bims-dicesi 2025-01-05 papers

bims-dicesi

Biomed News

on Diversification of cell signalling

Issue of 2025–01–05
35 papers selected by
Ashanika Karandawela

Diversity in Notch ligand-receptor signaling interactions.
Genetic variants of accessory proteins and G proteins in human genetic disease.
Photoswitch dissociation from a G protein-coupled receptor resolved by time-resolved serial crystallography.
Functional dynamics of G protein-coupled receptors reveal new routes for drug discovery.
Structural insights into the regulation of monomeric and dimeric apelin receptor.
Dimethyltryptamine (DMT) and ibogaine elicit membrane effects in HEK cells transiently transfected with the human 5-HT2A receptor.
The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling.
The β Common Cytokine Receptor Family Reveals New Functional Paradigms From Structural Complexities.
The Drosophila hematopoietic niche assembles through collective cell migration controlled by neighbor tissues and Slit-Robo signaling.
Higher-order transient structures and the principle of dynamic connectivity in membrane signaling.
Nicotine enhances the stemness and tumorigenicity in intestinal stem cells via Hippo-YAP/TAZ and Notch signal pathway.
Mms22-Rtt107 axis attenuates the DNA damage checkpoint and the stability of the Rad9 checkpoint mediator.
Chemically Induced Dimerization via Nanobody Binding Facilitates in Situ Ligand Assembly and On-Demand GPCR Activation.
Generic residue numbering of the GAIN domain of adhesion GPCRs.
Targeting the tumor immune microenvironment: GPCRs as key regulators in triple-negative breast cancer.
Familial Alzheimer's disease mutations in amyloid precursor protein impair calcineurin signaling to NMDA receptors.
Trace amine-associated receptor 1 (TAAR1): an emerging therapeutic target for neurodegenerative, neurodevelopmental, and neurotraumatic disorders.
The development of murine bone marrow-derived mast cells expressing functional human MRGPRX2 for ex vivo and in vivo studies.
Antiviral and Immunomodulatory Effects of Zafirlukast Against Flaviviruses via Inhibition of the TNF Signaling Pathway.
Role of the Notch signaling pathway in porcine oocyte maturation.
GRK6 palmitoylation dictates triple-negative breast cancer metastasis via recruiting the β-Arrestin 2/MAPKs/NF-κB signaling axis.
Identification and structure-guided development of triazole urea-based selective antagonists of Arabidopsis karrikin signaling.
Oncogenic RARγ isoforms promote head and neck cancer proliferation through vinexin-β-mediated cell cycle acceleration and autocrine activation of EGFR signal.
Tsg101 mimicry of canonical E2 enzymes underlies its role in ubiquitin signaling.
The importance of Fcγ and C-type lectin receptors in host immune responses during Pneumocystis pneumonia.
Role of GPR55 receptor in bovine sperm capacitation.
Homogeneous and bioluminescent biochemical and cellular assay for monitoring cGAMP and enzymes that generate and degrade cGAMP.
CDK5 interacts with MST2 and modulates the Hippo signalling pathway.
Structural Insights Into the Impact of the Glycine-Rich Loop Mutation in Noonan Syndrome on the ATP Binding Pocket of CRAF Kinase.
Inhibition of the MyD88 signaling pathway could upregulates Ghrelin expression to synergistically regulate hepatic Echinococcus multilocularis-infected progression.
Sleep drive is coupled to tissue damage via shedding of Caenorhabditis elegans EGFR ligand SISS-1.
Cell migration signaling through EGFR-VAV2-Rac1 pathway is sustained in endosomes.
RAP-2 and CNH-MAP4 Kinase MIG-15 confer resistance in bystander epithelium to cell-fate transformation by excess Ras or Notch activity.
Vacuolar H+-ATPase and Megalin-Mediated Prorenin Uptake: Focus on Elements Beyond the (Pro)Renin Receptor.
FGFR3 Upregulates Interferon-Stimulated Genes Via the JAK1-STAT1 Signaling Pathway in HPV2 E2 Stable Expressing Keratinocytes.

Elife. 2025 Jan 03. pii: RP91422. [Epub ahead of print]12

Diversity in Notch ligand-receptor signaling interactions.

Rachael Kuintzle, Leah A Santat, Michael B Elowitz.

  The Notch signaling pathway uses families of ligands and receptors to transmit signals to nearby cells. These components are expressed in diverse combinations in different cell types, interact in a many-to-many fashion, both within the same cell (in cis) and between cells (in trans), and their interactions are modulated by Fringe glycosyltransferases. A fundamental question is how the strength of Notch signaling depends on which pathway components are expressed, at what levels, and in which cells. Here, we used a quantitative, bottom-up, cell-based approach to systematically characterize trans-activation, cis-inhibition, and cis-activation signaling efficiencies across a range of ligand and Fringe expression levels in Chinese hamster and mouse cell lines. Each ligand (Dll1, Dll4, Jag1, and Jag2) and receptor variant (Notch1 and Notch2) analyzed here exhibited a unique profile of interactions, Fringe dependence, and signaling outcomes. All four ligands were able to bind receptors in cis and in trans, and all ligands trans-activated both receptors, although Jag1-Notch1 signaling was substantially weaker than other ligand-receptor combinations. Cis-interactions were predominantly inhibitory, with the exception of the Dll1- and Dll4-Notch2 pairs, which exhibited cis-activation stronger than trans-activation. Lfng strengthened Delta-mediated trans-activation and weakened Jagged-mediated trans-activation for both receptors. Finally, cis-ligands showed diverse cis-inhibition strengths, which depended on the identity of the trans-ligand as well as the receptor. The map of receptor-ligand-Fringe interaction outcomes revealed here should help guide rational perturbation and control of the Notch pathway.

Keywords:  Notch signaling; cell signaling; computational biology; developmental biology; mouse; quantitative biology; systems biology

DOI:  https://doi.org/10.7554/eLife.91422
Crit Rev Clin Lab Sci. 2025 Jan 01. 1-22

Genetic variants of accessory proteins and G proteins in human genetic disease.

Miles D Thompson, Peter Chidiac, Pedro A Jose, Alexander S Hauser, Caroline M Gorvin.

  We present a series of three articles on the genetics and pharmacogenetics of G protein- coupled receptors (GPCR). In the first article, we discuss genetic variants of the G protein subunits and accessory proteins that are associated with human phenotypes; in the second article, we build upon this to discuss "G protein-coupled receptor (GPCR) gene variants and human genetic disease" and in the third article, we survey "G protein-coupled receptor pharmacogenomics". In the present article, we review the processes of ligand binding, GPCR activation, inactivation, and receptor trafficking to the membrane in the context of human genetic disease resulting from pathogenic variants of accessory proteins and G proteins. Pathogenic variants of the genes encoding G protein α and β subunits are examined in diverse phenotypes. Variants in the genes encoding accessory proteins that modify or organize G protein coupling have been associated with disease; these include the contribution of variants of the regulator of G protein signaling (RGS) to hypertension; the role of variants of activator of G protein signaling type III in phenotypes such as hypoxia; the contribution of variation at the RGS10 gene to short stature and immunological compromise; and the involvement of variants of G protein-coupled receptor kinases (GRKs), such as GRK4, in hypertension. Variation in genes that encode proteins involved in GPCR signaling are outlined in the context of the changes in structure and function that may be associated with human phenotypes.

Keywords:  G protein; G protein-coupled receptor (GPCR); accessory protein; genetics; pharmacogenetics

DOI:  https://doi.org/10.1080/10408363.2024.2431853
Nat Commun. 2024 Dec 30. 15(1): 10837

Photoswitch dissociation from a G protein-coupled receptor resolved by time-resolved serial crystallography.

Hannah Glover, Torben Saßmannshausen, Quentin Bertrand, Matilde Trabuco, Chavdar Slavov, Arianna Bacchin, Fabio Andres, Yasushi Kondo, Robin Stipp, Maximilian Wranik, Georgii Khusainov, Melissa Carrillo, Demet Kekilli, Jie Nan, Ana Gonzalez, Robert Cheng, Werner Neidhart, Tobias Weinert, Filip Leonarski, Florian Dworkowski, Michal Kepa, Josef Wachtveitl, Michael Hennig, Joerg Standfuss.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in humans. The binding and dissociation of ligands tunes the inherent conformational flexibility of these important drug targets towards distinct functional states. Here we show how to trigger and resolve protein-ligand interaction dynamics within the human adenosine A2A receptor. For this, we designed seven photochemical affinity switches derived from the anti-Parkinson's drug istradefylline. In a rational approach based on UV/Vis spectroscopy, time-resolved absorption spectroscopy, differential scanning fluorimetry and cryo-crystallography, we identified compounds suitable for time-resolved serial crystallography. Our analysis of millisecond-scale dynamics revealed how trans-to-cis isomerization shifts selected istradefylline derivatives within the binding pocket. Depending on the chemical nature of the ligand, interactions between extracellular loops 2 and 3, acting as a lid on the binding pocket, are disrupted and rearrangement of the orthosteric binding pocket is invoked upon ligand dissociation. This innovative approach provides insights into GPCR dynamics at the atomic level, offering potential for developing novel pharmaceuticals.

DOI: https://doi.org/10.1038/s41467-024-55109-w
Nat Rev Drug Discov. 2025 Jan 02.

Functional dynamics of G protein-coupled receptors reveal new routes for drug discovery.

Paolo Conflitti, Edward Lyman, Mark S P Sansom, Peter W Hildebrand, Hugo Gutiérrez-de-Terán, Paolo Carloni, T Bertie Ansell, Shuguang Yuan, Patrick Barth, Anne S Robinson, Christopher G Tate, David Gloriam, Stephan Grzesiek, Matthew T Eddy, Scott Prosser, Vittorio Limongelli.

G protein-coupled receptors (GPCRs) are the largest human membrane protein family that transduce extracellular signals into cellular responses. They are major pharmacological targets, with approximately 26% of marketed drugs targeting GPCRs, primarily at their orthosteric binding site. Despite their prominence, predicting the pharmacological effects of novel GPCR-targeting drugs remains challenging due to the complex functional dynamics of these receptors. Recent advances in X-ray crystallography, cryo-electron microscopy, spectroscopic techniques and molecular simulations have enhanced our understanding of receptor conformational dynamics and ligand interactions with GPCRs. These developments have revealed novel ligand-binding modes, mechanisms of action and druggable pockets. In this Review, we highlight such aspects for recently discovered small-molecule drugs and drug candidates targeting GPCRs, focusing on three categories: allosteric modulators, biased ligands, and bivalent and bitopic compounds. Although studies so far have largely been retrospective, integrating structural data on ligand-induced receptor functional dynamics into the drug discovery pipeline has the potential to guide the identification of drug candidates with specific abilities to modulate GPCR interactions with intracellular effector proteins such as G proteins and β-arrestins, enabling more tailored selectivity and efficacy profiles.

DOI: https://doi.org/10.1038/s41573-024-01083-3
Nat Commun. 2025 Jan 02. 16(1): 310

Structural insights into the regulation of monomeric and dimeric apelin receptor.

Yang Yue, Lier Liu, Lijie Wu, Chanjuan Xu, Man Na, Shenhui Liu, Yuxuan Liu, Fei Li, Junlin Liu, Songting Shi, Hui Lei, Minxuan Zhao, Tianjie Yang, Wei Ji, Arthur Wang, Michael A Hanson, Raymond C Stevens, Jianfeng Liu, Fei Xu.

The apelin receptor (APJR) emerges as a promising drug target for cardiovascular health and muscle regeneration. While prior research unveiled the structural versatility of APJR in coupling to Gi proteins as a monomer or dimer, the dynamic regulation within the APJR dimer during activation remains poorly understood. In this study, we present the structures of the APJR dimer and monomer complexed with its endogenous ligand apelin-13. In the dimeric structure, apelin-13 binds exclusively to one protomer that is coupled with Gi proteins, revealing a distinct ligand-binding behavior within APJR homodimers. Furthermore, binding of an antagonistic antibody induces a more compact dimerization by engaging both protomers. Notably, structural analyses of the APJR dimer complexed with an agonistic antibody, with or without Gi proteins, suggest that G protein coupling may promote the dissociation of the APJR dimer during activation. These findings underscore the intricate interplay between ligands, dimerization, and G protein coupling in regulating APJR signaling pathways.

DOI: https://doi.org/10.1038/s41467-024-55555-6
Brain Res. 2024 Dec 26. pii: S0006-8993(24)00680-2. [Epub ahead of print] 149425

Dimethyltryptamine (DMT) and ibogaine elicit membrane effects in HEK cells transiently transfected with the human 5-HT2A receptor.

Jannik Nicklas Eliasen, Uffe Kristiansen, Kristi A Kohlmeier.

  Psychedelics show promise in treating psychiatric disorders. Therapeutic effects appear to involve activation of the 5-Hydroxytryptamine 2A receptor (5-HT2AR), a G protein-coupled receptor (GPCR). Several SNPs of the 5-HT2AR naturally occur, which are associated with differences in receptor function and altered responsiveness to treatments. New compounds suspected to act at the 5-HT2AR are actively being generated. HEK cells are not commonly used to study membrane effects induced by agonists of GPCRs. In this study, for the first time, membrane actions of two psychedelics, dimethyltryptamine (DMT) and ibogaine on HEK cells transiently transfected with either the human wildtype (WT) or the human I197V mutated 5-HT2AR were investigated using whole-cell electrophysiology. Membrane effects were observed in both genotypes and with both drugs in most cells, while no responses were observed in non-transfected HEK cells suggesting that responses were due to 5-HT2AR activation. In HEK cells transfected with the I197V SNP, a significantly shorter duration of the DMT response was observed, however there were no differences in drug-elicited amplitudes between drug or receptor genotype. I-V curves showed a significant effect of drug exposure for both DMT and ibogaine at the highest concentration evaluated. Taken together, our data show transfection of the 5-HT2AR, a GPCR, in HEK cells is able to activate downstream ion channels following exposure to two different 5-HT2AR agonists. Accordingly, investigations of novel compounds suspected to act at 5-HT2ARs can include examination of elicitation of ionic currents in 5-HT2AR transfected HEK cells, and drug effects at SNPs can also be evaluated.

Keywords:  Affective disorders; G Protein-coupled receptor; I–V curves; Membrane currents; Psychedelics; Single nucleotide polymorphism

DOI:  https://doi.org/10.1016/j.brainres.2024.149425
Elife. 2025 Jan 02. pii: RP96743. [Epub ahead of print]13

The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling.

Elise S Bruguera, Jacob P Mahoney, William I Weis.

  Wnt/β-catenin signaling directs animal development and tissue renewal in a tightly controlled, cell- and tissue-specific manner. In the mammalian central nervous system, the atypical ligand Norrin controls angiogenesis and maintenance of the blood-brain barrier and blood-retina barrier through the Wnt/β-catenin pathway. Like Wnt, Norrin activates signaling by binding and heterodimerizing the receptors Frizzled (Fzd) and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to membrane recruitment of the intracellular transducer Dishevelled (Dvl) and ultimately stabilizing the transcriptional coactivator β-catenin. Unlike Wnt, the cystine knot ligand Norrin only signals through Fzd4 and additionally requires the co-receptor Tetraspanin12 (Tspan12); however, the mechanism underlying Tspan12-mediated signal enhancement is unclear. It has been proposed that Tspan12 integrates into the Norrin-Fzd4 complex to enhance Norrin-Fzd4 affinity or otherwise allosterically modulate Fzd4 signaling. Here, we measure direct, high-affinity binding between purified Norrin and Tspan12 in a lipid environment and use AlphaFold models to interrogate this interaction interface. We find that Tspan12 and Fzd4 can simultaneously bind Norrin and that a pre-formed Tspan12/Fzd4 heterodimer, as well as cells co-expressing Tspan12 and Fzd4, more efficiently capture low concentrations of Norrin than Fzd4 alone. We also show that Tspan12 competes with both heparan sulfate proteoglycans and LRP6 for Norrin binding and that Tspan12 does not impact Fzd4-Dvl affinity in the presence or absence of Norrin. Our findings suggest that Tspan12 does not allosterically enhance Fzd4 binding to Norrin or Dvl, but instead functions to directly capture Norrin upstream of signaling.

Keywords:  Wnt; cell signaling; co-receptor; developmental biology; human; molecular biophysics; mouse; nanodiscs; specificity; structural biology

DOI:  https://doi.org/10.7554/eLife.96743
Immunol Rev. 2025 Jan;329(1): e13430

The β Common Cytokine Receptor Family Reveals New Functional Paradigms From Structural Complexities.

Winnie L Kan, Claire M Weekley, Tracy L Nero, Timothy R Hercus, Kwok Ho Yip, Damon J Tumes, Joanna M Woodcock, David M Ross, Daniel Thomas, David Terán, Catherine M Owczarek, Nora W Liu, Luciano G Martelotto, Jose M Polo, Harshita Pant, Denis Tvorogov, Angel F Lopez, Michael W Parker.

  Cytokines are small proteins that are critical for controlling the growth and activity of hematopoietic cells by binding to cell surface receptors and transmitting signals across membranes. The β common (βc) cytokine receptor family, consisting of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 cytokine receptors, is an architype of the heterodimeric cytokine receptor systems. We now know that signaling by cytokine receptors is not always an "all or none" phenomenon. Subtle alterations of the cytokine:receptor complex can result in differential or selective signaling and underpin a variety of diseases including chronic inflammatory conditions and cancers. Structural biology techniques, such as X-ray crystallography and cryo-electron microscopy alongside cell biology studies, are providing detailed insights into cytokine receptor signaling. Recently, we found that the IL-3 receptor ternary complex forms higher-order assemblies, like those found earlier for the GM-CSF receptor, and demonstrated that functionally distinct biological signals arise from different IL-3 receptor oligomeric assemblies. As we enhance our understanding of the structural nuances of cytokine-receptor interactions, we foresee a new era of theranostics whereby structurally guided mechanism-based manipulation of cytokine signaling through rational/targeted protein engineering will harness the full potential of cytokine biology for precision medicine.

Keywords:  crystal structures; cytokine; granulocyte‐macrophage colony‐stimulating factor; interleukin‐3; interleukin‐5; receptor signaling

DOI:  https://doi.org/10.1111/imr.13430
Elife. 2025 Jan 03. pii: RP100455. [Epub ahead of print]13

The Drosophila hematopoietic niche assembles through collective cell migration controlled by neighbor tissues and Slit-Robo signaling.

Kara A Nelson, Kari F Lenhart, Lauren Anllo, Stephen DiNardo.

  Niches are often found in specific positions in tissues relative to the stem cells they support. Consistency of niche position suggests that placement is important for niche function. However, the complexity of most niches has precluded a thorough understanding of how their proper placement is established. To address this, we investigated the formation of a genetically tractable niche, the Drosophila Posterior Signaling Center (PSC), the assembly of which had not been previously explored. This niche controls hematopoietic progenitors of the lymph gland (LG). PSC cells were previously shown to be specified laterally in the embryo, but ultimately reside dorsally, at the LG posterior. Here, using live-imaging, we show that PSC cells migrate as a tight collective and associate with multiple tissues during their trajectory to the LG posterior. We find that Slit emanating from two extrinsic sources, visceral mesoderm and cardioblasts, is required for the PSC to remain a collective, and for its attachment to cardioblasts during migration. Without proper Slit-Robo signaling, PSC cells disperse, form aberrant contacts, and ultimately fail to reach their stereotypical position near progenitors. Our work characterizes a novel example of niche formation and identifies an extrinsic signaling relay that controls precise niche positioning.

Keywords:  D. melanogaster; Drosophila; cell migration; developmental biology; hematopoietic niche; organogenesis; regenerative medicine; slit-robo; stem cells

DOI:  https://doi.org/10.7554/eLife.100455
Proc Natl Acad Sci U S A. 2025 Jan 07. 122(1): e2421280121

Higher-order transient structures and the principle of dynamic connectivity in membrane signaling.

Yuxi Zhang, Roderick MacKinnon.

  We examine the role of higher-order transient structures (HOTS) in M2R regulation of GIRK channels. Electron microscopic membrane protein location maps show that both proteins form HOTS that exhibit a statistical bias to be near each other. Theoretical calculations and electrophysiological measurements suggest that channel activity is isolated near larger M2R HOTS. By invoking weak interactions that permit transient binding of M2R to M2R and GIRK to GIRK (i-i interactions) and M2R to GIRK (i-j interactions), the distribution patterns and electrophysiological properties of HL-1 cells are replicated in a reaction-diffusion simulation. We propose the principle of dynamic connectivity to explain communication between protein components of a membrane signaling pathway. Dynamic connectivity is mediated by weak, transient interactions between proteins. HOTS created by weak i-i interactions, and statistical biases created by weak i-j interactions promoted by the multivalence of HOTS, are the key elements of dynamic connectivity.

Keywords:  GPCR; HOTS; higher-order transient structure; membrane signaling; self-assembly

DOI:  https://doi.org/10.1073/pnas.2421280121
Elife. 2025 Jan 03. pii: RP95267. [Epub ahead of print]13

Nicotine enhances the stemness and tumorigenicity in intestinal stem cells via Hippo-YAP/TAZ and Notch signal pathway.

Ryosuke Isotani, Masaki Igarashi, Masaomi Miura, Kyoko Naruse, Satoshi Kuranami, Manami Katoh, Seitaro Nomura, Toshimasa Yamauchi.

  Cigarette smoking is a well-known risk factor inducing the development and progression of various diseases. Nicotine (NIC) is the major constituent of cigarette smoke. However, knowledge of the mechanism underlying the NIC-regulated stem cell functions is limited. In this study, we demonstrate that NIC increases the abundance and proliferative activity of murine intestinal stem cells (ISCs) in vivo and ex vivo. Moreover, NIC induces Yes-associated protein (YAP) /Transcriptional coactivator with PDZ-binding motif (TAZ) and Notch signaling in ISCs via α7-nicotinic acetylcholine receptor (nAchR) and protein kinase C (PKC) activation; this effect was not detected in Paneth cells. The inhibition of Notch signaling by dibenzazepine (DBZ) nullified the effects of NIC on ISCs. NIC enhances in vivo tumor formation from ISCs after loss of the tumor suppressor gene Apc, DBZ inhibited NIC-induced tumor growth. Hence, this study identifies a NIC-triggered pathway regulating the stemness and tumorigenicity of ISCs and suggests the use of DBZ as a potential therapeutic strategy for treating intestinal tumors.

Keywords:  Notch; YAP/TAZ; intestinal stem cell; mouse; nAChR; nicotine; paneth cell; regenerative medicine; stem cells

DOI:  https://doi.org/10.7554/eLife.95267
Nat Commun. 2025 Jan 02. 16(1): 311

Mms22-Rtt107 axis attenuates the DNA damage checkpoint and the stability of the Rad9 checkpoint mediator.

Bingbing Wan, Danying Guan, Shibai Li, Tzippora Chwat-Edelstein, Xiaolan Zhao.

The DNA damage checkpoint is a highly conserved signaling pathway induced by genotoxin exposure or endogenous genome stress. It alters many cellular processes such as arresting the cell cycle progression and increasing DNA repair capacities. However, cells can downregulate the checkpoint after prolonged stress exposure to allow continued growth and alternative repair. Strategies that can dampen the DNA damage checkpoint are not well understood. Here, we report that budding yeast employs a pathway composed of the scaffold protein Rtt107, its binding partner Mms22, and an Mms22-associated ubiquitin ligase complex to downregulate the DNA damage checkpoint. Mechanistically, this pathway promotes the proteasomal degradation of a key checkpoint factor, Rad9. Furthermore, Rtt107 binding to Mms22 helps to enrich the ubiquitin ligase complex on chromatin for targeting the chromatin-bound form of Rad9. Finally, we provide evidence that the Rtt107-Mms22 axis operates in parallel with the Rtt107-Slx4 axis, which displaces Rad9 from chromatin. We thus propose that Rtt107 enables a bifurcated "anti-Rad9" strategy to optimally downregulate the DNA damage checkpoint.

DOI: https://doi.org/10.1038/s41467-024-54624-0
JACS Au. 2024 Dec 23. 4(12): 4780-4789

Chemically Induced Dimerization via Nanobody Binding Facilitates in Situ Ligand Assembly and On-Demand GPCR Activation.

Shubhra Jyoti Saha, Ross W Cheloha.

Methods that enable the on-demand synthesis of biologically active molecules offer the potential for a high degree of control over the timing and context of target activation; however, such approaches often require extensive engineering to implement. Tools to restrict the localization of assembly also remain limited. Here we present a new approach for stimulus-induced ligand assembly that helps to address these challenges. This methodology relies on the high affinity and specificity recognition exhibited by antibody fragments (nanobodies, Nbs). By using Nbs that recognize short peptide epitopes to create semisynthetic conjugates, we develop a bioengineering platform termed peptide epitope dimerization (PED) in which the addition of heterodimeric peptide composed of two Nb epitopes stimulates the proximity-induced synthesis of a functional ligand for the parathyroid hormone receptor-1, a G protein-coupled receptor. We further demonstrate that high efficiency assembly can be achieved on the cell surface via Nb-based delivery of template. This approach opens the door for the on-demand generation of bioactive receptor ligands preferentially at a desired biological niche.

DOI: https://doi.org/10.1021/jacsau.4c00711
Nat Commun. 2025 Jan 02. 16(1): 246

Generic residue numbering of the GAIN domain of adhesion GPCRs.

Florian Seufert, Guillermo Pérez-Hernández, Gáspár Pándy-Szekeres, Ramon Guixà-González, Tobias Langenhan, David E Gloriam, Peter W Hildebrand.

The GPCR autoproteolysis inducing (GAIN) domain is an ancient protein fold ubiquitous in adhesion G protein-coupled receptors (aGPCR). It contains a tethered agonist necessary and sufficient for receptor activation. The GAIN domain is a hotspot for pathological mutations. However, the low primary sequence conservation of GAIN domains has thus far hindered the knowledge transfer across different GAIN domains in human receptors as well as species orthologs. Here, we present a scheme for generic residue numbering of GAIN domains, based on structural alignments of over 14,000 modeled GAIN domain structures. This scheme is implemented in the GPCR database (GPCRdb) and elucidates the domain topology across different aGPCRs and their homologs in a large panel of species. We identify conservation hotspots and statistically cancer-enriched positions in human aGPCRs and show the transferability of positional and structural information between GAIN domain homologs. The GAIN-GRN scheme provides a robust strategy to allocate structural homologies at the primary and secondary levels also to GAIN domains of polycystic kidney disease 1/PKD1-like proteins, which now renders positions in both GAIN domain types comparable to one another. In summary, our work enables researchers to generate hypothesis and rationalize experiments related to GAIN domain function and pathology.

DOI: https://doi.org/10.1038/s41467-024-55466-6
Int Immunopharmacol. 2024 Dec 30. pii: S1567-5769(24)02452-4. [Epub ahead of print]147 113930

Targeting the tumor immune microenvironment: GPCRs as key regulators in triple-negative breast cancer.

Chengyi Wang, Yanyan Liu, Ru Zhang, Hao Gong, Xinnong Jiang, Shuai Xia.

  Triple-negative breast cancer (TNBC) poses a significant clinical challenge due to its aggressive nature and limited therapeutic options. Recent research underscores the pivotal role of G protein-coupled receptors (GPCRs) in shaping the tumor immune microenvironment (TIME) within TNBC. This review focuses on four principal GPCRs-chemokine receptors, sphingosine-1-phosphate receptors, prostaglandin E2 receptors, and lactate receptors-that have garnered substantial attention in TNBC studies. GPCRs modulate immune cell recruitment, polarization, and function, thereby fostering an immunosuppressive milieu conducive to tumor progression and metastasis. The review examines how alterations in GPCR expression on immune cells influence the pathogenesis and advancement of TNBC. Further, it discusses emerging therapeutic strategies targeting GPCR signaling pathways to remodel the immunosuppressive TIME in TNBC. These insights into GPCR-mediated immune regulation not only deepen our comprehension of TNBC's pathophysiology but also offer promising avenues for developing novel immunotherapies aimed at enhancing clinical outcomes for TNBC patients.

Keywords:  GPCR; Immune microenvironment; Triple-negative breast cancer; Tumor microenvironment

DOI:  https://doi.org/10.1016/j.intimp.2024.113930
J Biol Chem. 2024 Dec 26. pii: S0021-9258(24)02649-8. [Epub ahead of print] 108147

Familial Alzheimer's disease mutations in amyloid precursor protein impair calcineurin signaling to NMDA receptors.

Steven J Tavalin.

  Familial Alzheimer's disease (FAD) is frequently associated with mutations in the amyloid precursor protein (APP), which are thought to lead to cognitive deficits by impairing NMDA receptor (NMDAR)-dependent forms of synaptic plasticity. Given the reliance of synaptic plasticity on NMDAR-mediated Ca2+ entry, shaping of NMDAR activity by APP and/or its disease-causing variants could provide a basis for understanding synaptic plasticity impairments associated with FAD. A region of APP (residues 639-644 within APP695) processed by the γ-secretase complex, which generates amyloid β (Aβ) peptides, is a hotspot for FAD mutations. This region bears similarity to a binding motif for calcineurin (CaN), a Ca2+/calmodulin-dependent phosphatase. Interaction assays confirm that APP associates with CaN in native tissue as well as in a heterologous expression system. This capacity to bind CaN extends to APP family members amyloid precursor-like protein 1 and amyloid precursor-like protein 2 (APLP1 and APLP2, respectively). Electrophysiological analysis demonstrates that APP and its family members limit NMDAR activity, in a manner consistent with CaN-dependent regulation of NMDAR desensitization. FAD mutations, in this region of APP, impair this regulation and consequently enhance NMDAR activity. Thus, by altering the landscape for CaN regulation of NMDA receptors, FAD mutations in APP may contribute to faulty information processing by modifying the dynamic range and temporal window of a critical signal for synaptic plasticity.

Keywords:  Alzheimer’s disease; NMDA receptors; amyloid precursor protein; calcineurin; calcium; signal transduction; synaptic plasticity

DOI:  https://doi.org/10.1016/j.jbc.2024.108147
Naunyn Schmiedebergs Arch Pharmacol. 2024 Dec 30.

Trace amine-associated receptor 1 (TAAR1): an emerging therapeutic target for neurodegenerative, neurodevelopmental, and neurotraumatic disorders.

Saher Dalvi, Lokesh Kumar Bhatt.

  Trace amines are physiologically active amines present in all organisms. They are structurally identical to traditional monoamines and are rapidly metabolized by monoamine oxidases. The mammalian neurological system generates these molecules at rates equivalent to traditional monoamines, but because of their short half-life, they are only observable in trace quantities. Their receptors are G protein-coupled receptors present in both the CNS and peripheral locations, with trace amine-associated receptor 1 (TAAR1) being the most researched. TAAR1's capacity to regulate glutamatergic and monoaminergic neurotransmission has made it a viable therapeutic target for neuropsychiatric illnesses. Although the TAAR1 role in schizophrenia and other neuropsychiatric disorders is well established, its role in the pathology of neurodegenerative and neurotraumatic disorders recently got attention. This review discusses the role of TAAR1 in neurodegenerative, neurodevelopment, and neurotraumatic disorders and explores its potential to be a novel therapeutic target in these disorders.

Keywords:  Neurodegenerative disease; Neurotraumatic disorders; TAAR1; Trace amine-associated receptors; Trace amines

DOI:  https://doi.org/10.1007/s00210-024-03757-6
Front Immunol. 2024 ;15 1523393

The development of murine bone marrow-derived mast cells expressing functional human MRGPRX2 for ex vivo and in vivo studies.

Maram Bawazir, Saptarshi Roy, Hydar Ali.

   Introduction: A subtype of human mast cells (MCs) found in the skin and to a lesser extent in the lung and gut express a novel G protein-coupled receptor (GPCR) known as Mas-related GPCR-X2 (MRGPRX2, mouse counterpart MrgprB2). In addition to drug-induced pseudoallergy and cutaneous disorders, MrgprB2 contributes to ulcerative colitis, IgE-mediated lung inflammation and systemic anaphylaxis. Interestingly, most agonists activate MRGPRX2 with higher potency than MrgprB2. In this study, we sought to replace mouse MrgprB2 with human MRGPRX2 and to study receptor function ex vivo and in vivo.
Methods: MrgprB2-/- bone marrow (BM) cells were transduced with retrovirus encoding MRGPRX2 and differentiated into BMMCs (MRGRPX2-BMMCs) ex vivo. Cell surface MRGPRX2 expression was determined by flow cytometry. Effects of substance P (SP) and LL-37 on Ca2+ mobilization, degranulation and TNF-α generation were determined. MRGPRX2-BMMCs were engrafted intraperitoneally into MC-deficient Wsh/Wsh mice. After 6-8 weeks, immunofluorescence staining was performed on peritoneal lavage cells (PLCs), and sections of small intestine and colon with anti c-Kit and anti-MRGPRX2 antibodies. SP-induced degranulation in PLCs obtained from engrafted mice was determined.
Results: MRGPRX2-BMMCs expressed cell surface MRGPRX2 and responded to both SP and LL-37 for Ca2+ mobilization, degranulation and TNF-α generation. Furthermore, Wsh/Wsh mice engrafted with MRGPRX2-BMMCs expressed the receptor in peritoneal, intestinal and colonic MCs. In addition, PLCs from engrafted mice responded to SP for degranulation.
Conclusion: Replacing mouse MrgprB2 with functional human MRGPRX2 in primary BMMCs and their engraftment in MC-deficient mice demonstrated the expression of this receptor in different tissues, which provides unique opportunities to study receptor signaling ex vivo and disease phenotype in vivo.

Keywords:  LL-37; MRGPRX2; MrgprB2; mast cells; retrovirus; substance P

DOI:  https://doi.org/10.3389/fimmu.2024.1523393
J Med Virol. 2025 Jan;97(1): e70144

Antiviral and Immunomodulatory Effects of Zafirlukast Against Flaviviruses via Inhibition of the TNF Signaling Pathway.

Thamil Vaani Komarasamy, Javier Gilbert-Jaramillo, Nur Amelia Azreen Adnan, William S James, Vinod Rmt Balasubramaniam.

The two most clinically important members of the flavivirus genus, Zika virus (ZIKV) and dengue virus (DENV) pose a significant public health challenge. They cause a range of diseases in humans, from hemorrhagic to neurological manifestations, leading to economic and social burden worldwide. Nevertheless, there are no approved antiviral drugs to treat these infections. Zafirlukast is an orally available Food and Drug Administration (FDA) approved drug for the prophylaxis and treatment of chronic asthma. It is a leukotriene receptor antagonist (LTRA) with high selectivity of the cysteinyl leukotriene-1 receptor (CYSLTR1) that acts as an immune modulator. Thus, we evaluated the antiviral potential of Zafirlukast against ZIKV and DENV in SK-N-SH cells. We showed that Zafirlukast exhibited potent antiviral activity against ZIKV, which could be linked to Zafirlukast's immune blockade of TNF signaling pathways and its downstream signaling pathways such as MAPK and ERK1/2. In addition, our results showed that Zafirlukast also counteracts ZIKV-induced changes in key genes involved in cellular lipid metabolism. Thus, these findings highlight the translational potential of optimizing Zafirlukast as a therapeutic agent for the treatment of ZIKV and DENV.

DOI: https://doi.org/10.1002/jmv.70144
Cell Commun Signal. 2025 Jan 02. 23(1): 1

Role of the Notch signaling pathway in porcine oocyte maturation.

Pil-Soo Jeong, Hyo-Gu Kang, Dabin Cha, Se-Been Jeon, Min Ju Kim, Bong-Seok Song, Bo-Woong Sim, Sanghoon Lee.

   BACKGROUND: Although the Notch signaling pathway is known to play an important role in ovarian follicle development in mammals, whether it is involved in oocyte maturation remains unclear. Therefore, this study was performed to elucidate the existence and role of the Notch signaling pathway during oocyte maturation in a porcine model.
METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemical assays were used to determine the existence of Notch signaling pathway-related transcripts and proteins in porcine cumulus-oocyte complexes (COCs). In vitro maturation (IVM) and parthenogenetic activation of oocytes were employed to examine the effects of Notch signaling inhibition on meiotic progression and embryogenesis of COCs using RO4929097 (RO), an inhibitor of γ secretase. Various staining methods (TUNEL, Phalloidin-TRITC, MitoTracker, JC-1, BODIPY FL ATP, ER-Tracker, Fluo-3, and Rhod-2) and immunocytochemical and quantitative PCR assays were used to identify the effects of Notch signaling inhibition on meiotic progression, embryogenesis, cell cycle progression, spindle assembly, chromosome alignment, mitochondrial and endoplasmic reticulum distribution, and downstream pathway targets in COCs.
RESULTS: The RT-PCR and immunocytochemical analyses revealed the presence of Notch signaling-related receptors (NOTCH1-4) and ligands (JAG1 and 2 and DLL1, 3, and 4) at 0, 22, 28, and 44 h of IVM in the COCs. RO treatment during oocyte maturation markedly reduced meiotic maturation and embryogenesis, inhibiting the cell cycle progression, spindle assembly, and chromosome alignment processes that are important for meiotic maturation. Furthermore, RO significantly impaired the cellular distribution and functions of the mitochondria and endoplasmic reticula, which are important organelles for the cytoplasmic maturation of oocytes. Finally, the involvement of canonical Notch signaling in oocyte maturation was confirmed by the decreased expression of HES and HEY family transcripts and proteins in the RO-treated COCs.
CONCLUSIONS: It was first demonstrated that Notch signaling pathway-related transcripts and proteins were expressed during the meiotic maturation of porcine COCs. Furthermore, the inhibition of Notch signaling during IVM revealed the essential role of this signaling pathway during oocyte maturation in pigs.

Keywords:  Embryo development; Microtubule function; Notch signaling pathway; Oocyte maturation; Organelle function; Pig; γ secretase inhibitor

DOI:  https://doi.org/10.1186/s12964-024-01996-x
Breast Cancer Res. 2024 Dec 31. 26(1): 193

GRK6 palmitoylation dictates triple-negative breast cancer metastasis via recruiting the β-Arrestin 2/MAPKs/NF-κB signaling axis.

Wen-Ke Wang, Hui-Yu Lin, Che-Hsuan Lin, Hsun-Hua Lee, Yen-Lin Chen, Yu-Hsien Kent Lin, Hui-Wen Chiu, Shry-Ming Sheen-Chen, Yuan-Feng Lin.

   BACKGROUND: Triple negative breast cancer (TNBC) belongs to the worst prognosis of breast cancer subtype probably because of distant metastasis to other organs, e.g. lungs. However, the mechanism underlying TNBC metastasis remains largely unknown.
METHODS: Bioinformatics analysis was conducted to evaluate the mRNA/protein expression and prognostic significance of G protein-coupled receptor kinase 6 (GRK6) in BC subtypes. RT-PCR assays were used to test the GRK6 expression in human BC tissues and cell lines. The in vitro cellular migration and in vivo lung colony-forming assays were established to estimate the metastatic potentials of TNBC cells. Western blotting was employed to examine protein phosphorylation, translocation and expression in the designed experiments.
RESULTS: Here we show that GRK6 upregulation is extensively detected in TNBC compared to normal mammary tissues and other BC subtypes and correlates with an increased risk for distant metastasis in TNBC patients. GRK6 knockdown suppressed but overexpression potentiated the cellular migration and lung colony-forming abilities of TNBC cells. Moreover, our data demonstrated that the posttranslational palmitoylation of GRK6 is extremely critical for activating β-Arrestin 2/mitogen-activated protein kinases (MAPKs)/NF-κB signaling axis and fostering the metastatic potentials of TNBC cells. Accordingly, the pharmaceutical inhibition of GRK6 kinase activity dramatically suppressed the activation of β-Arrestin 2, MAPKs and NF-κB and the cellular migration ability of highly metastatic MDA-MB231 cells. Sequentially blocking the β-Arrestin 2/MAPKs/NF-κB axis with their inhibitors predominantly mitigated the GRK6-promoted migration ability of poorly metastatic HCC1937 cells.
CONCLUSION: Our results not only provide a novel mechanism for TNBC metastasis but also offer a new therapeutic strategy to combat metastatic TNBC via targeting GRK6 activity.

Keywords:  GRK6; MAPKs; Metastasis; NF-κB; Triple-negative breast cancer; β-Arrestin 2

DOI:  https://doi.org/10.1186/s13058-024-01953-z
Nat Commun. 2025 Jan 02. 16(1): 104

Identification and structure-guided development of triazole urea-based selective antagonists of Arabidopsis karrikin signaling.

Jianwen Wang, Ikuo Takahashi, Ko Kikuzato, Toshihiko Sakai, Zhangliang Zhu, Kai Jiang, Hidemitsu Nakamura, Takeshi Nakano, Masaru Tanokura, Takuya Miyakawa, Tadao Asami.

The smoke-derived butenolides, karrikins (KARs), regulate many aspects of plant growth and development. However, KARs and a plant hormone, strigolactones (SLs), have high resemblance in signal perception and transduction, making it hard to delineate KARs response due to the shortage of chemical-genetic tools. Here, we identify a triazole urea KK181N1 as an inhibitor of the KARs receptor KAI2. KK181N1 selectively depress the KAR-induced phenotypes in Arabidopsis. We further elucidate the antagonistic, KAI2 binding mechanism of KK181N1, showing that KK181N1 binds to the catalytic pockets of KAI2 in a non-covalent binding manner. Our experiments also demonstrate the binding affinity of triazole urea compounds are regulated by the structured water molecule networks. By fine-tuning this network, we successfully develop a more potent derivative of KK181N1. We anticipate that these chemicals will be applicable to the elucidation of KARs biology, especially for discriminating the molecular and physiological aspects of KARs and SL signaling.

DOI: https://doi.org/10.1038/s41467-024-54801-1
Int J Biol Sci. 2025 ;21(1): 1-16

Oncogenic RARγ isoforms promote head and neck cancer proliferation through vinexin-β-mediated cell cycle acceleration and autocrine activation of EGFR signal.

Yu-Chu Su, Shang-Yin Wu, Keng-Fu Hsu, Shih-Sheng Jiang, Ping-Chung Kuo, Ai-Li Shiau, Chao-Liang Wu, Yang-Kao Wang, Jenn-Ren Hsiao.

  Results of retinoid-based therapies in head and neck cancer (HNC) are generally disappointing, indicating a lack of understanding of retinoic acid signaling. The role of retinoic acid receptor gamma (RARγ) and its isoforms in HNC is yet to be established. In this study, we found that RARγ1, 2, 4 are the predominant RARγ isoforms expressed in various types of human cancers, including HNC. The mechanistic study revealed that RARγ1, 2, 4 enhanced the proliferation of HNC cells by accelerating cell cycle progression through interaction with vinexin-β, as well as by ligand-dependent activation of EGFR with downstream Akt, ERK, Src, and YAP signaling pathways. Retinoic acid binding and CDK7-dependent phosphorylation on specific serine residue at the AF-1 domain are mandatory for RARγ-mediated growth promotion of HNC. Knockdown of RARγ abolished proliferation of cultured HNC cells, and completely prevented tumor growth in xenografted nude mice. Similar effects were observed in various human cancer types other than HNC. Our results indicate that RARγ-targeting approach could be a promising therapeutic and chemopreventive strategy for human cancers.

Keywords:  Head and neck cancer (HNC); epidermal growth factor receptor (EGFR); isoform; retinoic acid receptor gamma (RARγ); vinexin-β

DOI:  https://doi.org/10.7150/ijbs.100351
Proc Natl Acad Sci U S A. 2025 Jan 07. 122(1): e2419542121

Tsg101 mimicry of canonical E2 enzymes underlies its role in ubiquitin signaling.

David A Nyenhuis, Susan M Watanabe, Nico Tjandra, Carol A Carter.

  Tsg101 is a highly conserved protein best known as an early-functioning component of cellular ESCRT machinery participating in recognition, sorting, and trafficking of cellular cargo to various intracellular destinations. It shares sequence and structural homology to canonical ubiquitin-conjugating (E2) enzymes and is linked to diverse events regulated by Ub signaling. How it might fulfill these roles is unclear. Here, we show that Tsg101 E2 mimicry permits interactions with diverse ubiquitin ligating (E3) enzymes and underlies its multifunctional capabilities. Coexpression of Tsg101 with the E3 ligase NNedd4-2s protected the enzyme from degradation and, remarkably, other widely divergent ligases as well. Structural alignment with UbcH5, a canonical E2 enzyme, revealed that recognition at the E2-E3 interface, a region broadly conserved despite sequence and structural differences in both E2 and E3 enzymes, was critical for protection. Nevertheless, UbcH5 failed to protect NNedd4-2s, indicating that the UEV chaperone function is unique to the variant. Studies using Cy5-Ub-VME showed that Tsg101-mediated protection reduced accessibility to Cys residues in the ligase. Access to Tsg101 Ub-binding sites was critical: Rabeprazole, which interferes with Tsg101 Ub-binding, diminished E3 ligase protection. Thus, E2 mimicry permitting control of E3 ligase ubiquitin signaling underlies Tsg101's broad ability to participate in multiple cellular functions. The study provides mechanistic insight into how Tsg101, by partnering with diverse E3 ligases, can contribute to a broad range of cellular activities.

Keywords:  E3 ligase; ESCRT; HIV-1; Tsg101; ubiquitin

DOI:  https://doi.org/10.1073/pnas.2419542121
Infect Immun. 2024 Dec 31. e0027624

The importance of Fcγ and C-type lectin receptors in host immune responses during Pneumocystis pneumonia.

Theodore J Kottom, Eva M Carmona, Kyle Schaefbauer, Kimberly E Stelzig, Madeline R Pellegrino, Marc Bindzus, Andrew H Limper.

  Pneumocystis jirovecii pneumonia (PJP) remains a significant cause of morbidity and mortality during AIDS. In AIDS, the absence of CD4 immunity results in exuberant and often fatal PJP. In addition, organism clearance requires a balanced macrophage response since excessive inflammation promotes lung injury and respiratory failure. Corticosteroids given in addition to antibiotics significantly improve outcomes during PJP. However, concerns exist that corticosteroids further suppress immunity and increase co-infections. New strategies to promote killing and clearance of Pneumocystis while balancing lung inflammation are required. Prior studies have shown that innate immunity to Pneumocystis is mediated by C-type lectin receptors (CLRs) on macrophages and involves downstream CARD9 activation. CARD9 can be targeted by a novel specific small molecule inhibitor (BRD5529) that significantly reduces inflammatory signaling by macrophages. CARD9 serves as the central intracellular molecule through which Dectin-1, Dectin-2, Mincle, and other CLRs signal. Dectin-1 CLR is activated through its own intracytoplasmic domain, whereas other innate CLRs (e.g., Dectin-2 and Mincle) require interactions with a common Fc-gamma receptor (FcγR) accessory chain to mediate responses. We now observe that mice double deficient in both Dectin-1 and Fcer1g (which lack the FcγR gamma chain) exhibit markedly reduced organism clearance compared with Card9-/- infected animals. These mice also possess deficiencies in immunoglobulin (Ig) Fc receptors directly mediating antibody responses, further implicating altered humoral responses in Pneumocystis killing. We further demonstrate in the Pneumocystis pneumonia (PCP) mouse model that BRD5529 administration successfully suppresses inflammatory cytokines. Our data support that innate immune responses through the CLR-CARD9 axis and humoral response act together to mediate effective responses resulting in optimal organism killing and generation of host inflammatory responses. Furthermore, host lung inflammation during PCP may be successfully reduced with a novel CARD9 small molecule inhibitor.IMPORTANCEPneumocystis pneumonia (PCP) causes severe respiratory impairment in hosts with suppressed immunity, particularly those with CD4 deficiencies, such as HIV. In addition to lymphocytic immunity, both innate and humoral immunities also participate in host defense against Pneumocystis. In the current studies, we defined the relative roles of CLR receptor-mediated inflammation, as well as FcgR-related inflammation and clearance of Pneumocystis organisms. Our studies reveal important roles for CLR activities for inducing lung inflammation, which can be ameliorated with a novel small molecule inhibitor of the CARD9 adaptor protein that is necessary for CLR signaling. In contrast, FcgR has a dominant role in organism clearance, underscoring an integral role of humoral responses for the elimination of this infection.

Keywords:  C-lectin receptor; Fc receptors; Pneumocystis carinii; clearance; inflammation

DOI:  https://doi.org/10.1128/iai.00276-24
Andrology. 2025 Jan 03.

Role of GPR55 receptor in bovine sperm capacitation.

Raquel Lottero-Leconte, Angela Lara, Jessica Plaza, Camila Arroyo-Salvo, María Eugenia Bogetti, Amada Eugenia Ynsaurralde Rivolta, Franco Dellavalle, Fiamma Sengiali, Pablo Cetica, Sofía Rio, Lucia Zalazar, Andreína Cesari, Marcelo Miragaya, Sergio Morado, Silvina Perez-Martinez.

   BACKGROUND: Endocannabinoids like anandamide (AEA), among other lipids, are recognized signaling molecules that participate in reproductive events.
OBJECTIVES: Our aims were to characterize orphan G protein-coupled receptor (GPR55) presence; investigate GPR55 activation by AEA and determine GPR55 role in the bovine sperm function.
MATERIALS AND METHODS: GPR55 presence was assessed by immunocytochemistry. Protein kinase A (pPKA) and PKC (pPKC) substrates, pERK1/2, G/F-actin were determined by Western blotting, activation of RAC-1 by pull-down assay, F-actin and acrosomal exocytosis by fluorescence microscopy, sperm motility by optic microscopy and computer-aided sperm analysis and fertilizing ability by in vitro fertilization (IVF).
RESULTS: We detected GPR55 in spermatozoa at T0, after incubation in non-capacitating and capacitating (presence of AEA) conditions and upon release from oviductal epithelia. AEA induced an increase in pPKA and pPKC, while CID16020046 (CID), selective GPR55 antagonist, prevented this effect. Incubation with H89, PKA inhibitor, significantly decreased pPKC, while Gö6983, a PKC inhibitor, partially reduced pPKA. pPKA remained elevated at 15- and 45-min incubation, while pPKC decreased at 15 and increased at 45 min. CID prevented pPKC increase at 5 and 45 min and decreased pPKA at 45 min. RAC-1 and F-actin increase induced by AEA was prevented by CID. Variations in two progressive motility kinematic parameters were observed with AEA and/or CID. Sperm pretreatment with AEA increased the rate of cleaved embryos and CID prevented this effect.
DISCUSSION: We demonstrated that GPR55 activation by AEA induces time-dependent signaling pathways involving pPKA and pPKC during bovine sperm capacitation. AEA regulates actin polymerization through GPR55 activation, suggesting the receptor participates in cytoskeleton remodeling, and yielded higher IVF rates. Also, sperm pre-incubation with molecules like AEA involved in capacitation could improve the embryo development.
CONCLUSION: We have demonstrated GPR55 presence in bovine spermatozoa. The regulation of PKA and PKC and of molecules associated with cytoskeletal dynamics, such as RAC-1 and actin, by GPR55 is closely related to sperm motility and acrosomal exocytosis.

Keywords:  GPR55 receptor; anandamide; bovine; sperm fertilizing ability

DOI:  https://doi.org/10.1111/andr.13823
Sci Rep. 2024 12 28. 14(1): 31165

Homogeneous and bioluminescent biochemical and cellular assay for monitoring cGAMP and enzymes that generate and degrade cGAMP.

Kevin Hsiao, Nathan H Murray, Dareen Mikheil, Matthew A Larsen, Hui Wang, Tim Ugo, Said A Goueli.

  The cyclic GMP-AMP synthase-stimulator of the interferon gene (cGAS-STING) signaling pathway is considered an essential pattern recognition and effector pathway in the natural immune system and is mainly responsible for recognizing DNA molecules present in the cytoplasm and activating downstream signaling pathways to generate type I interferons (IFN-I) and other inflammatory factors. STING, a crucial junction protein in the innate immune system, exerts an essential role in host resistance to external pathogen invasion. The DNA introduced by pathogens or tumors is recognized by the cytoplasmic nucleic acid receptor cGAS, and a second messenger, cGAMP, is generated using intracellular guanosine triphosphate (GTP) and adenosine triphosphate (ATP). Furthermore, cellular and extracellular cGAMP concentrations are also controlled by ENPP1, an enzyme that breaks down cGAMP to AMP and GMP. Therefore, the role of the cGAS-STING signaling pathway has generated great interest in inflammatory and cancer research. To advance our understanding of innate immune system and in particular the STING pathway, we have developed a homogeneous, bioluminescent cGAMP detection assay that is very sensitive and highly selective against other nucleotides, cyclic nucleotides, and dicyclic nucleotides. The assay can be also used to monitor the activity of cGAS and ENPP1 to enable the development of inhibitors of both enzymes which might be used for therapeutic applications.

Keywords:  Bioluminescence; ENPP1; Homogeneous; STING; cGAMP; cGAS

DOI:  https://doi.org/10.1038/s41598-024-82525-1
FEBS Open Bio. 2024 Dec 30.

CDK5 interacts with MST2 and modulates the Hippo signalling pathway.

Mehak Passi, Jan B Stöckl, Thomas Fröhlich, Simone Moser, Angelika M Vollmar, Stefan Zahler.

  MST2 (STK3) is a major upstream kinase in the Hippo signalling pathway, an evolutionary conserved pathway in regulation of organ size, self-renewal and tissue homeostasis. Its downstream effectors are the transcriptional regulators YAP and TAZ. This pathway is regulated by a variety of factors, such as substrate stiffness or cell-cell contacts. Using a yeast two-hybrid screen, we detected a novel interaction between the kinases MST2 and CDK5, which we further confirmed by co-immunoprecipitation experiments. Cyclin-dependent kinase 5 (CDK5) is an unusual member of the family of cyclin-dependent kinases, involved in tumour growth and angiogenesis. Although a link between CDK5 and Hippo has been previously postulated, the mode of action is still elusive. Here, we show that knockdown of CDK5 causes reduced transcriptional activity of YAP and that CDK5 influences the phosphorylation levels of the Hippo upstream kinase LATS1. Moreover, a phosphoproteomics approach revealed that CDK5 interferes with the phosphorylation of DLG5, another upstream kinase, which regulates the Hippo pathway. Hence, CDK5 seems to act as a signalling hub for integrating the Hippo pathway and other signalling cascades. These interactions might have important implications for the use of CDK5 inhibitors, which are already in clinical use for tumour diseases.

Keywords:  CDK5; Hippo; TAZ; YAP; yeast‐two‐hybrid

DOI:  https://doi.org/10.1002/2211-5463.13962
Proteins. 2024 Dec 30.

Structural Insights Into the Impact of the Glycine-Rich Loop Mutation in Noonan Syndrome on the ATP Binding Pocket of CRAF Kinase.

Fatemeh Janati-Fard, Mohammad R Housaindokht, Fatemeh Moosavi, Saeideh Nakhaei-Rad.

  The pathogenic G361A variant of CRAF, associated with increased intrinsic kinase activity in Noonan syndrome (NS), remains poorly understood in terms of its molecular and structural impact on kinase activity. To elucidate the mechanistic implications of the glycine to alanine substitution at residue 361 in CRAF, we employed molecular dynamics simulations. Our findings reveal that this mutation predominantly affects the ATP binding pocket and critical intermolecular interactions within the active cleft that favors the phosphate transfer reaction. Notably, our data highlight significant alterations in key interactions involving Lys470/Asp486 and ATP.Mg2+ in CRAFG361A that are absent in wild-type CRAF. Additionally, we identified a novel interaction mode between Lys431 and γ-phosphate in wild-type CRAF, a residue evolutionarily conserved in CRAFs but not in related kinases such as BRAF, ARAF, and KSR1/2. Furthermore, observed shifts in the αC-helix and G-loop relative to the wild-type correlate with an enlarged ATP-binding cavity in the mutant, reflecting structural adaptations due to these mutations. Overall, these structural insights underscore the elevated intrinsic kinase activity of the CRAFG361A variant and provide crucial mechanistic details that could inform the development of specific inhibitors targeting this variant.

Keywords:  ATP; CRAF; G‐loop; Noonan syndrome; active site; interactions; kinase; αC‐helix

DOI:  https://doi.org/10.1002/prot.26769
Front Immunol. 2024 ;15 1512180

Inhibition of the MyD88 signaling pathway could upregulates Ghrelin expression to synergistically regulate hepatic Echinococcus multilocularis-infected progression.

Jiang Zhu, Tanfang Zhou, Guangfeng Chen, Yuhui Wu, Xia Chen, Ya Song, Ayinula Tuohetali, Huijing Gao, Dongming Pang, Hao Wen, Kalibixiati Aimulajiang.

   Introduction: AE and whether the inhibition of the MyD88 inflammatory pathway can enhance Ghrelin expression to collaboratively modulate AE progression remains unclear.
Methods: In this study, we evaluated Ghrelin serum levels and changes in TLR4/MyD88/NF-κB pathway proteins and inflammatory factors in AE patients and E. multilocularis mouse models at different stages of infection (-4, -8, and -12 weeks). Additionally, we administered the MyD88 inhibitor TJ-M2010-5 intraperitoneally to infected mice to evaluate alterations in inflammation and Ghrelin levels, as well as disease progression.
Results: A decrease in serum Ghrelin levels in AE patients, whereas both Ghrelin and GHSR, along with TLR4/MyD88/NF-κB pathway proteins and markers of M1/M2 macrophage polarization, exhibited increased expression in the inflammatory cell zones surrounding hepatic lesions. Similar findings were observed in E. multilocularis-infected mice. M1-type inflammatory expression predominated throughout the infection's progression, with sustained high levels of Ghrelin counteracting inflammation. The TLR4/ MyD88/NF-κB pathway remained suppressed during the first 8 weeks, becoming activated only at 12 weeks. Inhibition of the MyD88 pathway resulted in reduced inflammation levels and upregulated Ghrelin expression, thereby collaboratively regulating the progression of hepatic infection.
Conclusion: These findings suggest an interactive regulation between the MyD88 inflammatory signaling pathway and Ghrelin, indicating that MyD88 inhibition could enhance Ghrelin expression to modulate the progression of E. multilocularis infection.

Keywords:  Echinococcus multilocularis; Ghrelin; MyD88; alveolar echinococcosis; hepatic

DOI:  https://doi.org/10.3389/fimmu.2024.1512180
Nat Commun. 2024 Dec 30. 15(1): 10886

Sleep drive is coupled to tissue damage via shedding of Caenorhabditis elegans EGFR ligand SISS-1.

Andrew J Hill, Bryan Robinson, Jesse G Jones, Paul W Sternberg, Cheryl Van Buskirk.

The benefits of sleep extend beyond the nervous system. Peripheral tissues impact sleep regulation, and increased sleep is observed in response to damaging conditions, even those that selectively affect non-neuronal cells. However, the 'sleep need' signal released by stressed tissues is not known. Sleep in the nematode C. elegans is independent of circadian cues and can be triggered rapidly by damaging conditions. This stress-induced sleep is mediated by neurons that require the Epidermal Growth Factor Receptor (EGFR) for their sleep-promoting function, but the only known C. elegans EGFR ligand, LIN-3, is not required for sleep. Here we describe SISS-1 (stress-induced sleepless), an EGF family ligand that is required for stress-induced sleep. We show that SISS-1 overexpression induces sleep in an EGFR-dependent, sleep neuron-dependent manner. We find that SISS-1 undergoes stress-responsive shedding by the ADM-4/ADAM17 metalloprotease, and that the ADM-4 site of action depends on the tissue specificity of the stressor. Our findings support a model in which SISS-1 is released from damaged tissues to activate EGFR in sleep neurons, identifying a molecular link between cellular stress and organismal sleep drive. Our data also point to a mechanism insulating this sleep signal from EGFR-mediated signaling during development.

DOI: https://doi.org/10.1038/s41467-024-55252-4
J Cell Sci. 2025 Jan 02. pii: jcs.263541. [Epub ahead of print]

Cell migration signaling through EGFR-VAV2-Rac1 pathway is sustained in endosomes.

Itziar Pinilla-Macua, Sachin Surve, Alexander Sorkin.

  Ligand binding to EGFR activates Rho family GTPases, triggering actin cytoskeleton reorganization, cell migration and invasion. Activated EGFR is also rapidly endocytosed but the role of EGFR endocytosis in cell motility is poorly understood. Hence, we used live-cell microscopy imaging to demonstrate that endogenous fluorescently labeled VAV2, a guanine nucleotide exchange factor for Rho GTPases, is co-endocytosed with EGFR in genome-edited human oral squamous cell carcinoma (HSC3) cells, an in vitro model for head-and-neck cancer where VAV2 is known to promote metastasis and associates with poor prognosis. Chemotactic migration of HSC3 cells toward an EGF gradient is found to require both VAV2 and clathrin-mediated endocytosis. Moreover, sustained activation of Rac1, a Rho family GTPase promoting cell migration and a major substrate of VAV2, also depends on clathrin. Endogenous fluorescently labeled Rac1 localizes to EGFR-containing endosomes. Altogether, our findings suggest that signaling through the EGFR-VAV2-Rac1 pathway persists in endosomes and that this endosomal signaling is required for EGFR-driven cell migration.

Keywords:  Cell motility; EGFR; Endocytosis; Rac1; VAV2

DOI:  https://doi.org/10.1242/jcs.263541
Proc Natl Acad Sci U S A. 2025 Jan 07. 122(1): e2414321121

RAP-2 and CNH-MAP4 Kinase MIG-15 confer resistance in bystander epithelium to cell-fate transformation by excess Ras or Notch activity.

Razan A Fakieh, David J Reiner.

  Induction of cell fates by growth factors impacts many facets of developmental biology and disease. LIN-3/EGF induces the equipotent vulval precursor cells (VPCs) in Caenorhabditis elegans to assume the 3˚-3˚-2˚-1˚-2˚-3˚ pattern of cell fates. 1˚ and 2˚ cells become specialized epithelia and undergo stereotyped series of cell divisions to form the vulva. Conversely, 3˚ cells are relatively quiescent and nonspecialized; they divide once and fuse with the surrounding epithelium. 3˚ cells have thus been characterized as passive, uninduced, or ground state. Based on our previous studies, we hypothesized that a 3˚-promoting program would confer resistance to cell fate-transformation by inappropriately activated 1˚ and 2˚ fate-promoting LET-60/Ras and LIN-12/Notch, respectively. Deficient MIG-15/CNH-MAP4 Kinase meets the expectations of genetic interactions for a 3˚-promoting protein. Moreover, endogenous MIG-15 is required for expression of a fluorescent biomarker of 3˚ cell fate, is expressed in VPCs, and functions cell autonomously in VPCs. The Ras family small GTPase RAP-2, orthologs of which activate orthologs of MIG-15 in other systems, emulates these functions of MIG-15. However, gain of RAP-2 function has no effect on patterning, suggesting its activity is constitutive in VPCs. The 3˚ biomarker is expressed independently of the AC, raising questions about the cellular origin of 3˚-promoting activity. Activated LET-60/Ras and LIN-12/Notch repress expression of the 3˚ biomarker, suggesting that the 3˚-promoting program is both antagonized by as well as antagonizes 1˚- and 2˚- promoting programs. This study provides insight into developmental properties of cells historically considered to be nonresponding to growth factor signals.

Keywords:  ACDS-10; LET-60; LIN-12; Rap2; citron NIK1 homology

DOI:  https://doi.org/10.1073/pnas.2414321121
J Cell Physiol. 2025 Jan;240(1): e31518

Vacuolar H+-ATPase and Megalin-Mediated Prorenin Uptake: Focus on Elements Beyond the (Pro)Renin Receptor.

Na Wang, Xifeng Lu, A H Jan Danser.

  Megalin is a multiple-ligand receptor that contributes to protein reabsorption in the kidney. Recently, megalin was found to act as a novel endocytic receptor for prorenin. Internalization depended on the (pro)renin receptor. This receptor is an accessory protein of vacuolar H+-ATPase (V-ATPase), a complex consisting of 14 subunits and two accessory proteins. Here we explored whether V-ATPase elements other than the (P)RR affect megalin-mediated prorenin uptake. Using RNAi technology, we inhibited each individual V-ATPase subunit in megalin-expressing BN16 cells. Subsequently, we quantified megalin expression and the uptake of prorenin. To unravel the underlying molecular mechanisms, we investigated the adaptor proteins autosomal recessive hypercholesterolemia (ARH) and Disabled-2 (Dab2), which are important for the endocytosis of megalin, glycogen synthase kinase 3β (GSK3β), a regulatory factor of megalin recycling, and endoplasmic reticulum stress factors (ERSF). Silencing subunit Atp6voa1 reduced prorenin uptake by 19%, while silencing accessory protein Atp6ap1 increased it by 15%. Silencing other subunits exerted a more modest or no effect. Silencing Atp6voa1 reduced surface megalin density, without altering its mRNA and protein levels, and this was associated with increased GSK3β phosphorylation and no change in ARH, Dab2, and ERSF. Silencing Atp6ap1 increased megalin mRNA and protein expression and this was accompanied by upregulation of ARH and ERSF, while Dab2 expression was unaltered. In conclusion, V-ATPase units differently affect megalin-mediated reabsorption of prorenin, thereby offering novel pharmacological targets to not only affect renal renin-angiotensin system activity, but also to treat renal diseases that are associated with disturbed protein reabsorption, like Dent's disease.

Keywords:  Atp6ap1; Atp6v0a1; albumin; megalin; prorenin; vacuolar H+‐ATPase

DOI:  https://doi.org/10.1002/jcp.31518
J Med Virol. 2025 Jan;97(1): e70147

FGFR3 Upregulates Interferon-Stimulated Genes Via the JAK1-STAT1 Signaling Pathway in HPV2 E2 Stable Expressing Keratinocytes.

Qingqing Guo, Xuanjin Wei, Jiayue Qi, Chengxin Li, Fang Xie.

  Human papillomavirus (HPV) infections are prevalent skin infectious diseases. While there are no specific anti-HPV drugs available, understanding the viral mechanisms could lead to novel therapeutic strategies. Verruca vulgaris, a common HPV infection, is frequently encountered in dermatological clinics. The HPV E2 protein, an early viral protein, has been implicated in high-risk HPV infections by interacting with fibroblast growth factor receptor 3 (FGFR3) to inhibit viral DNA replication. However, the role of HPV E2 and FGFR3 in low-risk HPV infections remains elusive. Our study takes HPV2, a common subtype of verruca vulgaris, to explore the proliferation and immune regulatory effects of HPV2 E2 on keratinocytes. By overexpressing FGFR3 in HPV2 E2 stable expressing keratinocytes, we assessed changes in interferon-stimulated genes (ISGs) level and cell proliferation. Our findings revealed that HPV2 E2 induced phosphorylation of FGFR3 could activate JAK1-STAT1 pathway, thereby enhancing antiviral immunity through the upregulation of ISGs. Furthermore, we observed co-localization and interaction between FGFR3 and HPV2 E2 in keratinocytes. In conclusion, our study underscores the crucial role of FGFR3 in innate antiviral immunity against HPV2 infection in keratinocytes. These findings may provide a potential therapeutic target for HPV infections.

Keywords:  FGFR3; HPV infection; HPV2 E2; JAK1‐STAT1; antiviral immunity

DOI:  https://doi.org/10.1002/jmv.70147