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Free Radical Biology & Medicine Jul 2024Bupivacaine (BUP) is an anesthetic commonly used in clinical practice that when used for spinal anesthesia, might exert neurotoxic effects. Thioredoxin-interacting...
Bupivacaine (BUP) is an anesthetic commonly used in clinical practice that when used for spinal anesthesia, might exert neurotoxic effects. Thioredoxin-interacting protein (TXNIP) is a member of the α-arrestin protein superfamily that binds covalently to thioredoxin (TRX) to inhibit its function, leading to increased oxidative stress and activation of apoptosis. The role of TXNIP in BUP-induced oxidative stress and apoptosis remains to be elucidated. In this context, the present study aimed to explore the effects of TXNIP knockdown on BUP-induced oxidative stress and apoptosis in the spinal cord of rats and in PC12 cells through the transfection of adeno-associated virus-TXNIP short hairpin RNA (AAV-TXNIP shRNA) and siRNA-TXNIP, respectively. In vivo, a rat model of spinal neurotoxicity was established by intrathecally injecting rats with BUP. The BUP + TXNIP shRNA and the BUP + Control shRNA groups of rats were injected with an AAV carrying the TXNIP shRNA and the Control shRNA, respectively, into the subarachnoid space four weeks prior to BUP treatment. The Basso, Beattie & Bresnahan (BBB) locomotor rating score, % MPE of TFL, H&E staining, and Nissl staining analyses were conducted. In vitro, 0.8 mM BUP was determined by CCK-8 assay to establish a cytotoxicity model in PC12 cells. Transfection with siRNA-TXNIP was carried out to suppress TXNIP expression prior to exposing PC12 cells to BUP. The results revealed that BUP effectively induced neurological behavioral dysfunction and neuronal damage and death in the spinal cord of the rats. Similarly, BUP triggered cytotoxicity and apoptosis in PC12 cells. In addition, treated with BUP both in vitro and in vivo exhibited upregulated TXNIP expression and increased oxidative stress and apoptosis. Interestingly, TXNIP knockdown in the spinal cord of rats through transfection of AAV-TXNIP shRNA exerted a protective effect against BUP-induced spinal neurotoxicity by ameliorating behavioral and histological outcomes and promoting the survival of spinal cord neurons. Similarly, transfection with siRNA-TXNIP mitigated BUP-induced cytotoxicity in PC12 cells. In addition, TXNIP knockdown mitigated the upregulation of ROS, MDA, Bax, and cleaved caspase-3 and restored the downregulation of GSH, SOD, CAT, GPX4, and Bcl2 induced upon BUP exposure. These findings suggested that TXNIP knockdown protected against BUP-induced spinal neurotoxicity by suppressing oxidative stress and apoptosis. In summary, TXNIP could be a central signaling hub that positively regulates oxidative stress and apoptosis during neuronal damage, which renders TXNIP a promising target for treatment strategies against BUP-induced spinal neurotoxicity.
Topics: Animals; Rats; Apoptosis; Bupivacaine; Carrier Proteins; Cell Cycle Proteins; Gene Knockdown Techniques; Injections, Spinal; Neurons; Neurotoxicity Syndromes; Oxidative Stress; PC12 Cells; Rats, Sprague-Dawley; RNA, Small Interfering; Spinal Cord; Thioredoxins
PubMed: 38614227
DOI: 10.1016/j.freeradbiomed.2024.04.223 -
British Journal of Pharmacology Aug 2024Pancreatic islets are modulated by cross-talk among different cell types and paracrine signalling plays important roles in maintaining glucose homeostasis. Urocortin 3...
BACKGROUND AND PURPOSE
Pancreatic islets are modulated by cross-talk among different cell types and paracrine signalling plays important roles in maintaining glucose homeostasis. Urocortin 3 (UCN3) secreted by pancreatic β cells activates the CRF receptor (CRFR) and downstream pathways mediated by different G protein or arrestin subtypes in δ cells to cause somatostatin (SST) secretion, and constitutes an important feedback circuit for glucose homeostasis.
EXPERIMENTAL APPROACH
Here, we used Arrb1, Arrb2, Gs and Gq knockout mice, the G-shRNA-GFP lentivirus, as well as functional assays and pharmacological characterization to study how the coupling of G, G and β-arrestin1 to CRFR contributed to UCN3-induced SST secretion in pancreatic δ cells.
KEY RESULTS
Our study showed that CRFR coupled to a panel of G protein and arrestin subtypes in response to UCN3 engagement. While RyR3 phosphorylation by PKA at the S156, S2706 and S4697 sites may underlie the Gs-mediated UCN3- CRFR axis for SST secretion, the interaction of SYT1 with β-arrestin1 is also essential for efficient SST secretion downstream of CRFR. The specific expression of the transcription factor Stat6 may contribute to G expression in pancreatic δ cells. Furthermore, we found that different UCN3 concentrations may have distinct effects on glucose homeostasis, and these effects may depend on different CRFR downstream effectors.
CONCLUSIONS AND IMPLICATIONS
Collectively, our results provide a landscape view of signalling mediated by different G protein or arrestin subtypes downstream of paracrine UCN3- CRFR signalling in pancreatic β-δ-cell circuits, which may facilitate the understanding of fine-tuned glucose homeostasis networks.
Topics: Animals; Urocortins; Mice; Signal Transduction; Somatostatin; Receptors, Corticotropin-Releasing Hormone; Mice, Knockout; Somatostatin-Secreting Cells; GTP-Binding Proteins; Mice, Inbred C57BL; Male
PubMed: 38613153
DOI: 10.1111/bph.16351 -
Cell Chemical Biology Apr 2024Adhesion G protein-coupled receptor (aGPCR) signaling influences development and homeostasis in a wide range of tissues. In the current model for aGPCR signaling, ligand...
Adhesion G protein-coupled receptor (aGPCR) signaling influences development and homeostasis in a wide range of tissues. In the current model for aGPCR signaling, ligand binding liberates a conserved sequence that acts as an intramolecular, tethered agonist (TA), yet this model has not been evaluated systematically for all aGPCRs. Here, we assessed the TA-dependent activities of all 33 aGPCRs in a suite of transcriptional reporter, G protein activation, and β-arrestin recruitment assays using a new fusion protein platform. Strikingly, only ∼50% of aGPCRs exhibited robust TA-dependent activation, and unlike other GPCR families, aGPCRs showed a notable preference for G signaling. AlphaFold2 predictions assessing TA engagement in the predicted intramolecular binding pocket aligned with the TA dependence of the cellular responses. This dataset provides a comprehensive resource to inform the investigation of all human aGPCRs and for targeting aGPCRs therapeutically.
PubMed: 38608683
DOI: 10.1016/j.chembiol.2024.03.004 -
Biochemical Pharmacology Jun 2024Arrestins are key negative regulators of G Protein-Coupled Receptors (GPCRs) through mediation of G protein desensitisation and receptor internalisation. Arrestins can...
Arrestins are key negative regulators of G Protein-Coupled Receptors (GPCRs) through mediation of G protein desensitisation and receptor internalisation. Arrestins can also contribute to signal transduction by scaffolding downstream signalling effectors for activation. GPCR kinase (GRK) enzymes phosphorylate the intracellular C-terminal domain, or intracellular loop regions of GPCRs to promote arrestin interaction. There are seven different GRK subtypes, which may uniquely phosphorylate the C-terminal tail in a type of 'phosphorylation barcode,' potentially differentially contributing to arrestin translocation and arrestin-dependent signalling. Such contributions may be exploited to develop arrestin-biased ligands. Here, we examine the effect of different GRK subtypes on the ability to promote translocation of arrestin-2 and arrestin-3 to the cannabinoid CB receptor (CB) with a range of ligands. We find that most GRK subtypes (including visual GRK1) can enhance arrestin-2 and -3 translocation to CB, and that GRK-dependent changes in arrestin-2 and arrestin-3 translocation were broadly shared for most agonists tested. GRK2/3 generally enhanced arrestin translocation more than the other GRK subtypes, with some small differences between ligands. We also explore the interplay between G protein activity and GRK2/3-dependent arrestin translocation, highlighting that high-efficacy G protein agonists will cause GRK2/3 dependent arrestin translocation. This study supports the hypothesis that arrestin-biased ligands for CB must engage GRK5/6 rather than GRK2/3, and G protein-biased ligands must have inherently low efficacy.
Topics: Humans; Receptor, Cannabinoid, CB1; Signal Transduction; HEK293 Cells; Arrestins; Protein Transport; GTP-Binding Proteins; G-Protein-Coupled Receptor Kinases; Animals; beta-Arrestin 2
PubMed: 38604257
DOI: 10.1016/j.bcp.2024.116190 -
Nature May 2024The µ-opioid receptor (µOR) is an important target for pain management and molecular understanding of drug action on µOR will facilitate the development of better...
The µ-opioid receptor (µOR) is an important target for pain management and molecular understanding of drug action on µOR will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance and single-molecule fluorescence resonance energy transfer, how ligand-specific conformational changes of µOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several conformations of the cytoplasmic face of the receptor that interconvert on different timescales, including a pre-activated conformation that is capable of G-protein binding, and a fully activated conformation that markedly reduces GDP affinity within the ternary complex. Interaction of β-arrestin-1 with the μOR core binding site appears less specific and occurs with much lower affinity than binding of G.
Topics: Humans; beta-Arrestin 1; Binding Sites; Fluorescence Resonance Energy Transfer; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine Diphosphate; Ligands; Models, Molecular; Protein Binding; Protein Conformation; Receptors, Opioid, mu; Single Molecule Imaging
PubMed: 38600384
DOI: 10.1038/s41586-024-07295-2 -
ACS Chemical Neuroscience May 2024ADB-HEXINACA has been recently reported as a synthetic cannabinoid receptor agonist (SCRA), one of the largest classes of new psychoactive substances (NPSs). This...
ADB-HEXINACA has been recently reported as a synthetic cannabinoid receptor agonist (SCRA), one of the largest classes of new psychoactive substances (NPSs). This compound marks the entry of the -hexyl tail group into the SCRA landscape, which has continued in the market with recent, newly detected SCRAs. As such, a proactive characterization campaign was undertaken, including the synthesis, characterization, and pharmacological evaluation of ADB-HEXINACA and a library of 41 closely related analogues. Two in vitro functional assays were employed to assess activity at CB and CB cannabinoid receptors, measuring G-coupled agonism through a fluorescence-based membrane potential assay (MPA) and β-arrestin 2 (βarr2) recruitment via a live cell-based nanoluciferase complementation reporter assay. ADB-HEXINACA was a potent and efficacious CB agonist (CB MPA pEC = 7.87 ± 0.12 M; = 124 ± 5%; βarr2 pEC = 8.27 ± 0.14 M; = 793 ± 42.5), as were most compounds assessed. Isolation of the heterocyclic core and alkyl tails allowed for the comprehensive characterization of structure-activity relationships in this compound class, which were rationalized in silico via induced fit docking experiments. Overall, most compounds assessed are possibly emerging NPSs.
Topics: Cannabinoid Receptor Agonists; Humans; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; HEK293 Cells; Structure-Activity Relationship; Animals
PubMed: 38597712
DOI: 10.1021/acschemneuro.3c00818 -
Clinical and Experimental Pharmacology... May 2024Lung adenocarcinoma (LUAD) is a serious threat to public health and is accompanied by increased morbidity and mortality worldwide. Neuronal PAS domain protein2 (NPAS2)...
Lung adenocarcinoma (LUAD) is a serious threat to public health and is accompanied by increased morbidity and mortality worldwide. Neuronal PAS domain protein2 (NPAS2) has been confirmed as an oncogene in LUAD; however, little is known about its molecular mechanism. Here, the expression level of NPAS2 was detected in LUAD cell lines and 16HBE cells. Gain- and loss-of-function experiments were performed. Cell Counting Kit-8, colony formation, flow cytometry, wound-healing and Transwell assays were conducted to assess cell proliferation, apoptosis, migration and invasion, respectively. Reprogramming of glucose metabolism was evaluated via oxygen consumption rate (OCR), complexes activities, lactic production and glucose consumption. The expression of critical proteins was examined by western blot. We demonstrated aberrant upregulation of NPAS2 and β-arrestin-1 (ARRB1) in LUAD cell lines. ARRB1 was found to be a critical transcription factor of NPAS2 with binding sites within the promoter region of NPAS2, thereby causing its transcriptional activation. Functional experiments revealed that NPAS2 depletion significantly inhibited the malignant behaviours of A549 cells by suppressing cell proliferation, migration, invasion and epithelial-mesenchymal transition and promoting cell apoptosis. Meanwhile, NPAS2 depletion increased OCR and activities of complexes (I, II, III and V), and reduced lactic acid production and glucose uptake in A549 cells, indicating that NPAS2 depletion inhibited aerobic glycolysis, accompanied by reduced expression of glycolytic enzymes. However, the changes caused by NPAS2 knockdown were partly restored by ARRB1 overexpression. In conclusion, our study suggests that ARRB1 could transcriptionally activate NPAS2, facilitating malignant activities and glycolysis, and ultimately promoting the progression of LUAD, proving a novel therapeutic strategy for the treatment of LUAD.
Topics: Humans; Carbohydrate Metabolism; Glycolysis; Adenocarcinoma of Lung; Cell Proliferation; Glucose; Lung Neoplasms; Cell Movement; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Nerve Tissue Proteins; Basic Helix-Loop-Helix Transcription Factors; beta-Arrestin 1
PubMed: 38584327
DOI: 10.1111/1440-1681.13860 -
Molecular Biotechnology Apr 2024Oral squamous cell carcinoma (OSCC) represents the primary subtype of head and neck squamous cell carcinoma (HNSCC), characterized by a high morbidity and mortality...
Oral squamous cell carcinoma (OSCC) represents the primary subtype of head and neck squamous cell carcinoma (HNSCC), characterized by a high morbidity and mortality rate. Although previous studies have established specific correlations between euchromatic histone lysine methyltransferase 2 (EHMT2), a histone lysine methyltransferase, and the malignant phenotype of OSCC cells, its biological functions in OSCC remain largely unknown. This study, grounded in bioinformatics predictions, aims to clarify the influence of EHMT2 on the malignant behavior of OSCC cells and delve into the underlying mechanisms. EHMT2 exhibited high expression in OSCC tissues and demonstrated an association with poor patient outcomes. Artificial EHMT2 silencing in OSCC cells, achieved through lentiviral vector infection, significantly inhibited colony formation, migration, invasion, and cell survival. Regarding the mechanism, EHMT2 was found to bind the promoter of arrestin beta 1 (ARRB1), thereby suppressing its transcription through H3K9me2 modification. ARRB1, in turn, was identified as a negative regulator of the Hedgehog pathway, leading to a reduction in the proteins GLI1 and PTCH1. Cancer stem cells (CSCs) were enriched through repeated sphere formation assays in two OSCC cell lines. EHMT2 was found to activate the Hedgehog pathway, thus promoting sphere formation, migration and invasion, survival, and tumorigenic activity of the OSCC-CSCs. Notably, these effects were counteracted by the additional overexpression of ARRB1. In conclusion, this study provides novel evidence suggesting that EHMT2 plays specific roles in enhancing stem cell properties in OSCC by modulating the ARRB1-Hedgehog signaling cascade.
PubMed: 38573544
DOI: 10.1007/s12033-024-01130-9 -
International Immunology Apr 2024Efficient induction of humoral immune responses depends on orchestrated migration of B cells within lymphoid organs, which is governed by G protein-coupled receptors...
Efficient induction of humoral immune responses depends on orchestrated migration of B cells within lymphoid organs, which is governed by G protein-coupled receptors (GPCRs) responding to chemoattractants, represented by chemokines. After ligand binding, GPCRs are phosphorylated by different GPCR kinases (GRKs) at distinct sites on the receptor C termini, which dictates functional outcomes of β-arrestin-mediated signaling, ranging from receptor inactivation to effector molecule activation. However, the molecular mechanisms by which individual GRKs are selectively targeted to GPCRs have been poorly understood. Our recent study revealed that a protein complex consisting of copper metabolism MURR1 domain-containing (COMMD) 3 and 8 (COMMD3/8 complex) functions as an adaptor that recruits a specific GRK to chemoattractant receptors and plays an important role in the control of B cell migration during humoral immune responses. In this review, we summarize the current understanding of chemoattractant receptor signaling in the context of humoral immunity and discuss the potential of the COMMD3/8 complex as a therapeutic target for autoimmune diseases.
PubMed: 38573198
DOI: 10.1093/intimm/dxae021 -
The Journal of Clinical Endocrinology... Apr 2024Melanocortin-4 receptor (MC4R) plays an important role in body weight regulation. Pathogenic MC4R variants are the most common cause of monogenic obesity.
CONTEXT
Melanocortin-4 receptor (MC4R) plays an important role in body weight regulation. Pathogenic MC4R variants are the most common cause of monogenic obesity.
OBJECTIVE
We have identified 17 MC4R variants in adult and pediatric patients with obesity. Here, we aimed to functionally characterize these variants by analyzing four different aspects of MC4R signaling. In addition, we aimed to analyze the effect of setmelanotide, a potent MC4R agonist, on these MC4R variants.
MATERIALS AND METHODS
Cell surface expression and α-MSH- or setmelanotide-induced cAMP response, β-arrestin-2 recruitment, and ERK activation were measured in cells expressing either wild type (WT) or variant MC4R.
RESULTS
We found a large heterogeneity in the function of these variants. We identified variants with a loss of response for all studied MC4R signaling, variants with no cAMP accumulation or ERK activation but normal β-arrestin-2 recruitment, and variants with normal cAMP accumulation and ERK activation but decreased β-arrestin-2 recruitment, indicating disrupted desensitization and signaling mechanisms. Setmelanotide displayed a greater potency and similar efficacy as α-MSH, and induced significantly increased maximal cAMP responses of several variants compared to α-MSH. Despite the heterogeneity in functional response, there was no apparent difference in the obesity phenotype in our patients.
DISCUSSION
We show that these obesity-associated MC4R variants affect MC4R signaling differently, yet leading to a comparable clinical phenotype. Our results demonstrate the clinical importance of assessing the effect of MC4R variants on a range of molecular signaling mechanisms to determine their association with obesity, which may aid in improving personalized treatment.
PubMed: 38567654
DOI: 10.1210/clinem/dgae210