bims-dicesi Biomed News
on Diversification of cell signalling
Issue of 2024–12–29
29 papers selected by
Ashanika Karandawela



  1. bioRxiv. 2024 Dec 09. pii: 2024.12.04.626681. [Epub ahead of print]
      The constitutive (ligand-independent) signaling of G protein-coupled receptors (GPCRs) is being increasingly appreciated as an integral aspect of their function; however, it can be technically hard to detect for poorly characterized, e.g. orphan, receptors of the cAMP-inhibitory Gi-coupled (GiPCR) family. In this study, we delineate the optimal strategies for the detection of such activity across several GiPCRs in two cell lines. As our study examples, we chose two canonical GiPCRs - the constitutively active Smoothened and the ligand-activated CXCR4,-and one atypical GPCRs, the chemokine receptor ACKR3. We verified the applicability of three Bioluminescence Resonance Energy Transfer (BRET)-based assays - one measuring changes in intracellular cAMP, another in Gβγ/GRK3ct association and third in Gαi-Gβγ dissociation, - for assessing both constitutive and ligand-modulated activity of these receptors. We also revealed the possible caveats and sources of false positives, and proposed optimization strategies. All three types of assays confirmed the ligand-dependent activity of CXCR4, the controversial G protein incompetence of ACKR3, the constitutive Gi-directed activity of SMO, and its modulation by PTCH1. We also demonstrated that PTCH1 promotes SMO localization to the cell surface, thus enhancing its responsiveness not only to agonists but also to antagonists, which is a novel mechanism of regulation of a Class F GiPCR Smoothened.
    DOI:  https://doi.org/10.1101/2024.12.04.626681
  2. Pharmacol Ther. 2024 Dec 23. pii: S0163-7258(24)00208-0. [Epub ahead of print] 108788
      G protein-coupled receptors (GPCRs), the largest family of membrane receptors in the mammalian genomes, regulate almost all known physiological processes by transducing numerous extracellular stimuli including almost two-thirds of endogenous hormones and neurotransmitters. The traditional view held that GPCR signaling occurs exclusively at the cell surface, where the receptors bind with the ligands and undergo conformational changes to recruit and activate heterotrimeric G proteins. However, with the application of advanced biochemical and biophysical techniques, this conventional model is challenged by the elucidation of spatiotemporal GPCR activation with the evidence that receptors can signal from subcellular compartments to exhibit various molecular and cellular responses with physiological and pathophysiological relevance. Thus, this 'location bias' of GPCR signaling has become another layer of complexity of GPCR signal transduction. In this review, we generally introduce the development of the concept of compartmentalized GPCR signaling and comprehensively summarize the receptors reported to be localized on the membranes of different intracellular organelles. We review the physiological functions of these compartmentalized GPCRs with emphasis on some well-characterized prototypical hormone/neurotransmitter-binding receptors, including β2-adrenergic receptor, opioid receptors, parathyroid hormone type 1 receptor, thyroid-stimulating hormone receptor, cannabinoid receptor type 1, and metabotropic glutamate receptor 5, as examples. In addition, the therapeutic implications of compartmentalized GPCR signaling by introducing lipophilic or hydrophilic ligands for intracellular targeting, lipid conjugation anchor drugs, and strategy to modulate receptor internalization/resensitization, are highlighted and open new avenues in GPCR pharmacology and therapeutics.
    Keywords:  GPCR; Hormone; Intracellular compartments; Location-bias; Therapeutics
    DOI:  https://doi.org/10.1016/j.pharmthera.2024.108788
  3. J Struct Biol. 2024 Dec 24. pii: S1047-8477(24)00104-7. [Epub ahead of print] 108164
      Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are bioactive lysophospholipids derived from cell membranes that activate the endothelial differentiation gene family of G protein-coupled receptors. Activation of these receptors triggers multiple downstream signaling cascades through G proteins such as Gi/o, Gq/11, and G12/13. Therefore, LPA and S1P mediate several physiological processes, including cytoskeletal dynamics, neurite retraction, cell migration, cell proliferation, and intracellular ion fluxes. The basis for the G-protein coupling selectivity of EGD receptors, however, remains unknown. Here, we present cryo-electron microscopy structures of LPA-activated LPA1 in complexes with Gi, Gq, and G13 heterotrimers. Comparison of the three LPA1-G protein structures shows clearly different conformations of intracellular loop 2 (ICL2) and ICL3 that are likely induced by the different Gα protein interfaces. Interestingly, this G-protein interface interaction is a common feature of LPA and S1P receptors. Our findings provide clues to understanding the promiscuity of G-protein coupling in the endothelial differentiation gene family.
    Keywords:  Cryoelectron microscopy; Endothelial differentiation gene family; G-protein-coupled receptor (GPCR); GTP-binding proteins; Lysophosphatidic acid (LPA); Receptors; Sphingosine-1-phosphate (S1P)
    DOI:  https://doi.org/10.1016/j.jsb.2024.108164
  4. Pharmacol Ther. 2024 Dec 22. pii: S0163-7258(24)00204-3. [Epub ahead of print] 108784
      Free-fatty acid receptor-4 (FFA4), previously known as GPR120, is a G protein-coupled receptor (GPCR) activated by medium-to-long chain free fatty acids (FFAs), including saturated, monounsaturated, and polyunsaturated fats, many of which (e.g., omega-3 fatty acids) are critical contributors to human health and disease. FFA4 is widely expressed across human tissues, and its activation supports a range of physiological functions, including the release of gastrointestinal incretin hormones like glucagon-like peptide-1 (GLP-1), regulation of pancreatic hormone secretion, peripheral glucose uptake, adipose regulation, and anti-inflammatory responses in macrophages. Due to its pivotal role in energy metabolism and inflammation, FFA4 has emerged as a major target in drug discovery. Historically, FFA4 signaling was linked to the Gαq/11 family of intracellular heterotrimeric G proteins, which mediate its GLP-1 releasing effects. However, emerging evidence indicates that FFA4 can signal through other Gα proteins in various cellular contexts. Notably, its anti-inflammatory effects are also dependent on interactions with β-arrestin proteins, further broadening the receptor's signaling versatility. This review explores the concept of biased agonism at FFA4, emphasizing how this receptor selectively signals through distinct transduction pathways, including Gα proteins and β-arrestins. We also examine the key structural elements of FFA4 that govern its interactions with different signaling partners, the elucidation of which has laid the groundwork for the development of biased agonists aimed at selectively modulating these FFA4-mediated pathways for therapeutic application.
    Keywords:  Biased agonism; FFA4; Free-fatty acid receptor 4; Free-fatty acids; GPR120; β-arrestin
    DOI:  https://doi.org/10.1016/j.pharmthera.2024.108784
  5. Bioessays. 2024 Dec 26. e202400233
      In this review, we introduce the concept of "dual thermosensing mechanisms," highlighting the functional collaboration between G protein-coupled receptors (GPCRs) and transient receptor potential (TRP) channels that enable sophisticated cellular thermal responsiveness. GPCRs have been implicated in thermosensory processes, with recent findings identifying several candidates across species, including mammals, fruit flies, and nematodes. In many cases, these GPCRs work in conjunction with another class of thermosensors, TRP channels, offering insights into the complex mechanisms underlying thermosensory signaling. We examine how GPCRs function as thermosensors and how their signaling regulates cellular thermosensation, illustrating the complexity of thermosensory systems. Understanding these dual thermosensory mechanisms would advance our comprehension of cellular thermosensation and its regulatory pathways.
    DOI:  https://doi.org/10.1002/bies.202400233
  6. bioRxiv. 2024 Dec 13. pii: 2024.12.12.628161. [Epub ahead of print]
      G protein-coupled receptor (GPCR) signaling is terminated by arrestin binding to a phosphorylated receptor. Binding propensity has been shown to be modulated by stabilizing the pre-activated state of arrestin through point mutations or C-tail truncation. Here, we hypothesize that pre-activated rotated states can be stabilized by small molecules, and this can promote binding to phosphorylation-deficient receptors, which underly a variety of human disorders. We performed virtual screening on druggable pockets identified on pre-activated conformations in Molecular Dynamics trajectories of arrestin-3, and found a compound targeting an activation switch, the back loop at the inter-domain interface. According to our model, consistent with available biochemical and structural data, the compound destabilized the ionic lock between the finger and the back loop, and enabled transition of the 'gate loop' towards the pre-activated state, which stabilizes pre-activated inter-domain rotation. The predicted binding pocket is consistent with saturation-transfer difference NMR data indicating close contact between the piperazine moiety of the compound and C/finger loops. The compound increases in-cell arrestin-3 binding to phosphorylation-deficient and wild-type β2-adrenergic receptor, but not to muscarinic M2 receptor, as verified by FRET and NanoBiT. This study demonstrates that the back loop can be targeted to modulate interaction of arrestin with phosphorylation-deficient GPCRs in a receptor-specific manner.
    DOI:  https://doi.org/10.1101/2024.12.12.628161
  7. Nat Commun. 2024 Dec 27. 15(1): 10714
      We describe a structural and functional study of the G protein-coupled apelin receptor, which binds two endogenous peptide ligands, apelin and Elabela/Toddler (ELA), to regulate cardiovascular development and function. Characterisation of naturally occurring apelin receptor variants from the UK Genomics England 100,000 Genomes Project, and AlphaFold2 modelling, identifies T892.64 as important in the ELA binding site, and R1684.64 as forming extensive interactions with the C-termini of both peptides. Base editing to introduce an R/H1684.64 variant into human stem cell-derived cardiomyocytes demonstrates that this residue is critical for receptor binding and function. Additionally, we present an apelin receptor crystal structure bound to the G protein-biased, small molecule agonist, CMF-019, which reveals a deeper binding mode versus the endogenous peptides at lipophilic pockets between transmembrane helices associated with GPCR activation. Overall, the data provide proof-of-principle for using genetic variation to identify key sites regulating receptor-ligand engagement.
    DOI:  https://doi.org/10.1038/s41467-024-55381-w
  8. Methods Mol Biol. 2025 ;2840 75-87
      YAP is a central regulator of the Hippo-YAP signaling axis, an evolutionarily conserved pathway that modulates organ growth and regeneration. Dysregulation of YAP signaling leads to uncontrolled proliferation, promoting epithelial-to-mesenchymal transition and invasion in cancer metastasis. Exogenous manipulation of YAP activity at the second-to-minute timescale is an important step in studying the signaling pathway. We present an optogenetic system to control the subcellular localization of YAP and therefore its activity as a transcriptional co-activator. We used the LOV2-Jα interacting domains to photocage a nuclear localization signal (NLS) attached to YAP. Under 488 nm light, the Jα helix unfolds and the interaction with LOV2 is disrupted, thereby exposing the NLS and allowing for the entire optogenetic construct to be shuttled into the nucleus. This nuclear translocation is reversible and tuneable and demonstrates functional activity after nuclear localization both in vitro and in vivo.
    Keywords:  Hippo-YAP; Optogenetics; Spatiotemporal dynamics; Zebrafish
    DOI:  https://doi.org/10.1007/978-1-0716-4047-0_6
  9. Protein Sci. 2025 Jan;34(1): e70013
      G protein Coupled Receptors (GPCRs) are the largest family of cell surface receptors in humans. Somatic mutations in GPCRs are implicated in cancer progression and metastasis, but mechanisms are poorly understood. Emerging evidence implicates perturbation of intra-receptor activation pathway motifs whereby extracellular signals are transmitted intracellularly. Recently, sufficiently sensitive methodology was described to calculate structural strain as a function of missense mutations in AlphaFold-predicted model structures, which was extensively validated on experimental and predicted structural datasets. When paired with Molecular Dynamics (MD) simulations, these tools provide a facile approach to screen mutations in silico. We applied this framework to calculate the structural and dynamic effects of cancer-associated mutations in the chemokine receptor CCR3, a Class A GPCR involved in cancer and autoimmune disorders. Residue-residue contact scoring refined effective strain results, highlighting significant remodeling of inter- and intra-motif contacts along the highly conserved GPCR activation pathway network. We then integrated AlphaFold-derived predicted Local Distance Difference Test scores with per-residue Root Mean Square Fluctuations and activation pathway Contact Analysis (CONAN) from coarse grain MD simulations to identify statistically significant changes in receptor dynamics upon mutation. Finally, analysis of negative control mutants suggests false positive results in AlphaFold pipelines should be considered but can be mitigated with stricter control of statistical analysis. Our results indicate selected mutants influence structural plasticity of CCR3 related to ligand interaction, activation, and G protein coupling, using a framework that could be applicable to a wide range of biochemically relevant protein targets following further validation.
    Keywords:  AlphaFold; CCR3; ColabFold; GPCR; cancer; molecular dynamics; pLDDT
    DOI:  https://doi.org/10.1002/pro.70013
  10. J Biol Chem. 2024 Dec 18. pii: S0021-9258(24)02614-0. [Epub ahead of print] 108112
      The formyl-peptide receptor 2 (FPR2) is a G-protein-coupled receptor (GPCR) that responds to pathogen-derived peptides and regulates both pro-inflammatory and pro-resolution cellular processes. While ligand selectivity and G-protein-signalling of FPR2 have been well characterized, molecular mechanisms controlling subsequent events such as endocytosis and recycling to the plasma membrane are less understood. Here we show the key role of the GPCR kinase 5 (GRK5) in facilitating FPR2 endocytosis and post-endocytic trafficking. We found, in response to activation by a synthetic peptide WKYMVm, the recruitment of β-Arrestins to the receptor requires both putative phosphorylation sites in the C-terminal of FPR2 and the presence of GRKs, predominantly GRK5. Furthermore, although GRKs are required for β-Arrestin recruitment and endocytosis, the recruitment of β-Arrestin is not itself essential for FPR2 endocytosis. Instead, β-Arrestin determines post-endocytic delivery of FPR2 to subcellular compartments and subsequent plasma membrane delivery and controls the magnitude of downstream signal transduction. Collectively, the newly characterized FPR2 molecular pharmacology will facilitate the design of more efficient therapeutics targeting chronic inflammation.
    DOI:  https://doi.org/10.1016/j.jbc.2024.108112
  11. J Biol Chem. 2024 Dec 24. pii: S0021-9258(24)02627-9. [Epub ahead of print] 108125
      Several peptides interact with phylogenetically unrelated G protein-coupled receptors (GPCRs); similarly, orthologous GPCRs interact with distinct ligands. The neuropeptide Substance P (SP) activates both NK1R and another unrelated primate-specific GPCR, MRGPRX2. Furthermore, MRGPRX 1, a paralog of MRGPRX2, recognizes BAM8-22, which has no evolutionary relatedness to SP. To elucidate the molecular basis and evolutionary history of this phylogenetically unrelated ligand selectivity, we developed a systematic procedure, the "interaction determinant likelihood (IDL) score" system, which estimates the amino acid residues responsible for peptide-GPCR interactions predicted by peptide descriptor (PD)-incorporated SVM, our original machine learning-based peptide-GPCR interaction predictor. An IDL score-based approach followed by pharmacological validation revealed the determinant residues for the ligand selectivity of SP-MRGPRX2 (F3.24 and G4.61) and BAM8-22-MRGPRX1 (L1.35). Molecular phylogenetic analysis revealed that the MRGPRX1 of common ancestral primates recognized BAM8-22, whereas the ancestral Cercopithecinae MRGPRX1 lost its interaction with BAM8-22 due to the loss of L1.35. The SP-MRGPRX2 interaction emerged in the common ancestors of Euarchonta, and, thereafter, the interaction of MRGPRX2 with both SP and BAM8-22 was acquired via substitution with L1.35 in several lineages. Collectively, the present study unraveled the molecular mechanisms and evolution of ligand specificity in evolutionary unrelated GPCRs.
    DOI:  https://doi.org/10.1016/j.jbc.2024.108125
  12. Epilepsia Open. 2024 Dec 27.
      Epilepsy is one of the common chronic neurological diseases, affecting more than 70 million people worldwide. The brains of people with epilepsy exhibit a pathological and persistent propensity for recurrent seizures. Epilepsy often coexists with cardiovascular disease, cognitive dysfunction, depression, etc., which seriously affects the patient's quality of life. Although our understanding of epilepsy has advanced, the pathophysiological mechanisms leading to epileptogenesis, drug resistance, and associated comorbidities remain largely unknown. The use of newer antiepileptic drugs has increased, but this has not improved overall outcomes. We need to deeply study the pathogenesis of epilepsy and find drugs that can not only prevent the epileptogenesis and interfere with the process of epileptogenesis but also treat epilepsy comorbidities. Sphingosine-1-phosphate (S1P) is an important lipid molecule. It not only forms the basis of cell membranes but is also an important bioactive mediator. It can not only act as a second messenger in cells to activate downstream signaling pathways but can also exert biological effects by being secreted outside cells and binding to S1P receptors on the cell membrane. Fingolimod (FTY720) is the first S1P receptor modulator developed and approved for the treatment of multiple sclerosis. More and more studies have proven that the S1P signaling pathway is closely related to epilepsy, drug-resistant epilepsy, epilepsy comorbidities, or other epilepsy-causing diseases. However, there is much controversy over the role of certain natural molecules in the pathway and receptor modulators (such as FTY720) in epilepsy. Here, we summarize and analyze the role of the S1P signaling pathway in epilepsy, provide a basis for finding potential therapeutic targets and/or epileptogenic biomarkers, analyze the reasons for these controversies, and put forward our opinions. PLAIN LANGUAGE SUMMARY: This article combines the latest research literature at home and abroad to review the sphingosine 1-phosphate signaling pathway and epileptogenesis, drug-resistant epilepsy, epilepsy comorbidities, other diseases that can cause epilepsy, as well as the sphingosine-1-phosphate signaling pathway regulators and epilepsy, with the expectation of providing a certain theoretical basis for finding potential epilepsy treatment targets and/or epileptogenic biomarkers in the sphingosine-1-phosphate signaling pathway.
    Keywords:  drug‐resistant epilepsy; epileptogenesis; fingolimod; sphingosine‐1‐phosphate; sphingosine‐1‐phosphate receptor
    DOI:  https://doi.org/10.1002/epi4.13112
  13. Int J Biol Macromol. 2024 Dec 19. pii: S0141-8130(24)09810-6. [Epub ahead of print]290 138999
      Insect gustatory receptors play a critical role in modulating feeding behaviors by detecting external nutritional cues through complex biochemical pathways. Bitter taste receptors are essential for insects to identify and avoid toxins. However, the detailed molecular and cellular mechanisms by which these receptors influence insect feeding behavior remain poorly understood. Our previous research identified the bitter taste receptor NlGr23a in the brown planthopper (BPH), which specifically binds to oxalic acid and elicits a significant feeding rejection response. In this study, using an Sf9 cell line stably expressing NlGr23a, we demonstrated that oxalic acid exposure significantly enhances phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB), a protein associated with BPH food consumption. Further analysis revealed the involvement of phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway in facilitating CREB phosphorylation upon activation by oxalic acid-NlGr23a binding. These in vitro findings were corroborated by in vivo experiments examining the expression profiles of relevant proteins and protein kinases in BPHs fed an oxalic acid-supplemented diet. Our results elucidate the biochemical cascades triggered by oxalic acid-NlGr23a interaction, advancing our understanding of insect gustatory receptor-mediated feeding behavior modulation and potentially informing novel strategies for integrated pest management.
    Keywords:  CREB; Feeding; Gustatory receptor; Nilaparvata lugens; Oxalic acid; PI3K/AKT
    DOI:  https://doi.org/10.1016/j.ijbiomac.2024.138999
  14. FASEB J. 2024 Dec 13. 38(24): e70233
      Decidualization of endometrial stromal cells is a prerequisite for successful embryo implantation and early pregnancy. Decidualization dysregulation results in implantation failure. In our previous study, we reported that PAI-1 is abnormally downregulated in the endometrial tissue samples of patients with recurrent implantation failure. This study will explore the dynamic expression changes of PAI-1 in the endometrium during the menstrual cycle and its molecular mechanism affecting endometrial decidualization. Our findings indicated that the abundance of PAI-1 increased in the mid-secretory phase and attached a peak in the decidual phase in the endometrium of women with regular menstrual cycles. In human endometrial stromal cells (HESCs), PAI-1 knockdown attenuated endometrial decidualization by upregulating VEGFR2/PI3K/AKT signaling pathway and impaired the F-actin reorganization. Furthermore, axitinib (a VEGFR2 inhibitor) was used to inhibit the VEGFR2 protein activity and the results suggested that it eliminated the effects of PAI-1 on PI3K/AKT signaling pathways and F-actin remodeling. In addition, the interaction between PAI-1 and KNG1 was confirmed by coimmunoprecipitation assay in HESCs. Altogether, PAI-1-KNG1 may enhance the decidualization of endometrium by inhibiting VEGFR2/PI3K/AKT signaling pathway-mediated F-actin reorganization in healthy females.
    Keywords:  F‐actin; PAI‐1; PI3K/AKT signaling pathway; VEGFR2; decidualization
    DOI:  https://doi.org/10.1096/fj.202401882R
  15. Nan Fang Yi Ke Da Xue Xue Bao. 2024 Dec 20. pii: 1673-4254(2024)12-2283-08. [Epub ahead of print]44(12): 2283-2290
       OBJECTIVES: To observe the role of miR-139-5p and Notch1 signaling pathway in regulation of homing of bone mesenchymal stem cells (BMSCs) of asthmatic rats.
    METHODS: Normal rat BMSCs were co-cultured with bronchial epithelial cells from normal or asthmatic rats, followed by transfection with miR-139-5p mimics or a negative control sequence. The changes in cell viability and cell cycle were analyzed, and the cellular expressions of CXCR4 and SDF-1 were detected using immunofluorescence staining. The changes of BMSC homing after the transfection were observed, and the expressions of Notch1, RBP-J, and Hes1 mRNAs and proteins and Th1/Th2 cytokines were detected with RT-qPCR, Western blotting or ELISA.
    RESULTS: The co-cultures of BMSCs and asthmatic bronchial epithelial cells showed significantly decreased expressions of miR-139-5p, IL-2 and IL-12 and increased expressions of CXCR4, SDF-1, IL-5, IL-9, Notch1, RBP-J, and Hes1. Transfection with miR-139-5p mimics significantly increased the expressions of miR-139-5p, IL-2, CXCR4 and SDF-1 and lowered the expression levels of IL-5, IL-9, Notch1, activated Notch1, and Hes1 in the co-cultured cells. Correlation analysis showed that BMSC homing was positively correlated with miR-139-5p and IL-12 and negatively correlated with IL-5 expression. The expression of CXCR4 was negatively correlated with activated Notch1, and SDF-1 was positively correlated with miR-139-5p but negatively correlated with Notch1 expression.
    CONCLUSIONS: High expression of miR-139-5p promotes homing of BMSCs in asthma by targeting the Notch1 signaling pathway to regulate the expressions of Th1/Th2 cytokines, thereby alleviating airway inflammation.
    Keywords:  Notch1 signaling pathway; asthma; bone mesenchymal stem cells; miR-139-5p
    DOI:  https://doi.org/10.12122/j.issn.1673-4254.2024.12.03
  16. bioRxiv. 2024 Dec 11. pii: 2024.12.10.627829. [Epub ahead of print]
       Background: Previously we found that increasing fibroblast growth factor (FGF) signaling in the neural crest cells within the frontonasal process (FNP) of the chicken embryo caused dysmorphology that was correlated with reduced proliferation, disrupted cellular orientation, and lower MAPK activation but no change in PLCy and PI3K activation. This suggests RTK signaling may drive craniofacial morphogenesis through specific downstream effectors that affect cellular activities. In this study we inhibited three downstream branches of RTK signaling to determine their role in regulating cellular activities and how these changes affect morphogenesis of the FNP.
    Results: Small molecule inhibitors of MEK1/2, PI3K, and PLCy were delivered individually and in tandem to the right FNP of chicken embryos. All treatments caused asymmetric proximodistal truncation on the treated side and a mild expansion on the untreated side compared to DMSO control treated FNPs. Inhibiting each pathway caused similar decreased proliferation and disrupted cellular orientation, but did not affect apoptosis.
    Conclusions: Since RTK signaling is a ubiquitous and tightly regulated biochemical system we conclude that the downstream pathways are robust to developmental perturbation through redundant signaling systems.
    Bullet points: Inhibiting three downstream effectors of receptor tyrosine kinase (RTK) signaling (MEK1/2, PLCy, and PI3K) in the frontonasal process of chicken embryos caused similar mild truncation of growth. Combining all three inhibitors had a slightly stronger effect on truncation.Individual inhibitors did not have specific effects on cellular proliferation, apoptosis, or cellular orientation.The downstream branches of RTK signaling likely have shared interdependent effects on cellular activities that contribute to morphogenesis.
    DOI:  https://doi.org/10.1101/2024.12.10.627829
  17. J Mol Histol. 2024 Dec 27. 56(1): 57
      Embryonic development during the preimplantation stages is highly sensitive and critically dependent on the reception of signaling cues. The precise coordination of diverse pathways and signaling factors is essential for successful embryonic progression. Even minor disruptions in these factors can result in physiological dysfunction, fetal malformations, or embryonic arrest. This issue is particularly evident in assisted reproductive technologies, such as in vitro fertilization, where embryonic arrest is frequently observed. A detailed understanding of these pathways enhances insight into the fundamental mechanisms underlying cellular processes and their contributions to embryonic development. The significance of elucidating signaling pathways and their regulatory factors in preimplantation development cannot be overstated. The application of this knowledge in laboratory settings has the potential to support strategies for modeling developmental stages and diseases, drug screening, therapeutic discovery, and reducing embryonic arrest. Furthermore, using various factors, small molecules, and pharmacological agents can enable the development or optimization of culture media for enhanced embryonic viability. While numerous pathways influence preimplantation development, this study examines several critical signaling pathways in this contex.
    Keywords:  Embryonic development; Preimplantation; Signaling pathway
    DOI:  https://doi.org/10.1007/s10735-024-10338-7
  18. Front Cell Dev Biol. 2024 ;12 1473210
      The activation of IP3 receptor (IP3R) Ca2+ channels generates agonist-mediated Ca2+ signals that are critical for the regulation of a wide range of biological processes. It is therefore surprising that CRISPR induced loss of all three IP3R isoforms (TKO) in HEK293 and HeLa cell lines yields cells that can survive, grow and divide, albeit more slowly than wild-type cells. In an effort to understand the adaptive mechanisms involved, we have examined the activity of key Ca2+ dependent transcription factors (NFAT, CREB and AP-1) and signaling pathways using luciferase-reporter assays, phosphoprotein immunoblots and whole genome transcriptomic studies. In addition, the diacylglycerol arm of the signaling pathway was investigated with protein kinase C (PKC) inhibitors and siRNA knockdown. The data showed that agonist-mediated NFAT activation was lost but CREB activation was maintained in IP3R TKO cells. Under base-line conditions transcriptome analysis indicated the differential expression of 828 and 311 genes in IP3R TKO HEK293 or HeLa cells, respectively, with only 18 genes being in common. Three main adaptations in TKO cells were identified in this study: 1) increased basal activity of NFAT, CREB and AP-1; 2) an increased reliance on Ca2+- insensitive PKC isoforms; and 3) increased production of reactive oxygen species and upregulation of antioxidant defense enzymes. We suggest that whereas wild-type cells rely on a Ca2+ and DAG signal to respond to stimuli, the TKO cells utilize the adaptations to allow key signaling pathways (e.g., PKC, Ras/MAPK, CREB) to transition to the activated state using a DAG signal alone.
    Keywords:  CREB; Ca2+ dependent transcription; IP3 receptor; NFAT; calcineurin; calcium signaling
    DOI:  https://doi.org/10.3389/fcell.2024.1473210
  19. J Fungi (Basel). 2024 Nov 27. pii: 827. [Epub ahead of print]10(12):
      Precocious sexual inducer (psi)-producing oxygenases (Ppos) participate in the production of C8 moldy volatile compounds (MVOCs), and these compounds could act as signal molecules modulating G protein signaling cascades, which participates in the growth and development, secondary metabolisms and pathogenicity of filamentous fungi. In this study, PePpoA and PePpoC proteins were identified in Penicillium expansum. The deletion of ppoA decreased C8 MVOC production in P. expansum, while they were not detected in the ΔppoC strain (p < 0.05). In addition, down-regulated cAMP/PKA and PKC/PLC signaling showed in the two mutants (p < 0.05). The two mutants showed slow colony growth and down-regulated expression of genes regulating spore development (abaA, wetA, brlA and vosA) with broken morphology of spore and hyphae. In addition, the two mutants had decreased pathogenicity on apple fruit and less patulin production in vitro and in vivo. Compared with ΔppoA strain, the deletion of ppoC inhibited G protein signaling pathways more, and the ΔppoC strain had more defective growth and development as well as reduced pathogenicity and patulin production (p < 0.05). Therefore, PePpoC proteins affect more growth and development, patulin biosynthesis and pathogenicity of P. expansum by regulating C8 MVOC-mediated G protein signaling transduction.
    Keywords:  C8 moldy VOCs; G protein signal transduction; Penicillium expansum; pathogenicity; patulin biosynthesis; psi-producing oxygenase
    DOI:  https://doi.org/10.3390/jof10120827
  20. Cardiorenal Med. 2024 Dec 20. 1-15
       BACKGROUND: Diabetes mellitus (DM) is a prevalent chronic disease that is becoming increasingly common worldwide and can lead to a number of dangerous complications. The Wnt signaling pathway is important for the onset and progression of diabetes. Wnt3a is a typical Wnt ligand that can increase the stability of β-catenin, control TCF7L2 expression, promote β-cell proliferation, and reduce apoptosis.
    SUMMARY: The involvement of the Wnt3a/β-catenin/TCF7L2 signaling pathway in the development of diabetes and associated problems related to the kidneys is reviewed in this article.
    KEY MESSAGE: We believe that a thorough comprehension of the molecular connections between diabetes and signaling pathways will eventually lead to improved diabetes management.
    DOI:  https://doi.org/10.1159/000543145
  21. J Biol Chem. 2024 Dec 18. pii: S0021-9258(24)02611-5. [Epub ahead of print] 108109
      ATP-activated P2X3 receptors play a pivotal role in chronic cough, affecting more than 10% of the population. Despite the challenges posed by the highly conserved structure of P2X receptors, efforts to develop selective drugs targeting P2X3 have led to the development of camlipixant, a potent, selective P2X3 antagonist. However, the mechanisms of receptor desensitization, ion permeation, and structural basis of camlipixant binding to P2X3 remain unclear. Here, we report a cryo-EM structure of camlipixant-bound P2X3, revealing a previously undiscovered selective drug-binding site in the receptor. Our findings also demonstrate that conformational changes in the upper-body domain, including the turret and camlipixant-binding pocket, play a critical role: turret opening facilitates P2X3 channel closure to a radius of 0.7 Å, hindering cation transfer, while turret closure leads to channel opening. Structural and functional studies combined with molecular dynamics simulations provide a comprehensive understanding of camlipixant's selective inhibition of P2X3, offering a foundation for future drug development targeting this receptor.
    Keywords:  Camlipixant; Ion channel; P2X3; Receptor; cryo-EM
    DOI:  https://doi.org/10.1016/j.jbc.2024.108109
  22. Elife. 2024 Dec 24. pii: RP100205. [Epub ahead of print]13
      The induction of adipose thermogenesis plays a critical role in maintaining body temperature and improving metabolic homeostasis to combat obesity. β3-adrenoceptor (β3-AR) is widely recognized as a canonical β-adrenergic G-protein-coupled receptor (GPCR) that plays a crucial role in mediating adipose thermogenesis in mice. Nonetheless, the limited expression of β3-AR in human adipocytes restricts its clinical application. The objective of this study was to identify a GPCR that is highly expressed in human adipocytes and to explore its potential involvement in adipose thermogenesis. Our research findings have demonstrated that the adhesion G-protein-coupled receptor A3 (ADGRA3), an orphan GPCR, plays a significant role in adipose thermogenesis through its constitutively active effects. ADGRA3 exhibited high expression levels in human adipocytes and mouse brown fat. Furthermore, the knockdown of Adgra3 resulted in an exacerbated obese phenotype and a reduction in the expression of thermogenic markers in mice. Conversely, Adgra3 overexpression activated the adipose thermogenic program and improved metabolic homeostasis in mice without exogenous ligand. We found that ADGRA3 facilitates the biogenesis of beige human or mouse adipocytes in vitro. Moreover, hesperetin was identified as a potential agonist of ADGRA3, capable of inducing adipocyte browning and ameliorating insulin resistance in mice. In conclusion, our study demonstrated that the overexpression of constitutively active ADGRA3 or the activation of ADGRA3 by hesperetin can induce adipocyte browning by Gs-PKA-CREB axis. These findings indicate that the utilization of hesperetin and the selective overexpression of ADGRA3 in adipose tissue could serve as promising therapeutic strategies in the fight against obesity.
    Keywords:  ADGRA3; GPCR; adipose; cell biology; hesperetin; human; mouse; obesity; thermogenesis
    DOI:  https://doi.org/10.7554/eLife.100205
  23. J Neuroinflammation. 2024 Dec 23. 21(1): 329
      Inflammation is a critical driver of the early stages of diabetic retinopathy (DR) and offers an opportunity for therapeutic intervention before irreversible damage and vision loss associated with later stages of DR ensue. Nonsteroidal anti-inflammatory drugs (NSAIDs) have shown mixed efficacy in slowing early DR progression, notably including severe adverse side effects likely due to their nonselective inhibition of all downstream signaling intermediates. In this study, we investigated the role of prostanoids, the downstream signaling lipids whose production is inhibited by NSAIDs, in promoting inflammation relevant to early-stage DR in two human retinal cell types: Müller glia and retinal microvascular endothelial cells. When cultured in multiple conditions modeling distinct aspects of systemic diabetes, Müller glia significantly increased production of prostaglandin E2 (PGE2), whereas retinal endothelial cells significantly increased production of prostaglandin F2α (PGF2α). Müller glia stimulated with PGE2 or PGF2α increased proinflammatory cytokine levels dose-dependently. These effects were blocked by selective antagonists to the EP2 receptor of PGE2 or the FP receptor of PGF2α, respectively. In contrast, only PGF2α stimulated adhesion molecule expression in retinal endothelial cells and leukocyte adhesion to cultured endothelial monolayers, effects that were fully prevented by FP receptor antagonist treatment. Together these results identify PGE2-EP2 and PGF2α-FP signaling as novel, selective targets for future studies and therapeutic development to mitigate or prevent retinal inflammation characteristic of early-stage DR.
    DOI:  https://doi.org/10.1186/s12974-024-03319-w
  24. Res Sq. 2024 Dec 13. pii: rs.3.rs-5442142. [Epub ahead of print]
      Activation of PLCβ enzymes by G iβγ and G αq/11 proteins is a common mechanism to trigger cytosolic Ca 2+ increase. We and others reported that G αq/11 inhibitor FR900358 (FR) can inhibit both and G αq - and, surprisingly, G iβγ -mediated intracellular Ca 2+ mobilization. Thus, the G αi -G βγ -PLCβ-Ca 2+ signaling axis depends entirely on the presence of active G αq , which reasonably explained FR-inhibited G iβγ -induced Ca 2+ release. However, the conclusion that G iβγ signaling is controlled by G αq derives mostly from HEK293 cells. Here we show that indeed in HEK293 cells both G αq/11 siRNA and G αq/11 inhibitors diminished Ca 2+ increase triggered by native G q -coupled P2Y 1 receptors, or by transfected G i -coupled A 1 - or G s -coupled A 2B adenosine receptors (ARs). However, in T24 bladder cancer cells, G i inhibitor PTX, but not G αq/11 inhibitors, FR, YM254890 (YM) or G q/11 siRNA, inhibited Ca 2+ increase triggered by native A 2B AR activation. Simultaneous inactivation of G i and G s further suppressed A 2B AR-triggered Ca 2+ increase in T24 cells. The G αq/11 inhibitor YM fully and partially inhibited endogenous P2Y 1 - and β 2 -adrenergic receptor-induced Ca 2+ increase in T24 cells, respectively. PKC activator PMA partially diminished A 2B AR-triggered but completely diminished β 2 -adrenergic receptor-triggered Ca 2+ increase in T24 cells. Neither β-arrestin1 nor β-arrestin2 siRNA affected A 2B AR-mediated Ca 2+ increase. Unlike in T24 cells, YM inhibited native A 2B AR-triggered calcium mobilization in MDA-MB-231 breast cancer cells. Thus, G αq/11 is vital for Ca 2+ increase in some cell types, but G iβγ -mediated Ca 2+ signaling can be Gα q/11 -dependent or independent based on cell type and receptor activated. Besides G proteins, PKC also modulates cytosolic Ca 2+ increase depending on cell type and receptor.
    DOI:  https://doi.org/10.21203/rs.3.rs-5442142/v1
  25. Front Mol Biosci. 2024 ;11 1514759
      Hemorphins are short atypical opioid peptide fragments embedded in the β-chain of hemoglobin. They have received considerable attention recently due to their interaction with opioid receptors. The affinity of hemorphins to opioid receptors μ-opioid receptor (MOR), δ-opioid receptor (DOR), and κ-opioid receptor (KOR) has been well established. However, the underlying binding mode and molecular interactions of hemorphins in opioid receptors remain largely unknown. Here, we report the pattern of interaction of camel and other mammalian hemorphins with DOR. Extensive in silico docking and molecular dynamics simulations were employed to identify intermolecular interactions and binding energies were calculated to determine the affinity of these peptides for DOR. Longer forms of hemorphins - hemorphin-7, hemorphin-6, camel hemorphin-7, and camel hemorphin-6 had strong interactions with DOR. However, camel hemorphin-7 and camel hemorphin-6 had high binding affinity towards DOR. Thus, the findings of this study provide molecular insights into how hemorphins, particularly camel hemorphin variants, could be a therapeutic agent for pain regulation, stress management, and analgesia.
    Keywords:  camel hemorphins; hemorphins; molecular docking; molecular simulations; opioid receptors
    DOI:  https://doi.org/10.3389/fmolb.2024.1514759
  26. J Pineal Res. 2024 Nov;76(8): e70022
      As a chronic gynecological disease, endometriosis is defined as the implantation of endometrial glands as well as stroma outside the uterine cavity. Proliferation is a major pathophysiology in endometriosis. Previous studies demonstrated a hormone named melatonin, which is mainly produced by the pineal gland, exerts a therapeutic impact on endometriosis. Despite that, the direct binding targets and underlying molecular mechanism have remained unknown. Our study revealed that melatonin treatment might be effective in inhibiting the growth of lesions in endometriotic mouse model as well as in human endometriotic cell lines. Additionally, the drug-disease protein-protein interaction (PPI) network was built, and epidermal growth factor receptor (EGFR) was identified as a new binding target of melatonin treatment in endometriosis. Computational simulation together with BioLayer interferometry was further applied to confirm the binding affinity. Our result also showed melatonin inhibited the phosphorylation level of EGFR not only in endometriotic cell lines but also in mouse models. Furthermore, melatonin inhibited the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-protein kinase B (Akt) pathway and arrested the cell cycle via inhibiting CyclinD1 (CCND1). In vitro and in vivo knockdown/restore assays further demonstrated the involvement of the binding target and signaling pathway that we found. Thus, melatonin can be applied as a novel therapy for the management of endometriosis.
    Keywords:  CCND1; EGFR; PI3K/AKT; cell cycle; endometriosis; melatonin; proliferation
    DOI:  https://doi.org/10.1111/jpi.70022
  27. Cell Death Dis. 2024 Dec 24. 15(12): 930
      Ovarian cancer (OC) is prone to adipose tissue metastasis. However, the underlying molecular mechanisms remain elusive. Here, we observed that omental adipocytes were induced into cancer-associated adipocytes (CAAs) by OC-derived TGF-β1 to establish a pre-metastatic niche (PMN) through collagen and fibronectin secretion. Mechanistically, OC-derived TGF-β1 binds to adipocyte membrane receptors and thus activates intracellular signaling by SMAD3 phosphorylation. The activation of TGF-β1/SMAD3 signaling pathway dedifferentiates adipocytes into CAAs by upregulating Tribbles homolog 3 (TRIB3), which suppresses the phosphorylation of CEBPβ. Additionally, CAAs secrete collagen I, collagen VI, and fibronectin to remodel the extracellular matrix and promote the adhesion of OC cells. Pharmacological inhibition of the TGF-β1/SMAD3 pathway significantly inhibits CAAs and PMN formation, thereby reducing the OC metastatic burden. Our findings indicate that the formation of CAAs and PMN in adipose tissues facilitates OC cell implantation and blocking the TGF-β1/SMAD3 signaling pathway could prevent OC omental metastasis.
    DOI:  https://doi.org/10.1038/s41419-024-07311-3
  28. Front Cardiovasc Med. 2024 ;11 1454302
       Introduction: The use of cardiopulmonary bypass (CPB) can induce sterile systemic inflammation that contributes to morbidity and mortality, especially in children. Patients have been found to have increased expression of cytokines and transmigration of leukocytes during and after CPB. Previous work has demonstrated that the supraphysiologic shear stresses existing during CPB are sufficient to induce proinflammatory behavior in non-adherent monocytes. The interactions between shear stimulated monocytes and vascular endothelial cells have not been well studied and have important translational implications. With these studies, we tested the hypothesis that non-physiological shear stress experienced by monocytes during CPB affects the integrity and function of the endothelial monolayer.
    Methods: We have used an in vitro CPB model to study the interaction between THP-1 monocyte-like cells and human neonatal dermal microvascular endothelial cells (HNDMVECs). THP-1 cells were sheared in polyvinyl chloride (PVC) tubing at 2.1 Pa, twice of the physiological shear stress, for 2 h. ELISA, adhesion and transmigration assays, qPCR, and RNA silencing were used to assess the interactions between THP-1 cells and HNDMVECs were characterized after co-culture.
    Results: We found that sheared THP-1 cells adhered to and transmigrated through the HNDMVEC monolayer more readily than static THP-1 controls. Sheared THP-1 cells disrupted the VE-cadherin and led to the reorganization of cytoskeletal F-actin of HNDMVECs. A higher level of IL-8 was detected in the sheared THP-1 and HNDMVEC co-culture medium compared to the static THP-1 and HNDMVEC medium. Further, treating HNDMVECs with IL-8 resulted in increased adherence of non-sheared THP-1 cells, and upregulation in HNDMVECs of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Finally, inhibition of HNDMVECs CXCR2/IL-8 receptor with Reparixin and of IL-8 expression with siRNA blocked sheared THP-1 cell adhesion to the endothelial monolayer.
    Conclusions: These results suggest that CPB-like sheared monocytes promote IL-8 production followed by increased endothelium permeability, and monocyte adhesion and transmigration. This study revealed a novel mechanism of post-CPB inflammation and will contribute to the development of targeted therapeutics to prevent and repair the damage to neonatal patients.
    Keywords:  IL-8; cardiopulmonary bypass; endothelial cells; monocytes; shear stress
    DOI:  https://doi.org/10.3389/fcvm.2024.1454302
  29. Environ Int. 2024 Dec 16. pii: S0160-4120(24)00802-X. [Epub ahead of print]195 109215
      Aryl organophosphorus flame retardants (aryl-OPFRs), commonly used product additives with close ties to daily life, have been regrettably characterized by multiple well-defined toxicity risks. Triphenyl phosphate (TPHP) and tri-o-cresyl phosphate (TOCP), two structurally similar aryl-OPFRs, were observed in our previous study to exhibit contrasting immunotoxic effects on THP-1 macrophages, yet the underlying mechanisms remain unclear. This study sought to address the knowledge gap by integrating transcriptomic and metabolomic analyses to elucidate the intricate mechanisms. During individual omics analyses, we unfortunately only obtained highly similar results for both TPHP and TOCP, failing to identify the key reasons for their differences. These results revealed comparable disturbances induced by both compounds, including disruptions in nucleic acid synthesis and energy metabolism, blocking ADP to ATP conversion by reducing TCA cycle intermediates, consequently leading to ATP depletion. However, through integrative analysis, specific pathways affected by each compound were successfully identified, shedding light on their unique effects. TPHP reduced GTP levels necessary for Rap1 activation, thereby inhibiting phagocytosis and adhesion of THP-1 macrophages. Conversely, TOCP stimulated the mTOR signaling pathway, enhancing phosphorylation of downstream proteins S6K, RHOA, and PKC, consequently promoting immune responses. This study not only clarified the distinct immunotoxic mechanisms of TPHP and TOCP but also provided critical insights into how structural variations in aryl-OPFRs can lead to markedly different immune responses, thereby informing future risk assessments and regulatory strategies for these compounds.
    Keywords:  Aryl-OPFRs; Immunotoxicity; Macrophage; TOCP; TPHP
    DOI:  https://doi.org/10.1016/j.envint.2024.109215