Adenylyl cyclases (ACs) generate the second messenger cAMP from ATP. Mammalian cells express nine transmembrane AC (mAC) isoforms (AC1-9) and a soluble AC (sAC, also referred to as AC10). This review ...will largely focus on mACs. mACs are activated by the G-protein G
and regulated by multiple mechanisms. mACs are differentially expressed in tissues and regulate numerous and diverse cell functions. mACs localize in distinct membrane compartments and form signaling complexes. sAC is activated by bicarbonate with physiologic roles first described in testis. Crystal structures of the catalytic core of a hybrid mAC and sAC are available. These structures provide detailed insights into the catalytic mechanism and constitute the basis for the development of isoform-selective activators and inhibitors. Although potent competitive and noncompetitive mAC inhibitors are available, it is challenging to obtain compounds with high isoform selectivity due to the conservation of the catalytic core. Accordingly, caution must be exerted with the interpretation of intact-cell studies. The development of isoform-selective activators, the plant diterpene forskolin being the starting compound, has been equally challenging. There is no known endogenous ligand for the forskolin binding site. Recently, development of selective sAC inhibitors was reported. An emerging field is the association of AC gene polymorphisms with human diseases. For example, mutations in the AC5 gene (
) cause hyperkinetic extrapyramidal motor disorders. Overall, in contrast to the guanylyl cyclase field, our understanding of the (patho)physiology of AC isoforms and the development of clinically useful drugs targeting ACs is still in its infancy.
It is widely accepted that cAMP signaling is compartmentalized within cells. However, our knowledge of how receptors, cAMP signaling enzymes, effectors, and other key proteins form specific signaling ...complexes to regulate specific cell responses is limited. The multicomponent nature of these systems and the spatiotemporal dynamics involved as proteins interact and move within a cell make cAMP responses highly complex. Adenylyl cyclases, the enzymatic source of cAMP production, are key starting points for understanding cAMP compartments and defining the functional signaling complexes. Three basic elements are required to form a signaling compartment. First, a localized signal is generated by a G protein-coupled receptor paired to one or more of the nine different transmembrane adenylyl cyclase isoforms that generate the cAMP signal in the cytosol. The diffusion of cAMP is subsequently limited by several factors, including expression of any number of phosphodiesterases (of which there are 24 genes plus spice variants). Finally, signal response elements are differentially localized to respond to cAMP produced within each locale. A-kinase-anchoring proteins, of which there are 43 different isoforms, facilitate this by targeting protein kinase A to specific substrates. Thousands of potential combinations of these three elements are possible in any given cell type, making the characterization of cAMP signaling compartments daunting. This review will focus on what is known about how cells organize cAMP signaling components as well as identify the unknowns. We make an argument for adenylyl cyclases being central to the formation and maintenance of these signaling complexes.
► Melatonin is produced in mammals, plants, unicellular eukaryotes, and bacteria. ► It acts as neurotransmitter, hormone, cytokine, metabolic modulator and antioxidant. ► Melatonin regulates ...functions of peripheral organs. ► Melatonin activates membrane bound or nuclear receptors.
Many of melatonin’s actions are mediated through interaction with the G-protein coupled membrane bound melatonin receptors type 1 and type 2 (MT1 and MT2, respectively) or, indirectly with nuclear orphan receptors from the RORα/RZR family. Melatonin also binds to the quinone reductase II enzyme, previously defined the MT3 receptor. Melatonin receptors are widely distributed in the body; herein we summarize their expression and actions in non-neural tissues. Several controversies still exist regarding, for example, whether melatonin binds the RORα/RZR family. Studies of the peripheral distribution of melatonin receptors are important since they are attractive targets for immunomodulation, regulation of endocrine, reproductive and cardiovascular functions, modulation of skin pigmentation, hair growth, cancerogenesis, and aging. Melatonin receptor agonists and antagonists have an exciting future since they could define multiple mechanisms by which melatonin modulates the complexity of such a wide variety of physiological and pathological processes.
Adenylyl cyclases (ACs) catalyze the conversion of ATP to the ubiquitous second messenger cAMP. Mammals possess nine isoforms of transmembrane ACs, dubbed AC1-9, that serve as major effector enzymes ...of G protein-coupled receptors (GPCRs). The transmembrane ACs display varying expression patterns across tissues, giving the potential for them to have a wide array of physiological roles. Cells express multiple AC isoforms, implying that ACs have redundant functions. Furthermore, all transmembrane ACs are activated by Gα
, so it was long assumed that all ACs are activated by Gα
-coupled GPCRs. AC isoforms partition to different microdomains of the plasma membrane and form prearranged signaling complexes with specific GPCRs that contribute to cAMP signaling compartments. This compartmentation allows for a diversity of cellular and physiological responses by enabling unique signaling events to be triggered by different pools of cAMP. Isoform-specific pharmacological activators or inhibitors are lacking for most ACs, making knockdown and overexpression the primary tools for examining the physiological roles of a given isoform. Much progress has been made in understanding the physiological effects mediated through individual transmembrane ACs. GPCR-AC-cAMP signaling pathways play significant roles in regulating functions of every cell and tissue, so understanding each AC isoform's role holds potential for uncovering new approaches for treating a vast array of pathophysiological conditions.
The ongoing COVID-19 outbreak has caused devastating mortality and posed a significant threat to public health worldwide. Despite the severity of this illness and 2.3 million worldwide deaths, the ...disease mechanism is mostly unknown. Previous studies that characterized differential gene expression due to SARS-CoV-2 infection lacked robust validation. Although vaccines are now available, effective treatment options are still out of reach.
To characterize the transcriptional activity of SARS-CoV-2 infection, a gene signature consisting of 25 genes was generated using a publicly available RNA-Sequencing (RNA-Seq) dataset of cultured cells infected with SARS-CoV-2. The signature estimated infection level accurately in bronchoalveolar lavage fluid (BALF) cells and peripheral blood mononuclear cells (PBMCs) from healthy and infected patients (mean 0.001 vs. 0.958; P < 0.0001). These signature genes were investigated in their ability to distinguish the severity of SARS-CoV-2 infection in a single-cell RNA-Sequencing dataset. TNFAIP3, PPP1R15A, NFKBIA, and IFIT2 had shown bimodal gene expression in various immune cells from severely infected patients compared to healthy or moderate infection cases. Finally, this signature was assessed using the publicly available ConnectivityMap database to identify potential disease mechanisms and drug repurposing candidates. Pharmacological classes of tricyclic antidepressants, SRC-inhibitors, HDAC inhibitors, MEK inhibitors, and drugs such as atorvastatin, ibuprofen, and ketoconazole showed strong negative associations (connectivity score < - 90), highlighting the need for further evaluation of these candidates for their efficacy in treating SARS-CoV-2 infection.
Thus, using the 25-gene SARS-CoV-2 infection signature, the SARS-CoV-2 infection status was captured in BALF cells, PBMCs and postmortem lung biopsies. In addition, candidate SARS-CoV-2 therapies with known safety profiles were identified. The signature genes could potentially also be used to characterize the COVID-19 disease severity in patients' expression profiles of BALF cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The many components of G‐protein‐coupled receptor (GPCR) signal transduction provide cells with numerous combinations with which to customize their responses to hormones, neurotransmitters, and ...pharmacologic agonists. GPCRs function as guanine nucleotide exchange factors for heterotrimeric (α, β, γ) G proteins, thereby promoting exchange of GTP for GDP and, in turn, the activation of ‘downstream’ signaling components. Recent data indicate that individual cells express mRNA for perhaps over 100 different GPCRs (out of a total of nearly a thousand GPCR genes), several different combinations of G‐protein subunits, multiple regulators of G‐protein signaling proteins (which function as GTPase activating proteins), and various isoforms of downstream effector molecules. The differential expression of such protein combinations allows for modulation of signals that are customized for a specific cell type, perhaps at different states of maturation or differentiation. In addition, in the linear arrangement of molecular interactions involved in a given GPCR–G‐protein–effector pathway, one needs to consider the localization of receptors and post‐receptor components in subcellular compartments, microdomains, and molecular complexes, and to understand the movement of proteins between these compartments. Co‐localization of signaling components, many of which are expressed at low overall concentrations, allows cells to tailor their responses by arranging, or spatially organizing in unique and kinetically favorable ways, the molecules involved in GPCR signal transduction. This review focuses on the role of lipid rafts and a subpopulation of such rafts, caveolae, as a key spatial compartment enriched in components of GPCR signal transduction. Recent data suggest cell‐specific patterns for expression of those components in lipid rafts and caveolae. Such domains likely define functionally important, cell‐specific regions of signaling by GPCRs and drugs active at those GPCRs.
British Journal of Pharmacology (2004) 143, 235–245. doi:10.1038/sj.bjp.0705930
The second messenger molecule 3′5′-cyclic adenosine monophosphate (cAMP) imparts several beneficial effects in lung diseases such as asthma, chronic obstructive pulmonary disease (COPD) and ...idiopathic pulmonary fibrosis (IPF). While cAMP is bronchodilatory in asthma and COPD, it also displays anti-fibrotic properties that limit fibrosis. Phosphodiesterases (PDEs) metabolize cAMP and thus regulate cAMP signaling. While some existing therapies inhibit PDEs, there are only broad family specific inhibitors. The understanding of cAMP signaling compartments, some centered around lipid rafts/caveolae, has led to interest in defining how specific PDE isoforms maintain these signaling microdomains. The possible altered expression of PDEs, and thus abnormal cAMP signaling, in obstructive lung diseases has been poorly explored. We propose that inhibition of specific PDE isoforms can improve therapy of obstructive lung diseases by amplifying specific cAMP signals in discreet microdomains.
Background and Purpose
Nicotinic ACh receptors containing the α7 sub‐unit (α7‐nAChRs) suppress inflammation through a wide range of pathways in immune cells. These receptors are thus potentially ...involved in a number of inflammatory diseases. However, the detailed mechanisms underlying the anti‐inflammatory effects of α7‐nAChRs remain to be described.
Experimental Approach
Anti‐inflammatory effects of α7‐nAChR agonists were assessed in both murine macrophages (RAW 264.7) and bone marrow‐derived macrophages (BMDM), stimulated with LPS, using immunoblotting, RT‐PCR and luciferase reporter assays. The role of adenylyl cyclase‐6 in the degradation of Toll‐like receptor 4 (TLR4) following endocytosis, was explored via overexpression and knockdown. A mouse model of chronic obstructive pulmonary disease (COPD) induced by porcine pancreatic elastase was used to confirm key findings.
Results
Anti‐inflammatory effects of α7‐nAChRs were largely dependent on adenylyl cyclase‐6 activation, as knockdown of adenylyl cyclase‐6 considerably reduced the effects of α7‐nAChR agonists while adenylyl cyclase‐6 overexpression promoted them. We found that α7‐nAChRs and adenylyl cyclase‐6 are co‐localized in lipid rafts of macrophages and directly interact. Activation of adenylyl cyclase‐6 led to increased degradation of TLR4. Administration of the α7‐nAChR agonist PNU‐282987 attenuated pathological and inflammatory end points in a mouse model of COPD.
Conclusion and Implications
The α7‐nAChRs inhibit inflammation through activating adenylyl cyclase‐6 and promoting degradation of TLR4. The use of α7‐nAChR agonists may represent a novel therapeutic approach for treating COPD and possibly other inflammatory diseases.
Human airway smooth muscle (HASM) is the primary target of ßAR agonists used to control airway hypercontractility in asthma and chronic obstructive pulmonary disease (COPD). ßAR agonists induce the ...production of cAMP by adenylyl cyclases (ACs), activate PKA and cause bronchodilation. Several other G-protein coupled receptors (GPCR) expressed in human airway smooth muscle cells transduce extracellular signals through cAMP but these receptors elicit different cellular responses. Some G-protein coupled receptors couple to distinct adenylyl cyclases isoforms with different localization, partly explaining this compartmentation, but little is known about the downstream networks that result. We used quantitative phosphoproteomics to define the downstream signaling networks emanating from cAMP produced by two adenylyl cyclases isoforms with contrasting localization in uman airway smooth muscle. After a short stimulus of adenylyl cyclases activity using forskolin, phosphopeptides were analyzed by LC-MS/MS and differences between cells overexpressing AC2 (localized in non-raft membranes) or AC6 (localized in lipid raft membranes) were compared to control human airway smooth muscle. The degree of AC2 and AC6 overexpression was titrated to generate roughly equal forskolin-stimulated cAMP production. 14 Differentially phosphorylated proteins (DPPs) resulted from AC2 activity and 34 differentially phosphorylated proteins resulted from AC6 activity. Analysis of these hits with the STRING protein interaction tool showed that AC2 signaling is more associated with modifications in RNA/DNA binding proteins and microtubule/spindle body proteins while AC6 signaling is associated with proteins regulating autophagy, calcium-calmodulin (Ca
/CaM) signaling, Rho GTPases and cytoskeletal regulation. One protein, OFD1, was regulated in opposite directions, with serine 899 phosphorylation increased in the AC6 condition 1.5-fold but decreased to 0.46-fold by AC2. In conclusion, quantitative phosphoproteomics is a powerful tool for deciphering the complex signaling networks resulting from discreet signaling events that occur in cAMP compartments. Our data show key differences in the cAMP pools generated from AC2 and AC6 activity and imply that distinct cellular responses are regulated by these two compartments.
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