TIR domains of plant immune receptors are 2',3'-cAMP/cGMP synthetases mediating cell death.

2',3'-cAMP is a positional isomer of the well-established second messenger 3',5'-cAMP, but little is known about the biology of this noncanonical cyclic nucleotide monophosphate (cNMP). Toll/interleukin-1 receptor (TIR) domains of nucleotide-binding leucine-rich repeat (NLR) immune receptors have the NADase function necessary but insufficient to activate plant immune responses. Here, we show that plant TIR proteins, besides being NADases, act as 2',3'-cAMP/cGMP synthetases by hydrolyzing RNA/DNA. Structural data show that a TIR domain adopts distinct oligomers with mutually exclusive NADase and synthetase activity. Mutations specifically disrupting the synthetase activity abrogate TIR-mediated cell death in Nicotiana benthamiana (Nb), supporting an important role for these cNMPs in TIR signaling. Furthermore, the Arabidopsis negative regulator of TIR-NLR signaling, NUDT7, displays 2',3'-cAMP/cGMP but not 3',5'-cAMP/cGMP phosphodiesterase activity and suppresses cell death activity of TIRs in Nb. Our study identifies a family of 2',3'-cAMP/cGMP synthetases and establishes a critical role for them in plant immune responses.

Putrescine and Its Metabolic Precursor Arginine Promote Biofilm and c-di-GMP Synthesis in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic bacterial pathogen, can synthesize and catabolize several small cationic molecules known as polyamines. In several clades of bacteria, polyamines regulate biofilm formation, a lifestyle-switching process that confers resistance to environmental stress. The polyamine putrescine and its biosynthetic precursors, l-arginine and agmatine, promote biofilm formation in Pseudomonas spp. However, it remains unclear whether the effect is a direct effect of polyamines or occurs through a metabolic derivative. Here, we used a genetic approach to demonstrate that putrescine accumulation, either through disruption of the spermidine biosynthesis pathway or the catabolic putrescine aminotransferase pathway, promoted biofilm formation in P. aeruginosa. Consistent with this observation, exogenous putrescine robustly induced biofilm formation in P. aeruginosa that was dependent on putrescine uptake and biosynthesis pathways. Additionally, we show that l-arginine, the biosynthetic precursor of putrescine, also promoted biofilm formation but did so by a mechanism independent of putrescine or agmatine conversion. We found that both putrescine and l-arginine induced a significant increase in the intracellular level of bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) (c-di-GMP), a bacterial second messenger widely found in Proteobacteria that upregulates biofilm formation. Collectively these data show that putrescine and its metabolic precursor, arginine, promote biofilm and c-di-GMP synthesis in P. aeruginosa. IMPORTANCE Biofilm formation allows bacteria to physically attach to a surface, confer tolerance to antimicrobial agents, and promote resistance to host immune responses. As a result, the regulation of biofilm formation is often crucial for bacterial pathogens to establish chronic infections. A primary mechanism of biofilm promotion in bacteria is the molecule c-di-GMP, which promotes biofilm formation. The level of c-di-GMP is tightly regulated by bacterial enzymes. In this study, we found that putrescine, a small molecule ubiquitously found in eukaryotic cells, robustly enhances P. aeruginosa biofilm and c-di-GMP. We propose that P. aeruginosa may sense putrescine as a host-associated signal that triggers a lifestyle switch that favors chronic infection.

CsrA Enhances Cyclic-di-GMP Biosynthesis and Yersinia pestis Biofilm Blockage of the Flea Foregut by Alleviating Hfq-Dependent Repression of the hmsT mRNA.

Plague-causing Yersinia pestis is transmitted through regurgitation when it forms a biofilm-mediated blockage in the foregut of its flea vector. This biofilm is composed of an extracellular polysaccharide substance (EPS) produced when cyclic-di-GMP (c-di-GMP) levels are elevated. The Y. pestis diguanylate cyclase enzymes HmsD and HmsT synthesize c-di-GMP. HmsD is required for biofilm blockage formation but contributes minimally to in vitro biofilms. HmsT, however, is necessary for in vitro biofilms and contributes to intermediate rates of biofilm blockage. C-di-GMP synthesis is regulated at the transcriptional and posttranscriptional levels. In this, the global RNA chaperone, Hfq, posttranscriptionally represses hmsT mRNA translation. How c-di-GMP levels and biofilm blockage formation is modulated by nutritional stimuli encountered in the flea gut is unknown. Here, the RNA-binding regulator protein CsrA, which controls c-di-GMP-mediated biofilm formation and central carbon metabolism responses in many Gammaproteobacteria, was assessed for its role in Y. pestis biofilm formation. We determined that CsrA was required for markedly greater c-di-GMP and EPS levels when Y. pestis was cultivated on alternative sugars implicated in flea biofilm blockage metabolism. Our assays, composed of mobility shifts, quantification of mRNA translation, stability, and abundance, and epistasis analyses of a csrA hfq double mutant strain substantiated that CsrA represses hfq mRNA translation, thereby alleviating Hfq-dependent repression of hmsT mRNA translation. Additionally, a csrA mutant exhibited intermediately reduced biofilm blockage rates, resembling an hmsT mutant. Hence, we reveal CsrA-mediated control of c-di-GMP synthesis in Y. pestis as a tiered, posttranscriptional regulatory process that enhances biofilm blockage-mediated transmission from fleas. IMPORTANCE Yersinia pestis, the bacterial agent of bubonic plague, produces a c-di-GMP-dependent biofilm-mediated blockage of the flea vector foregut to facilitate its transmission by flea bite. However, the intricate molecular regulatory processes that underlie c-di-GMP-dependent biofilm formation and thus, biofilm-mediated blockage in response to the nutritional environment of the flea are largely undefined. This study provides a novel mechanistic understanding of how CsrA transduces alternative sugar metabolism cues to induce c-di-GMP-dependent biofilm formation required for efficient Y. pestis regurgitative transmission through biofilm-mediated flea foregut blockage. The Y. pestis-flea interaction represents a unique, biologically relevant, in vivo perspective on the role of CsrA in biofilm regulation.

Elevated Extracellular cGMP Produced after Exposure to Enterotoxigenic Escherichia coli Heat-Stable Toxin Induces Epithelial IL-33 Release and Alters Intestinal Immunity.

Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries. Previous studies identified heat-stable enterotoxin (ST)-producing ETEC as a prevalent diarrheal pathogen in children younger than 5 years. While many studies have evaluated the interaction of ETEC heat-labile enterotoxin (LT) with host epithelium and immunity, few investigations have attempted similar studies with ST. To further understand ST pathogenesis, we examined the impact of ST on cGMP localization, epithelial cell cytokine production, and antibody development following immunization. In addition to robust intracellular cGMP in T84 cells in the presence of phosphodiesterase inhibitors (PDEis) that prevent the breakdown of cyclic nucleotides, we found that prolonged ST intoxication induced extracellular cGMP accumulation in the presence or absence of PDEis. Further, ST intoxication induced luminal cGMP in vivo in mice, suggesting that secreted cGMP may have other cellular functions. Using transcriptome sequencing (RNA-seq) and quantitative PCR (qPCR), we demonstrated that ST intoxication, or treatment with the clinically used ST mimic linaclotide, altered inflammatory cytokine gene expression, including the interleukin 1 (IL-1) family member IL-33, which could also be induced by cell-permeative 8-Br-cGMP. Finally, when present during immunization, ST suppressed induction of antibodies to specific antigens. In conclusion, our studies indicate that ST modulates epithelial cell physiology and the interplay between the epithelial and immune compartments.

Plasmodium falciparum Guanylyl Cyclase-Alpha and the Activity of Its Appended P4-ATPase Domain Are Essential for cGMP Synthesis and Blood-Stage Egress.

Guanylyl cyclases (GCs) synthesize cyclic GMP (cGMP) and, together with cyclic nucleotide phosphodiesterases, are responsible for regulating levels of this intracellular messenger which mediates myriad functions across eukaryotes. In malaria parasites (Plasmodium spp), as well as their apicomplexan and ciliate relatives, GCs are associated with a P4-ATPase-like domain in a unique bifunctional configuration. P4-ATPases generate membrane bilayer lipid asymmetry by translocating phospholipids from the outer to the inner leaflet. Here, we investigate the role of Plasmodium falciparum guanylyl cyclase alpha (GCα) and its associated P4-ATPase module, showing that asexual blood-stage parasites lacking both the cyclase and P4-ATPase domains are unable to egress from host erythrocytes. GCα-null parasites cannot synthesize cGMP or mobilize calcium, a cGMP-dependent protein kinase (PKG)-driven requirement for egress. Using chemical complementation with a cGMP analogue and point mutagenesis of a crucial conserved residue within the P4-ATPase domain, we show that P4-ATPase activity is upstream of and linked to cGMP synthesis. Collectively, our results demonstrate that GCα is a critical regulator of PKG and that its associated P4-ATPase domain plays a primary role in generating cGMP for merozoite egress.IMPORTANCE The clinical manifestations of malaria arise due to successive rounds of replication of Plasmodium parasites within red blood cells. Once mature, daughter merozoites are released from infected erythrocytes to invade new cells in a tightly regulated process termed egress. Previous studies have shown that the activation of cyclic GMP (cGMP) signaling is critical for initiating egress. Here, we demonstrate that GCα, a unique bifunctional enzyme, is the sole enzyme responsible for cGMP production during the asexual blood stages of Plasmodium falciparum and is required for the cellular events leading up to merozoite egress. We further demonstrate that in addition to the GC domain, the appended ATPase-like domain of GCα is also involved in cGMP production. Our results highlight the critical role of GCα in cGMP signaling required for orchestrating malaria parasite egress.

Translation of HDAC6 PET Imaging Using [18F]EKZ-001-cGMP Production and Measurement of HDAC6 Target Occupancy in Nonhuman Primates.

Histone deacetylase 6 (HDAC6) is a multifunctional cytoplasmic enzyme involved in diverse cellular processes such as intracellular transport and protein quality control. Inhibition of HDAC6 can alleviate defects in cell and rodent models of certain diseases, particularly neurodegenerative disorders, including Alzheimer's disease and amyotrophic lateral sclerosis. However, while HDAC6 represents a potentially powerful therapeutic target, development of effective brain-penetrant HDAC6 inhibitors remains challenging. Recently, [18F]EKZ-001 ([18F]Bavarostat), a brain-penetrant positron emission tomography (PET) radioligand with high affinity and selectivity toward HDAC6, was developed and evaluated preclinically for its ability to bind HDAC6. Herein, we describe the efficient and robust fully automated current Good Manufacturing Practices (cGMP) compliant production method. [18F]EKZ-001 quantification methods were validated in nonhuman primates (NHP) using full kinetic modeling, and [18F]EKZ-001 PET was applied to compare dose-occupancy relationships between two HDAC6 inhibitors, EKZ-317 and ACY-775. [18F]EKZ-001 is cGMP produced with an average decay-corrected radiochemical yield of 14% and an average molar activity of 204 GBq/µmol. We demonstrate that a two-tissue compartmental model and Logan graphical analysis are appropriate for [18F]EKZ-001 PET quantification in NHP brain. Blocking studies show that the novel compound EKZ-317 achieves higher target occupancy than ACY-775. This work supports the translation of [18F]EKZ-001 PET for first-in-human studies.

Structure of the active GGEEF domain of a diguanylate cyclase from Vibrio cholerae.

Cyclic-di-GMP (c-di-GMP) synthesized by diguanylate cyclases has been an important and ubiquitous secondary messenger in almost all bacterial systems. In Vibrio cholerae, c-di-GMP plays an intricate role in the production of the exopolysaccharide matrix, and thereby, in biofilm formation. The formation of the surface biofilm enables the bacteria to survive in aquatic bodies, when not infecting a human host. Diguanylate cyclases are the class of enzymes which synthesize c-di-GMP from two molecules of GTP and are endowed with a GGDEF or, a GGEEF signature domain. The VC0395_0300 protein from V. cholerae, has been established as a diguanylate cyclase with a necessary role in biofilm formation. Here we present the structure of an N-terminally truncated form of VC0395_0300, which retains the active GGEEF domain for diguanylate cyclase activity but lacks 160 residues from the poorly organized N-terminal domain. X-ray diffraction data was collected from a crystal of VC0395_0300(161-321) to a resolution of 1.9 Å. The structure displays remarkable topological similarity with diguanylate cyclases from other bacterial systems, but lacks the binding site for c-di-GMP present in its homologues. Finally, we demonstrate the ability of the truncated diguanylate cyclase VC0395_0300(161-321) to produce c-di-GMP, and its role in biofilm formation for the bacteria.

Natriuretic Peptides Attenuate Retinal Pathological Neovascularization Via Cyclic Guanosine Monophosphate Signaling in Pericytes and Astrocytes.

OBJECTIVE: In proliferative retinopathies, complications derived from neovascularization cause blindness. During early disease, pericyte's apoptosis contributes to endothelial dysfunction and leakage. Hypoxia then drives VEGF (vascular endothelial growth factor) secretion and pathological neoangiogenesis. Cardiac ANP (atrial natriuretic peptide) contributes to systemic microcirculatory homeostasis. ANP is also formed in the retina, with unclear functions. Here, we characterized whether endogenously formed ANP regulates retinal (neo)angiogenesis. Approach and Results: Retinal vascular development and ischemia-driven neovascularization were studied in mice with global deletion of GC-A (guanylyl cyclase-A), the cGMP (cyclic guanosine monophosphate)-forming ANP receptor. Mice with a floxed GC-A gene were interbred with Tie2-Cre, GFAP-Cre, or PDGF-Rß-CreERT2 lines to dissect the endothelial, astrocyte versus pericyte-mediated actions of ANP in vivo. In neonates with global GC-A deletion (KO), vascular development was mildly delayed. Moreover, such KO mice showed augmented vascular regression and exacerbated ischemia-driven neovascularization in the model of oxygen-induced retinopathy. Notably, absence of GC-A in endothelial cells did not impact retinal vascular development or pathological neovascularization. In vitro ANP/GC-A/cGMP signaling, via activation of cGMP-dependent protein kinase I, inhibited hypoxia-driven astrocyte's VEGF secretion and TGF-ß (transforming growth factor beta)-induced pericyte apoptosis. In neonates lacking ANP/GC-A signaling in astrocytes, vascular development and hyperoxia-driven vascular regression were unaltered; ischemia-induced neovascularization was modestly increased. Remarkably, inactivation of GC-A in pericytes retarded physiological retinal vascularization and markedly enhanced cell apoptosis, vascular regression, and subsequent neovascularization in oxygen-induced retinopathy. CONCLUSIONS: Protective pericyte effects of the ANP/GC-A/cGMP pathway counterregulate the initiation and progression of experimental proliferative retinopathy. Our observations indicate augmentation of endogenous pericyte ANP signaling as target for treatment of retinopathies associated with neovascularization.

Low-Dose Carbon Monoxide Inhibits Rhinovirus Replication in Human Alveolar and Airway Epithelial Cells.

Carbon monoxide (CO) and nitric oxide (NO) exhibit physiological properties that include the activation of guanylate cyclase. NO inhibits replication of rhinovirus (RV), a major cause of the common cold and exacerbation of bronchial asthma and chronic obstructive pulmonary disease. However, the anti-rhinoviral effects of CO remain unclear. This study investigated whether the exogenous application of low-dose CO could inhibit RV replication in human alveolar and airway epithelial cells. A549 human lung carcinoma cells with alveolar epithelial features and primary cultures of human tracheal epithelial (HTE) cells were pretreated with CO (100 ppm) and infected with a major group RV, type 14 RV (RV14). CO exposure reduced RV14 titers in the supernatants and RV RNA levels in A549 and HTE cells. The treatment with a guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, reversed the inhibitory effects of CO exposure on RV14 replication in A549 cells. Pretreatment of A549 cells with 8-Br-cGMP, a cell-permeable cGMP analog, caused the decrease in RV14 replication, while CO exposure increased cGMP production. CO exposure also increased the expression levels of interferon (IFN)-γ mRNA and protein. In contrast, pretreatment with CO did not increase DNA fragmentation and did not reduce the expression of intercellular adhesion molecule-1, the RV14 receptor, or the number of acidic endosomes, through which RV RNA enters the cytoplasm. These findings suggest that low-dose CO may decrease RV14 replication in alveolar and airway epithelial cells. IFN-γ production, which is induced by CO exposure via guanylate cyclase activation-mediated cGMP production, may be involved in RV14 replication inhibition.

CRRL269.

RATIONALE: Acute kidney injury (AKI) has a high prevalence and mortality in critically ill patients. It is also a powerful risk factor for heart failure incidence driven by hemodynamic changes and neurohormonal activation. However, no drugs have been approved by the Food and Drug Administration. Endogenous pGC-A (particulate guanylyl cyclase A receptor) activators were reported to preserve renal function and improve mortality in AKI patients, although hypotension accompanied by pGC-A activators have limited their therapeutic potential. OBJECTIVE: We investigated the therapeutic potential of a nonhypotensive pGC-A activator/designer natriuretic peptide, CRRL269, in a short-term, large animal model of ischemia-induced AKI and also investigated the potential of uCNP (urinary C-type natriuretic peptide) as a biomarker for AKI. METHODS AND RESULTS: We first showed that CRRL269 stimulated cGMP generation, suppressed plasma angiotensin II, and reduced cardiac filling pressures without lowering blood pressure in the AKI canine model. We also demonstrated that CRRL269 preserved glomerular filtration rate, increased renal blood flow, and promoted diuresis and natriuresis. Further, CRRL269 reduced kidney injury and apoptosis as evidenced by ex vivo histology and tissue apoptosis analysis. We also showed, compared with native pGC-A activators, that CRRL269 is a more potent inhibitor of apoptosis in renal cells and induced less decreases in intracellular Ca2+ concentration in vascular smooth muscle cells. The renal antiapoptotic effects were at least mediated by cGMP/PKG pathway. Further, CRRL269 inhibited proapoptotic genes expression using a polymerase chain reaction gene array. Additionally, we demonstrated that AKI increased uCNP levels. CONCLUSIONS: Our study supports developing CRRL269 as a novel renocardiac protective agent for AKI treatment.

Structural basis of DSF recognition by its receptor RpfR and its regulatory interaction with the DSF synthase RpfF.

The diffusible signal factors (DSFs) are a family of quorum-sensing autoinducers (AIs) produced and detected by numerous gram-negative bacteria. The DSF family AIs are fatty acids, differing in their acyl chain length, branching, and substitution but having in common a cis-2 double bond that is required for their activity. In both human and plant pathogens, DSFs regulate diverse phenotypes, including virulence factor expression, antibiotic resistance, and biofilm dispersal. Despite their widespread relevance to both human health and agriculture, the molecular basis of DSF recognition by their cellular receptors remained a mystery. Here, we report the first structure-function studies of the DSF receptor regulation of pathogenicity factor R (RpfR). We present the X-ray crystal structure of the RpfR DSF-binding domain in complex with the Burkholderia DSF (BDSF), which to our knowledge is the first structure of a DSF receptor in complex with its AI. To begin to understand the mechanistic role of the BDSF-RpfR contacts observed in the biologically important complex, we have also determined the X-ray crystal structure of the RpfR DSF-binding domain in complex with the inactive, saturated isomer of BDSF, dodecanoic acid (C12:0). In addition to these ligand-receptor complex structures, we report the discovery of a previously overlooked RpfR domain and show that it binds to and negatively regulates the DSF synthase regulation of pathogenicity factor F (RpfF). We have named this RpfR region the RpfF interaction (FI) domain, and we have determined its X-ray crystal structure alone and in complex with RpfF. These X-ray crystal structures, together with extensive complementary in vivo and in vitro functional studies, reveal the molecular basis of DSF recognition and the importance of the cis-2 double bond to DSF function. Finally, we show that throughout cellular growth, the production of BDSF by RpfF is post-translationally controlled by the RpfR N-terminal FI domain, affecting the cellular concentration of the bacterial second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Thus, in addition to describing the molecular basis for the binding and specificity of a DSF for its receptor, we describe a receptor-synthase interaction regulating bacterial quorum-sensing signaling and second messenger signal transduction.

Influence of the N-terminal segment and the PHY-tongue element on light-regulation in bacteriophytochromes.

Photoreceptors enable the integration of ambient light stimuli to trigger lifestyle adaptations via modulation of central metabolite levels involved in diverse regulatory processes. Red light-sensing bacteriophytochromes are attractive targets for the development of innovative optogenetic tools because of their natural modularity of coupling with diverse functionalities and the natural availability of the light-absorbing biliverdin chromophore in animal tissues. However, a rational design of such tools is complicated by the poor understanding of molecular mechanisms of light signal transduction over long distances-from the site of photon absorption to the active site of downstream enzymatic effectors. Here we show how swapping structural elements between two bacteriophytochrome homologs provides additional insight into light signal integration and effector regulation, involving a fine-tuned interplay of important structural elements of the sensor, as well as the sensor-effector linker. Facilitated by the availability of structural information of inhibited and activated full-length structures of one of the two homologs (Idiomarina species A28L phytochrome-activated diguanylyl cyclase (IsPadC)) and characteristic differences in photoresponses of the two homologs, we identify an important cross-talk between the N-terminal segment, containing the covalent attachment site of the chromophore, and the PHY-tongue region. Moreover, we highlight how these elements influence the dynamic range of photoactivation and how activation can be improved to light/dark ratios of ∼800-fold by reducing basal dark-state activities at the same time as increasing conversion in the light state. This will enable future optimization of optogenetic tools aiming at a direct allosteric regulation of enzymatic effectors.

Production of 8-nitro-cGMP in osteocytic cells and its upregulation by parathyroid hormone and prostaglandin E2.

Osteocytes regulate bone remodeling, especially in response to mechanical loading and unloading of bone, with nitric oxide reported to play an important role in that process. In the present study, we found that 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), a second messenger of nitric oxide in various types of cells, was produced by osteocytes in bone tissue as well as cultured osteocytic Ocy454 cells. The amount of 8-nitro-cGMP in Ocy454 cells increased during incubation with parathyroid hormone or prostaglandin E2, both of which are known to upregulate receptor activator of nuclear factor-κB ligand (RANKL) mRNA expression in osteocytes. On the other hand, exogenous 8-nitro-cGMP did not have effects on either the presence or absence of these bioactive substances. Furthermore, neither an inhibitor of nitric oxide synthase nor 8-bromo-cGMP, a cell-permeable analog of cGMP, showed remarkable effects on mRNA expression of sclerostin or RANKL. These results indicate that neither nitric oxide nor its downstream compounds, including 8-nitro-cGMP, alone are sufficient for induction of functional changes in osteocytes.

Scale-Down Model Development in ambr systems: An Industrial Perspective.

High-Throughput (HT) technologies such as miniature bioreactors (MBRs) are increasingly employed within the biopharmaceutical manufacturing industry. Traditionally, these technologies have been utilized for discrete screening approaches during pre-clinical development (e.g., cell line selection and process optimization). However, increasing interest is focused towards their use during late clinical phase process characterization studies as a scale-down model (SDM) of the cGMP manufacturing process. In this review, the authors describe a systematic approach toward SDM development in one of the most widely adopted MBRs, the ambr 15 and 250 mL (Sartorius Stedim Biotech) systems. Recent efforts have shown promise in qualifying ambr systems as SDMs to support more efficient, robust and safe biomanufacturing processes. The authors suggest that combinatorial improvements in process understanding (matching of mass transfer and cellular stress between scales through computational fluid dynamics and in vitro analysis), experimental design (advanced risk assessment and statistical design of experiments), and data analysis (combining uni- and multi-variate techniques) will ultimately yield ambr SDMs applicable for future regulatory submissions.

BUBR1 Insufficiency Is Correlated with eNOS Reduction Experimentally In Vitro and In Vivo, and in Gastric Cancer Tissue.

BACKGROUND/AIM: Budding uninhibited by benzimidazole-related 1 (BUBR1) and endothelial nitric oxide synthase (eNOS) are related to aging and angiogenesis. This study examined the effect of low BUBR1 expression on eNOS expression in vivo, in vitro, and human gastric cancer tissues. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were passaged to investigate the effect of aging on BUBR1 and eNOS expression; expression of eNOS and phospho-eNOS protein was assessed in BUBR1 siRNA-transfected HUVECs. Additionally, guanosine 3',5' cyclic monophosphate (cGMP) and eNOS protein levels were measured in BUBR1-insufficient mice (Bubr1L/-). BUBR1 and eNOS expression levels were also evaluated in human gastric cancer tissues. RESULTS: BUBR1 and eNOS, but not p-eNOS, levels were reduced significantly in aged and BUBR1 siRNA-transfected HUVECs. Additionally, cGMP production and the eNOS protein level were reduced in Bubr1L/- mice. Human gastric cancer tissues with low BUBR1 expression showed no eNOS expression. CONCLUSION: A decrease in BUBR1 reduced eNOS bioavailability through a pathway other than eNOS phosphorylation.

A novel p.(Glu111Val) missense mutation in GUCA1A associated with cone-rod dystrophy leads to impaired calcium sensing and perturbed second messenger homeostasis in photoreceptors.

Guanylate Cyclase-Activating Protein 1 (GCAP1) regulates the enzymatic activity of the photoreceptor guanylate cyclases (GC), leading to inhibition or activation of the cyclic guanosine monophosphate (cGMP) synthesis depending on its Ca2+- or Mg2+-loaded state. By genetically screening a family of patients diagnosed with cone-rod dystrophy, we identified a novel missense mutation with autosomal dominant inheritance pattern (c.332A>T; p.(Glu111Val); E111V from now on) in the GUCA1A gene coding for GCAP1. We performed a thorough biochemical and biophysical investigation of wild type (WT) and E111V human GCAP1 by heterologous expression and purification of the recombinant proteins. The E111V substitution disrupts the coordination of the Ca2+ ion in the high-affinity site (EF-hand 3, EF3), thus significantly decreasing the ability of GCAP1 to sense Ca2+ (∼80-fold higher Kdapp compared to WT). Both WT and E111V GCAP1 form dimers independently on the presence of cations, but the E111V Mg2+-bound form is prone to severe aggregation over time. Molecular dynamics simulations suggest a significantly increased flexibility of both the EF3 and EF4 cation binding loops for the Ca2+-bound form of E111V GCAP1, in line with the decreased affinity for Ca2+. In contrast, a more rigid backbone conformation is observed in the Mg2+-bound state compared to the WT, which results in higher thermal stability. Functional assays confirm that E111V GCAP1 interacts with the target GC with a similar apparent affinity (EC50); however, the mutant shifts the GC inhibition out of the physiological [Ca2+] (IC50E111V ∼10 µM), thereby leading to the aberrant constitutive synthesis of cGMP under conditions of dark-adapted photoreceptors.

Enhanced bacterial mutualism through an evolved biofilm phenotype.

Microbial communities primarily consist of multiple species that affect one another's fitness both directly and indirectly. This study showed that the cocultivation of Paenibacillus amylolyticus and Xanthomonas retroflexus exhibited facultative mutualistic interactions in a static environment, during the course of which a new adapted phenotypic variant of X. retroflexus appeared. Although the emergence of this variant was not directly linked to the presence of P. amylolyticus, its establishment in the coculture enhanced the productivity of both species due to mutations that stimulated biofilm formation. The mutations were detected in genes encoding a diguanylate cyclase predicted to synthesise cyclic-di-GMP. Examinations of the biofilm formed in cocultures of P. amylolyticus and the new variant of X. retroflexus revealed a distinct spatial organisation: P. amylolyticus only resided in biofilms in association with X. retroflexus and occupied the outer layers. The X. retroflexus variant therefore facilitated increased P. amylolyticus growth as it produced more biofilm biomass. The increase in X. retroflexus biomass was thus not at the expense of P. amylolyticus, demonstrating that interspecies interactions can shape diversification in a mutualistic coculture and reinforce these interactions, ultimately resulting in enhanced communal performance.

Measurement of cGMP-generating and -degrading activities and cGMP levels in cells and tissues: Focus on FRET-based cGMP indicators.

The intracellular messenger molecule cGMP has an established function in the regulation of numerous physiological events. Yet for the identification of further biological cGMP-mediated functions, precise information whether a cGMP response exists in a certain cell type or tissue is mandatory. In this review, the techniques to measure cGMP i.e. cGMP-formation, -degradation or levels are outlined and discussed. As a superior method to measure cGMP, the article focusses on FRET-based cGMP indicators, describes the different cGMP indicators and discusses their advantages and drawbacks. Finally, the successful applications of these cGMP indicators to measure cGMP responses in cells and tissues are outlined and summarized. Hopefully, with the availability of the FRET-based cGMP indicators, the knowledge about the cGMP responses in special cells or tissues is going to increase thereby allowing to assess further cGMP-mediated functional responses and possibly to address their pathophysiology with the available guanylyl cyclase activators, stimulators and PDE inhibitors.

Guanylate cyclase-activating protein 2 contributes to phototransduction and light adaptation in mouse cone photoreceptors.

Light adaptation of photoreceptor cells is mediated by Ca2+-dependent mechanisms. In darkness, Ca2+ influx through cGMP-gated channels into the outer segment of photoreceptors is balanced by Ca2+ extrusion via Na+/Ca2+, K+ exchangers (NCKXs). Light activates a G protein signaling cascade, which closes cGMP-gated channels and decreases Ca2+ levels in photoreceptor outer segment because of continuing Ca2+ extrusion by NCKXs. Guanylate cyclase-activating proteins (GCAPs) then up-regulate cGMP synthesis by activating retinal membrane guanylate cyclases (RetGCs) in low Ca2+ This activation of RetGC accelerates photoresponse recovery and critically contributes to light adaptation of the nighttime rod and daytime cone photoreceptors. In mouse rod photoreceptors, GCAP1 and GCAP2 both contribute to the Ca2+-feedback mechanism. In contrast, only GCAP1 appears to modulate RetGC activity in mouse cones because evidence of GCAP2 expression in cones is lacking. Surprisingly, we found that GCAP2 is expressed in cones and can regulate light sensitivity and response kinetics as well as light adaptation of GCAP1-deficient mouse cones. Furthermore, we show that GCAP2 promotes cGMP synthesis and cGMP-gated channel opening in mouse cones exposed to low Ca2+ Our biochemical model and experiments indicate that GCAP2 significantly contributes to the activation of RetGC1 at low Ca2+ when GCAP1 is not present. Of note, in WT mouse cones, GCAP1 dominates the regulation of cGMP synthesis. We conclude that, under normal physiological conditions, GCAP1 dominates the regulation of cGMP synthesis in mouse cones, but if its function becomes compromised, GCAP2 contributes to the regulation of phototransduction and light adaptation of cones.

The Discovery of ( S)-1-(6-(3-((4-(1-(Cyclopropanecarbonyl)piperidin-4-yl)-2-methylphenyl)amino)-2,3-dihydro-1 H-inden-4-yl)pyridin-2-yl)-5-methyl-1 H-pyrazole-4-carboxylic Acid, a Soluble Guanylate Cyclase Activator Specifically Designed for Topical Ocular Delivery as a Therapy for Glaucoma.

Soluble guanylate cyclase (sGC), the endogenous receptor for nitric oxide (NO), has been implicated in several diseases associated with oxidative stress. In a pathological oxidative environment, the heme group of sGC can be oxidized becoming unresponsive to NO leading to a loss in the ability to catalyze the production of cGMP. Recently a dysfunctional sGC/NO/cGMP pathway has been implicated in contributing to elevated intraocular pressure associated with glaucoma. Herein we describe the discovery of molecules specifically designed for topical ocular administration, which can activate oxidized sGC restoring the ability to catalyze the production of cGMP. These efforts culminated in the identification of compound (+)-23, which robustly lowers intraocular pressure in a cynomolgus model of elevated intraocular pressure over 24 h after a single topical ocular drop and has been selected for clinical evaluation.