NMR Structure of Retinal Guanylate Cyclase Activating Protein 5 (GCAP5) with R22A Mutation That Abolishes Dimerization and Enhances Cyclase Activation.

Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5R22A) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5WT). Here, we present ITC, NMR, and functional analysis of GCAP5R22A to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5R22A binds a total of 3 Ca2+, including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5WT were not detected by ITC, suggesting that R22A may affect the binding of Ca2+ to these sites. The NMR-derived structure of GCAP5R22A is overall similar to that of GCAP5WT (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5R22A lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5R22A with ∼25% higher affinity compared to GCAP5WT, consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.

CARD11 regulates the thymic Treg development in an NF-κB-independent manner.

Introduction: CARD11 is a lymphoid lineage-specific scaffold protein regulating the NF-κB activation downstream of the antigen receptor signal pathway. Defective CARD11 function results in abnormal development and differentiation of lymphocytes, especially thymic regulatory T cells (Treg). Method: In this study, we used patients' samples together with transgenic mouse models carrying pathogenic CARD11 mutations from patients to explore their effects on Treg development. Immunoblotting and a GFP receptor assay were used to evaluate the activation effect of CARD11 mutants on NF-κB signaling. Then the suppressive function of Tregs carrying distinct CARD11 mutations was measured by in vitro suppression assay. Finally, we applied the retroviral transduced bone marrow chimeras to rescue the Treg development in an NF-κB independent manner. Results and discuss: We found CARD11 mutations causing hyper-activated NF-κB signals also gave rise to compromised Treg development in the thymus, similar to the phenotype in Card11 deficient mice. This observation challenges the previous view that CARD11 regulates Treg lineage dependent on the NF-kB activation. Mechanistic investigations reveal that the noncanonical function CARD11, which negatively regulates the AKT/ FOXO1 signal pathway, is responsible for regulating Treg generation. Moreover, primary immunodeficiency patients carrying CARD11 mutation, which autonomously activates NF-κB, also represented the reduced Treg population in their peripheral blood. Our results propose a new regulatory function of CARD11 and illuminate an NF-κB independent pathway for thymic Treg lineage commitment.

Pericyte signaling via soluble guanylate cyclase shapes the vascular niche and microenvironment of tumors.

Pericytes and endothelial cells (ECs) constitute the fundamental components of blood vessels. While the role of ECs in tumor angiogenesis and the tumor microenvironment is well appreciated, pericyte function in tumors remains underexplored. In this study, we used pericyte-specific deletion of the nitric oxide (NO) receptor, soluble guanylate cyclase (sGC), to investigate via single-cell RNA sequencing how pericytes influence the vascular niche and the tumor microenvironment. Our findings demonstrate that pericyte sGC deletion disrupts EC-pericyte interactions, impairing Notch-mediated intercellular communication and triggering extensive transcriptomic reprogramming in both pericytes and ECs. These changes further extended their influence to neighboring cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) through paracrine signaling, collectively suppressing tumor growth. Inhibition of pericyte sGC has minimal impact on quiescent vessels but significantly increases the vulnerability of angiogenic tumor vessels to conventional anti-angiogenic therapy. In conclusion, our findings elucidate the role of pericytes in shaping the tumor vascular niche and tumor microenvironment and support pericyte sGC targeting as a promising strategy for improving anti-angiogenic therapy for cancer treatment.

Structural insight into hormone recognition by the natriuretic peptide receptor-A.

Atrial natriuretic peptide (ANP) plays a central role in the regulation of blood pressure and volume. ANP activities are mediated by natriuretic peptide receptor-A (NPR-A), a single-pass transmembrane receptor harboring intrinsic guanylate cyclase activity. This study investigated the mechanism underlying NPR-A-dependent hormone recognition through the determination of the crystal structures of the NPR-A extracellular hormone-binding domain complexed with full-length ANP, truncated mutants of ANP, and dendroaspis natriuretic peptide (DNP) isolated from the venom of the green Mamba snake, Dendroaspis angusticeps. The bound peptides possessed pseudo-two-fold symmetry, despite the lack of two-fold symmetry in the primary sequences, which enabled the tight coupling of the peptide to the receptor, and evidently contributes to guanylyl cyclase activity. The binding of DNP to the NPR-A was essentially identical to that of ANP; however, the affinity of DNP for NPR-A was higher than that of ANP owing to the additional interactions between distinctive sequences in the DNP and NPR-A. Consequently, our findings provide valuable insights that can be applied to the development of novel agonists for the treatment of various human diseases.

A 2-week treatment with 5-azacytidine improved the hypercontractility state in prostate from obese mice: Role of the nitric oxide-cyclic guanosine monophosphate signalling pathway.

Benign prostatic hyperplasia (BPH) is characterised by increases in prostate volume and contraction. Downregulation of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signalling pathway contributes to prostate dysfunctions. Previous studies in cancer cells or vessels have shown that the epigenetic mechanisms control the gene and protein expression of the enzymes involved in the production of NO and cGMP. This study is aimed to evaluate the effect of a 2-week treatment of 5-azacytidine (5-AZA), a DNA-methyltransferase inhibitor, in the prostate function of mice fed with a high-fat diet. Functional, histological, biochemical and molecular assays were carried out. Obese mice presented greater prostate weight, α-actin expression and contractile response induced by the α-1adrenoceptors agonist. The relaxation induced by the NO-donor and the protein expression of endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) were significantly decreased in the prostate of obese mice. The treatment with 5-AZA reverted the higher expression of α-actin, reduced the hypercontractility state of the prostate and increased the expression of eNOS and sGC and intraprostatic levels of cGMP. When prostates from obese mice treated with 5-AZA were incubated in vitro with inhibitors of the NOS or sGC, the inhibitory effect of 5-AZA was reverted, therefore, showing the involvement of NO and cGMP. In conclusion, our study paves the way to develop or repurpose therapies that recover the expression of eNOS and sGC and, hence, to improve prostate function in BPH.

The nitric oxide-soluble guanylate cyclase-cGMP pathway in pulmonary hypertension: from PDE5 to soluble guanylate cyclase.

The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway plays a key role in the pathogenesis of pulmonary hypertension (PH). Targeted treatments include phosphodiesterase type 5 inhibitors (PDE5i) and sGC stimulators. The sGC stimulator riociguat is approved for the treatment of pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH). sGC stimulators have a dual mechanism of action, enhancing the sGC response to endogenous NO and directly stimulating sGC, independent of NO. This increase in cGMP production via a dual mechanism differs from PDE5i, which protects cGMP from degradation by PDE5, rather than increasing its production. sGC stimulators may therefore have the potential to increase cGMP levels under conditions of NO depletion that could limit the effectiveness of PDE5i. Such differences in mode of action between sGC stimulators and PDE5i could lead to differences in treatment efficacy between the classes. In addition to vascular effects, sGC stimulators have the potential to reduce inflammation, angiogenesis, fibrosis and right ventricular hypertrophy and remodelling. In this review we describe the evolution of treatments targeting the NO-sGC-cGMP pathway, with a focus on PH.

Development and characterization of a Gucy2d-cre mouse to selectively manipulate a subset of inhibitory spinal dorsal horn interneurons.

Recent transcriptomic studies identified Gucy2d (encoding guanylate cyclase D) as a highly enriched gene within inhibitory dynorphin interneurons in the mouse spinal dorsal horn. To facilitate investigations into the role of the Gucy2d+ population in somatosensation, Gucy2d-cre transgenic mice were created to permit chemogenetic or optogenetic manipulation of this subset of spinal neurons. Gucy2d-cre mice created via CRISPR/Cas9 genomic knock-in were bred to mice expressing a cre-dependent reporter (either tdTomato or Sun1.GFP fusion protein), and the resulting offspring were characterized. Surprisingly, a much wider population of spinal neurons was labeled by cre-dependent reporter expression than previous mRNA-based studies would suggest. Although the cre-dependent reporter expression faithfully labeled ~75% of cells expressing Gucy2d mRNA in the adult dorsal horn, it also labeled a substantial number of additional inhibitory neurons in which no Gucy2d or Pdyn mRNA was detected. Moreover, cre-dependent reporter was also expressed in various regions of the brain, including the spinal trigeminal nucleus, cerebellum, thalamus, somatosensory cortex, and anterior cingulate cortex. Injection of AAV-CAG-FLEX-tdTomato viral vector into adult Gucy2d-cre mice produced a similar pattern of cre-dependent reporter expression in the spinal cord and brain, which excludes the possibility that the unexpected reporter-labeling of cells in the deep dorsal horn and brain was due to transient Gucy2d expression during early stages of development. Collectively, these results suggest that Gucy2d is expressed in a wider population of cells than previously thought, albeit at levels low enough to avoid detection with commonly used mRNA-based assays. Therefore, it is unlikely that these Gucy2d-cre mice will permit selective manipulation of inhibitory signaling mediated by spinal dynorphin interneurons, but this novel cre driver line may nevertheless be useful to target a broader population of inhibitory spinal dorsal horn neurons.

Naturally occurring T cell mutations enhance engineered T cell therapies.

Adoptive T cell therapies have produced exceptional responses in a subset of patients with cancer. However, therapeutic efficacy can be hindered by poor T cell persistence and function1. In human T cell cancers, evolution of the disease positively selects for mutations that improve fitness of T cells in challenging situations analogous to those faced by therapeutic T cells. Therefore, we reasoned that these mutations could be co-opted to improve T cell therapies. Here we systematically screened the effects of 71 mutations from T cell neoplasms on T cell signalling, cytokine production and in vivo persistence in tumours. We identify a gene fusion, CARD11-PIK3R3, found in a CD4+ cutaneous T cell lymphoma2, that augments CARD11-BCL10-MALT1 complex signalling and anti-tumour efficacy of therapeutic T cells in several immunotherapy-refractory models in an antigen-dependent manner. Underscoring its potential to be deployed safely, CARD11-PIK3R3-expressing cells were followed up to 418 days after T cell transfer in vivo without evidence of malignant transformation. Collectively, our results indicate that exploiting naturally occurring mutations represents a promising approach to explore the extremes of T cell biology and discover how solutions derived from evolution of malignant T cells can improve a broad range of T cell therapies.

Role of the NO-GC/cGMP signaling pathway in platelet biomechanics.

Cyclic guanosine monophosphate (cGMP) is a second messenger produced by the NO-sensitive guanylyl cyclase (NO-GC). The NO-GC/cGMP pathway in platelets has been extensively studied. However, its role in regulating the biomechanical properties of platelets has not yet been addressed and remains unknown. We therefore investigated the stiffness of living platelets after treatment with the NO-GC stimulator riociguat or the NO-GC activator cinaciguat using scanning ion conductance microscopy (SICM). Stimulation of human and murine platelets with cGMP-modulating drugs decreased cellular stiffness and downregulated P-selectin, a marker for platelet activation. We also quantified changes in platelet shape using deep learning-based platelet morphometry, finding that platelets become more circular upon treatment with cGMP-modulating drugs. To test for clinical applicability of NO-GC stimulators in the context of increased thrombogenicity risk, we investigated the effect of riociguat on platelets from human immunodeficiency virus (HIV)-positive patients taking abacavir sulfate (ABC)-containing regimens. Our results corroborate a functional role of the NO-GC/cGMP pathway in platelet biomechanics, indicating that biomechanical properties such as stiffness or shape could be used as novel biomarkers in clinical research.

Increased platelet activation and development of thrombosis has been linked to a dysfunctional NO-GC/cGMP signaling pathway. How this pathway affects platelet stiffness, however, has not been studied yet. For the first time, we used novel microscopy techniques to investigate stiffness and shape of platelets in human and murine blood samples treated with cGMP modifying drugs. Stiffness contains information about biomechanical properties of the cytoskeleton, and shape quantifies the spreading behavior of platelets. We showed that the NO-GC/cGMP signaling pathway affects platelet stiffness, shape, and activation in human and murine blood. HIV-positive patients are often treated with medication that may disrupt the NO-GC/cGMP signaling pathway, leading to increased cardiovascular risk. We showed that treatment with cGMP-modifying drugs altered platelet shape and aggregation in blood from HIV-negative volunteers but not from HIV-positive patients treated with medication. Our study suggests that platelet stiffness and shape can be biomarkers for estimating cardiovascular risk.

Identification of new multi-substituted 1H-pyrazolo[3,4-c]pyridin-7(6H)-ones as soluble guanylyl cyclase (sGC) stimulators with vasoprotective and anti-inflammatory activities.

Herein, we describe the rational design, synthesis and in vitro functional characterization of new heme-dependent, direct soluble guanylyl cyclase (sGC) agonists. These new compounds bear a 1H-pyrazolo[3,4-c]pyridin-7(6H)-one skeleton, modified to enable efficient sGC binding and stimulation. To gain insights into structure-activity relationships, the N6-alkylation of the skeleton was explored, while a pyrimidine ring, substituted with various C5'-polar groups, was installed at position C3. Among the newly synthesized 1H-pyrazolo[3,4-c]pyridin-7(6H)-ones, derivatives 14b, 15b and 16a display characteristic features of sGC "stimulators" in A7r5 vascular smooth muscle cells in vitro. They strongly synergize with the NO donor, sodium nitroprusside (SNP) in inducing cGMP generation in a manner that requires the presence of a reduced heme moiety associated with sGC, and elevate the cGMP-responsive phosphorylation of the protein VASP at Ser239. In line with their sGC stimulating capacity, docking calculations of derivatives 16a, 15(a-c) on a cryo-EM structure of human sGC (hsGC) in an ΝΟ-activated state indicated the implication of 1H-pyrazolo[3,4-c]pyridin-7(6H)-one skeleton in efficient bonding interactions with the recently identified region that binds known sGC stimulators, while the presence of either a N6-H or N6-methyl group pointed to enhanced binding affinity. Moreover, the in vitro functional effects of our newly identified sGC stimulators were compatible with a beneficial role in vascular homeostasis. Specifically, derivative 14b reduced A7r5 cell proliferation, while 16a dampened the expression of adhesion molecules ICAM-1 and P/E-Selectin in Human Umbilical Vein Endothelial Cells (HUVECs), as well as the subsequent adhesion of U937 leukocytes to the HUVECs, triggered by tumor necrosis factor alpha (TNF-α) or interleukin-1 beta (IL-1ß). The fact that these compounds elevate cGMP only in the presence of NO may indicate a novel way of interaction with the enzyme and may make them less prone than other direct sGC agonists to induce characteristic hypotension in vivo.

Simulated Microgravity and Hypergravity Affect the Expression Level of Soluble Guanylate Cyclase, Adenylate Cyclase, and Phosphodiesterase Genesin Rat Ventricular Cardiomyocytes.

Ion channels activity is regulated through soluble guanylate cyclase (sGC) and adenylate cyclase (AC) pathways, while phosphodiesterases (PDE) control the intracellular levels of cAMP and cGMP. Here we applied RNA transcriptome sequencing to study changes in the gene expression of the sGC, AC, and PDE isoforms in isolated rat ventricular cardiomyocytes under conditions of microgravity and hypergravity. Our results demonstrate that microgravity reduces the expression of sGC isoform genes, while hypergravity increases their expression. For a subset of AC isoforms, gene expression either increased or decreased under both microgravity and hypergravity conditions. The expression of genes encoding 10 PDE isoforms decreased under microgravity, but increased under hypergravity. However, under both microgravity and hypergravity, the gene expression increased for 7 PDE isoforms and decreased for 3 PDE isoforms. Overall, our findings indicate specific gravity-dependent changes in the expression of genes of isoforms associated with the studied enzymes.

Updates on Pityriasis Rubra Pilaris: A Scoping Review.

Pityriasis rubra pilaris (PRP) is a rare, inflammatory papulosquamous skin disease with unknown exact etiology. Historically, PRP has been challenging to diagnose, especially during the acute phase, and to treat, due to its unclear pathogenesis. To better inform clinical practice, a literature review was conducted employing a broad search strategy to capture PRP-related published studies between January 1, 2012 to October 31, 2022. Two hundred twenty-one studies were identified, which were categorized into 9 themes: (1) potential causes and triggering factors, (2) comorbidities, (3) diagnostic difficulties, (4) genetics, (5) clinical manifestations and laboratory values, (6) treatment, (7) treatment-related adverse events, (8) quality of life, and (9) other. COVID-19 infection, COVID-19 vaccination, and malignancy were the most commonly reported potential triggering factors. Misdiagnosis is very common during the early acute stages. Pathogenesis and genetic studies have further implicated caspase recruitment domain family member 14 (CARD14) mutations in the development of familial PRP (Type V) and have underlined the overlap between psoriasis and PRP. To date, there are currently no specific and validated scoring systems or tools to assess the severity of PRP. While large, randomized trials are still lacking, biologic agents remain the most effective therapy.

Homozygous CARD14 variant presenting as infantile erythroderma.

A wide range of inherited and acquired conditions can manifest as infantile erythroderma, among which CARD14-associated papulosquamous eruption (CAPE) is a rare cause. An infant boy presented with a psoriasiform rash that progressed to erythroderma and was unresponsive to topical steroids and cyclosporine. The early onset of the disease, its severity and resistance to conventional treatment were suggestive of a genetic cause. Genetic evaluation revealed a homozygous CARD14 variant of uncertain significance establishing the diagnosis of CAPE, and his parents were heterozygous carriers. There was only minimal improvement in the condition with supportive management and treatment with acitretin. Unfortunately, the child succumbed to sepsis and metabolic complications following a sudden worsening of skin disease. This case highlights the significance of genetic studies in diagnosing treatment-refractory cases of infantile erythroderma and emphasises the importance of early recognition of this rare condition.

The Functionality of the DC Pair in a Rhodopsin Guanylyl Cyclase from Catenaria anguillulae.

Rhodopsin guanylyl cyclases (RGCs) belong to the class of enzymerhodopsins catalyzing the transition from GTP into the second messenger cGMP, whereas light-regulation of enzyme activity is mediated by a membrane-bound microbial rhodopsin domain, that holds the catalytic center inactive in the dark. Structural determinants for activation of the rhodopsin moiety eventually leading to catalytic activity are largely unknown. Here, we investigate the mechanistic role of the D283-C259 (DC) pair that is hydrogen bonded via a water molecule as a crucial functional motif in the homodimeric C. anguillulae RGC. Based on a structural model of the DC pair in the retinal binding pocket obtained by MD simulation, we analyzed formation and kinetics of early and late photocycle intermediates of the rhodopsin domain wild type and specific DC pair mutants by combined UV-Vis and FTIR spectroscopy at ambient and cryo-temperatures. By assigning specific infrared bands to S-H vibrations of C259 we are able to show that the DC pair residues are tightly coupled. We show that deprotonation of D283 occurs already in the inactive L state as a prerequisite for M state formation, whereas structural changes of C259 occur in the active M state and early cryo-trapped intermediates. We propose a comprehensive molecular model for formation of the M state that activates the catalytic moiety. It involves light induced changes in bond strength and hydrogen bonding of the DC pair residues from the early J state to the active M state and explains the retarding effect of C259 mutants.

Roles of Protein S-Nitrosylation in Endothelial Homeostasis and Dysfunction.

Significance: Nitric oxide (NO) plays several distinct roles in endothelial homeostasis. Except for activating the guanylyl cyclase enzyme-dependent cyclic guanosine monophosphate signaling pathway, NO can bind reactive cysteine residues in target proteins, a process known as S-nitrosylation (SNO). SNO is proposed to explain the multiple biological functions of NO in the endothelium. Investigating the targets and mechanism of protein SNO in endothelial cells (ECs) can provide new strategies for treating endothelial dysfunction-related diseases. Recent Advances: In response to different environments, proteomics has identified multiple SNO targets in ECs. Functional studies confirm that SNO regulates NO bioavailability, inflammation, permeability, oxidative stress, mitochondrial function, and insulin sensitivity in ECs. It also influences EC proliferation, migration, apoptosis, and transdifferentiation. Critical Issues: Single-cell transcriptomic analysis of ECs isolated from different mouse tissues showed heterogeneous gene signatures. However, litter research focuses on the heterogeneous properties of SNO proteins in ECs derived from different tissues. Although metabolism reprogramming plays a vital role in endothelial functions, little is known about how protein SNO regulates metabolism reprogramming in ECs. Future Directions: Precisely deciphering the effects of protein SNO in ECs isolated from different tissues under different conditions is necessary to further characterize the relationship between protein SNO and endothelial dysfunction-related diseases. In addition, identifying SNO targets that can influence endothelial metabolic reprogramming and the underlying mechanism can offer new views on the crosstalk between metabolism and post-translational protein modification. Antioxid. Redox Signal. 40, 186-205.