The human skeleton is renewed and regenerated throughout life, by a cellular process known as bone remodeling. Stem cells are clono-genic cells that are capable of differentiation into multiple mature cell types (multipotency), and simultaneously replenishing stem cell pool (self-renewal), which allows them to sustain tissue development and maintenance. Circulating mesenchymal stromal/stem cells (MSCs), are mobile adult stem cells with specific gene expression profiling, as well as enhanced mitochondrial remodeling as a promising source for personalized cell and gene therapy. A global LGR5-associated genetic interaction network highlights the functional organization and molecular phenotype of circulating MSCs.

In this issue, Guo et al.1 and Chen et al.2 resolved the cryo-EM structures of G protein-coupled receptor (GPR) 4 and GPR68, unveiling that histidine protonation initiates conformational rearrangements and dictates G protein coupling bias, illuminating pH-sensing mechanisms.

Maintaining pH at cellular, tissular, and systemic levels is essential for human health. Proton-sensing GPCRs regulate physiological and pathological processes by sensing the extracellular acidity. However, the molecular mechanism of proton sensing and activation of these receptors remains elusive. Here, we present cryoelectron microscopy (cryo-EM) structures of human GPR4, a prototypical proton-sensing GPCR, in its inactive and active states. Our studies reveal that three extracellular histidine residues are crucial for proton sensing of human GPR4. The binding of protons induces substantial conformational changes in GPR4's ECLs, particularly in ECL2, which transforms from a helix-loop to a β-turn-β configuration. This transformation leads to the rearrangements of H-bond network and hydrophobic packing, relayed by non-canonical motifs to accommodate G proteins. Furthermore, the antagonist NE52-QQ57 hinders human GPR4 activation by preventing hydrophobic stacking rearrangement. Our findings provide a molecular framework for understanding the activation mechanism of a human proton-sensing GPCR, aiding future drug discovery.

Proton sensing by G protein-coupled receptors (GPCRs) is crucial in many life activities. However, its underlying mechanism remains unclear. Here, we report 8 cryoelectron microscopy (cryo-EM) structures of human GPR4 and GPR68 at different pH values and in complex with Gs or Gq trimers or in apo state. Structural inspection, structure-based pKa calculations, and mutational and computational analyses revealed that protonation of two conserved extracellular histidines induced polar network formation and other conformational changes to tether 7-transmembrane (TM7) to second extracellular loop (ECL2), and these changes constitute the central mechanisms of proton-induced activation of GPR4 and GPR68. Unexpectedly, proton sensation by specific extracellular histidine determined biased G protein coupling of GPR4. Moreover, GPR68's additional pH-sensing H842.67 enhances its function in a more acidic optimal pH range. The propagation path connecting proton-sensing histidines to the toggle switch was characterized. Collectively, we provide structural insights into the proton sensing, activation, and downstream effector coupling mechanisms of proton-sensing GPCRs.

The regulation of pH homeostasis is crucial in many biological processes vital for survival, growth, and function of life. The pH-sensing G protein-coupled receptors (GPCRs), including GPR4, GPR65 and GPR68, play a pivotal role in detecting changes in extracellular proton concentrations, impacting both physiological and pathological states. However, comprehensive understanding of the proton sensing mechanism is still elusive. Here, we determined the cryo-electron microscopy structures of GPR4 and GPR65 in various activation states across different pH levels, coupled with Gs, Gq or G13 proteins, as well as a small molecule NE52-QQ57-bound inactive GPR4 structure. These structures reveal the dynamic nature of the extracellular loop 2 and its signature conformations in different receptor states, and disclose the proton sensing mechanism mediated by networks of extracellular histidine and carboxylic acid residues. Notably, we unexpectedly captured partially active intermediate states of both GPR4-Gs and GPR4-Gq complexes, and identified a unique allosteric binding site for NE52-QQ57 in GPR4. By integrating prior investigations with our structural analysis and mutagenesis data, we propose a detailed atomic model for stepwise proton sensation and GPCR activation. These insights may pave the way for the development of selective ligands and targeted therapeutic interventions for pH sensing-relevant diseases.

Cell senescence-associated endothelia dysfunction is a vital point in the pathological progression of atherosclerosis (AS). G-protein coupled receptor 4 (GPR4) is a proton-sensing receptor involved in developing endothelial dysfunction.

In this study, we investigated the protective role of NE 52-QQ57, a GPR4 inhibitor in endothelial cell senescence induced using an oxidized low-density lipoprotein (ox-LDL). We also unravel the underlying molecular mechanism of NE 52-QQ57 as a therapeutic agent.

Endothelial cell senescence model was established using human aortic endothelial cells (HAECs) stimulated with ox-LDL. The expression levels of GPR4, p53, p16, and sirtuin1 (SIRT1) were evaluated using real-time PCR and western blot assays. ROS production was determined using dihydroethidium (DHE) staining. Further, interleukin-6 (IL-6) and monocyte chemotactic protein 1 (MCP-1) secretion and expression were determined using ELISA and real-time PCR analysis, respectively. Finally, β-galactosidase (SA-β-Gal) staining associated with cell senescence, telomerase activity, and cell cycle assay were used to determine the state of cell senescence.

Firstly, GPR4 was found to be upregulated in the ox-LDL-stimulated HAECs. We also identified elevated ROS, IL-6, and MCP-1 levels induced by ox-LDL and significantly abrogated by NE 52-QQ57 treatment. Second, a reversal in SA-β-Gal activity, telomerase activity, and G0/G1 proportion, with an upregulation in p53 and p16 expressions was observed on NE 52-QQ57 treatment in the ox-LDL induced model. Lastly, the decreased expression level of SIRT1 was extremely elevated by NE 52-QQ57. Notably, the inhibitory effect of NE 52-QQ57 against ox-LDL-induced cell senescence was abolished by the SIRT1 inhibitor EX-527.

The GPR4 antagonist NE 52-QQ57 might prevent cellular senescence by promoting the expression of SIRT1.

Olfactory receptors (ORs), taste receptors and opsins are well-known for their pivotal roles in mediating the senses of smell, taste and sight, respectively. However, in the past two decades, research has shown that these sensory receptors also regulate physiological processes in a variety of non-sensory tissues. Although ORs, taste receptors and opsins have all been shown to have physiological roles beyond their traditional locations, most work in the kidney has focused on ORs. To date, renal ORs have been shown to have roles in blood pressure regulation (OLFR78 and OLFR558) and glucose homeostasis (OLFR1393). However, sensory receptors remain drastically understudied outside of traditional sensory systems, in part because of inherent challenges in studying these receptors. Increased knowledge of the physiological and pathophysiological roles of sensory receptors has the potential to substantially improve understanding of the function of numerous organs and systems, including the kidney. In addition, most sensory receptors are G protein-coupled receptors, which are considered to be the most druggable class of proteins, and thus could potentially be exploited as future therapeutic targets.

Lung bacterial infections, including hospital-acquired pneumonia, remain a serious problem for public health. Endothelial cell (EC) exposure to heat-killed Staphylococcus aureus (HKSA) represents a clinical scenario of high titers of killed bacterial particles present in the host after antibiotic therapy, which triggers inflammatory cascades, cytokine storms, and EC dysfunction leading to acute lung injury (ALI). GPR68 is a member of the proton-sensing G protein-coupled receptor family. Acting as a pH sensor, GPR68 becomes activated upon pH reduction and contributes to pathologic cell responses by activating ER stress and unfolded protein response. This study investigated the role of GPR68 in HKSA-induced EC dysfunction and HKSA-induced ALI. HKSA robustly increased GPR68 mRNA levels in human pulmonary EC and directly stimulated GPR68 activity. A selective GPR68 small molecule inhibitor, OGM-8345, attenuated HKSA-induced EC permeability and protected cell junction integrity. OGM-8345 inhibited HKSA-induced activation of inflammatory genes TNF-α, IL-6, IL-8, IL-1β, and CXCL5 and decreased cytokine secretion by HKSA-challenged EC. Co-treatment with the GPR68 activator Ogerin or medium acidification to pH 6.5 augmented HKSA-induced EC dysfunction, which was rescued by OGM-8345. Intratracheal HKSA injection increased vascular leak and lung inflammation in mice which were monitored by lung Evans blue extravasation, increased cell and protein count in bronchoalveolar lavage, and mRNA expression of inflammatory genes. ALI and barrier dysfunction was attenuated by OGM-8345. We show for the first time the role of GPR68 in mediating HKSA-induced lung injury and the strong potential for OGM-8345 as a therapeutic treatment of bacterial pathogen-induced ALI associated with tissue acidification.

G protein-coupled receptors (GPRs), including pro-inflammatory GPR4 and ovarian cancer GPR1 (OGR1/GPR68), are involved in the pH sensing of the extracellular space and have been implicated in inflammatory bowel disease (IBD). Previous data show that a loss of GPR4 or OGR1 independently is associated with reduced intestinal inflammation in mouse models of experimental colitis. In the present manuscript, we investigated the impact of the simultaneous loss of GPR4 and OGR1 in animal models of IBD. To study the effects of combined loss of Gpr4 Ogr1 in IBD we used the well-established acute dextran sodium sulfate (DSS) and spontaneous Il10-/- murine colitis models. Disease severity was assessed using multiple clinical scores (e.g., body weight loss, disease activity score, murine endoscopic index of colitis severity (MEICS) and histological analyses). Real-time quantitative polymerase chain reaction (qPCR), Western blot, and flow cytometry were used to investigate changes in pro-inflammatory cytokines expression and immune cells infiltration. We found that a combined loss of GPR4 and OGR1 significantly reduces colon inflammation in IBD relative to single deficiencies as evidenced by reduced body weight loss, disease score, CD4/CD8 ratio, and Il1β, Il6, and Tnf in the colon. Similarly, in the II10 deficiency model, the inflammation was significantly ameliorated upon the simultaneous deletion of GPR4 and OGR1, evidenced by a reduction in the MEICS score, colon length, Tnf and Il1β measurements, and a decrease in the number of macrophages in the colon, as compared to single deletions. Importantly, hydroxyproline levels were decreased close to baseline in Il10-/- × Gpr4-/- × Ogr1-/- mice. Our findings demonstrate that the simultaneous loss of GRP4 and OGR1 functions exerts an additive effect on multiple parameters associated with colonic inflammation. These results further reinforce the hypothesis that chronic inflammatory acidosis is a driver of fibrosis and is dependent on GPR4 and OGR1 signaling. The inhibition of both GPR4 and OGR1 by pH-sensing receptor modulators may constitute as a potential therapeutic option for IBD, as both pH-sensing receptors appear to sustain inflammation by acting on complementary pro-inflammatory pathways.

Local microcurrent monitoring is of great significance for biological and battery systems, yet it poses a formidable challenge. The current measurement techniques rely on electromagnetic materials which inevitably introduce interference to the system under examination. To address this issue, a promising approach based on a dielectric fiber-optic sensor is demonstrated. The microfiber is capable of detecting microcurrent through monitoring the localized proton concentration signal with a pH resolution of 0.0052 pH units. By sensing the refractive index variation surrounding the sensor induced by the interaction between local proton concentration changes and oxidizer-treated microfiber surface through the evanescent field, this sensing mechanism effectively avoids the interference of the electromagnetic material on the performance of the tested system. This sensor exhibits a limit of detection for microcurrent of 1 μA. The sensing region is a microfiber with a diameter of 8.8 μm. It can get invaluable information that cannot be obtained through conventional electrochemical methods. Examples include photocurrent attenuation in photogenerated carrier materials during illumination, electrical activation in nerve cells, and fluctuations in the efficiency of electrical energy generation during battery discharge. This approach provides a powerful complement to electrochemical methods for the elucidation of microscale reaction mechanisms. The information provided by the prepared dielectric fiber-optic sensor will shed more light on proton kinetics and electrochemical and electrobiological mechanisms, which may fill an important gap in the current bioelectricity and battery monitoring methods.

Animals have evolved pH-sensing membrane receptors, such as G-protein-coupled receptor 4 (GPR4), to monitor pH changes related to their physiology and generate adaptive reactions. However, the evolutionary trajectory and structural mechanism of proton sensing by GPR4 remain unresolved. Here, we observed a positive correlation between the optimal pH of GPR4 activity and the blood pH range across different species. By solving 7-cryoelectron microscopy (cryo-EM) structures of Xenopus tropicalis GPR4 (xtGPR4) and Mus musculus GPR4 (mmGPR4) under varying pH conditions, we identified that protonation of HECL2-45.47 and H7.36 enabled polar network establishment and tighter association between the extracellular loop 2 (ECL2) and 7 transmembrane (7TM) domain, as well as a conserved propagating path, which are common mechanisms underlying protonation-induced GPR4 activation across different species. Moreover, protonation of distinct extracellular HECL2-45.41 contributed to the more acidic optimal pH range of xtGPR4. Overall, our study revealed common and distinct mechanisms of proton sensing by GPR4, from a structural, functional, and evolutionary perspective.

Three proton-sensing G protein-coupled receptors (GPCRs)-GPR4, GPR65, and GPR68-respond to extracellular pH to regulate diverse physiology. How protons activate these receptors is poorly understood. We determined cryogenic-electron microscopy (cryo-EM) structures of each receptor to understand the spatial arrangement of proton-sensing residues. Using deep mutational scanning (DMS), we determined the functional importance of every residue in GPR68 activation by generating ∼9,500 mutants and measuring their effects on signaling and surface expression. Constant-pH molecular dynamics simulations provided insights into the conformational landscape and protonation patterns of key residues. This unbiased approach revealed that, unlike other proton-sensitive channels and receptors, no single site is critical for proton recognition. Instead, a network of titratable residues extends from the extracellular surface to the transmembrane region, converging on canonical motifs to activate proton-sensing GPCRs. Our approach integrating structure, simulations, and unbiased functional interrogation provides a framework for understanding GPCR signaling complexity.

Radioresistance is thought to be a major consequence of tumor milieu acidification resulting from the Warburg effect. Previously, using ogremorphin (OGM), a small molecule inhibitor of GPR68, an extracellular proton sensing receptor, we demonstrated that GPR68 is a key pro-survival pathway in glioblastoma cells. Here, we demonstrate that GPR68 inhibition also induces ferroptosis in lung cell carcinoma (A549) and pancreatic ductal adenocarcinoma (Panc02) cells. Moreover, OGM synergized with ionizing radiation to induce lipid peroxidation, a hallmark of ferroptosis, as well as reduce colony size in 2D and 3D cell culture. GPR68 inhibition is not acutely detrimental but increases intracellular free ferrous iron, which is known to trigger reactive oxygen species (ROS) generation. In summary, GPR68 inhibition induces lipid peroxidation in cancer cells and sensitizes them to ionizing radiation in part through the mobilization of intracellular free ferrous iron. Our results suggest that GPR68 is a key mediator of cancer cell radioresistance activated by acidic tumor microenvironment.

The aim of the present study was to explore the role of ovarian cancer G protein-coupled receptor 1 (OGR1) in osteoclast differentiation and activity induced by extracellular acid. The impact of extracellular acidification on osteoclasts was investigated. Briefly, osteoclasts were generated from RAW 264.7 cells using 100 ng/ml receptor activator of nuclear factor-κB ligand in cell culture media at pH 6.8 or 7.4. Tartrate-resistant acid phosphatase (TRAP) staining and the bone resorption pit assay were used to detect the effects of extracellular acid on the number and absorptive capacity of osteoclasts. Intracellular Ca2+ levels were analyzed using laser scanning confocal microscopy. Reverse transcription-quantitative PCR was used to detect the expression levels of genes associated with osteoclast formation and bone erosion. The role of OGR1 in the acid-stimulated formation and bone resorption of osteoclasts was also investigated. The results showed that in the pH 6.8 medium group the number of osteoclasts was 511.2±54.72 and the area of bone absorption was 4,184.88±277.14 µm2; both were significantly higher than those in the pH 7.4 medium group (all P<0.01). Inhibition of OGR1 using copper ion (Cu2+) reduced the number of osteoclasts and the area of bone resorption in the pH 6.8 medium group (all P<0.05). Furthermore, extracellular acid (pH 6.8) was able to induce a transient increase of Ca2+ levels in osteoclasts; however, inhibition of OGR1 using Cu2+ effectively attenuated the acid-induced increase of Ca2+ in osteoclasts. In addition, the elevation in Ca2+ levels was inhibited when BAPTA, a cytoplasmic Ca2+ chelator with cellular permeability, was added to the cells; however, the extracellular Ca2+-chelating agent ethylene glycol tetraacetic acid did not inhibit the acid-stimulated increase in Ca2+. Treatment with the phospholipase C inhibitor U73122 also inhibited the acid-stimulated increase of Ca2+ in osteoclasts. Furthermore, the mRNA expression levels of TRAP, matrix metalloproteinase-9, osteoclast-related receptor, nuclear factor-activated T cell 1 (NFATc1), cathepsin K and integrin β3 were elevated in the pH 6.8 medium group compared with those in the pH 7.4 medium group (all P<0.05). By contrast, the inhibition of OGR1 using Cu2+ partially reduced the effects of the acidic environment on osteoclast differentiation and activity-related gene expression (all P<0.05). In addition, the mRNA and protein expression levels of calcineurin were increased in osteoclasts in the pH 6.8 group compared with those in the pH 7.4 group (P<0.05), whereas blocking OGR1 suppressed the expression of acid-induced calcineurin. The mRNA expression levels of NFATc1 in osteoclasts were also increased in the pH 6.8 medium group compared with those in the pH 7.4 medium group (P<0.05). By contrast, the specific calcineurin inhibitor cyclosporine A significantly inhibited the acid-induced expression of NFATc1 in osteoclasts. In conclusion, the present study revealed that extracellular acidification may increase osteoclast differentiation and bone resorption activity. Furthermore, OGR1-mediated Ca2+ elevation could have a crucial role in osteoclasts by regulating the Ca2+-calcineurin-NFATc1 signaling pathway and downstream signaling.

In severe asthma, symptoms are unstable despite intensive treatment based on high doses of inhaled corticosteroids and on-demand use of oral corticosteroids. Although, recently, various biological agents related to Th2 cytokines have been added to intensive controller medications for severe asthma, a significant progress has not been observed in the management for symptoms (dyspnea, wheezing and cough). Medical treatment focused on Type 2 inflammation is probably insufficient to maintain good long-term management for severe asthma. Airway eosinophilia and decreased reversibility in forced expiratory volume in 1 second (FEV1) are listed as major predictors for exacerbation-prone asthma. However, it is generally considered that asthma is complex and heterogeneous. It is necessary to establish precision medicine using treatable traits based on a multidimensional approach related to asthma. Since phospholipids generate lysophospholipids and arachidonic acid by phospholipases, lysophospholipids can be associated with the pathogenesis of this disease via action on smooth muscle, endothelium, and epithelium in the airways. Lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), and sphingosine 1-phosphate (S1P) are increased in bronchoalveolar fluid after allergen challenge. LPA, LPC, and S1P recruit eosinophils to the lungs and cause β2-adrenergic desensitization. LAP and S1P cause contraction and hyperresponsiveness in airway smooth muscle. Moreover, lysophosphatidylserine and S1P are associated with the allergic reaction related to IgE/FcεRI in mast cells. Lysophospholipid action is probably comprised of corticosteroid resistance and is independent of Type 2 inflammation, and may be corelated with oxidative stress. Lysophospholipids may be a novel molecular target in advancing the management and treatment of asthma. This review discusses the clinical relevance of lysophospholipids in asthma.

G protein-coupled receptor 4 (GPR4) belongs to the subfamily of proton-sensing GPCRs (psGPCRs), which detect pH changes in extracellular environment and regulate diverse physiological responses. GPR4 was found to be overactivated in acidic tumor microenvironment as well as inflammation sites, with a triad of acidic residues within the transmembrane domain identified as crucial for proton sensing. However, the 3D structure remains unknown, and the roles of other conserved residues within psGPCRs are not well understood. Here we report cryo-electron microscopy (cryo-EM) structures of active zebrafish GPR4 at both pH 6.5 and 8.5, each highlighting a distribution of histidine and acidic residues at the extracellular region. Cell-based assays show that these ionizable residues moderately influence the proton-sensing capacity of zebrafish GPR4, compared to the more significant effects of the triad residues. Furthermore, we reveal a cluster of aromatic residues within the orthosteric pocket that may propagate the signaling to the intercellular region via repacking the aromatic patch at the central region. This study provides a framework for future signaling and functional investigation of psGPCRs.

Atomic-level simulations are widely used to study biomolecules and their dynamics. A common goal in such studies is to compare simulations of a molecular system under several conditions-for example, with various mutations or bound ligands-in order to identify differences between the molecular conformations adopted under these conditions. However, the large amount of data produced by simulations of ever larger and more complex systems often renders it difficult to identify the structural features that are relevant to a particular biochemical phenomenon. We present a flexible software package named Python ENSemble Analysis (PENSA) that enables a comprehensive and thorough investigation into biomolecular conformational ensembles. It provides featurization and feature transformations that allow for a complete representation of biomolecules such as proteins and nucleic acids, including water and ion binding sites, thus avoiding the bias that would come with manual feature selection. PENSA implements methods to systematically compare the distributions of molecular features across ensembles to find the significant differences between them and identify regions of interest. It also includes a novel approach to quantify the state-specific information between two regions of a biomolecule, which allows, for example, tracing information flow to identify allosteric pathways. PENSA also comes with convenient tools for loading data and visualizing results, making them quick to process and easy to interpret. PENSA is an open-source Python library maintained at https://github.com/drorlab/pensa along with an example workflow and a tutorial. We demonstrate its usefulness in real-world examples by showing how it helps us determine molecular mechanisms efficiently.

Accumulating evidence demonstrates that the "Warburg effect" that glycolysis is enhanced even in the presence of oxygen existed in hematopoietic malignancies, contributing to extracellular acidosis. G-protein coupled receptor 68 (GPR68), as a proton sensing GPCR responding to extracellular acidosis, is expected to play a critical role in hematopoietic malignancies. In the present study, we found that GPR68 was overexpressed in acute myeloid leukemia (AML) cells, and GPR68 deficiency impaired AML cell survival in vitro and cell engraftment in vivo. Mechanistic studies revealed that unlike GPR68 regulates Calpain1 in myelodysplastic syndromes (MDS) cells, GPR68 deficiency reduced cytosolic Ca2+ levels and calcineurin (CaN) activity in AML cells through an NFAT-independent mechanism. Moreover, the decreased Ca2+ levels disturbed cellular respiration (i.e., oxidative phosphorylation, OxPhos) by inhibiting isocitrate dehydrogenase (IDH) activity; this was more pronounced when BCL2 was inhibited simultaneously. Interestingly, GPR68 inhibition also decreased aerobic glycolysis in AML cells in a Ca2+-independent manner, suggesting that GPR68 mediated glucose metabolic symbiosis. As glucose metabolic symbiosis and the heterogeneous dependencies on aerobic glycolysis and cellular respiration tremendously impact chemosensitivity, the inhibition of GPR68 potentiated the tumoricidal effect of first-line chemotherapeutic agents, including BCL-2 inhibitors targeting OxPhos and cytarabine (Ara-C) targeting glycolysis. Consistent with these in vitro observations, higher levels of GPR68 were associated with inferior clinical outcomes in AML patients who received chemotherapies. In short, GPR68 drives the Ca2+/CaN pro-survival pathway and mediates glucose metabolic pathways in AML cells. Targeting GPR68 eradicates AML cells and alleviates chemoresistance, which could be exploited as a therapeutic target.

Metabolites can double as a signaling modality that initiates physiological adaptations. Metabolism, a chemical language encoding biological information, has been recognized as a powerful principle directing inflammatory responses. Cytosolic pH is a regulator of inflammatory response in macrophages. Here, we found that L-malate exerts anti-inflammatory effect via BiP-IRF2BP2 signaling, which is a sensor of cytosolic pH in macrophages. First, L-malate, a TCA intermediate upregulated in pro-inflammatory macrophages, was identified as a potent anti-inflammatory metabolite through initial screening. Subsequent screening with DARTS and MS led to the isolation of L-malate-BiP binding. Further screening through protein‒protein interaction microarrays identified a L-malate-restrained coupling of BiP with IRF2BP2, a known anti-inflammatory protein. Interestingly, pH reduction, which promotes carboxyl protonation of L-malate, facilitates L-malate and carboxylate analogues such as succinate to bind BiP, and disrupt BiP-IRF2BP2 interaction in a carboxyl-dependent manner. Both L-malate and acidification inhibit BiP-IRF2BP2 interaction, and protect IRF2BP2 from BiP-driven degradation in macrophages. Furthermore, both in vitro and in vivo, BiP-IRF2BP2 signal is required for effects of both L-malate and pH on inflammatory responses. These findings reveal a previously unrecognized, proton/carboxylate dual sensing pathway wherein pH and L-malate regulate inflammatory responses, indicating the role of certain carboxylate metabolites as adaptors in the proton biosensing by interactions between macromolecules.

Tissue acidification resulting from dysregulated cellular bioenergetics accompanies various inflammatory states. GPR68, along with other members of proton-sensing G protein-coupled receptors, responds to extracellular acidification and has been implicated in chronic inflammation-related diseases such as ischemia, cancer, and colitis. The present study examined the role of extracellular acidification on human pulmonary endothelial cell (EC) permeability and inflammatory status per se and investigated potential synergistic effects of acidosis on endothelial dysfunction caused by bacterial lipopolysaccharide (LPS, Klebsiella pneumoniae). Results showed that medium acidification to pH 6.5 caused a delayed increase in EC permeability illustrated by a decrease in transendothelial electrical resistance and loss of continuous VE-cadherin immunostaining at cell junctions. Likewise, acidic pH induced endothelial inflammation reflected by increased mRNA and protein expression of EC adhesion molecules VCAM-1 and ICAM-1, upregulated mRNA transcripts of tumor necrosis factor-α, IL-6, IL-8, IL-1β, and CXCL5, and increased secretion of ICAM-1, IL-6, and IL-8 in culture medium monitored by ELISA. Among the GPCRs tested, acidic pH selectively increased mRNA and protein expression of GPR68, and only the GPR68-specific small molecule inhibitor OGM-8345 rescued acidosis-induced endothelial permeability and inflammation. Furthermore, acidic pH exacerbated LPS-induced endothelial permeability and inflammatory response in cultured lung macrovascular as well as microvascular endothelial cells. These effects were suppressed by OGM-8345 in both EC types. Altogether, these results suggest that GPR68 is a critical mediator of acidic pH-induced dysfunction of human pulmonary vascular endothelial cells and mediates the augmenting effect of tissue acidification on LPS-induced endothelial cell injury.

Microvascular brain endothelial cells tightly limit the entry of blood components and peripheral cells into the brain by forming the blood-brain barrier (BBB). The BBB is regulated by a cascade of mechanical and chemical signals including shear stress and elasticity of the adjacent endothelial basement membrane (BM). During physiological aging, but especially in neurological diseases including multiple sclerosis (MS), stroke, small vessel disease, and Alzheimer's disease (AD), the BBB is exposed to inflammation, rigidity changes of the BM, and disturbed cerebral blood flow (CBF). These altered forces lead to increased vascular permeability, reduced endothelial reactivity to vasoactive mediators, and promote leukocyte transmigration. Whereas the molecular players involved in leukocyte infiltration have been described in detail, the importance of mechanical signalling throughout this process has only recently been recognized. Here, we review relevant features of mechanical forces acting on the BBB under healthy and pathological conditions, as well as the endothelial mechanosensory elements detecting and responding to altered forces. We demonstrate the underlying complexity by focussing on the family of transient receptor potential (TRP) ion channels. A better understanding of these processes will provide insights into the pathogenesis of several neurological disorders and new potential leads for treatment.

G-protein-coupled receptors are integral membrane proteins that transduce chemical signals from the extracellular matrix into the cell. Traditional drug design has considered ligand-receptor interactions only under normal conditions. However, studies on opioids indicate that such interactions are very different in diseased tissues. In such microenvironments, protons play an important role in structural and functional alterations of both ligands and receptors. The pertinent literature strongly suggests that future drug design should take these aspects into account in order to reduce adverse side effects while preserving desired effects of novel compounds.

Phenotypic transformation of vascular smooth muscle (VSM) from a contractile state to a synthetic, proliferative state is a hallmark of cardiovascular disease (CVD). In CVD, diseased tissue often becomes acidic from altered cellular metabolism secondary to compromised blood flow, yet the contribution of local acid/base imbalance to the disease process has been historically overlooked. In this study, we examined the regulatory impact of the pH-sensing G protein-coupled receptor GPR68 on vascular smooth muscle (VSM) proliferation in vivo and in vitro in wild-type (WT) and GPR68 knockout (KO) male and female mice. Arterial injury reduced GPR68 expression in WT vessels and exaggerated medial wall remodeling in GPR68 KO vessels. In vitro, KO VSM cells showed increased cell-cycle progression and proliferation compared with WT VSM cells, and GPR68-inducing acidic exposure reduced proliferation in WT cells. mRNA and protein expression analyses revealed increased Rap1A in KO cells compared with WT cells, and RNA silencing of Rap1A reduced KO VSM cell proliferation. In sum, these findings support a growth-inhibitory capacity of pH-sensing GPR68 and suggest a mechanistic role for the small GTPase Rap1A in GPR68-mediated VSM growth control. These results shed light on GPR68 and its effector Rap1A as potential targets to combat pathological phenotypic switching and proliferation in VSM.NEW & NOTEWORTHY Extracellular acidosis remains an understudied feature of many pathologies. We examined a potential regulatory role for pH-sensing GPR68 in vascular smooth muscle (VSM) growth in the context of CVD. With in vivo and in vitro growth models with GPR68-deficient mice and GPR68 induction strategies, novel findings revealed capacity of GPR68 to attenuate growth through the small GTPase Rap1A. These observations highlight GPR68 and its effector Rap1A as possible therapeutic targets to combat pathological VSM growth.

This chapter presents an overview of the emerging strategies to address the unmet needs in the management of inflammatory bowel diseases (IBD). IBD poses significant challenges, as over half of patients experience disease progression despite interventions, leading to irreversible complications, and a substantial proportion do not respond to existing therapies, such as biologics. To overcome these limitations, we describe a diverse array of novel therapeutic approaches. In the area of immune homeostasis restoration, the focus is on targeting cytokine networks, leukocyte trafficking, novel immune pathways, and cell therapies involving regulatory T cells and mesenchymal stem cells (MSC). Recognizing the critical role of impaired intestinal barrier integrity in IBD, we highlight therapies aimed at restoring barrier function and promoting mucosal healing, such as those targeting cell proliferation, tight junctions, and lipid mediators. Addressing the challenges posed by fibrosis and fistulas, we describe emerging targets for reversing fibrosis like kinase and cytokine inhibitors and nuclear receptor agonists, as well as the potential of MSC for fistulas. The restoration of a healthy gut microbiome, through strategies like fecal microbiota transplantation, rationally defined bacterial consortia, and targeted antimicrobials, is also highlighted. We also describe innovative approaches to gut-targeted drug delivery to enhance efficacy and minimize side effects. Reinforcing these advancements is the critical role of precision medicine, which emphasizes the use of multiomics analysis for the discovery of biomarkers to enable personalized IBD care. Overall, the emerging landscape of therapeutic opportunities for IBD holds great potential to surpass the therapeutic ceiling of current treatments.

Metabolic rewiring of cancer cells is one of the hallmarks of cancer. As a consequence, the metabolic landscape of the tumour microenvironment (TME) differs compared to correspondent healthy tissues. Indeed, due to the accumulation of acid metabolites, such as lactate, the pH of the TME is generally acidic with a pH drop that can be as low as 5.6. Disruptions in the acid-base balance and elevated lactate levels can drive malignant progression not only through cell-intrinsic mechanisms but also by impacting the immune response. Generally, acidity and lactate dampen the anti-tumour response of both innate and adaptive immune cells favouring tumour progression and reducing the response to immunotherapy. In this review, we summarize the current knowledge on the functional, metabolic and epigenetic effects of acidity and lactate on the cells of the immune system. In particular, we focus on the role of monocarboxylate transporters (MCTs) and other solute carrier transporters (SLCs) that, by mediating the exchange of lactate (among other metabolites) and bicarbonate, participate in pH regulation and lactate transport in the cancer context. Finally, we discuss advanced approaches to target pH or lactate in the TME to enhance the anti-tumour immune response.

Metabolic acidosis (MAc)-an extracellular pH (pHo) decrease caused by a [HCO3 -]o decrease at constant [CO2]o-usually causes intracellular pH (pHi) to fall. Here we determine the extent to which the pHi decrease depends on the pHo decrease vs the concomitant [HCO3 -]o decrease. We use rapid-mixing to generate out-of-equilibrium CO2/HCO3 - solutions in which we stabilize [CO2]o and [HCO3 -]o while decreasing pHo (pure acidosis, pAc), or stabilize [CO2]o and pHo while decreasing [HCO3 -]o (pure metabolic/down, pMet↓). Using the fluorescent dye 2',7'-bis-2-carboxyethyl)-5(and-6)carboxyfluorescein (BCECF) to monitor pHi in rat hippocampal neurons in primary culture, we find that-in naïve neurons-the pHi decrease caused by MAc is virtually the sum of those caused by pAc (∼70%) + pMet↓ (∼30%). However, if we impose a first challenge (MAc1, pAc1, or pMet↓1), allow the neurons to recover, and then impose a second challenge (MAc2, pAc2, or pMet↓2), we find that pAc/pMet↓ additivity breaks down. In a twin-challenge protocol in which challenge #2 is MAc, the pHo and [HCO3 -]o decreases during challenge #1 must be coincident in order to mimic the effects of MAc1 on MAc2. Conversely, if challenge #1 is MAc, then the pHo and [HCO3 -]o decreases during challenge #2 must be coincident in order for MAc1 to produce its physiological effects during the challenge #2 period. We conclude that the history of challenge #1 (MAc1, pAc1, or pMet↓1)-presumably as detected by one or more acid-base sensors-has a major impact on the pHi response during challenge #2 (MAc2, pAc2, or pMet↓2).

This review article investigates the relationship between mitochondrial dysfunction and cancer progression, emphasizing the metabolic shifts that promote tumor growth. Mitochondria are crucial for cellular energy production, but they also play a significant role in cancer progression by promoting glycolysis even under oxygen-rich conditions, a phenomenon known as the Warburg effect. This metabolic reprogramming enables cancer cells to maintain an alkaline internal pH and an acidic external environment, which are critical for their proliferation and survival in hypoxic conditions. The article also explores the acidic tumor microenvironment (TME), a consequence of intensive glycolytic activity and proton production by cancer cells. This acidic milieu enhances the invasiveness and metastatic potential of cancer cells and contributes to increased resistance to chemotherapy. Alkalization therapy, which involves neutralizing this acidity through dietary modifications and the administration of alkalizing agents such as sodium bicarbonate, is highlighted as an effective strategy to counteract these adverse conditions and impede cancer progression. Integrating insights from science-based medicine, the review evaluates the effectiveness of alkalization therapy across various cancer types through clinical assessments. Science-based medicine, which utilizes inductive reasoning from observed clinical outcomes, lends support to the hypothesis of metabolic reprogramming in cancer treatment. By addressing both metabolic and environmental disruptions, this review suggests that considering cancer as primarily a metabolic disorder could lead to more targeted and effective treatment strategies, potentially improving outcomes for patients with advanced-stage cancers.

Glycosaminoglycans (GAGs) and proteoglycans (PGs) are essential components of the extracellular matrix (ECM) with pivotal roles in cellular mechanosensing pathways. GAGs, such as heparan sulfate (HS) and chondroitin sulfate (CS), interact with various cell surface receptors, including integrins and receptor tyrosine kinases, to modulate cellular responses to mechanical stimuli. PGs, comprising a core protein with covalently attached GAG chains, serve as dynamic regulators of tissue mechanics and cell behavior, thereby playing a crucial role in maintaining tissue homeostasis. Dysregulation of GAG/PG-mediated mechanosensing pathways is implicated in numerous pathological conditions, including cancer and inflammation. Understanding the intricate mechanisms by which GAGs and PGs modulate cellular responses to mechanical forces holds promise for developing novel therapeutic strategies targeting mechanotransduction pathways in disease. This comprehensive overview underscores the importance of GAGs and PGs as key mediators of mechanosensing in maintaining tissue homeostasis and their potential as therapeutic targets for mitigating mechano-driven pathologies, focusing on cancer and inflammation.

An interoceptive homeostatic reflex monitors levels of CO2/H+ to maintain blood gas homeostasis and rapidly regulate tissue acid-base balance by driving lung ventilation and CO2 excretion-this CO2-evoked increase in respiration is the hypercapnic ventilatory reflex (HCVR). Retrotrapezoid nucleus (RTN) neurons provide crucial excitatory drive to downstream respiratory rhythm/pattern-generating circuits, and their activity is directly modulated by changes in CO2/H+ RTN neurons express GPR4 and TASK-2, global deletion of which abrogates CO2/H+ activation of RTN neurons and the HCVR. It has not been determined if the intrinsic pH sensitivity of these proton detectors is required for these effects. We used CRISPR/Cas9 genome editing to generate mice with mutations in either of two pH-sensing histidine residues in GPR4 to determine effects on RTN neuronal CO2/H+ sensitivity and the HCVR. In global GPR4(H81F) and GPR4(H167F) mice, CO2-stimulated breathing and CO2-induced RTN neuronal activation were strongly blunted, with no effect on hypoxia-stimulated breathing. In brainstem slices from GPR4(H81F) mice, peak firing of RTN neurons during bath acidification was significantly reduced compared with GPR4 wild-type mice, and a subpopulation of RTN neurons was rendered pH-insensitive, phenocopying previous results from GPR4-deleted mice. These effects were independent of changes in RTN number/distribution, neuronal excitability or transcript levels for GPR4 and TASK-2. CO2-stimulated breathing was reduced to a similar extent in GPR4(H81F) and TASK-2-deleted mice, with combined mutation yielding no additional deficit in the HCVR. Together, these data demonstrate that the intrinsic pH sensitivity of GPR4 is necessary for full elaboration of the HCVR.

The precise regulation of pH homeostasis is crucial for normal physiology. However, in tissue microenvironments, it can be impacted by pathological conditions such as inflammation and cancer. Due to the overproduction and accumulation of acids (protons), the extracellular pH is characteristically more acidic in inflamed tissues and tumors in comparison to normal tissues. A family of proton-sensing G-protein-coupled receptors (GPCRs) has been identified as molecular sensors for cells responding to acidic tissue microenvironments. Herein, we review the current research progress pertaining to these proton-sensing GPCRs, including GPR4, GPR65 (TDAG8), and GPR68 (OGR1), in inflammation and cancer. Growing evidence suggests that GPR4 and GPR68 are mainly pro-inflammatory, whereas GPR65 is primarily anti-inflammatory, in various inflammatory disorders. Both anti- and pro-tumorigenic effects have been reported for this family of receptors. Moreover, antagonists and agonists targeting proton-sensing GPCRs have been developed and evaluated in preclinical models. Further research is warranted to better understand the roles of these proton-sensing GPCRs in pathophysiology and is required in order to exploit them as potential therapeutic targets for disease treatment.

Significance: Chronic diabetic wounds on the lower extremities (diabetic foot ulcers, DFU) are one of the most prevalent and life-threatening complications of diabetes, responsible for significant loss of quality of life and cost to the health care system. Available pharmacologic treatments fail to achieve complete healing in many patients. Recent studies and investigational treatments have highlighted the potential of modulating wound pH in DFU. Recent Advances: Data from in vitro, preclinical, and clinical studies highlight the role of pH in the pathophysiology of DFU, and topical administration of pH-lowering agents have shown promise as a therapeutic strategy for diabetic wounds. In this critical review, we describe the role of pH in DFU pathophysiology and present selected low-molecular-weight and hydrogel-based pH-modulating systems for wound healing and infection control in diabetic wounds. Critical Issues: The molecular mechanisms leading to pH alterations in diabetic wounds are complex and may differ between in vitro models, animal models of diabetes, and the human pathophysiology. Wound pH-lowering bandages for DFU therapy must be tested in established animal models of diabetic wound healing and patients with diabetes to establish a comprehensive benefit-risk profile. Future Directions: As our understanding of the role of pH in the pathophysiology of diabetic wounds is deepening, new treatments for this therapeutic target are being developed and will be tested in preclinical and clinical studies. These therapeutic systems will establish a target product profile for pH-lowering treatments such as an optimal pH profile for each wound healing stage. Thus, controlling wound bed pH could become a powerful tool to accelerate chronic diabetic wound healing.

GPR68 is a proton-sensing G-protein Coupled Receptor (GPCR) involved in a variety of physiological processes and disorders including neoplastic pathologies. While GPR68 and few other GPCRs have been shown to be activated by a decrease in the extracellular pH, the molecular mechanism of their activation remains largely unknown. In this work, we used a combined computational and in vitro approach to provide new insight into the activation mechanism of the receptor. Molecular Dynamics simulations of GPR68 were used to model the changes in residue interactions and motions triggered by pH. Global and local rearrangements consistent with partial activation were observed upon protonation of the inactive state. Selected extracellular histidine and transmembrane acidic residues were found to have significantly upshifted pKa values during the simulations, consistently with their previously hypothesised role in activation through changes in protonation state. Moreover, a novel pairing between histidine and acidic residues in the extracellular region was highlighted by both sequence analyses and simulation data and tested through site-directed mutagenesis. At last, we identified a previously unknown hydrophobic lock in the extracellular region that might stabilise the inactive conformation and regulate the transition to the active state.

To identify the genetic cause of a Chinese family with hypomaturation amelogenesis imperfecta (AI) and to characterize the structure of GPR68 mutated enamel in order to develop a deeper understanding of the role of the GPR68 protein during the intricate process of amelogenesis.

One Chinese family with generalized hypomaturation AI was recruited. Two of the third molars from the proband were subjected to scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). Whole exome sequencing (WES) was performed, and the identified mutation was confirmed by Sanger sequencing. Bioinformatics studies were further conducted to analyze the potential deleterious effects of the mutation.

The proband presented with a hypomaturation AI phenotype, characterized by fragile and discolored enamel surface. The AI enamel showed prismatic structure, which was sporadically obscured by areas of amorphous material and porous structure. EDX analysis showed the proband's enamel demonstrated a significant decrease in calcium and phosphorus content and a significant increase in oxygen compared with normal enamel. A novel homozygous mutation of G protein-coupled receptor 68 (GPR68) (c .149 T > A, p.Ile50Asn) was identified in the proband. Bioinformatics analysis indicated that the mutation site displayed a high level of evolutionary conservation among species, and the mutation might impact the stability and conformation of the protein.

The novel homozygous GPR68 mutation resulted in hypomaturation AI. We first described the effect of GPR68 mutation on enamel structure. Our results provide new genetic evidence that mutations involved in GPR68 contribute to hypomaturation AI.

Although growing evidence suggests close correlations between Alzheimer's disease (AD) and circadian rhythm disruption (CRD), few studies have focused on the influence of circadian rhythm on levels of immune cells in AD. We aimed to delineate the mechanism underlying the effects of circadian related genes on T cell immune function in AD. A total of 112 brain samples were used to construct the CRD-related model by performing weighted gene co-expression network analysis and machine learning algorithms (LASSO, SVM-RFE, and RF). The ssGSEA method was used to calculate the CRDscore in order to quantify CRD status. Using single-cell transcriptome data of CSF cells, we investigated the CD4+ T cell metabolism and cell-cell communication in high- and low-risk CRD groups. Connectivity map (CMap) was applied to explore small molecule drugs targeting CRD, and the expression of the signature gene GPR4 was further validated in AD. The CRDscore algorithm, which is based on 23 circadian-related genes, can effectively classify the CRD status in AD datasets. The single-cell analysis revealed that the CD4+ T cells with high CRDscore were characterized by hypometabolism. Cell communication analysis revealed that CD4+ T cells might be involved in promoting CD8+ T cell adhesion under CRD, which may facilitate T cell infiltration into the brain parenchyma. Overall, this study indicates the potential connotation of circadian rhythm in AD, providing insights into understanding T cell metabolic reprogramming under CRD.

The proton-sensing heterotrimeric guanine nucleotide-binding protein-coupled receptor GPR65 is expressed in immune cells and regulates tissue homeostasis in response to decreased extracellular pH, which occurs in the context of inflammation and tumorigenesis. Genome-wide association studies linked GPR65 to several autoimmune and inflammatory diseases such as multiple sclerosis and inflammatory bowel disease (IBD). The loss-of-function GPR65 I231L IBD risk variant alters cellular metabolism, impairs protective tissue functions, and increases proinflammatory cytokine production. Hypothesizing that a small molecule designed to potentiate GPR65 at subphysiological pH could decrease inflammatory responses, we found positive allosteric modulators of GPR65 that engage and activate both human and mouse orthologs of the receptor. We observed that the chemical probe BRD5075 alters cytokine and chemokine programs in dendritic cells, establishing that immune signaling can be modulated by targeting GPR65. Our investigation offers improved chemical probes to further interrogate the biology of human GPR65 and its clinically relevant genetic variants.

G protein-coupled receptor G2A was postulated to be a promising target for the development of new therapeutics in neuropathic pain, acute myeloid leukemia, and inflammation. However, there is still a lack of potent, selective, and drug-like G2A agonists to be used as a chemical tool or as the starting matter for the development of drugs. In this work, we present the discovery and structure-activity relationship elucidation of a new potent and selective G2A agonist scaffold. Systematic optimization resulted in (3-(pyridin-3-ylmethoxy)benzoyl)-d-phenylalanine (T-10418) exhibiting higher potency than the reference and natural ligand 9-HODE and high selectivity among G protein-coupled receptors. With its favorable activity, a clean selectivity profile, excellent solubility, and high metabolic stability, T-10418 qualifies as a pharmacological tool to investigate the effects of G2A activation.