptor-specific ligand, as an AT1-R-biased agonist, selectively promoting ß-arrestin activation while blocking the detrimental Ang II/AT1-R/Gq axis. This original pharmacological profile of Ang-(1-7) at AT1-R, similar to that of synthetic AT1-R-biased agonists, could, in part, contribute to its cardiovascular benefits. Accordingly, in vivo, Ang-(1-7) counteracts the phenylephrine-induced aorta contraction, which was blunted in AT1-R knockout mice. Collectively, these data suggest that Ang-(1-7) natural-biased agonism at AT1-R could fine-tune the physiology of the renin-angiotensin-aldosterone system.

Cardiac cAMP: production, hydrolysis, modulation and detection.

Cyclic adenosine 3',5'-monophosphate (cAMP) modulates a broad range of biological processes including the regulation of cardiac myocyte contractile function where it constitutes the main second messenger for ß-adrenergic receptors' signaling to fulfill positive chronotropic, inotropic and lusitropic effects. A growing number of studies pinpoint the role of spatial organization of the cAMP signaling as an essential mechanism to regulate cAMP outcomes in cardiac physiology. Here, we will briefly discuss the complexity of cAMP synthesis and degradation in the cardiac context, describe the way to detect it and review the main pharmacological arsenal to modulate its availability.

Structure-activity relationships and structural conformation of a novel urotensin II-related peptide.

Urotensin II (UII) has been described as the most potent vasoconstrictor peptide and recognized as the endogenous ligand of the orphan G protein-coupled receptor GPR14. Recently, a UII-related peptide (URP) has been isolated from the rat brain and its sequence has been established as H-Ala-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH. In order to study the structure-function relationships of URP, we have synthesized a series of URP analogs and measured their binding affinity on hGPR14-transfected cells and their contractile activity in a rat aortic ring bioassay. Alanine substitution of each residue of URP significantly reduced the binding affinity and the contractile activity of the peptides, except for the Ala8-substituted analog that retained biological activity. Most importantly, D-scan of URP revealed that [D-Trp4]URP abrogated and [D-Tyr6]URP partially suppressed the UII-evoked contractile response. [Orn5]URP, which had very low agonistic efficacy, was the most potent antagonist in this series. The solution structure of URP has been determined by 1H NMR spectroscopy and molecular dynamics. URP exhibited a single conformation characterized by an inverse gamma-turn comprising residues Trp-Lys-Tyr which plays a crucial role in the biological activity of URP. These pharmacological and structural data should prove useful for the rational design of non-peptide ligands as potential GPR14 agonists and antagonists.