bims-dicesi 2024-12-22 papers

bims-dicesi

Biomed News

on Diversification of cell signalling

Issue of 2024–12–22
24 papers selected by
Ashanika Karandawela

Intracellular pocket conformations determine signaling efficacy through the μ opioid receptor.
Development of a Class A/B Hybrid GPCR System for the Proximity-Assisted Screening of GPCR Ligands.
Casein kinase 1 controls components of a TORC2 signaling network in budding yeast.
Functional crosstalk of sucrose and G protein signaling in maize thermotolerance by modulating osmoregulation system.
Roles of metabotropic signaling of nicotine receptors in the development and maintenance of nicotine reward through regulation of dopamine D3 receptor expression.
Exploring G Protein-Coupled Receptors in Hematological Cancers.
Synaptic Gα12/13 signaling establishes hippocampal PV inhibitory circuits.
Retromer Opposes Opioid-Induced Downregulation of the Mu Opioid Receptor.
G protein-coupled receptors: a gateway to targeting oncogenic EVs?
Structural Diversity and Mutational Challenges of Toll-Like Receptor 4 Antagonists as Inflammatory Pathway Blocker.
Conformational dynamics of a nicotinic receptor neurotransmitter site.
Unravelling the druggability and immunological roles of the SOCS-family proteins.
Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells.
Impact of Intracellular Proteins on μ-Opioid Receptor Structure and Ligand Binding.
A Novel 14mer Peptide Inhibits Autophagic Flux via Selective Activation of the mTORC1 Signalling Pathway: Implications for Alzheimer's Disease.
Bridging molecular to cellular scales for models of membrane receptor signaling.
Macrophage-like Blood Cells Are Involved in Inter-Tissue Communication to Activate JAK/STAT Signaling, Inducing Antitumor Turandot Proteins in Drosophila Fat Body via the TNF-JNK Pathway.
The Remarkable Diversity of Vertebrate Bitter Taste Receptors: Recent Advances in Genomic and Functional Studies.
The activation of the G-protein-coupled estrogen receptor promotes the aggressiveness of MDA-MB231 cells by targeting the IRE1α/TXNIP pathway.
Oxylipin Receptors and Their Role in Inter-Partner Signalling in a Model Cnidarian-Dinoflagellate Symbiosis.
G-Protein-Coupled Receptor Surface Creates a Favorable Pathway for Membrane Permeation of Drug Molecules.
Pharmacology, Signaling and Therapeutic Potential of Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators.
CEP signaling coordinates plant immunity with nitrogen status.
Evidence for functional interaction between the CB1 and the mGlu7 receptors mediated signaling in modulation of anxiety behavior and cognition.

bioRxiv. 2024 Dec 07. pii: 2024.04.03.588021. [Epub ahead of print]

Intracellular pocket conformations determine signaling efficacy through the μ opioid receptor.

David A Cooper, Joseph DePaolo-Boisvert, Stanley A Nicholson, Barien Gad, David D L Minh.

It has been challenging to determine how a ligand that binds to a receptor activates downstream signaling pathways and to predict the strength of signaling. The challenge is compounded by functional selectivity, in which a single ligand binding to a single receptor can activate multiple signaling pathways at different levels. Spectroscopic studies show that in the largest class of cell surface receptors, 7 transmembrane receptors (7TMRs), activation is associated with ligand-induced shifts in the equilibria of intracellular pocket conformations in the absence of transducer proteins. We hypothesized that signaling through the μ opioid receptor, a prototypical 7TMR, is linearly proportional to the equilibrium probability of observing intracellular pocket conformations in the receptor-ligand complex. Here we show that a machine learning model based on this hypothesis accurately calculates the efficacy of both G protein and β-arrestin-2 signaling. Structural features that the model associates with activation are intracellular pocket expansion, toggle switch rotation, and sodium binding pocket collapse. Distinct pathways are activated by different arrangements of the ligand and sodium binding pockets and the intracellular pocket. While recent work has categorized ligands as active or inactive (or partially active) based on binding affinities to two conformations, our approach accurately computes signaling efficacy along multiple pathways.

DOI: https://doi.org/10.1101/2024.04.03.588021
ACS Chem Biol. 2024 Dec 16.

Development of a Class A/B Hybrid GPCR System for the Proximity-Assisted Screening of GPCR Ligands.

Monika T Gnatzy, Steffen Hartmann, Felix Hausch.

Class A G protein-coupled receptors (GPCRs) are key mediators in numerous signaling pathways and important drug targets for several diseases. A major shortcoming in GPCR ligand screening is the detection limit for weak binding molecules, which is especially critical for poorly druggable GPCRs. Here, we present a proximity-based screening system for class A GPCRs, which adopts the natural two-step activation mechanism of class B GPCRs. In this approach, class A/B chimeras with the extracellular domain of the class B receptor CRF1R grafted to the transmembrane domain of target class A receptors are stimulated with hybrid ligands. These ligands contain a high-affinity peptide derived from CRF, which recruits the hybrid ligands to the engineered target GPCR, dramatically increasing the local concentration of the test substances. We exemplified this method for neurotensin receptor 1 (NTR1) and endothelin receptor B (ETB), two important class A GPCR drug targets for pulmonary arterial hypertension or psychological disorders and neurodegenerative diseases. We observed >20× activity enhancement by the directed proximity approach, enabling the detection of weakly activating sequences that would have otherwise remained undetected. Our approach allows to probe GPCR activation in the membrane of living cells and may be especially useful for GPCRs for which it has been difficult to generate small drug-like molecules.

DOI: https://doi.org/10.1021/acschembio.4c00658
J Cell Sci. 2024 Dec 15. pii: jcs262036. [Epub ahead of print]137(24):

Casein kinase 1 controls components of a TORC2 signaling network in budding yeast.

Rafael Lucena, Akshi Jasani, Steph Anastasia, Douglas Kellogg, Maria Alcaide-Gavilan.

  Tor kinases play diverse and essential roles in control of nutrient signaling and cell growth. These kinases are assembled into two multiprotein complexes known as TORC1 and TORC2. In budding yeast, TORC2 relays nutrient-dependent signals that strongly influence growth rate and cell size. However, the mechanisms that control TORC2 signaling are poorly understood. Activation of TORC2 requires Mss4, a phosphatidylinositol 4-phosphate 5-kinase that recruits and activates downstream targets of TORC2. Localization of Mss4 to the plasma membrane is thought to be controlled by phosphorylation, and previous work has suggested that yeast homologs of casein kinase 1, Yck1 and Yck2 (referred to here collectively as Yck1/2), Control phosphorylation of Mss4. Here, we generated a new analog-sensitive allele of YCK2 and used it to test whether Yck1/2 influence localization of Mss4 or signaling in the TORC2 network. We found that Yck1/2 strongly influence Mss4 phosphorylation and localization, as well as influencing regulation of multiple components of the TORC2 network. However, inhibition of Yck1/2 causes mild effects on the best-characterized signaling axis in the TORC2 pathway, suggesting that Yck1/2 might play a larger role in influencing less well-understood aspects of TORC2 signaling.

Keywords:  Casein kinase 1; Nutrients; PP2A; TORC2 signaling

DOI:  https://doi.org/10.1242/jcs.262036
Protoplasma. 2024 Dec 19.

Functional crosstalk of sucrose and G protein signaling in maize thermotolerance by modulating osmoregulation system.

Hong-Yan Chen, Zhong-Guang Li.

  Sucrose (SUC) is a signaling molecule with multiple physiological functions. G protein is a kind of receptor that converts extracellular first messenger into intracellular second messenger. However, it is little known that SUC interplays with G protein signaling in maize thermotolerance. In this work, using maize seedlings as materials, the interplay between SUC and G protein signaling in maize thermotolerance was investigated. The results indicate that heat stress-decreased survival percentage and tissue viability of the seedlings was mitigated by SUC. Similarly, heat stress-increased malondialdehyde content and electrolyte leakage also was reduced by SUC. These findings show that SUC can potentially enhance thermotolerance in maize seedlings. Also, SUC-enhanced thermotolerance was abolished by suramin (G protein inhibitor) and N-ethylmaleimide (SUC transport inhibitor), but enhanced by 3-O-methyl-D-glucose (G protein activator), indicating the interplay of SUC and G protein signaling in maize thermotolerance. To investigate the possible mechanism behind SUC-G protein interaction in enhancing maize thermotolerance, osmoregulation in mesocotyls of seedlings were evaluated before and after heat stress. The results suggest that osmolytes (SUC, glucose, fructose, total soluble sugar, proline, and glycine betaine) contents in mesocotyls under non-heat and heat stress were increased by SUC in varying degrees. Likewise, the osmolyte-metabolizing enzymes (sucrose-phosphate synthase, sucrose synthase, pyrroline-5-carboxylate synthase, ornithine aminotransferase, betaine-aldehyde dehydrogenase, and trehalase) activities were enhanced by SUC. Analogously, ZmSPS1, ZmSUS6, ZmP5CS, ZmOAT, ZmBADH, and ZmTRE1 expression in mesocotyls was up-regulated by SUC to different extent. These findings illustrate that the functional crosstalk of sucrose and G protein signaling in maize thermotolerance by modulating osmoregulation system.

Keywords:  G protein; Maize; Osmoregulation system; Sucrose; Thermotolerance

DOI:  https://doi.org/10.1007/s00709-024-02020-2
J Neurochem. 2025 Jan;169(1): e16271

Roles of metabotropic signaling of nicotine receptors in the development and maintenance of nicotine reward through regulation of dopamine D3 receptor expression.

Dooti Kundu, Srijan Acharya, Shujie Wang, Yongkai Cao, Hee Jin Kim, Jae Hoon Cheong, Kyeong-Man Kim.

  The α4β2 nicotinic acetylcholine receptor (nAChR), an ionophore, has been suggested to signal through metabotropic pathways and interact with other receptor families, such as dopamine receptors. In this study, the interaction between α4β2 nAChR and dopamine receptors was investigated through in vivo and in vitro studies. Nicotine exposure in adolescent rats is known to induce a sustained increase in nicotine's rewarding effects which was assessed by conditioned place preference (CPP) assay. The expression levels of α4β2 nAChR and dopamine D2/D3 receptors (D2R, D3R) increased after nicotine treatment. To determine which of these two dopamine receptors was increased by nicotine treatment, a newly developed ligand with high selectivity for D3R was used in the radioligand binding assay. Although the expression of both α4β2 nAChR and D3R was enhanced by nicotine exposure during adolescence, only the elevated level of D3R persisted into adulthood. In experiments conducted on mice, D3R knockout mice showed significantly lower CPP scores in adulthood compared to wild-type mice. Cellular studies showed that an increase in D3R expression was attributed to enhanced D3R promoter activity, regulated by a signaling cascade composed of Src, Syk, PKC, and NF-κB. These results demonstrate that the metabotropic signaling pathway is involved in the interaction between α4β2 nAChR and D3R, and also suggest how nicotine reward initiated in adolescence could relapse after a long abstinence period. Given the significance of adolescent nicotine exposure on nicotine addiction, this study is thought to offer a novel mechanistic perspective for understanding nicotine reward and relapse.

Keywords:  NF‐κB; conditioned place preference; dopamine D3 receptor; ligand; nicotine reward; α4β2 nicotinic receptors

DOI:  https://doi.org/10.1111/jnc.16271
ACS Pharmacol Transl Sci. 2024 Dec 13. 7(12): 4000-4009

Exploring G Protein-Coupled Receptors in Hematological Cancers.

Choi Har Tsang, Pawel Kozielewicz.

Hematological cancers, such as lymphomas and leukemias, pose significant challenges in oncology, necessitating a deeper understanding of their molecular landscape to enhance therapeutic strategies. This article critically examines and discusses recent research on the roles of G protein-coupled receptors (GPCRs) in myeloma, lymphomas, and leukemias with a particular focus on pediatric acute lymphoblastic (lymphocytic) leukemia (ALL). By utilizing RNA sequencing (RNA-seq), we analyzed GPCR expression patterns in pediatric ALL samples (aged 3-12 years old), with a further focus on Class A orphan GPCRs. Our analysis revealed distinct GPCR expression profiles in pediatric ALL, identifying several candidates with aberrant upregulated expression compared with healthy counterparts. Among these GPCRs, GPR85, GPR65, and GPR183 have varying numbers of studies in the field of hematological cancers and pediatric ALL. Furthermore, we explored missense mutations of pediatric ALL in relation to the RNA gene expression findings, providing insights into the genetic underpinnings of this disease. By integrating both RNA-seq and missense mutation data, this article aims to provide an insightful and broader perspective on the potential correlations between specific GPCR and their roles in pediatric ALL.

DOI: https://doi.org/10.1021/acsptsci.4c00473
Proc Natl Acad Sci U S A. 2024 Dec 24. 121(52): e2407828121

Synaptic Gα12/13 signaling establishes hippocampal PV inhibitory circuits.

Krassimira Garbett, Baris Tosun, Jaybree M Lopez, Cassandra M Smith, Kelly Honkanen, Richard C Sando.

  Combinatorial networks of cell adhesion molecules and cell surface receptors drive fundamental aspects of neural circuit establishment and function. However, the intracellular signals orchestrated by these cell surface complexes remain less understood. Here, we report that the Gα12/13 pathway lies downstream of several GPCRs with critical synaptic functions. Impairment of the Gα12/13 pathway in postnatal hippocampal neurons diminishes inhibitory inputs without altering neuronal morphology or excitatory transmission. Gα12/13 signaling in hippocampal CA1 neurons in vivo selectively regulates PV interneuron synaptic connectivity, supporting an inhibitory synapse subtype-specific function of this pathway. Our studies establish Gα12/13 as a signaling node that shapes inhibitory hippocampal circuitry.

Keywords:  G protein signaling; G protein-coupled receptors; cell signaling; interneurons; synapse

DOI:  https://doi.org/10.1073/pnas.2407828121
bioRxiv. 2024 Dec 02. pii: 2024.12.02.626482. [Epub ahead of print]

Retromer Opposes Opioid-Induced Downregulation of the Mu Opioid Receptor.

Aleksandra Dagunts, Hayden Adoff, Brandon Novy, Monica De Maria, Braden T Lobingier.

The mu opioid receptor (MOR) is protected from opioid-induced trafficking to lysosomes and proteolytic downregulation by its ability to access the endosomal recycling pathway through its C-terminal recycling motif, LENL. MOR sorting towards the lysosome results in downregulation of opioid signaling while recycling of MOR to the plasma membrane preserves signaling function. However, the mechanisms by which LENL promotes MOR recycling are unknown, and this sequence does not match any known consensus recycling motif. Here we took a functional genomics approach with a comparative genome-wide screen design to identify genes which control opioid receptor expression and downregulation. We identified 146 hits including all three subunits of the endosomal Retromer complex. We show that the LENL motif in MOR is a novel Retromer recycling motif and that LENL is a necessary, sufficient, and conserved mechanism to give MOR access to the Retromer recycling pathway and protect MOR from agonist-induced downregulation to multiple clinically relevant opioids including fentanyl and methadone.

DOI: https://doi.org/10.1101/2024.12.02.626482
Extracell Vesicles Circ Nucl Acids. 2024 ;5(2): 233-248

G protein-coupled receptors: a gateway to targeting oncogenic EVs?

Lotte Di Niro, Amber C Linders, Thomas Glynn, D Michiel Pegtel, Marco Siderius, Caitrin Crudden, Martine J Smit.

  Dysregulated intercellular communication is a key feature driving cancer progression. Recently, extracellular vesicles (EVs) have added a new channel to this dense communication network. Despite solid evidence that EVs are central mediators of dysregulated signaling in onco-pathological settings, this has yet to be translated into clinically actionable strategies. The heterogeneity of EV cargo molecules, plasticity of biogenesis routes, and large overlap with their role in physiological communication, complicate a potential targeting strategy. However, recent work has linked EV biology to perhaps the "most druggable" proteins - G protein-coupled receptors (GPCRs). GPCR targeting accounts for ~60% of drugs in development and more than a third of all currently approved drugs, spanning almost all areas of medicine. Although several GPCRs have been linked to cancer initiation and progression, relatively few agents have made it into oncological regimes, suggesting that their potential is underexploited. Herein, we examine the molecular mechanisms linking GPCRs to EV communication in cancer settings. We propose that GPCRs hold potential in the search for EV-targeting in oncology.

Keywords:  G protein-coupled receptors (GPCRs); extracellular vesicles (EVs); oncogenic signaling; therapeutic targeting

DOI:  https://doi.org/10.20517/evcna.2024.10
Drug Dev Res. 2025 Feb;86(1): e70031

Structural Diversity and Mutational Challenges of Toll-Like Receptor 4 Antagonists as Inflammatory Pathway Blocker.

S K Batin Rahaman, Sandip K Nandi, Sudip Kumar Mandal, Utsab Debnath.

  Toll-like receptor 4 (TLR4) is an important mediator that activates bacterial inflammation through its signaling pathway. It binds lipopolysaccharide (LPS) in the presence of myeloid differentiation protein 2 (MD2) to dimerise the TLR4-MD2-LPS complex. The TLR4 mediated signaling pathway stimulates cytokine production in humans, initiating inflammatory responses. Overactivation of the TLR4 pathway can trigger binding of LPS to the TLR4-MD2 complex, which may lead to the development of several inflammatory disorders. Therefore, the TLR4-MD2 complex is a potential therapeutic target for the identification of new and effective anti-inflammatory agents. Various biologically active TLR4 and MD2 targeting natural and synthetic molecules are explored with anti-inflammatory activity in micromolar ranges. But no FDA-approved drugs are available in the market as of now, and some are discontinued in clinical trials due to drug resistance and severe side effects. In this review, we have assessed recent molecular advancements in TLR4-MD2 antagonists which are showing direct inhibition in lower micro and nanomolar levels. Along with it, protein informatics analysis of the binding pockets of wild type and mutated TLR4-MD2 proteins are also discussed here to give a new insight about the changes in physicochemical properties of the ligand binding area. We have also pointed out several important residues in three different sites of the large LPS binding pocket of TLR4-MD2 complex to understand probable binding affinity of small molecule inhibitors (SMIs). In addition, the present status of clinical trials for TLR4 antagonists is also reviewed. The current assessment will pave a future perspective to design different small molecules as a direct inhibitor of TLR4-MD2 complex for anti-inflammatory activities.

Keywords:  MD2 complex; TLR‐4 antagonist; clinical trials; protein informatics; signaling pathway analysis; toll‐like receptor 4

DOI:  https://doi.org/10.1002/ddr.70031
Elife. 2024 Dec 18. pii: RP92418. [Epub ahead of print]13

Conformational dynamics of a nicotinic receptor neurotransmitter site.

Mrityunjay Singh, Dinesh C Indurthi, Lovika Mittal, Anthony Auerbach, Shailendra Asthana.

  Agonists enhance receptor activity by providing net-favorable binding energy to active over resting conformations, with efficiency (η) linking binding energy to gating. Previously, we showed that in nicotinic receptors, η-values are grouped into five structural pairs, correlating efficacy and affinity within each class, uniting binding with allosteric activation (Indurthi and Auerbach, 2023). Here, we use molecular dynamics (MD) simulations to investigate the low-to-high affinity transition (L→H) at the Torpedo α-δ nicotinic acetylcholine receptor neurotransmitter site. Using four agonists spanning three η-classes, the simulations reveal the structural basis of the L→H transition where: the agonist pivots around its cationic center ('flip'), loop C undergoes staged downward displacement ('flop'), and a compact, stable high-affinity pocket forms ('fix'). The η derived from binding energies calculated in silico matched exact values measured experimentally in vitro. Intermediate states of the orthosteric site during receptor activation are apparent only in simulations, but could potentially be observed experimentally via time-resolved structural studies.

Keywords:  E. coli; allostery; ion channel; molecular biophysics; nicotinic receptor; structural biology

DOI:  https://doi.org/10.7554/eLife.92418
Front Immunol. 2024 ;15 1449397

Unravelling the druggability and immunological roles of the SOCS-family proteins.

Dylan M Lynch, Beth Forrester, Thomas Webb, Alessio Ciulli.

  The Suppressor of Cytokine Signalling (SOCS) protein family play a critical role in cytokine signalling and regulation of the JAK/STAT pathway with functional consequences to the immune response. Members of this family are implicated in multiple different signalling cascades that drive autoimmune diseases and cancer, through their binding to phosphotyrosine modified proteins as well as ubiquitination activity as part of Cullin5 RING E3 ligases. Here we review the SOCS family members CISH and SOCS1-SOCS7, with a focus on their complex role in immunity. The interactome and signalling network of this protein family is discussed, and the intricate mechanisms through which SOCS proteins alter and manage the immune system are assessed. We offer structural insights into how SOCS proteins engage their interacting partners and native substrates at the protein-protein interaction level. We describe how this knowledge has enabled drug discovery efforts on SOCS proteins to date and propose strategies for therapeutic intervention using small molecules, either via direct inhibition or leveraging their E3 ligase activity for targeted protein degradation.

Keywords:  Cullin RING E3 ligases; E3 ligases; SH2 domains; SOCS proteins; cell signalling; phosphotyrosine (pTyr); small molecule inhibitors; targeted protein degradation

DOI:  https://doi.org/10.3389/fimmu.2024.1449397
Cells. 2024 Nov 28. pii: 1967. [Epub ahead of print]13(23):

Sprouty2 Regulates Endocytosis and Degradation of Fibroblast Growth Factor Receptor 1 in Glioblastoma Cells.

Barbara Hausott, Lena Pircher, Michaela Kind, Jong-Whi Park, Peter Claus, Petra Obexer, Lars Klimaschewski.

  The Sprouty (SPRY) proteins are evolutionary conserved modulators of receptor tyrosine kinase (RTK) signaling. SPRY2 inhibits fibroblast growth factor (FGF) signaling, whereas it enhances epidermal growth factor (EGF) signaling through inhibition of EGF receptor (EGFR) endocytosis, ubiquitination, and degradation. In this study, we analyzed the effects of SPRY2 on endocytosis and degradation of FGF receptor 1 (FGFR1) using two human glioblastoma (GBM) cell lines with different endogenous SPRY2 levels. SPRY2 overexpression (SPRY2-OE) inhibited clathrin- and caveolae-mediated endocytosis of FGFR1, reduced the number of caveolin-1 vesicles and the uptake of transferrin. Furthermore, FGFR1 protein was decreased by SPRY2-OE, whereas EGFR protein was increased. SPRY2-OE enhanced FGFR1 degradation by increased c-casitas b-lineage lymphoma (c-CBL)-mediated ubiquitination, but it diminished binding of phospholipase Cγ1 (PLCγ1) to FGFR1. Consequently, SPRY2-OE inhibited FGF2-induced activation of PLCγ1, whereas it enhanced EGF-induced PLCγ1 activation. Despite the reduction of FGFR1 protein and the inhibition of FGF signaling, SPRY2-OE increased cell viability, and knockdown of SPRY2 enhanced the sensitivity to cisplatin. These results demonstrate that the inhibitory effect of SPRY2-OE on FGF signaling is at least in part due to the reduction in FGFR1 levels and the decreased binding of PLCγ1 to the receptor.

Keywords:  c-casitas b-lineage lymphoma; caveolin-1; clathrin; extracellular signal-regulated kinase; phospholipase Cγ1; ubiquitin

DOI:  https://doi.org/10.3390/cells13231967
J Phys Chem B. 2024 Dec 19.

Impact of Intracellular Proteins on μ-Opioid Receptor Structure and Ligand Binding.

Caitlin E Scott, Leah A Juechter, Josephine Rocha, Lauren D Jones, Brenna Outten, Taylor D Aishman, Alaina R Ivers, George C Shields.

Chronic pain is a prevalent problem affecting approximately one out of every five adults in the U.S. The most effective way to treat chronic pain is with opioids, but they cause dangerous side effects such as tolerance, addiction, and respiratory depression, which makes them quite deadly. Opioids, such as fentanyl, target the μ-opioid receptor (MOR), which can then bind to the intracellular Gi protein or the β-arrestin protein. The Gi pathway is primarily responsible for pain relief and potential side effects, but the β-arrestin pathway is chiefly responsible for the unwanted side effects. Ideally, an effective pain medication without side effects would bind to MOR, which would bias signaling solely through the Gi pathway. We used the Bio3D library to conduct principal component analysis to compare the cryo-electron microscopy MOR structures in complex with the Gi versus an X-ray crystallography MOR structure with a nanobody acting as a Gi mimic. Our results agree with a previous study by Munro, which concluded that nanobody-bound MOR is structurally different than Gi-bound MOR. Furthermore, we investigated the structural diversity of opioids that can bind to MOR. Quantum mechanical calculations show that the low energy solution structures of fentanyl differ from the one bound to MOR in the experimental structure, and pKa calculations reveal that fentanyl is protonated in aqueous solution. Glide docking studies show that higher energy structures of fentanyl in solution form favorable docking complexes with MOR. Our calculations show the relative abundance of each fentanyl conformation in solution as well as the energetic barriers that need to be overcome to bind to MOR. Docking studies confirm that multiple fentanyl conformations can bind to the receptor. Perhaps a variety of conformations of fentanyl can stabilize multiple conformations of the MOR, which can explain why fentanyl can induce different intracellular signaling and multiple physiological effects.

DOI: https://doi.org/10.1021/acs.jpcb.4c05214
Int J Mol Sci. 2024 Nov 29. pii: 12837. [Epub ahead of print]25(23):

A Novel 14mer Peptide Inhibits Autophagic Flux via Selective Activation of the mTORC1 Signalling Pathway: Implications for Alzheimer's Disease.

Cloe García Porta, Kashif Mahfooz, Joanna Komorowska, Sara Garcia-Rates, Susan Greenfield.

  During development, a 14mer peptide, T14, modulates cell growth via the α-7 nicotinic acetylcholine receptor (α7 nAChR). However, this process could become excitotoxic in the context of the adult brain, leading to pathologies such as Alzheimer's disease (AD). Recent work shows that T14 acts selectively via the mammalian target of rapamycin complex 1 (mTORC1). This pathway is essential for normal development but is overactive in AD. The triggering of mTORC1 has also been associated with the suppression of autophagy, commonly observed in ageing and neurodegeneration. We therefore investigated the relationship between T14 and autophagic flux in tissue cultures, mouse brain slices, and human Alzheimer's disease hippocampus. Here, we demonstrate that T14 and p-mTOR s2448 expression significantly increases in AD human hippocampus, which was associated with the gradual decrease in the autophagosome number across Braak stages. During development, the reduction in T14 positively correlated with pTau (Ser202, Thr205) and two selective autophagy receptors: p62 and optineurin. In vitro studies also indicated that T14 increases p-mTOR s2448 expression, resulting in the aggregation of polyubiquinated substances. The effective blockade of T14 via its cyclic variant, NBP14, has been validated in vitro, in vivo, and ex vivo. In this study, NBP14 significantly attenuated p-mTOR s2448 expression and restored normal autophagic flux, as seen with rapamycin. We conclude that T14 acts at the α-7 receptor to selectively activate the mTORC1 pathway and consequently inhibit autophagic flux. Hence, this study describes a further step in the process by which T14 could drive neurodegeneration.

Keywords:  Alzheimer’s disease; NBP14; T14; autophagy; mTORC1; rapamycin

DOI:  https://doi.org/10.3390/ijms252312837
bioRxiv. 2024 Dec 05. pii: 2024.12.04.626844. [Epub ahead of print]

Bridging molecular to cellular scales for models of membrane receptor signaling.

Kelvin J Peterson, Boris M Slepchenko, Leslie M Loew.

Biochemical interactions at membranes are the starting points for cell signaling networks. But bimolecular reaction kinetics are difficult to experimentally measure on 2-dimensional membranes and are usually measured in volumetric in vitro assays. Membrane tethering produces confinement and steric effects that will significantly impact binding rates in ways that are not readily estimated from volumetric measurements. Also, there are situations when 2D reactions do not conform to simple kinetics. Here we show how highly coarse-grained molecular simulations using the SpringSaLaD software can be used to estimate membrane-tethered rate constants from experimentally determined volumetric kinetics. The approach is validated using an analytical solution for dimerization of binding sites anchored via stiff linkers. This approach can provide 2-dimensional bimolecular rate constants to parameterize cell-scale models of receptor-mediated signaling. We explore how factors such as molecular reach, steric effects, disordered domains, local concentration and diffusion affect the kinetics of binding. We find that for reaction-limited cases, the key determinant in converting 3D to 2D rate constant is the distance of the binding sites from the membrane. On the other hand, the mass action rate law may no longer be obeyed for diffusion-limited reaction on surfaces; the simulations reveal when this situation pertains. We then apply our approach to epidermal growth factor receptor (EGFR) mediated activation of the membrane-bound small GTPase Ras. The analysis reveals how prior binding of Ras to the allosteric site of SOS, a guanine nucleotide exchange factor (GEF) that is recruited to EGFR, significantly accelerates its catalytic activity.
SIGNIFICANCE STATEMENT: In cell signaling, the activation of a surface receptor leads to a cascade of intracellular biochemical events. Many of these occur near the inner plasma membrane surface. However, accurate rate parameters for these initial steps in models of signaling are rarely available because membrane-tethered reaction kinetics are difficult to experimentally measure. Here, we use a highly coarse-grained molecular simulator to model the kinetics of reactions between binding sites that are tethered to a membrane. We can fit these simulation outputs to 2-dimensional rate laws to obtain rate constants that can be used to build complex models of cell signaling. These rate constants can also be compared to understand the key biophysical features controlling the kinetics of bimolecular membrane reactions.

DOI: https://doi.org/10.1101/2024.12.04.626844
Int J Mol Sci. 2024 Dec 06. pii: 13110. [Epub ahead of print]25(23):

Macrophage-like Blood Cells Are Involved in Inter-Tissue Communication to Activate JAK/STAT Signaling, Inducing Antitumor Turandot Proteins in Drosophila Fat Body via the TNF-JNK Pathway.

Juri Kinoshita, Yuriko Kinoshita, Tadashi Nomura, Yoshihiro H Inoue.

   Turandot (Tot) family proteins, which are induced via the JAK/STAT pathway after infection, also suppress lymph gland tumors in Drosophila mxcmbn1 mutant larvae. We investigated the potential role of hemocytes in Tot induction in tumor-bearing mutants via immunostaining and RNAi experiments. Normal hemocytes transplanted into mutant larvae were recruited to the tumor and fat body (FB), suggesting that these cells transmit tumor-related information. The transplanted hemocytes ectopically expressed Unpaired3 (Upd3), which is necessary for the activation of JAK/STAT. Eiger, a Drosophila tumor necrosis factor (TNF) ortholog, was highly expressed in tumors. Depletion of the Eiger receptor in hemocytes reduced Tot levels and eventually enhanced tumor growth. The c-Jun N-terminal kinase (JNK) pathway, acting downstream of the receptor, was also activated in the hemocytes of mutants. Downregulation of the JNK pathway in hemocytes inhibited Tot induction, leading to enhanced tumor growth. These results suggest that upd3 expression in hemocytes depends on the Eiger-JNK pathway. We propose that after Eiger activates the JNK pathway in hemocytes present on the tumor, cells expressing Upd3 are recruited to the FB. Upd3 then activates JAK/STAT to induce the expression of antitumor proteins. This study highlights the intricate communication between tissues via blood cells during tumor suppression.

Keywords:  Drosophila; JAK-STAT pathway; JNK pathway; TNF family; cytokines; hematopoietic cell tumor; innate immune system

DOI:  https://doi.org/10.3390/ijms252313110
Int J Mol Sci. 2024 Nov 25. pii: 12654. [Epub ahead of print]25(23):

The Remarkable Diversity of Vertebrate Bitter Taste Receptors: Recent Advances in Genomic and Functional Studies.

Akihiro Itoigawa, Tomoya Nakagita, Yasuka Toda.

  Bitter taste perception is crucial for animal survival. By detecting potentially harmful substances, such as plant secondary metabolites, as bitter, animals can avoid ingesting toxic compounds. In vertebrates, this function is mediated by taste receptors type 2 (T2Rs), a family of G protein-coupled receptors (GPCRs) expressed on taste buds. Given their vital roles, T2Rs have undergone significant selective pressures throughout vertebrate evolution, leading to frequent gene duplications and deletions, functional changes, and intrapopulation differentiation across various lineages. Recent advancements in genomic and functional research have uncovered the repertoires and functions of bitter taste receptors in a wide range of vertebrate species, shedding light on their evolution in relation to dietary habits and other ecological factors. This review summarizes recent research on bitter taste receptors and explores the mechanisms driving the diversity of these receptors from the perspective of vertebrate ecology and evolution.

Keywords:  G protein-coupled receptors; bitter taste perception; molecular evolution

DOI:  https://doi.org/10.3390/ijms252312654
Res Pharm Sci. 2024 Oct;19(5): 606-621

The activation of the G-protein-coupled estrogen receptor promotes the aggressiveness of MDA-MB231 cells by targeting the IRE1α/TXNIP pathway.

Maryam Mohammad-Sadeghipour, Mohammad Hadi Nematollahi, Hassan Ahmadinia, Mohammad Reza Hajizadeh, Mehdi Mahmoodi.

   Background and purpose: This study investigated modulating the G protein-coupled estrogen receptor (GPER) on the IRElα/TXNIP pathway and its role in drug resistance in MDA-MB231 cells.
Experimental approach: To determine the optimal concentrations of G1 and 4-hydroxytamoxifen (TAM), GPER expression and ERK1/2 phosphorylation were analyzed using qRT-PCR and western blotting, respectively. Cells were treated with individual concentrations of G1 (1000 nM), G15 (1000 nM), and TAM (2000 nM), as well as combinations of these treatments (G1 + G15, TAM + G15, and G1 + TAM) for 24 and 48 h. The expression levels of GPER, IRE1α, miR-17-5p, TXNIP, ABCB1, and ABCC1 genes and TXNIP protein expression were evaluated. Finally, apoptosis and cell migration were examined using flow cytometry and the wound-healing assay, respectively.
Findings/Results: Activating GPER with its specific agonist G1 and TAM significantly increased IRE1α levels in MDA-MB231 cells. IRE1α through splicing XBP1 led to unfolded protein response. In addition, decreased TXNIP gene and protein expression reduced apoptosis, increased migration, and upregulated the genes associated with drug resistance.
Conclusion and implication: Our investigation revealed that blocking the GPER/IRE1α/TXNIP pathway in MDA-MB231 cells could enhance treatment efficacy and improve chemotherapy responsiveness. The distinct unfolded protein response observed in MDA-MB231 cells may stem from the unique characteristics of these cells, which lack receptors for estrogen, progesterone, and HER2/neu hormones, possessing only the GPER receptor (ER-/PR-/HER2-/GPER+). This study introduced a new pathway in TNBC cells, indicating that targeting GPER could be crucial in comprehensive therapeutic strategies in TNBC cells.

Keywords:  Breast cancer; Drug resistance; G protein-coupled estrogen receptor; Thioredoxin interacting protein; Unfolded protein response; miR-17-5P

DOI:  https://doi.org/10.4103/RPS.RPS_96_24
Environ Microbiol. 2024 Dec;26(12): e70015

Oxylipin Receptors and Their Role in Inter-Partner Signalling in a Model Cnidarian-Dinoflagellate Symbiosis.

Andrea G Gamba, Clinton A Oakley, Immy A Ashley, Arthur R Grossman, Virginia M Weis, David J Suggett, Simon K Davy.

  Oxylipin signalling is central in biology, mediating processes such as cellular homeostasis, inflammation and molecular signalling. It may also facilitate inter-partner communication in the cnidarian-dinoflagellate symbiosis, though this aspect remains understudied. In this study, four oxylipin receptors were characterised using immunohistochemistry and immunoblotting in the sea anemone Exaiptasia diaphana ('Aiptasia'): Prostaglandin E2 receptor 2 (EP2) and 4 (EP4), Transient Receptor Potential cation channel A1 (TRPA1) and Glutamate Receptor Ionotropic, Kainate 2 (GRIK2). Receptor abundance and localisation were compared between aposymbiotic anemones and symbiotic anemones hosting either native Breviolum minutum or non-native Durusdinium trenchii. All receptors were localised to the putative symbiosome of freshly isolated symbionts, suggesting a role in host-symbiont crosstalk. EP2, EP4 and TRPA1 abundance decreased in the gastrodermis of anemones hosting B. minutum, indicating potential downregulation of pathways mediated by these receptors. In contrast, GRIK2 abundance increased in anemones hosting D. trenchii in both the epidermis and gastrodermis; GRIK2 acts as a chemosensor of potential pathogens in other systems and could play a similar role here given D. trenchii's reputation as a sub-optimal partner for Aiptasia. This study contributes to the understanding of oxylipin signalling in the cnidarian-dinoflagellate symbiosis and supports further exploration of host-symbiont molecular signalling.

Keywords:   Exaiptasia diaphana ; Aiptasia; Symbiodiniaceae; immunocytochemistry; lipid signalling

DOI:  https://doi.org/10.1111/1462-2920.70015
J Phys Chem Lett. 2024 Dec 17. 12643-12651

G-Protein-Coupled Receptor Surface Creates a Favorable Pathway for Membrane Permeation of Drug Molecules.

Cristina Gil Herrero, Sebastian Thallmair.

G-protein-coupled receptors (GPCRs) play a crucial role in modulating physiological responses and serve as the main drug target. Specifically, salmeterol and salbutamol, which are used for the treatment of pulmonary diseases, exert their effects by activating the GPCR β2-adrenergic receptor (β2AR). In our study, we employed coarse-grained molecular dynamics simulations with the Martini 3 force field to investigate the dynamics of drug molecules in membranes in the presence and absence of β2AR. Our simulations reveal that, in more than 50% of the flip-flop events, the drug molecules use the β2AR surface to permeate the membrane. The pathway along the GPCR surface is significantly more energetically favorable for the drug molecules, which was revealed by umbrella sampling simulations along spontaneous flip-flop pathways. Furthermore, we assessed the behavior of drugs with intracellular targets, such as kinase inhibitors, whose therapeutic efficacy could benefit from this observation. In summary, our results show that β2AR surface interactions can significantly enhance the membrane permeation of drugs, emphasizing their potential for consideration in future drug development strategies.

DOI: https://doi.org/10.1021/acs.jpclett.4c02875
ACS Pharmacol Transl Sci. 2024 Dec 13. 7(12): 3671-3690

Pharmacology, Signaling and Therapeutic Potential of Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators.

Jackson A Kos, Monica Langiu, Shane D Hellyer, Karen J Gregory.

Metabotropic glutamate receptors are a family of eight class C G protein-coupled receptors regulating higher order brain functions including cognition and motion. Metabotropic glutamate receptors have thus been heavily investigated as potential drug targets for treating neurological disorders. Drug discovery efforts directed toward metabotropic glutamate receptor subtype 5 (mGlu5) have been particularly fruitful, with a wealth of drug candidates and pharmacological tools identified. mGlu5 negative allosteric modulators (NAMs) are promising novel therapeutics for developmental, neuropsychiatric and neurodegenerative disorders (e.g., Alzheimer's Disease, Huntington's Disease, Parkinson's Disease, amyotrophic lateral sclerosis, autism spectrum disorders, substance use disorders, stroke, anxiety and depression) and show promise in ameliorating adverse effects induced by other medications (e.g., L-dopa induced dyskinesia in Parkinson's Disease). However, despite preclinical success, mGlu5 NAMs are yet to reach the market due to poor safety and efficacy profiles in clinical trials. Herein, we review the physiology and signal transduction of mGlu5. We provide a comprehensive critique of therapeutic options with respect to mGlu5 inhibitors, spanning from orthosteric antagonists to NAMs. Finally, we address the challenges associated with drug development and highlight future directions to guide rational drug discovery of safe and effective novel therapeutics.

DOI: https://doi.org/10.1021/acsptsci.4c00213
Nat Commun. 2024 Dec 16. 15(1): 10686

CEP signaling coordinates plant immunity with nitrogen status.

Jakub Rzemieniewski, Henriette Leicher, Hyun Kyung Lee, Caroline Broyart, Shahran Nayem, Christian Wiese, Julian Maroschek, Zeynep Camgöz, Vilde Olsson Lalun, Michael Anthony Djordjevic, A Corina Vlot, Ralph Hückelhoven, Julia Santiago, Martin Stegmann.

Plant endogenous signaling peptides shape growth, development and adaptations to biotic and abiotic stress. Here, we identify C-TERMINALLY ENCODED PEPTIDEs (CEPs) as immune-modulatory phytocytokines in Arabidopsis thaliana. Our data reveals that CEPs induce immune outputs and are required to mount resistance against the leaf-infecting bacterial pathogen Pseudomonas syringae pv. tomato. We show that effective immunity requires CEP perception by tissue-specific CEP RECEPTOR 1 (CEPR1) and CEPR2. Moreover, we identify the related RECEPTOR-LIKE KINASE 7 (RLK7) as a CEP4-specific CEP receptor contributing to CEP-mediated immunity, suggesting a complex interplay of multiple CEP ligands and receptors in different tissues during biotic stress. CEPs have a known role in the regulation of root growth and systemic nitrogen (N)-demand signaling. We provide evidence that CEPs and their receptors promote immunity in an N status-dependent manner, suggesting a previously unknown molecular crosstalk between plant nutrition and cell surface immunity. We propose that CEPs and their receptors are central regulators for the adaptation of biotic stress responses to plant-available resources.

DOI: https://doi.org/10.1038/s41467-024-55194-x
Life Sci. 2024 Dec 12. pii: S0024-3205(24)00903-2. [Epub ahead of print]361 123313

Evidence for functional interaction between the CB1 and the mGlu7 receptors mediated signaling in modulation of anxiety behavior and cognition.

Barbara Chruścicka-Smaga, Magdalena Sowa-Kućma, Patrycja Pańczyszyn-Trzewik, Bartosz Bobula, Agata Korlatowicz, Katarzyna Latocha, Paulina Pabian, Ewelina Czechowska, Tomasz Lenda, Agata Faron-Górecka, Katarzyna Stachowicz.

  Anxiety is a severe social problem. It is a disease entity that occurs alone or accompanies other diseases such as depression, phobia, or post-traumatic stress disorder. Our earlier studies demonstrated that blockage of arachidonic acid (AA) pathway via inhibition of cyclooxygenase-2 (COX-2) enzyme can modulate mGluRs-induced anxiety-like behavior. Here, we hypothesized that modulation of 2-arachidoglycerol (2-AG), a component of the AA pathway, concomitantly with modulation of mGluR7 signaling, should be adequate to trigger a similar response from the test organism. Since 2-AG is an endogenous agonist for CB1 receptors, we used a CB1/GPR55/μ-opioid receptor antagonist (AM251) alone and in combination with mGluR7 allosteric agonist (AMN082). Stress-induced hyperthermia (SIH) test was performed as a behavioral readout. AM251 has a dual mode on AMN082-mediated effects in SIH in CD-1 mice. Furthermore, the CB1 receptor ligand influenced adaptation to stress in repeated SIH procedures and learning possibilities of mice in the Barnes maze. We also found changes in mGluR7 protein expression levels in the prefrontal cortex (PFC) after mice were exposed to AM251, which showed the potential to attenuate the AMN082-induced decline in mGluR7 levels. The changes induced by AM251 on AMN082-mediated behavioral and biochemical effects were confirmed in electrophysiological experiments in which AM251 abolished AMN082-mediated LTP escalation in PFC. The mGluR7 overexpressed cell line was used to exclude the direct involvement of mGluR7 in AM251 activity. All the above results and the co-localization of CB1 and mGlu7 receptors detected in specific brain regions strongly suggest the specific interaction between CB1 and mGlu7 receptors and their signaling.

Keywords:  AM251; Barnes maze; CB1 receptor; Stress-induced hyperthermia; mGluR4; mGluR7

DOI:  https://doi.org/10.1016/j.lfs.2024.123313