cGAS-STING, an important pathway in cancer immunotherapy.

Cytosolic DNA sensing, the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, is an important novel role in the immune system. Multiple STING agonists were developed for cancer therapy study with great results achieved in pre-clinical work. Recent progress in the mechanical understanding of STING pathway in IFN production and T cell priming, indicates its promising role for cancer immunotherapy. STING agonists co-administrated with other cancer immunotherapies, including cancer vaccines, immune checkpoint inhibitors such as anti-programmed death 1 and cytotoxic T lymphocyte-associated antigen 4 antibodies, and adoptive T cell transfer therapies, would hold a promise of treating medium and advanced cancers. Despite the applications of STING agonists in cancer immunotherapy, lots of obstacles remain for further study. In this review, we mainly examine the biological characters, current applications, challenges, and future directions of cGAS-STING in cancer immunotherapy.

Genetic manipulation of cyclic nucleotide signaling during hippocampal neuroplasticity and memory formation.

Decades of research have underscored the importance of cyclic nucleotide signaling in memory formation and synaptic plasticity. In recent years, several new genetic techniques have expanded the neuroscience toolbox, allowing researchers to measure and modulate cyclic nucleotide gradients with high spatiotemporal resolution. Here, we will provide an overview of studies using genetic approaches to interrogate the role cyclic nucleotide signaling plays in hippocampus-dependent memory processes and synaptic plasticity. Particular attention is given to genetic techniques that measure real-time changes in cyclic nucleotide levels as well as newly-developed genetic strategies to transiently manipulate cyclic nucleotide signaling in a subcellular compartment-specific manner with high temporal resolution.

Nucleotide second messengers in bacterial decision making.

Since the initial discovery of bacterial nucleotide second messengers (NSMs), we have made huge progress towards understanding these complex signalling networks. Many NSM networks contain dozens of metabolic enzymes and binding targets, whose activity is tightly controlled at every regulatory level. They function as global regulators and in specific signalling circuits, controlling multiple aspects of bacterial behaviour and development. Despite these advances there is much still to discover, with current research focussing on the molecular mechanisms of signalling circuits, the role of the environment in controlling NSM pathways and attempts to understand signalling at the whole cell/community level. Here we examine recent developments in the NSM signalling field and discuss their implications for understanding this important driver of microbial behaviour.

A STING to inflammation and autoimmunity.

Various intracellular pattern recognition receptors (PRRs) recognize cytosolic pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Cyclic GMP-AMP synthase (cGAS), a cytosolic PRR, recognizes cytosolic nucleic acids including dsDNAs. The recognition of dsDNA by cGAS generates cyclic GMP-AMP (GAMP). The cGAMP is then recognized by STING generating type 1 IFNs and NF-κB-mediated generation of pro-inflammatory cytokines and molecules. Thus, cGAS-STING signaling mediated recognition of cytosolic dsDNA causing the induction of type 1 IFNs plays a crucial role in innate immunity against cytosolic pathogens, PAMPs, and DAMPs. The overactivation of this system may lead to the development of autoinflammation and autoimmune diseases. The article opens with the introduction of different PRRs involved in the intracellular recognition of dsDNA and gives a brief introduction of cGAS-STING signaling. The second section briefly describes cGAS as intracellular PRR required to recognize intracellular nucleic acids (dsDNA and CDNs) and the formation of cGAMP. The cGAMP acts as a second messenger to activate STING- and TANK-binding kinase 1-mediated generation of type 1 IFNs and the activation of NF-κB. The third section of the article describes the role of cGAS-STING signaling in the induction of autoinflammation and various autoimmune diseases. The subsequent fourth section describes both chemical compounds developed and the endogenous negative regulators of cGAS-STING signaling required for its regulation. Therapeutic targeting of cGAS-STING signaling could offer new ways to treat inflammatory and autoimmune diseases.

New approaches regarding the in vitro maturation of oocytes: manipulating cyclic nucleotides and their partners in crime.

Several discoveries have been described recently (5-10 years) about the biology of ovarian follicles (oocyte, cumulus cells and granulosa cells), including new aspects of cellular communication, the control of oocyte maturation and the acquisition of oocyte competence for fertilization and further embryo development. These advances are nourishing assisted reproduction techniques (ART) with new possibilities, in which novel culture systems are being developed and tested to improve embryo yield and quality. This mini-review aims to describe how the recent knowledge on the physiological aspects of mammalian oocyte is reflecting as original or revisited approaches into the context of embryo production. These new insights include recent findings on the mechanisms that control oocyte maturation, especially modulating intraoocyte levels of cyclic nucleotides during in vitro maturation using endogenous or exogenous agents. In this mini-review we also discuss the positive and negative effects of these manipulations on the outcoming embryo.

cGAS-cGAMP-STING: The three musketeers of cytosolic DNA sensing and signaling.

Innate immunity is the first line of host defense against invading pathogens. The detection of aberrant nucleic acids which represent some conserved PAMPs triggers robust type I IFN-mediated innate immune responses. Host- or pathogen-derived cytosolic DNA binds and activates the DNA sensor cGAS, which synthesizes the second messenger 2'3'-cGAMP and triggers STING-dependent downstream signaling. Here, we highlight recent progress in cGAS-cGAMP-STING, the Three Musketeers of cytosolic DNA sensing and signaling, and their essential roles in infection, autoimmune diseases, and cancer. We also focus on the regulation of these critical signal components by variant host/pathogen proteins and update our understanding of this indispensable pathway to provide new insights for drug discovery. © 2016 IUBMB Life, 68(11):858-870, 2016.

Heterogeneity of pulmonary endothelial cyclic nucleotide response to Pseudomonas aeruginosa ExoY infection.

Here, we tested the hypothesis that a promiscuous bacterial cyclase synthesizes purine and pyrimidine cyclic nucleotides in the pulmonary endothelium. To test this hypothesis, pulmonary endothelial cells were infected with a strain of the Gram-negative bacterium Pseudomonas aeruginosa that introduces only exoenzyme Y (PA103 ΔexoUexoT::Tc pUCPexoY; ExoY(+)) via a type III secretion system. Purine and pyrimidine cyclic nucleotides were simultaneously detected using mass spectrometry. Pulmonary artery (PAECs) and pulmonary microvascular (PMVECs) endothelial cells both possess basal levels of four different cyclic nucleotides in the following rank order: cAMP > cUMP ≈ cGMP ≈ cCMP. Endothelial gap formation was induced in a time-dependent manner following ExoY(+) intoxication. In PAECs, intercellular gaps formed within 2 h and progressively increased in size up to 6 h, when the experiment was terminated. cGMP concentrations increased within 1 h postinfection, whereas cAMP and cUMP concentrations increased within 3 h, and cCMP concentrations increased within 4 h postinfection. In PMVECs, intercellular gaps did not form until 4 h postinfection. Only cGMP and cUMP concentrations increased at 3 and 6 h postinfection, respectively. PAECs generated higher cyclic nucleotide levels than PMVECs, and the cyclic nucleotide levels increased earlier in response to ExoY(+) intoxication. Heterogeneity of the cyclic nucleotide signature in response to P. aeruginosa infection exists between PAECs and PMVECs, suggesting the intracellular milieu in PAECs is more conducive to cNMP generation.

The cGAS-cGAMP-STING pathway of cytosolic DNA sensing and signaling.

The innate immune system deploys a variety of sensors to detect signs of infection. Nucleic acids represent a major class of pathogen signatures that can trigger robust immune responses. The presence of DNA in the cytoplasm of mammalian cells is a danger signal that activates innate immune responses; however, how cytosolic DNA triggers these responses remained unclear until recently. In this review, we focus on the mechanism of DNA sensing by the newly discovered cGAS-cGAMP-STING pathway and highlight recent progress in dissecting the in vivo functions of this pathway in immune defense as well as autoimmunity.

[Role of T lymphocyte cyclic nucleotides and G protein in sarcoidosis]. / Quels rôles les anomalies de la voie des nucléotides cycliques et des protéines G jouent-elles dans le désordre biochimique des lymphocytes T dans la sarcoïdose ?

CD4+ T lymphocytes play a major role in the pathophysiology of sarcoidosis. Many studies have investigated the immunological and genetic abnormalities in this disease. There are few studies concerning the metabolic pathways. Essentially they concern the pathways: STAT1, MAPK38, NF-κB, Galphai, cAMP and cGMP PDE and PEMT1. Using studies in the literature and results of our own work concerning some metabolic aspects of T lymphocytes in sarcoidosis, we present a revue of the various hypotheses, which involve dysfunction of cAMP signaling pathways, such as RAS/RAF/MEK/ERK in T lymphocytes, leading to a disorder of immunity.

Novel signaling mechanisms in the ovary during oocyte maturation and ovulation.

During the peri-ovulatory period, the gonadotropin LH triggers major changes in both the somatic and germ cell compartments of the ovarian follicle. The oocyte completes the meiotic cell cycle to become a fertilizable egg, and dramatic changes in gene expression and secretion take place in the somatic compartment of the follicle in preparation for follicular rupture and oocyte release. The concerted changes are regulated by activation of intracellular signaling pathways as well as paracrine and autocrine regulatory loops. This review will provide a summary of the current knowledge of the molecular events triggered by LH focusing mostly on the signaling pathways required for oocyte maturation.

Cyclic nucleotide-dependent relaxation pathways in vascular smooth muscle.

Vascular smooth muscle tone is controlled by a balance between the cellular signaling pathways that mediate the generation of force (vasoconstriction) and release of force (vasodilation). The initiation of force is associated with increases in intracellular calcium concentrations, activation of myosin light-chain kinase, increases in the phosphorylation of the regulatory myosin light chains, and actin-myosin crossbridge cycling. There are, however, several signaling pathways modulating Ca(2+) mobilization and Ca(2+) sensitivity of the contractile machinery that secondarily regulate the contractile response of vascular smooth muscle to receptor agonists. Among these regulatory mechanisms involved in the physiological regulation of vascular tone are the cyclic nucleotides (cAMP and cGMP), which are considered the main messengers that mediate vasodilation under physiological conditions. At least four distinct mechanisms are currently thought to be involved in the vasodilator effect of cyclic nucleotides and their dependent protein kinases: (1) the decrease in cytosolic calcium concentration ([Ca(2+)]c), (2) the hyperpolarization of the smooth muscle cell membrane potential, (3) the reduction in the sensitivity of the contractile machinery by decreasing the [Ca(2+)]c sensitivity of myosin light-chain phosphorylation, and (4) the reduction in the sensitivity of the contractile machinery by uncoupling contraction from myosin light-chain phosphorylation. This review focuses on each of these mechanisms involved in cyclic nucleotide-dependent relaxation of vascular smooth muscle under physiological conditions.

Subcellular signaling in the endothelium: cyclic nucleotides take their place.

When lecturing on the topic of cellular signaling I have had occasion to ask the class for examples of cellular processes NOT impacted by cyclic AMP (cAMP) and am struck by how few examples exist. Indeed, studies spanning the past 60 years have detailed how this ubiquitous second messenger impacts virtually all cellular processes, including intermediary metabolism, contractility, motility, proliferation, and gene expression in most mammalian cells. Since the hydrophobic cAMP could in principle diffuse rapidly throughout the cell once formed, the remarkable spatial and temporal specificity of its numerous actions in cells is truly impressive. Herein I introduce the main players involved in coordinating actions of cAMP in vascular endothelial cells (VECs), and focus on the increasing awareness of the dominant role that cyclic nucleotide phosphodiesterases (PDEs), the sole cellular enzymes capable of hydrolytically inactivating cAMP, play in fostering this specificity.

Role of phosphodiesterases in adult-onset pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterized by increased mean pulmonary artery pressure (mPAP) due to vasoconstriction and structural changes in the small pulmonary arteries (PAs); proliferation of pulmonary artery smooth muscle cells (PASMCs) contributes to the remodeling. The abnormal pathophysiology in the pulmonary vasculature relates to decreased cyclic nucleotide levels in PASMCs. Phosphodiesterases (PDEs) catalyze the hydrolysis of cAMP and cGMP, thereby PDE inhibitors are effective in vasodilating the PA and decreasing PASMC proliferation. Experimental studies support the use of PDE3, PDE5, and PDE1 inhibitors in PAH. PDE5 inhibitors such as sildenafil are clinically approved to treat different forms of PAH and lower mPAP, increase functional capacity, and decrease right ventricular hypertrophy, without decreasing systemic arterial pressure. New evidence suggests that the combination of PDE inhibitors with other therapies for PAH may be beneficial in treating the disease. Furthermore, inhibiting PDEs in the heart and the inflammatory cells that infiltrate the PA may offer new targets to reduce right ventricular hypertrophy and inhibit inflammation that is associated with and contributes to the development of PAH. This chapter summarizes the advances in the area and the future of PDEs in PAH.

Cyclic nucleotides and phosphodiesterases in monocytic differentiation.

Monocytes are immune cells that can differentiate into a number of cell types including macrophages, dendritic cells, and osteoclasts upon exposure to various cytokines. The phenotypes of these differentiated cells are highly heterogeneous and their differentiation can be affected by the cyclic nucleotides, 3'-5'-cyclic adenosine monophosphate (cAMP) and 3'-5'-cyclic guanosine monophosphate (cGMP). The intracellular levels of cAMP and cGMP are controlled through regulation of production by adenylyl and guanylyl cyclases and through degradation by cyclic nucleotide phosphodiesterases (PDEs). PDE inhibition and subsequent changes in cyclic nucleotide levels can alter the final phenotype of a differentiating monocyte with regards to surface marker expression, gene expression, or changes in secreted chemokine and cytokine levels. The differentiation process itself can also be either inhibited or augmented by changes in cyclic nucleotide levels, depending on the system being studied and the timing of cyclic nucleotide elevation. This chapter explores the effects of PDE inhibition and increases in cGMP and cAMP on monocytic differentiation into osteoclasts, dendritic cells, and macrophages.

Phosphodiesterases in the central nervous system: implications in mood and cognitive disorders.

Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes that are involved in the regulation of the intracellular second messengers cyclic AMP (cAMP) and cyclic GMP (cGMP) by controlling their rates of hydrolysis. There are 11 different PDE families and each family typically has multiple isoforms and splice variants. The PDEs differ in their structures, distribution, modes of regulation, and sensitivity to inhibitors. Since PDEs have been shown to play distinct roles in processes of emotion and related learning and memory processes, selective PDE inhibitors, by preventing the breakdown of cAMP and/or cGMP, modulate mood and related cognitive activity. This review discusses the current state and future development in the burgeoning field of PDEs in the central nervous system. It is becoming increasingly clear that PDE inhibitors have therapeutic potential for the treatment of neuropsychiatric disorders involving disturbances of mood, emotion, and cognition.

Therapeutic potential of phosphodiesterase inhibitors in parasitic diseases.

Protozoan parasites of the order kinetoplastida are the causative agents of three of the world's most important neglected human diseases: African trypanosomiasis, American trypanosomiasis, and leishmaniasis. Current therapies are limited, with some treatments having serious and sometimes lethal side effects. The growing number of cases that are refractory to treatment is also of concern. With few new drugs in development, there is an unmet medical need for new, more effective, and safer medications. Recent studies employing genetic and pharmacological techniques have begun to shed light on the role of the cyclic nucleotide phosphodiesterases in the life cycle of these pathogens and suggest that these important regulators of cyclic nucleotide signaling may be promising new targets for the treatment of parasitic diseases.

Dependence of multidrug resistance protein-mediated cyclic nucleotide efflux on the background sodium conductance.

Anterior pituitary cells fire action potentials and release cyclic nucleotides both spontaneously and in response to agonist stimulation, but the relationship between electrical activity and cyclic nucleotide efflux has not been studied. In these cells, a tetrodotoxin-resistant background N(+) conductance is critical for firing of action potentials, and multidrug resistance proteins (MRPs) MRP4 and MRP5 contribute to cyclic nucleotide efflux. Here, we show that abolition of the background Na(+) conductance in rat pituitary cells by complete or partial replacement of extracellular Na(+) with organic cations or sucrose induced a rapid and reversible hyperpolarization of cell membranes and inhibition of action potential firing, accompanied by a rapid inhibition of cyclic nucleotide efflux. Valinomycin-induced hyperpolarization of plasma membranes also inhibited cyclic nucleotide efflux, whereas depolarization of cell membranes induced by the inhibition of Ca(2+) influx or stimulation of Na(+) influx by gramicidin was accompanied by a facilitation of cyclic nucleotide efflux. In contrast, inhibition of cyclic nucleotide efflux by probenecid did not affect the background Na(+) conductance. In human embryonic kidney 293 cells stably transfected with human MRP4 or MRP5, replacement of bath Na(+) with organic cations also hyperpolarized the cell membranes and inhibited cyclic nucleotide efflux. In these cells, the Na(+)/H(+) antiporter monensin did not affect the membrane potential and was practically ineffective in altering cyclic nucleotide efflux. In both pituitary and MRP4- and MRP5-expressing cells, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571) inhibited cyclic nucleotide efflux. These results indicate that the MRP4/5-mediated cyclic nucleotide efflux can be rapidly modulated by membrane potential determined by the background Na(+) conductance.

Revealing promiscuous drug-target interactions by chemical proteomics.

The (poly-)pharmacological activities of a drug can only be understood if its interactions with cellular components are comprehensively characterized. Mass spectrometry-based chemical proteomics approaches have recently emerged as powerful tools for the characterization of drug-target interactions in samples from cell lines and tissues. At the same time, off-target activities can be identified. This information can contribute toward optimization of candidate drug molecules and reduction of side effects. In this review, we describe recent advances in chemical proteomics and outline potential applications in drug discovery.

The analysis of desensitizing CNGA1 channels reveals molecular interactions essential for normal gating.

The pore region of cyclic nucleotide-gated (CNG) channels acts as the channel gate. Therefore, events occurring in the cyclic nucleotide-binding (CNB) domain must be coupled to the movements of the pore walls. When Glu363 in the pore region, Leu356 and Thr355 in the P helix, and Phe380 in the upper portion of the S6 helix are mutated into an alanine, gating is impaired: mutant channels E363A, L356A, T355A, and F380A desensitize in the presence of a constant cGMP concentration, contrary to what can be observed in wild-type (WT) CNGA1 channels. Similarly to C-type inactivation of K(+) channels, desensitization in these mutant channels is associated with rearrangements of residues in the outer vestibule. In the desensitized state, Thr364 residues in different subunits become closer and Pro366 becomes more accessible to extracellular reagents. Desensitization is also observed in the mutant channel L356C, but not in the double-mutant channel L356C+F380C. Mutant channels L356F and F380K did not express, but cGMP-gated currents with a normal gating were observed in the double-mutant channels L356F+F380L and L356D+F380K. Experiments with tandem constructs with L356C, F380C, and L356C+F380C and WT channels indicate that the interaction between Leu356 and Phe380 is within the same subunit. These results show that Leu356 forms a hydrophobic interaction with Phe380, coupling the P helix with S6, whereas Glu363 could interact with Thr355, coupling the pore wall to the P helix. These interactions are essential for normal gating and underlie the transduction between the CNB domain and the pore.

Phosphodiesterases link the aryl hydrocarbon receptor complex to cyclic nucleotide signaling.

The aryl hydrocarbon receptor (AHR) is a major transcription factor regulated by different mechanisms. The classical view of AHR activation by xenobiotics needs to be amended by recent findings on the regulation of AHR by endogenous ligands and by crosstalk with other signaling pathways. In the cytosol the AHR recruits a large number of binding partners, including HSP90, p23, XAP2 and the ubiquitin ligases cullin 4B and CHIP. Furthermore, XAP2 binds the cyclic nucleotide phosphodiesterases PDE2A and PDE4A5. PDE2A inhibits nuclear translocation of AHR suggesting an important regulatory role of cyclic nucleotides in AHR trafficking. Signaling involving cAMP is organized in subcellular compartments and a distinct cAMP compartment might be required for proper AHR mobility and function. We conclude that the AHR complex integrates ligand binding and cyclic nucleotide signaling to generate an adequate transcriptional response.