Damage Signaling by Extracellular Nucleotides: A Role for Cyclic Nucleotides in Elevating Cytosolic Free Calcium?

Extracellular ATP (eATP) is now held to be a constitutive damage-associated molecular pattern (DAMP) that is released by wounding, herbivory or pathogen attack. The concentration of eATP must be tightly regulated as either depletion or overload leads to cell death. In Arabidopsis thaliana, sensing of eATP is by two plasma membrane legume-like lectin serine–threonine receptor kinases (P2K1 and P2K2), although other receptors are postulated. The transcriptional response to eATP is dominated by wound- and defense-response genes. Wounding and pathogen attack can involve the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) which, in common with eATP, can increase cytosolic-free Ca2+ as a second messenger. This perspective on DAMP signaling by eATP considers the possibility that the eATP pathway involves production of cyclic nucleotides to promote opening of cyclic nucleotide-gated channels and so elevates cytosolic-free Ca2+. In silico analysis of P2K1 and P2K2 reveals putative adenylyl and guanylyl kinase sequences that are the hallmarks of “moonlighting” receptors capable of cAMP and cGMP production. Further, an Arabidopsis loss of function cngc mutant was found to have an impaired increase in cytosolic-free Ca2+ in response to eATP. A link between eATP, cyclic nucleotides, and Ca2+ signaling therefore appears credible.

Cyclic nucleotide-regulated channels (CNG) in GtoPdb v.2021.3

Cyclic nucleotide-gated (CNG) channels are responsible for signalling in the primary sensory cells of the vertebrate visual and olfactory systems. CNG channels are voltage-independent cation channels formed as tetramers. Each subunit has 6TM, with the pore-forming domain between TM5 and TM6. CNG channels were first found in rod photoreceptors [83, 120], where light signals through rhodopsin and transducin to stimulate phosphodiesterase and reduce intracellular cyclic GMP level. This results in a closure of CNG channels and a reduced ‘dark current’. Similar channels were found in the cilia of olfactory neurons [181] and the pineal gland [71]. The cyclic nucleotides bind to a domain in the C terminus of the subunit protein: other channels directly binding cyclic nucleotides include hyperolarisation-activated, cyclic nucleotide-gated channels (HCN), ether-a-go-go and certain plant potassium channels.The HCN channels are cation channels that are activated by hyperpolarisation at voltages negative to ~-50 mV. The cyclic nucleotides cyclic AMP and cyclic GMP directly bind to the cyclic nucleotide-binding domain of HCN channels and shift their activation curves to more positive voltages, thereby enhancing channel activity. HCN channels underlie pacemaker currents found in many excitable cells including cardiac cells and neurons [64, 192]. In native cells, these currents have a variety of names, such as Ih, Iq and If. The four known HCN channels have six transmembrane domains and form tetramers. It is believed that the channels can form heteromers with each other, as has been shown for HCN1 and HCN4 [2]. High resolution structural studies of CNG and HCN channels has provided insight into the the gating processes of these channels [139, 146, 140]. A standardised nomenclature for CNG and HCN channels has been proposed by the NC-IUPHAR Subcommittee on voltage-gated ion channels [108].

Questions and Answers Related to the Prebiotic Production of Oligonucleotide Sequences from 3′,5′ Cyclic Nucleotide Precursors

Template-free nonenzymatic polymerization of 3′,5′ cyclic nucleotides is an emerging topic of the origin of life research. In the last ten years, a number of papers have been published addressing various aspects of this process. These works evoked a vivid discussion among scientists working in the field of prebiotic chemistry. The aim of the current review is to answer the most frequently raised questions related to the detection and characterization of oligomeric products as well as to the geological context of this chemistry.

Development of Phosphodiesterase–Protein-Kinase Complexes as Novel Targets for Discovery of Inhibitors with Enhanced Specificity

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (protein kinases, PKs) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PKs generates an expanded active site that enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PKs and aid in signal termination. PDEs are important drug targets, and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targeted PDE–PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay was adapted to identify inhibitors that block cyclic nucleotide pockets in PDE–PK complexes in one mode and disrupt protein-protein interactions between PDEs and PKs in a second mode. We tested this approach with three different systems—cAMP-specific PDE8–PKAR, cGMP-specific PDE5–PKG, and dual-specificity RegA–RD complexes—and ranked inhibitors according to their inhibition potency. Targeting PDE–PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.

Development of Phosphodiesterase-Protein Kinase Complexes as Novel Targets for Discovery of Inhibitors with Enhanced Specificity

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (Protein Kinases, PK) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PK generates an expanded active site which enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PK, and aid in signal termination. PDEs are important drug targets and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targets PDE-PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay has been adapted to identify inhibitors that block cyclic nucleotide pockets in PDE-PK complexes in one mode, and disrupt protein-protein interactions between PDEs and cyclic nucleotide activating protein kinases in a second mode. We tested this approach with three different systems: cAMP-specific PDE8-PKAR, cGMP-specific PDE5-PKG and dual-specificity RegA-RD complexes and ranked inhibitors according to their inhibition potency. Targeting PDE-PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.

A metabolomics insight into the Cyclic Nucleotide Monophosphate signaling cascade in tomato under non-stress and salinity conditions

ABSTRACTCyclic Nucleotides Monophosphate (cNMP) are key signalling compounds whose role in plant cell signal transduction is till poorly understood. In this work we used sildenafil, a phosphodiesterase (PDE) inhibitor used in human, to amplify the signal cascade triggered by cNMP using tomato as model plant. Metabolomics was then used, together with plant growth and root architecture parameters, to unravel the changes elicited by PDE inhibition either under non-stress and 100 mM NaCl salinity conditions.The PDE inhibitor elicited a significant increase in biomass (+62%) and root length (+56%) under no stress conditions, and affected root architecture in terms of distribution over diameter classes. Together with cGMP, others cNMP were modulated by the treatment. Moreover, PDE inhibition triggered a broad metabolic reprogramming involving photosynthesis and secondary metabolism. A complex crosstalk network of phytohormones and other signalling compounds could be observed in treated plants. Nonetheless, metabolites related to redox imbalance processes and NO signalling could be highlighted in tomato following PDE application. Despite salinity damped down the growth-promoting effects of sildenafil, interesting implications in plant mitigation to stress-related detrimental effects could be observed.HIGHLIGHTThe role of Cyclic Nucleotides Monophosphate in plant cell signal transduction involves regulation of plant growth and architecture, together with a broad biochemical reprogramming of metabolism.