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Phosphodiesterases (PDEs) are a superfamily of enzymes that hydrolyze cyclic nucleotides. While the 11 PDE subfamilies share common features, key differences confer signaling specificity. The differences include substrate selectivity, enzymatic activity regulation, tissue expression, and subcellular localization. Selective inhibitors of each subfamily have elucidated the protean role of PDEs in normal cell function. PDEs are also linked to diseases, some of which affect the immune, cardiac, and vascular systems. Selective PDE inhibitors are clinically used to treat these specific disorders. Ongoing preclinical studies and clinical trials are likely to lead to the approval of additional PDE-targeting drugs for therapy in human disease. In this review, we discuss the structure and function of PDEs and examine current and evolving therapeutic uses of PDE inhibitors, highlighting their mechanisms and innovative applications that could further leverage this crucial family of enzymes in clinical settings. [ABSTRACT FROM AUTHOR]

Cells interpret a variety of signals through G-protein-coupled receptors (GPCRs) and stimulate the generation of second messengers such as cyclic adenosine monophosphate (cAMP). A long-standing puzzle is deciphering how GPCRs elicit different physiological responses despite generating similar levels of cAMP. We previously showed that some GPCRs generate cAMP from both the plasma membrane and the Golgi apparatus. Here we demonstrate that cardiomyocytes distinguish between subcellular cAMP inputs to elicit different physiological outputs. We show that generating cAMP from the Golgi leads to the regulation of a specific protein kinase A (PKA) target that increases the rate of cardiomyocyte relaxation. In contrast, cAMP generation from the plasma membrane activates a different PKA target that increases contractile force. We further validated the physiological consequences of these observations in intact zebrafish and mice. Thus, we demonstrate that the same GPCR acting through the same second messenger regulates cardiac contraction and relaxation dependent on its subcellular location.

High-molecular-weight hyaluronan (HMWH) is an agonist at cluster of differentiation (CD)44, the cognate hyaluronan receptor, on nociceptors, where it acts to induce antihyperalgesia in preclinical models of inflammatory and neuropathic pain. In the present experiments, we studied the CD44 second messengers that mediate HMWH-induced attenuation of pain associated with oxaliplatin and paclitaxel chemotherapy-induced peripheral neuropathy (CIPN). While HMWH attenuated CIPN only in male rats, after ovariectomy or intrathecal administration of an oligodeoxynucleotide (ODN) antisense to G protein-coupled estrogen receptor (GPR30) mRNA, female rats were also sensitive to HMWH. Intrathecal administration of an ODN antisense to CD44 mRNA markedly attenuated HMWH-induced antihyperalgesia in male rats with CIPN induced by oxaliplatin or paclitaxel. Intradermal administration of inhibitors of CD44 second messengers, RhoA (member of the Rho family of GTPases), phospholipase C, and phosphatidylinositol (PI) 3-kinase gamma (PI3Kγ), attenuated HMWH-induced antihyperalgesia as does intrathecal administration of an ODN antisense to PI3Kγ. Our results demonstrated that HMWH induced antihyperalgesia in CIPN, mediated by its action at CD44 and downstream signaling by RhoA, phospholipase C, and PI3Kγ.

cAMP is a ubiquitous second messenger involved in the regulation of diverse cellular processes. Spatiotemporal regulation of cAMP through compartmentalization within various subcellular microdomains is essential to ensure specific signaling. In the following protocol, we describe a method for directly visualizing signaling dynamics within cAMP microdomains using fluorescent sensors targeted to endogenous proteins (FluoSTEPs). Instead of overexpressing a biosensor-tagged protein of interest to target a microdomain, FluoSTEP Indicator of cAMP using Epac (FluoSTEP-ICUE) utilizes spontaneously complementing split GFP and CRISPR-Cas9 genome editing to localize a FRET-based cAMP biosensor to an endogenously expressed protein of interest. Utilizing this approach, FluoSTEP-ICUE can be used to measure cAMP levels within endogenous signaling compartments, thus providing a powerful tool for studying the spatiotemporal regulation of cAMP signaling.

There are several endogenous molecules that can trigger migraine attacks when administered to humans. Notably, calcitonin gene-related peptide (CGRP) has been identified as a key player in a signalling cascade involved in migraine attacks, acting through the second messenger cyclic adenosine monophosphate (cAMP) in various cells, including intracranial vascular smooth muscle cells. However, it remains unclear whether intracellular cAMP signalling requires CGRP receptor activation during a migraine attack in humans. To address this question, we conducted a randomized, double-blind, placebo-controlled, parallel trial using a human provocation model involving the administration of CGRP and cilostazol in individuals with migraine pretreated with erenumab or placebo. Our study revealed that migraine attacks can be provoked in patients by cAMP-mediated mechanisms using cilostazol, even when the CGRP receptor is blocked by erenumab. Furthermore, the dilation of cranial arteries induced by cilostazol was not influenced by the CGRP receptor blockade. These findings provide clinical evidence that cAMP-evoked migraine attacks do not require CGRP receptor activation. This discovery opens up new possibilities for the development of mechanism-based drugs for the treatment of migraine. [ABSTRACT FROM AUTHOR]

Type III clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems (type III CRISPR-Cas systems) use guide RNAs to recognize RNA transcripts of foreign genetic elements, which triggers the generation of cyclic oligoadenylate (cOA) second messengers by the Cas10 subunit of the type III effector complex. In turn, cOAs bind and activate ancillary effector proteins to reinforce the host immune response. Type III systems utilize distinct cOAs, including cyclic tri- (cA3), tetra- (cA4) and hexa-adenylates (cA6). However, the molecular mechanisms dictating cOA product identity are poorly understood. Here we used cryoelectron microscopy to visualize the mechanism of cA6 biosynthesis by the Csm effector complex from Enterococcus italicus (EiCsm). We show that EiCsm synthesizes oligoadenylate nucleotides in 3'-5' direction using a set of conserved binding sites in the Cas10 Palm domains to determine the size of the nascent oligoadenylate chain. Our data also reveal that conformational dynamics induced by target RNA binding results in allosteric activation of Cas10 to trigger oligoadenylate synthesis. Mutations of a key structural element in Cas10 perturb cOA synthesis to favor cA3 and cA4 formation. Together, these results provide comprehensive insights into the dynamics of cOA synthesis in type III CRISPR-Cas systems and reveal key determinants of second messenger product selectivity, thereby illuminating potential avenues for their engineering., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Phosphodiesterase (PDE) enzymes regulate intracellular signaling pathways crucial for brain development and the pathophysiology of neurological disorders. Among the 11 PDE subtypes, PDE4 and PDE5 are particularly significant due to their regulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) signaling, respectively, which are vital for learning, memory, and neuroprotection. This review synthesizes current evidence on the roles of PDE4 and PDE5 in neurological health and disease, focusing on their regulation of second messenger pathways and their implications for brain function. Elevated PDE4 activity impairs synaptic plasticity by reducing cAMP levels and protein kinase A (PKA) activity, contributing to cognitive decline, acute brain injuries, and neuropsychiatric conditions such as bipolar disorder and schizophrenia. Similarly, PDE5 dysregulation disrupts nitric oxide (NO) signaling and protein kinase G (PKG) pathways, which are involved in cerebrovascular homeostasis, recovery after ischemic events, and neurodegenerative processes in Alzheimer's, Parkinson's, and Huntington's diseases. PDE4 and PDE5 are promising therapeutic targets for neurological disorders. Pharmacological modulation of these enzymes offers potential to enhance cognitive function and mitigate pathological mechanisms underlying brain injuries, neurodegenerative diseases, and psychiatric disorders. Further research into the regulation of PDE4 and PDE5 will advance therapeutic strategies for these conditions.

Cyclic di-adenylate monophosphate (cyclic di-AMP) is an important second messenger in microorganisms. Cyclic di-AMP regulates bacterial cell volume and turgor via control of potassium and compatible solute transport but is also involved in many other processes, including the activation of the metazoan innate immune response to bacterial infections. We compare the activity of full-length membrane-embedded CdaA, the enzyme that synthesizes cyclic di-AMP, with the water-soluble catalytic domain CdaA-DAC. Purified CdaA from L. lactis was studied in the detergent-solubilized state, and in lipid nanodiscs and vesicles. We show that CdaA is tetrameric and the membrane-bound complex has more than 2-orders of magnitude higher activity than soluble CdaA-DAC. CdaA activity increases with pH but does not strongly depend on the salt or lipid content, factors that are crucial for the control of osmoregulatory transporters. Cryo-EM and in-silico structure prediction of CdaA show that the two DAC dimers engage in a head-to-head interaction, leading to cyclic-di-AMP formation. The inhibitor phosphoglucomutase prevents this active conformation. We observe dynamic flexibility between the catalytic and membrane domains, even in the presence of ATP or non-hydrolyzable substrate ApCpp. This is the first comprehensive functional and structural characterization of a full-length cyclic di-AMP-specific cyclase., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The bacterial pathogen Staphylococcus aureus forms multicellular communities known as biofilms in which cells are held together by an extracellular matrix principally composed of repurposed cytoplasmic proteins and extracellular DNA. These biofilms assemble during infections or under laboratory conditions by growth on medium containing glucose, but the intracellular signal for biofilm formation and its downstream targets were unknown. Here, we present evidence that biofilm formation is triggered by a drop in the levels of the second messenger cyclic-di-AMP. Previous work identified genes needed for the release of extracellular DNA, including genes for the cyclic-di-AMP phosphodiesterase GdpP, the transcriptional regulator XdrA, and the purine salvage enzyme Apt. Using a cyclic-di-AMP riboswitch biosensor and mass spectrometry, we show that the second messenger drops in abundance during biofilm formation in a glucose-dependent manner. Mutation of these three genes elevates cyclic-di-AMP and prevents biofilm formation in a murine catheter model. Supporting the generality of this mechanism, we found that gdpP was required for biofilm formation by diverse strains of S. aureus . We additionally show that the downstream consequence of the drop in cyclic-di-AMP is inhibition of the "accessory gene regulator" operon agr , which is known to suppress biofilm formation through phosphorylation of the transcriptional regulator AgrA by the histidine kinase AgrC. Consistent with this, an agr mutation bypasses the block in biofilm formation and eDNA release caused by a gdpP mutation. Finally, we report the unexpected observation that GdpP inhibits phosphotransfer from AgrC to AgrA, revealing a direct connection between the phosphodiesterase and agr ., Competing Interests: Competing interests statement:The authors declare no competing interest.

In this issue of Molecular Cell, Luo et al. 1 identify a signaling pathway, OSM1-COLD6, that induces cold tolerance in rice by promoting production of the non-canonical cyclic nucleotide 2',3'-cAMP. The study opens new avenues for enhancing cold tolerance in rice breeding., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Prokaryotic anti-phage immune systems use TIR and cGAS-like enzymes to produce 1''-3'-glycocyclic ADP-ribose (1''-3'-gcADPR) and cyclic dinucleotide (CDN) and cyclic trinucleotide (CTN) signalling molecules, respectively, which limit phage replication 1-3 . However, how phages neutralize these distinct and common systems is largely unclear. Here we show that the Thoeris anti-defence proteins Tad1 4 and Tad2 5 both achieve anti-cyclic-oligonucleotide-based anti-phage signalling system (anti-CBASS) activity by simultaneously sequestering CBASS cyclic oligonucleotides. Apart from binding to the Thoeris signals 1''-3'-gcADPR and 1''-2'-gcADPR, Tad1 also binds to numerous CBASS CDNs and CTNs with high affinity, inhibiting CBASS systems that use these molecules in vivo and in vitro. The hexameric Tad1 has six binding sites for CDNs or gcADPR, which are independent of the two high-affinity binding sites for CTNs. Tad2 forms a tetramer that also sequesters various CDNs in addition to gcADPR molecules, using distinct binding sites to simultaneously bind to these signals. Thus, Tad1 and Tad2 are both two-pronged inhibitors that, alongside anti-CBASS protein 2 (Acb2 6-8 ), establish a paradigm of phage proteins that use distinct binding sites to flexibly sequester a considerable breadth of cyclic nucleotides., Competing Interests: Competing interests: J.B.-D. is a scientific advisory board member of SNIPR Biome and Excision Biotherapeutics, a consultant to LeapFrog Bio and BiomX, and a scientific advisory board member and co-founder of Acrigen Biosciences and ePhective Therapeutics. The J.B.-D. laboratory received prior research support from Felix Biotechnology., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Upon stimulation of membrane receptors, nicotinic acid adenine dinucleotide phosphate (NAADP) is formed as second messenger within seconds and evokes Ca 2+ signaling in many different cell types. Here, to directly stimulate NAADP signaling, MASTER-NAADP, a Membrane permeAble, STabilized, bio-rEversibly pRotected precursor of NAADP is synthesized and release of its active NAADP mimetic, benzoic acid C-nucleoside, 2'-phospho-3'F-adenosine-diphosphate, by esterase digestion is confirmed. In the presence of NAADP receptor HN1L/JPT2 (hematological and neurological expressed 1-like protein, HN1L, also known as Jupiter microtubule-associated homolog 2, JPT2), this active NAADP mimetic releases Ca 2+ and increases the open probability of type 1 ryanodine receptor. When added to intact cells, MASTER-NAADP initially evokes single local Ca 2+ signals of low amplitude. Subsequently, also global Ca 2+ signaling is observed in T cells, natural killer cells, and Neuro2A cells. In contrast, control compound MASTER-NADP does not stimulate Ca 2+ signaling. Likewise, in cells devoid of HN1L/JPT2, MASTER-NAADP does not affect Ca 2+ signaling, confirming that the product released from MASTER-NAADP is a bona fide NAADP mimetic., (© 2024. The Author(s).)

The rhizosphere system of plants hosts a diverse consortium of bacteria that confer beneficial effects on plant, such as plant growth-promoting rhizobacteria (PGPR), biocontrol agents with disease-suppression activities, and symbiotic nitrogen fixing bacteria with the formation of root nodule. Efficient colonization in planta is of fundamental importance for promoting of these beneficial activities. However, the process of root colonization is complex, consisting of multiple stages, including chemotaxis, adhesion, aggregation, and biofilm formation. The secondary messenger, c-di-GMP (cyclic bis-(3'-5') dimeric guanosine monophosphate), plays a key regulatory role in a variety of physiological processes. This paper reviews recent progress on the actions of c-di-GMP in plant beneficial bacteria, with a specific focus on its role in chemotaxis, biofilm formation, and nodulation., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Aims: Ischaemia/reperfusion (I/R) injury is an important complication of reperfusion therapy for acute myocardial infarction, extremely compromising the cardiac benefits of revascularization; however, specific and efficient treatment for cardiac I/R injury is still lacking. Isthmin-1 (ISM1) is a novel adipokine and plays indispensable roles in regulating glycolipid metabolism and cell survival. The present study aims to investigate the potential role and molecular mechanism of ISM1 in cardiac I/R injury using gain- and loss-of-function approaches., Methods and Results: Cardiac-specific ISM1 overexpression and silence were achieved using an adeno-associated virus serotype 9 system, and then these mice were subjected to I/R surgery, followed by biochemical test, echocardiography and histopathologic examinations, etc. Meanwhile, neonatal rat cardiomyocytes (NRCMs) with ISM1 silence or overexpression also received simulated I/R (sI/R) injury to further verify its role in vitro. The potential downstream pathways and molecular targets of ISM1 were screened by RNA sequencing. We also treated injured mice and NRCMs with recombinant ISM1 (rISM1) to explore whether supplementation with ISM1 was sufficient to protect against I/R injury. Furthermore, acute myocardial infarction patients with percutaneous coronary intervention (PCI) and paired healthy controls were included to reveal the clinical relevance of circulating ISM1. Cardiac-specific ISM1 silencing aggravated while ISM1 overexpression alleviated I/R-induced acute cardiac injury and cardiac remodelling and dysfunction. Mechanistically, ISM1 targeted αvβ5 integrin to facilitate the nuclear accumulation of nuclear transcription factor Y subunit alpha, transcriptionally increased soluble guanylyl cyclase beta subunit expression, and eventually enhanced cGMP generation. Besides, we confirmed that treatment with rISM1 before or after reperfusion could confer cardioprotective effects in mice. Clinically, lower ISM1 levels post-PCI was associated with worse outcome in patients., Conclusion: ISM1 can protect against cardiac I/R injury through cGMP-PKG signalling pathway, and it is a promising therapeutic and predictive target of cardiac I/R injury., Competing Interests: Conflict of interest: On behalf of all authors, the corresponding author states that there is no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Purines and their derivatives control intracellular energy homeostasis and nucleotide synthesis, and act as signaling molecules. Here, we combine structural and sequence information to define a purine-binding motif that is present in sensor domains of thousands of bacterial receptors that modulate motility, gene expression, metabolism, and second-messenger turnover. Microcalorimetric titrations of selected sensor domains validate their ability to specifically bind purine derivatives, and evolutionary analyses indicate that purine sensors share a common ancestor with amino-acid receptors. Furthermore, we provide experimental evidence of physiological relevance of purine sensing in a second-messenger signaling system that modulates c-di-GMP levels., (© 2024. The Author(s).)

Plants adapt to fluctuating environmental conditions by adjusting their metabolism and gene expression to maintain fitness 1 . In legumes, nitrogen homeostasis is maintained by balancing nitrogen acquired from soil resources with nitrogen fixation by symbiotic bacteria in root nodules 2-8 . Here we show that zinc, an essential plant micronutrient, acts as an intracellular second messenger that connects environmental changes to transcription factor control of metabolic activity in root nodules. We identify a transcriptional regulator, FIXATION UNDER NITRATE (FUN), which acts as a sensor, with zinc controlling the transition between an inactive filamentous megastructure and an active transcriptional regulator. Lower zinc concentrations in the nodule, which we show occur in response to higher levels of soil nitrate, dissociates the filament and activates FUN. FUN then directly targets multiple pathways to initiate breakdown of the nodule. The zinc-dependent filamentation mechanism thus establishes a concentration readout to adapt nodule function to the environmental nitrogen conditions. In a wider perspective, these results have implications for understanding the roles of metal ions in integration of environmental signals with plant development and optimizing delivery of fixed nitrogen in legume crops., (© 2024. The Author(s).)

SUMMARYNucleotide-derived second messengers are present in all domains of life. In prokaryotes, most of their functionality is associated with general lifestyle and metabolic adaptations, often in response to environmental fluctuations of physical parameters. In the last two decades, cyclic di-AMP has emerged as an important signaling nucleotide in many prokaryotic lineages, including Firmicutes, Actinobacteria, and Cyanobacteria. Its importance is highlighted by the fact that both the lack and overproduction of cyclic di-AMP affect viability of prokaryotes that utilize cyclic di-AMP, and that it generates a strong innate immune response in eukaryotes. In bacteria that produce the second messenger, most molecular targets of cyclic di-AMP are associated with cell volume control. Besides, other evidence links the second messenger to cell wall remodeling, DNA damage repair, sporulation, central metabolism, and the regulation of glycogen turnover. In this review, we take a biochemical, quantitative approach to address the main cellular processes that are directly regulated by cyclic di-AMP and show that these processes are very connected and require regulation of a similar set of proteins to which cyclic di-AMP binds. Altogether, we argue that cyclic di-AMP is a master regulator of cell volume and that other cellular processes can be connected with cyclic di-AMP through this core function. We further highlight important directions in which the cyclic di-AMP field has to develop to gain a full understanding of the cyclic di-AMP signaling network and why some processes are regulated, while others are not., Competing Interests: The authors declare no conflict of interest.

Monitoring changes of signaling molecules and metabolites with high temporal resolution is key to understanding dynamic biological systems. Here, we use directed evolution to develop a genetically encoded ratiometric biosensor for c-di-GMP, a ubiquitous bacterial second messenger regulating important biological processes like motility, surface attachment, virulence and persistence. The resulting biosensor, cdGreen2, faithfully tracks c-di-GMP in single cells and with high temporal resolution over extended imaging times, making it possible to resolve regulatory networks driving bimodal developmental programs in different bacterial model organisms. We further adopt cdGreen2 as a simple tool for in vitro studies, facilitating high-throughput screens for compounds interfering with c-di-GMP signaling and biofilm formation. The sensitivity and versatility of cdGreen2 could help reveal c-di-GMP dynamics in a broad range of microorganisms with high temporal resolution. Its design principles could also serve as a blueprint for the development of similar, orthogonal biosensors for other signaling molecules, metabolites and antibiotics., (© 2024. The Author(s).)

Cytosolic double-stranded DNA (dsDNA) is frequently accumulated in cancer cells due to chromosomal instability or exogenous stimulation. Cyclic GMP-AMP synthase (cGAS) acts as a cytosolic DNA sensor, which is activated upon binding to dsDNA to synthesize the crucial second messenger 2'3'-cyclic GMP-AMP (2'3'-cGAMP) that in turn triggers stimulator of interferon genes (STING) signaling. The canonical role of cGAS-cGAMP-STING pathway is essential for innate immunity and viral defense. Recent emerging evidence indicates that 2'3'-cGAMP plays an important role in cancer progression via cell autonomous and non-autonomous mechanisms. Beyond its role as an intracellular messenger to activate STING signaling in tumor cells, 2'3'-cGAMP also serves as an immunotransmitter produced by cancer cells to modulate the functions of non-tumor cells especially immune cells in the tumor microenvironment by activating STING signaling. In this review, we summarize the synthesis, transmission, and degradation of 2'3'-cGAMP as well as the dual functions of 2'3'-cGAMP in a STING-dependent manner. Additionally, we discuss the potential therapeutic strategies that harness the cGAMP-mediated antitumor response for cancer therapy., (© 2023. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

CRISPR-Cas provides adaptive immunity in prokaryotes. Type III CRISPR systems detect invading RNA and activate the catalytic Cas10 subunit, which generates a range of nucleotide second messengers to signal infection. These molecules bind and activate a diverse range of effector proteins that provide immunity by degrading viral components and/or by disturbing key aspects of cellular metabolism to slow down viral replication. Here, we focus on the uncharacterised effector Csx23, which is widespread in Vibrio cholerae. Csx23 provides immunity against plasmids and phage when expressed in Escherichia coli along with its cognate type III CRISPR system. The Csx23 protein localises in the membrane using an N-terminal transmembrane α-helical domain and has a cytoplasmic C-terminal domain that binds cyclic tetra-adenylate (cA4), activating its defence function. Structural studies reveal a tetrameric structure with a novel fold that binds cA4 specifically. Using pulse EPR, we demonstrate that cA4 binding to the cytoplasmic domain of Csx23 results in a major perturbation of the transmembrane domain, consistent with the opening of a pore and/or disruption of membrane integrity. This work reveals a new class of cyclic nucleotide binding protein and provides key mechanistic detail on a membrane-associated CRISPR effector., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Efficient quorum sensing (QS) response is the premise for recovering the activities of stored aerobic granular sludge (AGS). This study aims to explore the crosstalk between the secondary messenger and the N-acylated-homoserine lactones (AHLs) to yield protein-rich granules efficiently from stored AGS by enhancing its QS efficiency selectively. 80 nmol/L cyclic diguanylate (c-di-GMP) with 20 nmol/L AHLs could increase the activity of isocitrate lyase activity (ICD) by 89 % and isocitrate dehydrogenase activity (ICDHc) by 113.5 %, to accelerate the tricarboxylic acid (TCA) cycle for yielding excess proteins by 166.4 %. In contrast, 80 nmol/L autoinducer-2 (AI-2) with 20 nmol/L AHLs could increase the activities of ICD and ICDHc by 485 % and 54.5 %, respectively, accelerating the glyoxylate (GCA) cycle to activate fat acid synthesis for stimulating polysaccharides (PS) secretion by 137.9 %. The strategy with c-di-GMP successfully recovers the refrigerated-stored and dried-stored AGS into proteins-rich AGS, with enriched functional strains for the PN secretion., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: DJL is the editorial board member of this journal., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bis (3',5')-cyclic diguanylic acid (c-di-GMP) is a ubiquitous second messenger that controls several metabolic pathways in bacteria. In Streptomyces , c-di-GMP is associated with morphological differentiation, which is related to secondary metabolite production. In this study, we identified and characterized a diguanylate cyclase (DGC), CdgB, from Streptomyces diastatochromogenes 1628, which may be involved in c-di-GMP synthesis, through genetic and biochemical analyses. To further investigate the role of CdgB, the cdgB -deleted mutant strain Δ- cdgB and the cdgB -overexpressing mutant strain O- cdgB were constructed by genetic engineering. A phenotypic analysis revealed that the O- cdgB colonies exhibited reduced mycelium formation, whereas the Δ-cdgB colonies displayed wrinkled surfaces and shriveled mycelia. Notably, O- cdgB demonstrated a significant increase in the toyocamycin (TM) yield by 47.3%, from 253 to 374 mg/L, within 10 days. This increase was accompanied by a 6.7% elevation in the intracellular concentration of c-di-GMP and a higher transcriptional level of the toy cluster within four days. Conversely, Δ- cdgB showed a lower c-di-GMP concentration (reduced by 6.2%) in vivo and a reduced toyocamycin production (decreased by 28.9%, from 253 to 180 mg/L) after 10 days. In addition, S. diastatochromogenes 1628 exhibited a slightly higher inhibitory effect against Fusarium oxysporum f. sp. cucumerinum and Rhizoctonia solani compared to Δ- cdgB , but a lower inhibition rate than that of O- cdgB . The results imply that CdgB provides a foundational function for metabolism and the activation of secondary metabolism in S. diastatochromogenes 1628.

Cyclic dinucleotides (CDNs) are cyclic molecules consisting of two nucleoside monophosphates linked by two phosphodiester bonds, which act as a second messenger and bind to the interferon gene stimulating factor (STING) to activate the downstream signaling pathway and ultimately induce interferon secretion, initiating an anti-infective immune response. Cyclic dinucleotides and their analogs are lead compounds in the immunotherapy of infectious diseases and tumors, as well as immune adjuvants with promising applications. Many agonists of pathogen recognition receptors have been developed as effective adjuvants to optimize vaccine immunogenicity and efficacy. In this work, the binding mechanism of human-derived interferon gene-stimulating protein and its isoforms with cyclic dinucleotides and their analogs was theoretically investigated using computer simulations and combined with experimental results in the hope of providing guidance for the subsequent synthesis of cyclic dinucleotide analogs.

Type VI secretion systems (T6SS) are bacterial macromolecular complexes that secrete effectors into target cells or the extracellular environment, leading to the demise of adjacent cells and providing a survival advantage. Although studies have shown that the T6SS in Pseudomonas aeruginosa is regulated by the Quorum Sensing system and second messenger c-di-GMP, the underlying molecular mechanism remains largely unknown. In this study, we discovered that the c-di-GMP-binding adaptor protein PA0012 has a repressive effect on the expression of the T6SS HSI-I genes in P. aeruginosa PAO1. To probe the mechanism by which PA0012 (renamed TssZ, Type Six Secretion System -associated PilZ protein) regulates the expression of HSI-I genes, we conducted yeast two-hybrid screening and identified HinK, a LasR-type transcriptional regulator, as the binding partner of TssZ. The protein-protein interaction between HinK and TssZ was confirmed through co-immunoprecipitation assays. Further analysis suggested that the HinK-TssZ interaction was weakened at high c-di-GMP concentrations, contrary to the current paradigm wherein c-di-GMP enhances the interaction between PilZ proteins and their partners. Electrophoretic mobility shift assays revealed that the non-c-di-GMP-binding mutant TssZ R5A/R9A interacts directly with HinK and prevents it from binding to the promoter of the quorum-sensing regulator pqsR. The functional connection between TssZ and HinK is further supported by observations that TssZ and HinK impact the swarming motility, pyocyanin production, and T6SS-mediated bacterial killing activity of P. aeruginosa in a PqsR-dependent manner. Together, these results unveil a novel regulatory mechanism wherein TssZ functions as an inhibitor that interacts with HinK to control gene expression., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger known to orchestrate a myriad of cellular functions over a wide range of timescales. In the last 20 years, a variety of single-cell sensors have been developed to measure second messenger signals including cAMP, Ca2+, and the balance of kinase and phosphatase activities. These sensors utilize changes in fluorescence emission of an individual fluorophore or Förster resonance energy transfer (FRET) to detect changes in second messenger concentration. cAMP and kinase activity reporter probes have provided powerful tools for the study of localized signals. Studies relying on these and related probes have the potential to further revolutionize our understanding of G protein-coupled receptor signaling systems. Unfortunately, investigators have not been able to take full advantage of the potential of these probes due to the limited signal-to-noise ratio of the probes and the limited ability of standard epifluorescence and confocal microscope systems to simultaneously measure the distributions of multiple signals (e.g. cAMP, Ca2+, and changes in kinase activities) in real time. In this review, we focus on recently implemented strategies to overcome these limitations: hyperspectral imaging and adaptive thresholding approaches to track dynamic regions of interest (ROI). This combination of approaches increases signal-to-noise ratio and contrast, and allows identification of localized signals throughout cells. These in turn lead to the identification and quantification of intracellular signals with higher effective resolution. Hyperspectral imaging and dynamic ROI tracking approaches offer investigators additional tools with which to visualize and quantify multiplexed intracellular signaling systems., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Rapid conversion of force into a biological signal enables living cells to respond to mechanical forces in their environment. The force is believed to initially affect the plasma membrane and then alter the behavior of membrane proteins. Phospholipase D2 (PLD2) is a mechanosensitive enzyme that is regulated by a structured membrane-lipid site comprised of cholesterol and saturated ganglioside (GM1). Here we show stretch activation of TWIK-related K + channel (TREK-1) is mechanically evoked by PLD2 and spatial patterning involving ordered GM1 and 4,5-bisphosphate (PIP 2 ) clusters in mammalian cells. First, mechanical force deforms the ordered lipids, which disrupts the interaction of PLD2 with the GM1 lipids and allows a complex of TREK-1 and PLD2 to associate with PIP 2 clusters. The association with PIP 2 activates the enzyme, which produces the second messenger phosphatidic acid (PA) that gates the channel. Co-expression of catalytically inactive PLD2 inhibits TREK-1 stretch currents in a biological membrane. Cellular uptake of cholesterol inhibits TREK-1 currents in culture and depletion of cholesterol from astrocytes releases TREK-1 from GM1 lipids in mouse brain. Depletion of the PLD2 ortholog in flies results in hypersensitivity to mechanical force. We conclude PLD2 mechanosensitivity combines with TREK-1 ion permeability to elicit a mechanically evoked response., Competing Interests: EP, MP, SH, MG, HW, ZY, KM, WJ, HF, EJ, SH No competing interests declared, (© 2023, Petersen et al.)

Tissue infiltration by circulating leukocytes via directed migration (also referred to as chemotaxis) is a common pathogenic mechanism of inflammatory diseases. G protein-coupled receptors (GPCRs) are essential for sensing chemokine gradients and directing the movement of leukocytes during immune responses. The tumor necrosis factor α-induced protein 8-like (TIPE or TNFAIP8L) family of proteins are newly described pilot proteins that control directed migration of murine leukocytes. However, how leukocytes integrate site-specific directional cues, such as chemokine gradients, and utilize GPCR and TIPE proteins to make directional decisions are not well understood. Using both gene knockdown and biochemical methods, we demonstrated here that 2 human TIPE family members, TNFAIP8 and TIPE2, were essential for directed migration of human CD4+ T cells. T cells deficient in both of these proteins completely lost their directionality. TNFAIP8 interacted with the Gαi subunit of heterotrimeric (α, β, γ) G proteins, whereas TIPE2 bound to PIP2 and PIP3 to spatiotemporally control immune cell migration. Using deletion and site-directed mutagenesis, we established that Gαi interacted with TNFAIP8 through its C-terminal amino acids, and that TIPE2 protein interacted with PIP2 and PIP3 through its positively charged amino acids on the α0 helix and at the grip-like entrance. We also discovered that TIPE protein membrane translocation (i.e. crucial for sensing chemokine gradients) was dependent on PIP2. Collectively, our work describes a new mechanistic paradigm for how human T cells integrate GPCR and phospholipid signaling pathways to control directed migration. These findings have implications for therapeutically targeting TIPE proteins in human inflammatory and autoimmune diseases., Competing Interests: Conflict of interest. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Oligoribonuclease (orn) of P. aeruginosa is a highly conserved exonuclease, which can regulate the global gene expression levels of bacteria through regulation of both the nanoRNA and c-di-GMP. NanoRNA can regulate the expression of the bacterial global genome as a transcription initiator, and c-di-GMP is the most widely second messenger in bacterial cells., Objective: This study seeks to elucidate on the regulation by orn on pathogenicity of P. aeruginosa., Methods: P. aeruginosa with orn deletion was constructed by suicide plasmid homologous recombination method. The possible regulatory process of orn was analyzed by TMT quantitative labeling proteomics. Then experiments were conducted to verify the changes of Δorn on bacterial motility, virulence and biofilm formation. Bacterial pathogenicity was further detected in cell and animal skin trauma models. ELISA detection c-di-GMP concentration and colony aggregation and biofilm formation were observed by scanning electron microscope., Results: orn deletion changed the global metabolism of P. aeruginosa and reduced intracellular energy metabolism. It leads to the disorder of the quorum sensing system, the reduction of bacterial motility and virulence factors pyocyanin and rhamnolipids. But, orn deletion enhanced pathogenicity in vitro and in vivo, a high level of c-di-GMP and biofilm development of P. aeruginosa., Conclusion: orn regulates the ability of P. aeruginosa to adapt to the external environment., (© 2024. The Author(s).)

Type III CRISPR-Cas systems provide adaptive immunity against foreign mobile genetic elements through RNA-guided interference. Sequence-specific recognition of RNA targets by the type III effector complex triggers the generation of cyclic oligoadenylate (cOA) second messengers that activate ancillary effector proteins, thus reinforcing the host immune response. The ancillary nuclease Can2 is activated by cyclic tetra-AMP (cA4); however, the mechanisms underlying cA4-mediated activation and substrate selectivity remain elusive. Here we report crystal structures of Thermoanaerobacter brockii Can2 (TbrCan2) in substrate- and product-bound complexes. We show that TbrCan2 is a single strand-selective DNase and RNase that binds substrates via a conserved SxTTS active site motif, and reveal molecular interactions underpinning its sequence preference for CA dinucleotides. Furthermore, we identify a molecular interaction relay linking the cA4 binding site and the nuclease catalytic site to enable divalent metal cation coordination and catalytic activation. These findings provide key insights into the molecular mechanisms of Can2 nucleases in type III CRISPR-Cas immunity and may guide their technological development for nucleic acid detection applications., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

One of the debilitating causes of high mortality in the case of tuberculosis and other bacterial infections is the resistance development against standard drugs. There are limited studies so far to describe how a bacterial second messenger molecule can directly participate in distinctive antibiotic tolerance characteristics of a cell in a mechanism-dependent manner. Here we show that intracellular cyclic di-AMP (c-di-AMP) concentration can modulate drug sensitivity of Mycobacterium smegmatis by interacting with an effector protein or interfering with the 5'-UTR regions in mRNA of the genes and thus causing transcriptional downregulation of important genes in the pathways. We studied four antibiotics with different mechanisms of action: rifampicin, ciprofloxacin, erythromycin, and tobramycin and subsequently found that the level of drug sensitivity of the bacteria is directly proportional to the c-di-AMP concentration inside the cell. Further, we unraveled the underlying molecular mechanisms to delineate the specific genes and pathways regulated by c-di-AMP and hence result in differential drug sensitivity in M. smegmatis., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Pseudomonas aeruginosa is a cause of chronic respiratory tract infections in people with cystic fibrosis (CF), non-CF bronchiectasis, and chronic obstructive pulmonary disease. Prolonged infection allows the accumulation of mutations and horizontal gene transfer, increasing the likelihood of adaptive phenotypic traits. Adaptation is proposed to arise first in bacterial populations colonizing upper airway environments. Here, we model this process using an experimental evolution approach. Pseudomonas aeruginosa PAO1, which is not airway adapted, was serially passaged, separately, in media chemically reflective of upper or lower airway environments. To explore whether the CF environment selects for unique traits, we separately passaged PAO1 in airway-mimicking media with or without CF-specific factors. Our findings demonstrated that all airway environments-sinus and lungs, under CF and non-CF conditions-selected for loss of twitching motility, increased resistance to multiple antibiotic classes, and a hyper-biofilm phenotype. These traits conferred increased airway colonization potential in an in vivo model. CF-like conditions exerted stronger selective pressures, leading to emergence of more pronounced phenotypes. Loss of twitching was associated with mutations in type IV pili genes. Type IV pili mediate surface attachment, twitching, and induction of cAMP signalling. We additionally identified multiple evolutionary routes to increased biofilm formation involving regulation of cyclic-di-GMP signalling. These included the loss of function mutations in bifA and dipA phosphodiesterase genes and activating mutations in the siaA phosphatase. These data highlight that airway environments select for traits associated with sessile lifestyles and suggest upper airway niches support emergence of phenotypes that promote establishment of lung infection., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

The Ca 2+ ion is an important second messenger in lymphocytes, similarly to its function in other mammalian cells. The generation of long-lasting intracellular Ca 2+ elevations is essential for Ca 2+ -dependent gene transcription, proliferation, differentiation, and cytokine production in lymphocytes. Since store-operated Ca 2+ entry (SOCE) is considered the predominant mode of Ca 2+ influx in lymphocytes, the activation and function of lymphocytes can be generally predicted by monitoring SOCE. A method suitable for dynamic monitoring of Ca 2+ influx using fura-2 labeling in lymphocytes is introduced in this chapter. Using this technique, large-scale screening of the activation status of primary or cultured lymphocytes can be realized., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The ability of Pseudomonas aeruginosa to establish chronic infections is associated with an effective switch from a motile to a sessile lifestyle. This proficiency is controlled by intracellular levels of the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Targeting the c-di-GMP network could be a strategy to interfere with P. aeruginosa pathogenicity. Therefore, the development of tools to profile c-di-GMP intracellular levels is crucial. Here, we describe a protocol for the in vivo measurement of c-di-GMP levels in P. aeruginosa., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Prokaryotic type III CRISPR-Cas systems provide immunity against viruses and plasmids using CRISPR-associated Rossman fold (CARF) protein effectors 1-5 . Recognition of transcripts of these invaders with sequences that are complementary to CRISPR RNA guides leads to the production of cyclic oligoadenylate second messengers, which bind CARF domains and trigger the activity of an effector domain 6,7 . Whereas most effectors degrade host and invader nucleic acids, some are predicted to contain transmembrane helices without an enzymatic function. Whether and how these CARF-transmembrane helix fusion proteins facilitate the type III CRISPR-Cas immune response remains unknown. Here we investigate the role of cyclic oligoadenylate-activated membrane protein 1 (Cam1) during type III CRISPR immunity. Structural and biochemical analyses reveal that the CARF domains of a Cam1 dimer bind cyclic tetra-adenylate second messengers. In vivo, Cam1 localizes to the membrane, is predicted to form a tetrameric transmembrane pore, and provides defence against viral infection through the induction of membrane depolarization and growth arrest. These results reveal that CRISPR immunity does not always operate through the degradation of nucleic acids, but is instead mediated via a wider range of cellular responses., (© 2024. The Author(s).)

In adrenal fasciculata cells stimulated by ACTH, Ca 2+ and cAMP play indispensable roles as second messengers in cortisol production. However, whether their second messengers cooperatively or independently participate in steroid production remains unclear. We focused on the roles of Ca 2+ and cAMP in cortisol production in bovine adrenal fasciculata cells stimulated by ACTH for a relatively short period (1 h). Incubation of the cells with 100 pM ACTH in Ca 2+ -containing (normal) medium for 1 h increased cortisol production without affecting cAMP content. In contrast, treatment of the cells with the peptide at a higher concentration (1 nM) significantly augmented both cortisol production and cAMP content. However, ACTH did not increase either of them in the Ca 2+ -free medium. ACTH rapidly increased the intracellular free Ca 2+ concentration ([Ca 2+ ] i ) in the normal medium, but did not influence [Ca 2+ ] i in the Ca 2+ -free medium, indicating that ACTH caused Ca 2+ influx into the cells. ACTH-induced Ca 2+ influx and cortisol production were suppressed by a voltage-sensitive L-type Ca 2+ channel blocker but not by a T-type, N-type, or P-type Ca 2+ channel blocker. In contrast, dibutyryl cAMP, a cell-permeable cAMP analog, greatly enhanced cortisol production in the normal or Ca 2+ -free medium and slowly caused Ca 2+ influx into the cells. These results strongly suggest that Ca 2+ , as a second messenger, is more critical than cAMP for cortisol production. However, both second messengers jointly participate in the production in adrenal fasciculata cells stimulated by ACTH.

Climate change and the increasing frequency of high temperature (HT) events are significant threats to global crop yields. To address this, a comprehensive understanding of how plants respond to heat shock (HS) is essential. Signaling pathways involving calcium (Ca 2+ ), a versatile second messenger in plants, encode information through temporal and spatial variations in ion concentration. Ca 2+ is detected by Ca 2+ -sensing effectors, including channels and binding proteins, which trigger specific cellular responses. At elevated temperatures, the cytosolic concentration of Ca 2+ in plant cells increases rapidly, making Ca 2+ signals the earliest response to HS. In this review, we discuss the crucial role of Ca 2+ signaling in raising plant thermotolerance, and we explore its multifaceted contributions to various aspects of the plant HS response (HSR).

Coordinated events of calcium (Ca 2+ ) released from the endoplasmic reticulum (ER) are key second messengers in excitable cells. In pain-sensing dorsal root ganglion (DRG) neurons, these events can be observed as Ca 2+ sparks, produced by a combination of ryanodine receptors (RyR) and inositol 1,4,5-triphosphate receptors (IP3R1). These microscopic signals offer the neuronal cells with a possible means of modulating the subplasmalemmal Ca 2+ handling, initiating vesicular exocytosis. With super-resolution dSTORM and expansion microscopies, we visualised the nanoscale distributions of both RyR and IP3R1 that featured loosely organised clusters in the subplasmalemmal regions of cultured rat DRG somata. We adapted a novel correlative microscopy protocol to examine the nanoscale patterns of RyR and IP3R1 in the locality of each Ca 2+ spark. We found that most subplasmalemmal sparks correlated with relatively small groups of RyR whilst larger sparks were often associated with larger groups of IP3R1. These data also showed spontaneous Ca 2+ sparks in <30% of the subplasmalemmal cell area but consisted of both these channel species at a 3.8-5 times higher density than in nonactive regions of the cell. Taken together, these observations reveal distinct patterns and length scales of RyR and IP3R1 co-clustering at contact sites between the ER and the surface plasmalemma that encode the positions and the quantity of Ca 2+ released at each Ca 2+ spark.

Cyclic dimeric adenosine monophosphate (c-di-AMP) has been well studied in bacteria, including those of the genus Streptococcus, since the first recognition of this dinucleotide in 2008. Streptococci possess a sole diadenylate cyclase, CdaA, and distinct c-di-AMP phosphodiesterases. Interestingly, cdaA is required for viability of some streptococcal species but not all when streptococci are grown in standard laboratory media. Bacteria of this genus also have distinct c-di-AMP effector proteins, diverse c-di-AMP-signaling pathways, and subsequent biological outcomes. In streptococci, c-di-AMP may influence bacterial growth, morphology, biofilm formation, competence program, drug resistance, and bacterial pathogenesis. c-di-AMP secreted by streptococci has also been shown to interact with the mammalian host and induces immune responses including type I interferon production. In this review, we summarize the reported c-di-AMP networks in seven species of the genus Streptococcus, which cause diverse clinical manifestations, and propose future perspectives to investigate the signaling molecule in these streptococcal pathogens., (© 2023 John Wiley & Sons Ltd.)

3',5'-cyclic adenosine monophosphate (cAMP) is a second messenger critically involved in the control of a myriad of processes with significant implications for vascular and cardiac cell function. The temporal and spatial compartmentalization of cAMP is governed by the activity of phosphodiesterases (PDEs), a superfamily of enzymes responsible for the hydrolysis of cyclic nucleotides. Through the fine-tuning of cAMP signaling, PDE4 enzymes could play an important role in cardiac hypertrophy and arrhythmogenesis, while it decisively influences vascular homeostasis through the control of vascular smooth muscle cell proliferation, migration, differentiation and contraction, as well as regulating endothelial permeability, angiogenesis, monocyte/macrophage activation and cardiomyocyte function. This review summarizes the current knowledge and recent advances in understanding the contribution of the PDE4 subfamily to cardiovascular function and underscores the intricate challenges associated with targeting PDE4 enzymes as a therapeutic strategy for the management of cardiovascular diseases.

Previous studies have demonstrated that bis-(3',5')-cyclic diguanosine monophosphate (bis-3',5'-c-di-GMP) is a ubiquitous second messenger employed by bacteria. Here, we report that 2',3'-cyclic guanosine monophosphate (2',3'-cGMP) controls the important biological functions, quorum sensing (QS) signaling systems and virulence in Ralstonia solanacearum through the transcriptional regulator RSp0980. This signal specifically binds to RSp0980 with high affinity and thus abolishes the interaction between RSp0980 and the promoters of target genes. In-frame deletion of RSp0334, which contains an evolved GGDEF domain with a LLARLGGDQF motif required to catalyze 2',3'-cGMP to (2',5')(3',5')-cyclic diguanosine monophosphate (2',3'-c-di-GMP), altered the abovementioned important phenotypes through increasing the intracellular 2',3'-cGMP levels. Furthermore, we found that 2',3'-cGMP, its receptor and the evolved GGDEF domain with a LLARLGGDEF motif also exist in the human pathogen Salmonella typhimurium. Together, our work provides insights into the unusual function of the GGDEF domain of RSp0334 and the special regulatory mechanism of 2',3'-cGMP signal in bacteria., (© 2023. The Author(s).)

Cyclic di-GMP (c-di-GMP) is a second messenger that transduces extracellular stimuli into cellular responses and regulates various biological processes in bacteria. H-NS is a global regulatory protein that represses expression of many genes, but how H-NS activity is modulated by environmental signals remains largely unclear. Here, we show that high intracellular c-di-GMP levels, induced by environmental cues, relieve H-NS-mediated transcriptional silencing in Salmonella enterica serovar Typhimurium. We find that c-di-GMP binds to the H-NS protein to inhibit its binding to DNA, thus derepressing genes silenced by H-NS. However, c-di-GMP is unable to displace H-NS from DNA. In addition, a K107A mutation in H-NS abolishes response to c-di-GMP but leaves its DNA binding activity unaffected in vivo. Our results thus suggest a mechanism by which H-NS acts as an environment-sensing regulator in Gram-negative bacteria., (© 2023. The Author(s).)

The mycobacterial repressor, DarR, a TetR family regulator (TFR), was the first transcription regulator shown to bind c-di-AMP. However, the molecular basis for this interaction and the mechanism involved in DNA binding by DarR remain unknown. Here we describe DarR-c-di-AMP and DarR-DNA structures and complementary biochemical assays. The DarR-c-di-AMP structure reveals a unique effector binding site for a TFR, located between DarR dimer subunits. Strikingly, we show this motif also binds cAMP. The location of the adenine nucleotide binding site between subunits suggests this interaction may facilitate dimerization and hence DNA binding. Indeed, biochemical assays show cAMP enhances DarR DNA binding. Finally, DarR-DNA structures reveal a distinct TFR DNA-binding mechanism involving two interacting dimers on the DNA. Thus, the combined data unveil a newly described second messenger binding motif and DNA binding mode for this important family of regulators., (© 2023. The Author(s).)

Diuretic hormones (DHs) bind to G protein-coupled receptors (GPCRs), regulating water and ion balance to maintain homeostasis in animals. Two distinct DHs are known in insects: calcitonin (CT)-like DH31 and corticotropin-releasing factor (CRF)-like DH44. In this study, we identified and characterized DH31 and two DH31 GPCR variants, DH31-Ra and DH31-Rb, from spotted-wing drosophila, Drosophila suzukii, a globally prevalent vinegar fly causing severe damage to small fruits. Both GPCRs are active, but DH31-Ra is the dominant receptor based on gene expression analyses and DH31 peptide binding affinities. A notable difference between the two variants lies in 1) the GPCR structures of their C-termini and 2) the utilization of second messengers, and the amino acid sequences of the two variants are identical. DH31-Ra contains 12 additional amino acids, providing different intracellular C-terminal configurations. DH31-Ra utilizes both cAMP and Ca 2+ as second messengers, whereas DH31-Rb utilizes only cAMP; this is the first time reported for an insect CT-like DH31 peptide. DH31 stimulated fluid secretion in D. suzukii adults, and secretion increased in a dose-dependent manner. However, when the fly was injected with a mixture of DH31 and CAPA, an anti-diuretic hormone, fluid secretion was suppressed. Here, we discuss the structures of the DH31 receptors and the differential signaling pathways, including second messengers, involved in fly diuresis. These findings provide fundamental insights into the characterization of D. suzukii DH31 and DH31-Rs, and facilitate the identification of potential biological targets for D. suzukii management., (Published by Elsevier Ltd.)