bims-dicesi 2024-12-15 papers

bims-dicesi

Biomed News

on Diversification of cell signalling

Issue of 2024–12–15
33 papers selected by
Ashanika Karandawela

An atomic look at the interface of GHSR and its partners.
AlphaFold3 versus experimental structures: assessment of the accuracy in ligand-bound G protein-coupled receptors.
Regulation of the chemokine receptors CXCR4 and ACKR3 by receptor activity-modifying proteins.
G protein-coupled receptor-receptor interactions in gonadal physiology.
Optimized vector for functional expression of the human bitter taste receptor TAS2R14 in HEK293 cells.
Corticotropin-releasing factor-like diuretic hormone 44 and five corresponding GPCRs in Drosophila suzukii: Structural and functional characterization.
More and more pleiotropy within the IL-6 family of cytokines.
The G protein inhibitor YM-254890 is an allosteric glue.
Molecular basis of lipid and ligand regulation of prostaglandin receptor DP2.
Novel determinants of NOTCH1 trafficking and signaling in breast epithelial cells.
Structure-Activity Relationships and Molecular Pharmacology of Positive Allosteric Modulators of the Mu-Opioid Receptor.
Taurine stimulation of planarian motility: a role for the dopamine receptor pathway.
Ion mobility mass spectrometry unveils conformational effects of drug lead EPI-001 on the intrinsically disordered N-terminal domain of the androgen receptor.
Toll-like receptors as a missing link in Notch signaling cascade during neurodevelopment.
An activating CaSR variant with biased signaling reveals a critical residue for Gα11 coupling.
Decoding a Cell's Fate: How Notch and Receptor Tyrosine Kinase Signals Specify the Drosophila R7 Photoreceptor.
Physiological and Pathological Role of mTOR Signaling in Astrocytes.
The Rho effector ARHGAP18 coordinates a Hippo pathway feedback loop through YAP and Merlin to regulate the cytoskeleton and epithelial cell polarity.
Ubiquitin E3 ligases in the plant Arg/N-degron pathway.
Prolonged cell cycle arrest in response to DNA damage in yeast requires the maintenance of DNA damage signaling and the spindle assembly checkpoint.
Exploring key features of selectivity in somatostatin receptors through molecular dynamics simulations.
Integrins and Integrin-Driven Secretory Pathways as Multi-dimensional Regulators of Tumor-Associated Macrophage Recruitment and Reprogramming in Tumor Microenvironment.
Regulation of NHE3 subcellular localization in epididymal principal cells: pH, cyclic adenosine 3,5 monophosphate (cAMP), and adenosine signaling.
Regulating IL-2 immune signaling function via a core allosteric structural network.
Endogenous oligomer formation underlies DVL2 condensates and promotes Wnt/β-catenin signaling.
Membrane progesterone receptor e (paqr9) is necessary for chorion elevation in zebrafish.
LNS8801: An enantiomerically pure agonist of the G protein-coupled estrogen receptor suitable for clinical development.
Characteristics of Gracilariopsis lemaneiformis hydrocolloids and their effects on intestine PPAR signaling and liver lipid metabolism in Oreochromis niloticus: A multiomics analysis.
Juvenile hormone induces phosphorylation of insulin/insulin-like growth factor signaling proteins in previtellogenic Aedes aegypti mosquitoes.
Multiomics unraveled that gibberellin signaling underlies adaptation of rice to ciprofloxacin stress: Calling for concerns on the adverse effects of pharmaceutical residues in water during agricultural irrigations.
Third intracellular loop of HCMV US28 is necessary for signaling and viral reactivation.
Comprehensive host cell proteins profiling in biopharmaceuticals by a sensitivity enhanced mass spectrometry strategy using TMT-labeling and signal boosting.
Equine lentivirus Gag protein degrades mitochondrial antiviral signaling protein via the E3 ubiquitin ligase Smurf1.

Comput Struct Biotechnol J. 2024 Dec;23 4242-4251

An atomic look at the interface of GHSR and its partners.

Carlos A V Barreto, Irina S Moreira.

  G protein-coupled receptors (GPCRs) regulate cellular activity by transducing external signals and selectively coupling them to intracellular partners. Ghrelin receptor (GHSR) has garnered significant interest over the past decade owing to its diverse functional roles. In this study, we simulated five distinct GHSR-partner complexes, including Gq, Gi, and arrestin in two conformational states, to investigate the structural determinants of partner coupling. Interface and contact analyses revealed conserved interaction sites and novel interactions that were specific to each partner family. Molecular dynamics simulations provided insights into GHSR conformational dynamics, highlighting notable differences in key structural regions across complexes, such as the TM5 bulge. Our findings underscore the structural diversity of GHSR coupling mechanisms and contribute to a deeper understanding of their functional versatility.

Keywords:  Arrestin; G protein; G protein-coupled receptor (GPCRs); Ghrelin receptor (GHSR); Selectivity

DOI:  https://doi.org/10.1016/j.csbj.2024.11.035
Acta Pharmacol Sin. 2024 Dec 06.

AlphaFold3 versus experimental structures: assessment of the accuracy in ligand-bound G protein-coupled receptors.

Xin-Heng He, Jun-Rui Li, Shi-Yi Shen, H Eric Xu.

  G protein-coupled receptors (GPCRs) are critical drug targets involved in numerous physiological processes, yet many of their structures remain unresolved due to inherent flexibility and diverse ligand interactions. This study systematically evaluates the accuracy of AlphaFold3-predicted GPCR structures compared to experimentally determined structures, with a primary focus on ligand-bound states. Our analysis reveals that while AlphaFold3 shows improved performance over AlphaFold2 in predicting overall GPCR backbone architecture, significant discrepancies persist in ligand-binding poses, particularly for ions, peptides, and proteins. Despite advancements, these limitations constrain the utility of AlphaFold3 models in functional studies and structure-based drug design, where high-resolution details of ligand interactions are crucial. We assess the accuracy of predicted structures across various ligand types, quantifying deviations in binding pocket geometries and ligand orientations. Our findings highlight specific challenges in the computational prediction of ligand-bound GPCR structures, emphasizing areas where further refinement is needed. This study provides valuable insights for researchers using AlphaFold3 in GPCR studies, underscores the ongoing necessity for experimental structure determination, and offers direction for improving protein-ligand interaction predictions in future computational models.

Keywords:  AlphaFold; GPCR; artificial intelligence; structural biology; structure-based drug design

DOI:  https://doi.org/10.1038/s41401-024-01429-y
J Biol Chem. 2024 Dec 09. pii: S0021-9258(24)02557-2. [Epub ahead of print] 108055

Regulation of the chemokine receptors CXCR4 and ACKR3 by receptor activity-modifying proteins.

Fabian Pfersdorf, Lucas Romanazzi, Mette Marie Rosenkilde, Martin Gustavsson.

  The chemokine CXCL12 and its two cognate receptors - CXCR4 and ACKR3 - are key players in various homeostatic and pathophysiological processes, including embryonic development, autoimmune diseases, tissue repair and cancer. Recent reports identified an interaction of CXCR4 and ACKR3 with receptor activity-modifying proteins (RAMPs), and RAMP3 has been shown to facilitate ACKR3's recycling properties. Yet, the functional effects of RAMPs on the CXCL12 signalling axis remain largely elusive. Here, we characterize the effects of RAMPs on CXCR4 and ACKR3 function. We show that, in the absence of a ligand, RAMPs do not affect the cell membrane localization or constitutive internalization of the two receptors. RAMP3 inhibits ligand-stimulated internalization of ACKR3, which retains the receptor at the membrane and inhibits its ability to scavenge CXCL12. In addition, while cAMP inhibition by CXCR4 is unaffected by RAMPs, basal and ligand-stimulated β-arrestin recruitment to both CXCR4 and ACKR3 is reduced in the presence of RAMP3 due to complex formation at the cell surface. The effects on ACKR3 are observed for chemokine, small molecule and peptide agonists as well as for a N-terminal truncated receptor variant, suggesting that RAMP regulation involves contacts with the transmembrane domain of the receptor. Taken together, our results show that RAMPs regulate the CXCL12 signalling axis by directly interfering with receptor function. These findings could have direct implications for the interplay between receptors in vivo as well as future drug design in the therapeutic targeting of the CXCL12 signalling axis.

Keywords:  ACKR3; CXCL12; CXCR4; G protein-coupled receptor (GPCR); arrestin recruitment; chemokine receptor; protein-protein interaction; receptor activity-modifying proteins (RAMPs); receptor internalization; signal transduction

DOI:  https://doi.org/10.1016/j.jbc.2024.108055
Andrology. 2024 Dec 09.

G protein-coupled receptor-receptor interactions in gonadal physiology.

Clara Lazzaretti, Mohammed Akli Ayoub, Livio Casarini.

   BACKGROUND: Gonadotropins are glycoprotein hormones fundamental in the endocrine regulation of reproduction. They act on structurally similar members of G protein-coupled receptors (GPCRs) expressed exclusively in the gonads and support gametogenesis, sex steroid synthesis, and pregnancy. While it is a common opinion that the gonadotropin receptors act as a single molecule entity (monomer), increasing evidence underlines the formation of molecular complexes involving multiple receptors.
OBJECTIVES: To review current knowledge of membrane receptor-receptor interactions in reproduction.
RESULTS AND DISCUSSION: Homo/heteromers of gonadotropin receptors may act as allosteric modulators, act as biased agonist and/or cooperate in sustaining intracellular signals fundamental to support reproduction. Technical limitations lead to in vitro data that require to be confirmed in vivo to figure out the physiological impact of gonadotropin receptor assemblies.
CONCLUSIONS: Gonadotropin receptor homo/heteromers provide a new field of research that deserves attention for possible clinical and therapeutic implications in physiology and pathophysiology.

Keywords:  FSHR; LHCGR; dimers; gametogenesis; heteromers

DOI:  https://doi.org/10.1111/andr.13821
Protein Expr Purif. 2024 Dec 10. pii: S1046-5928(24)00215-8. [Epub ahead of print] 106643

Optimized vector for functional expression of the human bitter taste receptor TAS2R14 in HEK293 cells.

Christine Belloir, Adèle Gautier, Adeline Karolkowski, Thomas Delompré, Mathilde Jeannin, Lucie Moitrier, Fabrice Neiers, Loïc Briand.

  Bitter is one of the five basic taste qualities, along with salty, sour, sweet and umami, used by mammals to access the quality of their food and orient their eating behaviour. Bitter taste detection prevents the ingestion of food potentially contaminated by bitter-tasting toxins. Bitter taste perception is mediated by a family of G protein-coupled receptors (GPCRs) called TAS2Rs. Humans possess 25 TAS2Rs (human type II taste receptors), enabling the detection of thousands of chemically diverse bitter compounds. The identification of agonists/antagonists and molecular mechanisms that govern receptor-ligand interaction has been primarily achieved through functional expression of TAS2Rs in heterologous cells. However, TAS2R receptors, like many other GPCRs, suffer from marginal cell surface expression. In this study, we compared the functionality of 9 engineered chimeric receptors, focusing our experiments on TAS2R14, a broadly tuned receptor that recognizes over 151 identified compounds. Among the different tested signal peptides, rat somatostatin receptor subtype 3 results in higher potency of aristolochic acid-induced calcium signalling than other tested export tags, such as bovine rhodopsin, murine Igκ-chain or human mGluR5. The addition of a MAX sequence enhances both TAS2R14 potency and efficacy. We also confirm that the FLAG epitope, when located at the C-terminal, interferes less with the TAS2R14 functionality, enabling reliable evaluation of this receptor at the cell surface using immunohistochemistry. Finally, these observations are also confirmed for TAS2R14 and TAS1R2/TAS1R3 (the sweet taste receptor) stimulated by 12 bitter compounds and by sucralose and neotame, respectively.

Keywords:  GPCR; TAS2R; bitter; calcium imaging; taste perception; taste receptor

DOI:  https://doi.org/10.1016/j.pep.2024.106643
J Insect Physiol. 2024 Dec 06. pii: S0022-1910(24)00128-8. [Epub ahead of print]161 104740

Corticotropin-releasing factor-like diuretic hormone 44 and five corresponding GPCRs in Drosophila suzukii: Structural and functional characterization.

Hojung Yoon, Briana Price, Ryssa Parks, Hyo Sang Jang, Muhammad Hafeez, Jacob Corcoran, Seung-Joon Ahn, Man-Yeon Choi.

  Diuretic hormones (DHs) activate corresponding G protein-coupled receptors (GPCRs), mediating the water and ion homeostasis in arthropods. There are two different DHs known to be expressed in insects, calcitonin (CT)-like DH31 and corticotropin-releasing factor (CRF)-like DH44. In this study, we identified and characterized a DH44 and five GPCR variants, DH44-R1 and DH44-R2a/b/c/d, in Drosophila suzukii (spotted-wing drosophila), causing detrimental damage to fresh and soft-skinned fruits. Among the five DH44 receptors, DH44-R1 was the longest GPCR and most strongly responded to DH44, and the other DH44-R2 splicing variants were relatively shorter and over 90 % similar to each other. Some DH44-Rs including DH44-R1 utilized both cAMP and Ca2+ as second messengers. Interestingly, DH44-R1 was dominantly expressed in the brain, whereas DH44-R2 variants were dominant in the digestive organs, particularly the Malpighian tubules (MTs) by their gene expressions. The results suggest that DH44 may have multiple physiological functions, including the regulation of the sleep-wake cycle and diuretic activity. Injection of DH44 stimulated fluid secretion in adults, and the rate of the excretion increased in a dose-dependent manner. Moreover, when the flies were injected with a mixture of DH31 and DH44, a high mortality rate was observed. Here, we demonstrate the gene structures, expressions, characterization of DH44 and five GPCRs, their second messengers, and the effects of DH peptides on the fly. These investigations offer molecular insights into the physiological roles of the DH system and may assist in the fundamental aspects of developing D. suzukii management in the field.

Keywords:  Diuretic hormone; Drosophila suzukii; G protein-coupled receptor; Malpighian tubules; Neuropeptide; cAMP

DOI:  https://doi.org/10.1016/j.jinsphys.2024.104740
FEBS J. 2024 Dec 13.

More and more pleiotropy within the IL-6 family of cytokines.

Stefan Rose-John, Simon A Jones.

  Historically, cytokines belonging to the gp130 family bind to specific ligand-binding receptors that stimulate cell signaling through a receptor complex comprising gp130 or gp130 together with another structurally related signaling receptor. However, recent findings increasingly dispel these stereotypes and suggest that the receptor specificity of gp130-activating cytokines is less strict than originally assumed. Weitz et al. now provide the latest example of this pleiotropy and report that human interleukin-6 can bind and stimulate signaling via the interleukin-11 receptor. Possible biological and therapeutic consequences of these findings are discussed.

Keywords:  binding specificity; cytokine receptors; cytokines; gp130; interleukin‐6 family; pleiotropy

DOI:  https://doi.org/10.1111/febs.17355
bioRxiv. 2024 Nov 28. pii: 2024.11.25.625299. [Epub ahead of print]

The G protein inhibitor YM-254890 is an allosteric glue.

Tony Trent, Justin J Miller, Gregory R Bowman.

Given the prominence of G protein coupled receptors (GPCRs) as drug targets, targeting their immediate downstream effectors, G proteins, could be of immense therapeutic value. The discovery that the natural product YM-254890 (YM) can arrest uveal melanoma by specifically inhibiting constitutively active Gq/11without impacting other G protein families demonstrates the potential of this approach. However, efforts to find other G protein family-specific inhibitors have had limited success. Better understanding the mechanism of YM could facilitate efforts to develop other highly specific G protein inhibitors. We hypothesized that differences between the conformational distributions of various G proteins play an important role in determining he specificity of inhibitors like YM. To explore this hypothesis, we built Markov state models (MSMs) from molecular dynamics simulations of the Gα subunits of three different G proteins, as YM predominantly contacts Gα. We also modeled the heterotrimeric versions of these proteins where Gα is bound to the Gβγ heterodimer. We find that YM-sensitive Gα proteins have a higher probability of adopting YM-bound-like conformations than insensitive variants. There is also strong allosteric coupling between the YM- and Gβγ-binding interfaces of Gα. This allostery gives rise to positive cooperativity, wherein the presence of Gβγ enhances preorganization for YM binding. We predict that YM acts as an "allosteric" glue that allosterically stabilizes the complex between Gα and Gβγ despite the minimal contacts between YM and Gβγ.

DOI: https://doi.org/10.1101/2024.11.25.625299
Proc Natl Acad Sci U S A. 2024 Dec 17. 121(51): e2403304121

Molecular basis of lipid and ligand regulation of prostaglandin receptor DP2.

Jiuyin Xu, Youwei Xu, Li Hou, Xinheng He, Yang Li, Jing Zhao, Xue Meng, James Jiqi Wang, Yanli Wu, Heng Zhang, Yunhai Li, Wen Hu, Qingning Yuan, Kai Wu, Xi Cheng, Yi Jiang, Yu Xia, H Eric Xu, Canrong Wu.

  Prostaglandin D2 receptor 2 (DP2) is an important anti-inflammatory and antiallergic drug target. While inactive DP2 structures are known, its activation mechanisms and biased signaling remain unclear. Here, we report cryo-EM structures of an apo DP2-Gi complex, a DP2-Gi complex bound to the endogenous ligand Prostaglandin D2 (PGD2), and a DP2-Gi complex bound to indomethacin, an arrestin-biased ligand, at resolutions of 2.5 Å, 2.8Å, and 2.3 Å, respectively. These structures reveal a distinct binding pose of PGD2 and indomethacin and provide key insights into receptor activation and transducer coupling. Combining the structural data with functional studies, we uncover the molecular basis for biased signaling of indomethacin toward β-arrestin over G proteins. Notably, a phospholipid binding site was identified at the DP2-G protein interface that modulates DP2-G protein interactions. Together, our functional and structural findings provide insights into DP2 activation, biased signaling, drug interactions, and lipid regulation, enabling rational design of safer antiallergy therapeutics targeting this key immune receptor.

Keywords:  DP2; lipid regulation; prostaglandin receptor; receptor activation; signal bias

DOI:  https://doi.org/10.1073/pnas.2403304121
Life Sci Alliance. 2025 Mar;pii: e202403122. [Epub ahead of print]8(3):

Novel determinants of NOTCH1 trafficking and signaling in breast epithelial cells.

Francis M Kobia, Luis Castro E Almeida, Alyssa Jj Paganoni, Francesca Carminati, Adrian Andronache, Francesco Lavezzari, Mark Wade, Thomas Vaccari.

The evolutionarily conserved Notch signaling pathway controls cell-cell communication, enacting cell fate decisions during development and tissue homeostasis. Its dysregulation is associated with a wide range of diseases, including congenital disorders and cancers. Signaling outputs depend on maturation of Notch receptors and trafficking to the plasma membrane, endocytic uptake and sorting, lysosomal and proteasomal degradation, and ligand-dependent and independent proteolytic cleavages. We devised assays to follow quantitatively the trafficking and signaling of endogenous human NOTCH1 receptor in breast epithelial cells in culture. Based on such analyses, we executed a high-content screen of 2,749 human genes to identify new regulators of Notch that might be amenable to pharmacologic intervention. We uncovered 39 new NOTCH1 modulators for NOTCH1 trafficking and signaling. Among them, we find that PTPN23 and HCN2 act as positive NOTCH1 regulators by promoting endocytic trafficking and NOTCH1 maturation in the Golgi apparatus, respectively, whereas SGK3 serves as a negative regulator that can be modulated by pharmacologic inhibition. Our findings might be relevant in the search of new strategies to counteract pathologic Notch signaling.

DOI: https://doi.org/10.26508/lsa.202403122
ACS Chem Neurosci. 2024 Dec 11.

Structure-Activity Relationships and Molecular Pharmacology of Positive Allosteric Modulators of the Mu-Opioid Receptor.

Mengchu Li, Xinmin Gan, Kun Liu, Rajeswaran Walajapet, M Alex Stanczyk, Hannah C Stewart, Jason C Rech, Andrew D White, John R Traynor.

  Positive allosteric modulation of the mu-opioid receptor is a promising strategy to address the ever-growing problem of acute and chronic pain management. Positive allosteric modulators (PAMs) of the mu-opioid receptor could be employed to enhance the efficacy of endogenous opioid peptides to a degree that provides pain relief without the need for traditional opioid drugs. Alternatively, PAMs might be used to enhance the action of opioid drugs and so provide an opioid-sparing effect, allowing for the use of lower doses of opioid agonists and potentially decreasing associated side effects. BMS-986122 (2-(3-bromo-4-methoxyphenyl)-3-[(4-chlorophenyl)-sulfonyl]-thiazolidine) has been previously identified as a PAM of the mu-opioid receptor. In the present work, we have designed and synthesized 33 analogs of BMS-986122 to explore the structure-activity relationships of this scaffold and confirm its allosteric mechanism of action. Among several newly identified modulators, the most promising compound (14b) had improved activity to increase the in vitro potency of the standard mu-opioid agonist DAMGO and showed in vivo activity in mice to enhance the antinociceptive action of morphine.

Keywords:  BMS-986122; G protein; analgesia; mouse; mu-opioid receptor; positive allosteric modulation; structure−activity relationships; thiazolidines; β-arrestin

DOI:  https://doi.org/10.1021/acschemneuro.4c00541
PeerJ. 2024 ;12 e18671

Taurine stimulation of planarian motility: a role for the dopamine receptor pathway.

Elisa J Livengood, Robyn A M V Fong, Angela M Pratt, Veronika O Alinskas, Grace Van Gorder, Michael Mezzio, Margaret E Mulligan, Evelyn B Voura.

  Taurine, a normal dietary component that is found in many tissues, is considered important for a number of physiological processes. It is thought to play a particular role in eye development and in the maturation of both the muscular and nervous systems, leading to its suggested use as a therapeutic for Alzheimer's and Parkinson's diseases. Taurine increases metabolism and has also been touted as a weight loss aid. Due to its possible benefits to health and development, taurine is added as a supplement to a wide array of products, including infant formula and energy drinks. Despite its pervasive use as a nutritional additive and implied physiological actions, there is little consensus on how taurine functions. This is likely because, mechanistically, taurine has been demonstrated to affect multiple metabolic pathways. Simple models and straightforward assay systems are required to make headway in understanding this complexity. We chose to begin this work using the planarian because these animals have basic, well-understood muscular and nervous systems and are the subjects of many well-tested assays examining how their physiology is influenced by exposure to various environmental, nutritional, and therapeutic agents. We used a simple behavioral assay, the planarian locomotor velocity test (pLmV), to gain insight into the stimulant properties of taurine. Using this assay, we observed that taurine is a mild stimulant that is not affected by sugars or subject to withdrawal. We also provide evidence that taurine makes use of the dopamine D1 receptor to mediate this stimulant effect. Given the pervasiveness of taurine in many commercial products, our findings using the planarian system provide needed insight into the stimulant properties of taurine that should be considered when adding it to the diet.

Keywords:  Dopamine; Energy drinks; Glucose; Planarian; Planarian locomotor velocity test; Stimulant; Taurine; Withdrawal; sucrose

DOI:  https://doi.org/10.7717/peerj.18671
Protein Sci. 2025 Jan;34(1): e5254

Ion mobility mass spectrometry unveils conformational effects of drug lead EPI-001 on the intrinsically disordered N-terminal domain of the androgen receptor.

Ikhlas M M Ahmed, Adam Rofe, Martyn C Henry, Eric West, Craig Jamieson, Iain J McEwan, Rebecca Beveridge.

  Intrinsically disordered proteins (IDPs) are important drug targets as they are key actors within cell signaling networks. However, the conformational plasticity of IDPs renders them challenging to characterize, which is a bottleneck in developing small molecule drugs that bind to IDPs and modulate their behavior. In relation to this, ion mobility mass spectrometry (IM-MS) is a useful tool to investigate IDPs, as it can reveal their conformational preferences. It can also offer important insights in drug discovery, as it can measure binding stoichiometry and unveil conformational shifts of IDPs exerted by the binding of small drug-like molecules. Herein, we have used IM-MS to investigate the effect of drug lead EPI-001 on the disordered N-terminal domain of the androgen receptor (AR-NTD). Despite structural heterogeneity rendering the NTD a challenging region of the protein to drug, this domain harbors most, if not all, of the transcriptional activity. We quantify the stoichiometry of EPI-001 binding to various constructs corresponding to functional domains of AR-NTD and show that it binds to separate constructs containing transactivation unit (TAU)-1 and TAU-5, respectively, and that 1-2 molecules bind to a larger construct containing both sequences. We also identify a conformational shift upon EPI-001 binding to the TAU-5, and to a much lesser extent with TAU-1 containing constructs. This work provides novel insight on the interactions of EPI-001 with the AR-NTD, and the structural alterations that it exerts, and positions IM-MS as an informative tool that will enhance the tractability of IDPs, potentially leading to better therapies.

Keywords:  EPI‐001; androgen receptor; intrinsically disordered proteins; ion mobility mass spectrometry; native mass spectrometry; prostate cancer; protein–drug interactions

DOI:  https://doi.org/10.1002/pro.5254
Front Mol Neurosci. 2024 ;17 1465023

Toll-like receptors as a missing link in Notch signaling cascade during neurodevelopment.

Mario Stojanovic, Svjetlana Kalanj-Bognar.

  Neurodevelopment encompasses a complex series of molecular events occuring at defined time points distinguishable by the specific genetic readout and active protein machinery. Due to immense intricacy of intertwined molecular pathways, extracting and describing all the components of a single pathway is a demanding task. In other words, there is always a risk of leaving potential transient molecular partners unnoticed while investigating signaling cascades with core functions-and the very neglected ones could be the turning point in understanding the context and regulation of the signaling events. For example, signaling pathways of Notch and Toll-like receptors (TLRs) have been so far unrelated in the vast body of knowledge about neurodevelopment, however evidence from available literature points to their remarkable overlap in influence on identical molecular processes and reveals their potential functional links. Based on data demonstrating Notch and TLR structural engagement and functions during neurodevelopment, along with our description of novel molecular binding models, here we hypothesize that TLR proteins act as likely crucial components in the Notch signaling cascade. We advocate for the hypothesized role of TLRs in Notch signaling by: elaborating components and features of their pathways; reviewing their effects on fates of neural progenitor cells during neurodevelopment; proposing molecular and functional aspects of the hypothesis, along with venues for testing it. Finally, we discuss substantial indications of environmental influence on the proposed Notch-TLR system and its impact on neurodevelopmental outcomes.

Keywords:  behavior pattern; cell–cell communication; neural progenitor cells; neurodevelopment; signaling pathways

DOI:  https://doi.org/10.3389/fnmol.2024.1465023
J Bone Miner Res. 2024 Dec 10. pii: zjae199. [Epub ahead of print]

An activating CaSR variant with biased signaling reveals a critical residue for Gα11 coupling.

Matthew R Benson, Rachael A Wyatt, Michael A Levine, Caroline M Gorvin.

  Autosomal dominant hypocalcemia (ADH) is due to enhanced calcium-dependent signaling caused by heterozygous gain-of-function (GOF) variants in the CASR gene (ADH1) or in the GNA11 gene, encoding Gα11 (ADH2). Both ADH1 and ADH2 are associated with hypocalcemia and normal or inappropriately low levels of circulating PTH. ADH1 patients typically manifest hypercalciuria, while ADH2 is associated with short stature in approximately 42% of cases. We evaluated a 10-year-old boy with hypoparathyroidism and short stature. Biochemical analyses revealed hypocalcemia, hyperphosphatemia and inconsistent hypercalciuria. Genetic analyses revealed a de novo heterozygous p.Leu723Arg variant in CASR. We characterized the expression of recombinant wild type and Leu723Arg CaSR proteins in HEK293 cells and assessed G protein activation in vitro by CaSR using Bioluminescence Resonance Energy Transfer (BRET). Transient expression studies showed the Leu723Arg variant was normally expressed but resulted in a significantly lower EC50 for extracellular calcium activation of G11 but not other G proteins (i.e. Gi, Gq, Gs). The Leu723Arg substitution has a novel GOF phenotype that leads to biased CaSR activation of G11 signaling, suggesting that residue 723 specifies activation of G11 but not other G proteins. Similar studies of a previously described CaSR variant associated with hypoparathyroidism and short stature, Leu616Val, showed no changes in any G protein pathways, indicating it is likely to be a benign variant. Given the preferential activation of G11 by the Leu723Arg CaSR variant, we propose that the patient's short stature shares a similar basis to that in patients with ADH2 due to GOF variants in GNA11.

Keywords:  Calcium homeostasis; Calcium-sensing receptor; G protein coupling; Hypoparathyroidism; Short stature

DOI:  https://doi.org/10.1093/jbmr/zjae199
Dev Biol. 2024 Dec 07. pii: S0012-1606(24)00273-2. [Epub ahead of print]

Decoding a Cell's Fate: How Notch and Receptor Tyrosine Kinase Signals Specify the Drosophila R7 Photoreceptor.

Ronald A Arias, Andrew Tomlinson.

  The process by which the Drosophila R7 photoreceptor is specified has become a classic model for understanding how cell-cell signals direct cell fates. In the R7 precursor cell, both the Notch and receptor tyrosine kinase (RTK) signaling pathways are active, and the information they encode directs the specification of the R7 photoreceptor identity. In this process, Notch performs three distinct functions: it both opposes and promotes the actions of the RTK pathway to specify the photoreceptor fate, and it determines the type of photoreceptor that is specified. The RTK pathway drives transcription of phyl - a gene expression necessary for photoreceptor specification. We show that Notch activity induces transcription of the yan gene which encodes a transcriptional repressor of phyl. This defines an antagonism between the two pathways, with RTK promoting and Notch opposing phyl transcription. We previously showed that Notch activity supplies Sevenless to the R7 precursor to allow the RTK pathway hyperactivation required to overcome the Notch repression, and we now identify the regulation of Yan activity as a site of integration of RTK and Notch signaling pathways. Once the cell is specified as a photoreceptor, the third Notch function then prevents seven-up (svp) transcription. The Svp transcription factor directs the R1/6 photoreceptor fate, and the prevention of its expression ensures the default R7 specification.

Keywords:  Drosophila R7 photoreceptor; Notch; RTK; cell fate specification; signal integration

DOI:  https://doi.org/10.1016/j.ydbio.2024.12.001
Neurochem Res. 2024 Dec 09. 50(1): 53

Physiological and Pathological Role of mTOR Signaling in Astrocytes.

Luise Hochmuth, Johannes Hirrlinger.

  The mammalian target of rapamycin (mTOR) signaling pathway is one of the key regulators of cellular energy metabolism. It senses diverse alterations in the extracellular environment such as availability of nutrients and growth factors, and mediates the corresponding intracellular response. In the brain, astrocytes crucially contribute to energy and neurotransmitter metabolism, and numerous other functions. However, the relevance of physiological, astrocytic mTOR signaling in maintaining brain homeostasis and function is not well understood. Pathophysiological mTOR signaling is involved in manifold diseases in the central nervous system and most of the knowledge about astrocytic mTOR signaling has been derived from observations on these disorders. Dysregulation of the mTOR signaling pathway impairs important functions of astrocytes including neurotransmitter uptake and -signaling as well as energy metabolism. Some of these alterations could trigger neuropathological conditions such as epilepsy. This review focuses on how mTOR signaling regulates properties of astrocytes, and how these signaling events might contribute to the physiological function of the brain.

Keywords:  Astrocyte; Glutamate; Mitochondria; mTOR

DOI:  https://doi.org/10.1007/s11064-024-04306-6
bioRxiv. 2024 Nov 28. pii: 2024.11.26.625473. [Epub ahead of print]

The Rho effector ARHGAP18 coordinates a Hippo pathway feedback loop through YAP and Merlin to regulate the cytoskeleton and epithelial cell polarity.

Emma C Murray, Gilian M Hodge, Leighton S Lee, Cameron A R Mitchell, Andrew T Lombardo.

The organization of the cell's cytoskeletal filaments is coordinated through a complex symphony of signaling cascades originating from internal and external cues. Two major actin regulatory pathways are signal transduction through Rho family GTPases and growth and proliferation signaling through the Hippo pathway. These two pathways act to define the actin cytoskeleton, controlling foundational cellular attributes such as morphology and polarity. In this study, we use human epithelial cells to investigate the interplay between the Hippo and Rho Family signaling pathways, which have predominantly been characterized as independent actin regulatory mechanisms. We identify that the RhoA effector, ARHGAP18, forms a complex with the Hippo pathway transcription factor YAP to address a long-standing enigma in the field. Using super resolution STORM microscopy, we characterize the changes in the actin cytoskeleton, on the single filament level, that arise from CRISPR/Cas9 knockout of ARHGAP18. We report that the loss of ARHGAP18 results in alterations of the cell that derive from both aberrant RhoA signaling and inappropriate nuclear localization of YAP. These findings indicate that the Hippo and Rho family GTPase signaling cascades are coordinated in their temporal and spatial control of the actin cytoskeleton.

DOI: https://doi.org/10.1101/2024.11.26.625473
Biochem J. 2024 Dec 18. 481(24): 1949-1965

Ubiquitin E3 ligases in the plant Arg/N-degron pathway.

Keely E A Oldham, Peter D Mabbitt.

  Regulation of protein longevity via the ubiquitin (Ub) - proteasome pathway is fundamental to eukaryotic biology. Ubiquitin E3 ligases (E3s) interact with substrate proteins and provide specificity to the pathway. A small subset of E3s bind to specific exposed N-termini (N-degrons) and promote the ubiquitination of the bound protein. Collectively these E3s, and other N-degron binding proteins, are known as N-recognins. There is considerable functional divergence between fungi, animal, and plant N-recognins. In plants, at least three proteins (PRT1, PRT6, and BIG) participate in the Arg/N-degron pathway. PRT1 has demonstrated E3 ligase activity, whereas PRT6 and BIG are candidate E3s. The Arg/N-degron pathway plays a central role in plant development, germination, and submersion tolerance. The pathway has been manipulated both to improve crop performance and for conditional protein degradation. A more detailed structural and biochemical understanding of the Arg/N-recognins and their substrates is required to fully realise the biotechnological potential of the pathway. This perspective focuses on the structural and molecular details of substrate recognition and ubiquitination in the plant Arg/N-degron pathway. While PRT1 appears to be plant specific, the PRT6 and BIG proteins are similar to UBR1 and UBR4, respectively. Analysis of the cryo-EM structures of Saccharomyces UBR1 suggests that the mode of ubiquitin conjugating enzyme (E2) and substrate recruitment is conserved in PRT6, but regulation of the two N-recognins may be significantly different. The structurally characterised domains from human UBR4 are also likely to be conserved in BIG, however, there are sizeable gaps in our understanding of both proteins.

Keywords:  plant signal transduction; protein turnover; structural biology; ubiquitin signalling

DOI:  https://doi.org/10.1042/BCJ20240132
Elife. 2024 Dec 10. pii: RP94334. [Epub ahead of print]13

Prolonged cell cycle arrest in response to DNA damage in yeast requires the maintenance of DNA damage signaling and the spindle assembly checkpoint.

Felix Y Zhou, David P Waterman, Marissa Ashton, Suhaily Caban-Penix, Gonen Memisoglu, Vinay V Eapen, James E Haber.

  Cells evoke the DNA damage checkpoint (DDC) to inhibit mitosis in the presence of DNA double-strand breaks (DSBs) to allow more time for DNA repair. In budding yeast, a single irreparable DSB is sufficient to activate the DDC and induce cell cycle arrest prior to anaphase for about 12-15 hr, after which cells 'adapt' to the damage by extinguishing the DDC and resuming the cell cycle. While activation of the DNA damage-dependent cell cycle arrest is well understood, how it is maintained remains unclear. To address this, we conditionally depleted key DDC proteins after the DDC was fully activated and monitored changes in the maintenance of cell cycle arrest. Degradation of Ddc2ATRIP, Rad9, Rad24, or Rad53CHK2 results in premature resumption of the cell cycle, indicating that these DDC factors are required both to establish and maintain the arrest. Dun1 is required for the establishment, but not the maintenance, of arrest, whereas Chk1 is required for prolonged maintenance but not for initial establishment of the mitotic arrest. When the cells are challenged with two persistent DSBs, they remain permanently arrested. This permanent arrest is initially dependent on the continuous presence of Ddc2, Rad9, and Rad53; however, after 15 hr these proteins become dispensable. Instead, the continued mitotic arrest is sustained by spindle assembly checkpoint (SAC) proteins Mad1, Mad2, and Bub2 but not by Bub2's binding partner Bfa1. These data suggest that prolonged cell cycle arrest in response to 2 DSBs is achieved by a handoff from the DDC to specific components of the SAC. Furthermore, the establishment and maintenance of DNA damage-induced cell cycle arrest require overlapping but different sets of factors.

Keywords:  DDC; DNA damage checkpoint; DSB; HO endonuclease; S. cerevisiae; S. cerevisiae budding yeast; SAC; auxin-inducible degron; chromosomes; double-strand break; gene expression; spindle assembly 3 checkpoint

DOI:  https://doi.org/10.7554/eLife.94334
Comput Struct Biotechnol J. 2024 Dec;23 1311-1319

Exploring key features of selectivity in somatostatin receptors through molecular dynamics simulations.

C Guccione, S Gervasoni, I Öztürk, A Bosin, P Ruggerone, G Malloci.

  Somatostatin receptors (SSTRs) are widely distributed throughout the human body and play crucial roles in various physiological processes. They are recognized as key targets for both radiotherapy and radiodiagnosis due to their overexpression in several cancer types. However, the discovery and design of selective drugs for each of the five isoforms have been significantly hindered by the lack of complete structural information. In this study, we conducted a systematic computational analysis of all five SSTRs in complex with the endogenous ligand somatostatin to elucidate their structural and dynamic features. We thoroughly characterized each isoform using available experimental structures for SSTR2 and SSTR4, as well as AlphaFold2 models for SSTR1, SSTR3, and SSTR5. By performing multi-copy μs-long molecular dynamics simulations, we examined the differences and similarities in dynamical behavior and somatostatin binding among all SSTRs. Our analysis focused on understanding the opening and closing movements of the extracellular loop 2, which are crucial for ligand binding and recognition. Interestingly, we observed a unique conformation of somatostatin within the binding pocket of SSTR5 in which the loop can partially close, as compared to the other isoforms. Fingerprint analyses provided distinct interaction patterns of somatostatin with all receptors, thus enabling precise guidelines for the discovery and development of more selective somatostatin-based pharmaceuticals tailored for precision medicine therapies.

Keywords:  Molecular dynamics simulations; Precision medicine; Somatostatin receptors; Structure-based drug discovery; Theranostics

DOI:  https://doi.org/10.1016/j.csbj.2024.03.005
Matrix Biol. 2024 Dec 05. pii: S0945-053X(24)00145-8. [Epub ahead of print]

Integrins and Integrin-Driven Secretory Pathways as Multi-dimensional Regulators of Tumor-Associated Macrophage Recruitment and Reprogramming in Tumor Microenvironment.

Nibedita Dalpati, Shubham Kumar Rai, Prerna Sharma, Pranita P Sarangi.

  Integrins, a group of transmembrane receptors, play a crucial role in mediating the interactions between cells and extracellular matrix (ECM) proteins. The intracellular signaling initiated by these cell-matrix interactions in leukocytes mediates many essential cellular processes such as survival, migration, metabolism, and other immunological functions. Macrophages, as phagocytes, participate in both proinflammatory and anti-inflammatory processes, including progression. Numerous reports have shown that the integrin-regulated secretome, comprising cytokines, chemokines, growth factors, proteases, and other bioactive molecules, is a crucial modulator of macrophage functions in tumors, significantly influencing macrophage programming and reprogramming within the tumor microenvironment (TME) in addition to driving their step-by-step entry process into tumor tissue spaces. Importantly, studies have demonstrated a pivotal role for integrin receptor-mediated secretome and associated signaling pathways in functional reprogramming from anti-tumorigenic to pro-tumorigenic phenotype in tumor-associated macrophages (TAMs). In this comprehensive review, we have provided an in-depth analysis of the latest findings of various key pathways, mediators, and signaling cascades associated with integrin-driven polarization of macrophages in tumors. This manuscript will provide an updated understanding of the modulation of inflammatory monocytes/ macrophages and TAMs by integrin-driven secretory pathways in various functions such as migration, differentiation, and their role in tumor progression, angiogenesis, and metastasis.

Keywords:  Integrins; Monocytes/macrophages; Polarization; Secretome; Tissue-specific infiltration; Tumor-Associated Macrophages; and Tumor microenvironment

DOI:  https://doi.org/10.1016/j.matbio.2024.12.003
Andrology. 2024 Dec 12.

Regulation of NHE3 subcellular localization in epididymal principal cells: pH, cyclic adenosine 3,5 monophosphate (cAMP), and adenosine signaling.

Larissa Berloffa Belardin, Kéliane Brochu, Christine Légaré, Sylvie Breton.

   INTRODUCTION: The epididymis creates an optimal acidic luminal environment for sperm maturation and storage. In epididymal principal cells (PCs), proton secretion is activated by the accumulation of the sodium-proton exchanger type 3, NHE3 (SLC9A3), in apical stereocilia. PCs also secrete ATP, which is hydrolyzed into adenosine by ectonucleotidases. Adenosine has opposite effects depending on which purinergic receptors it activates. Activation of ADORA1 (A1) and ADORA3 (A3) receptors decreases intracellular cAMP (cAMP), while activation of ADORA2A (A2A) and ADORA2B (A2B) receptors increases cAMP. In other epithelia, cAMP triggers NHE3 internalization from the apical membrane. Here, we examined the roles of pH, cAMP, and adenosine (via A3, A2A, and A2B receptors) in the subcellular localization of NHE3 in PCs.
METHODS: 3D immunofluorescence confocal microscopy was used to visualize NHE3 in stereocilia or intracellular vesicles. Single confocal microscopy images superimposed with bright-field imaging was used to quantify NHE3 subcellular localization. The lumen of the cauda (Cd) epididymis of C57Bl/6Ncrl mice was perfused in vivo at pH 6.0 and 7.8. The effect of a permeant analog of cAMP (cpt-cAMP) was studied at pH 7.8, while the effect of adenosine was investigated at pH 6.0. Expression of A2A, A2B, and A3 was examined by immunofluorescence, and their respective role was evaluated by using specific agonists and antagonists at different luminal pH. Immunofluorescence for clathrin, an endosomal marker, was examined at pH 7.8 with and without an A2B agonist.
RESULTS: At an acidic pH perfusion solution of 6.0, NHE3 was predominantly localized intracellularly, whereas an alkaline pH of 7.8 promoted its accumulation in apical stereocilia. Perfusion with cpt-cAMP at pH 7.8 reduced the amount of NHE3 in stereocilia. Immunolabeling showed the localization of A3, A2A, and A2B receptors in the apical membrane of epithelial cells in the Cd epididymis. Adenosine and an A3 agonist increased NHE3 stereocilia accumulation at pH 6.0, and the adenosine effect was abolished with an A3 antagonist. An A2A agonist had no effect on NHE3 localization, while an A2B agonist decreased the amount of NHE3 in stereocilia observed at pH 7.8. A concomitant increase in intracellular labeling for clathrin was induced by the A2B agonist at pH 7.8.
CONCLUSIONS: Our study indicates that in the Cd epididymis, NHE3 localization in PCs is modulated by luminal pH, cAMP, and adenosine receptor signaling. Acidic pH promotes NHE3 internalization, while alkaline pH facilitates its accumulation in stereocilia. Activation of A3 by luminal adenosine maintains NHE3 on the cell surface. Conversely, A2B activation by adenosine induces NHE3 internalization. We propose that the distinct effects mediated by these receptors are the consequence of their opposite effect on cAMP signaling. This intricate interplay of pH and adenosine highlights some of the regulatory mechanisms influencing the establishment of an optimal acidic environment for sperm maturation and storage in the epididymis.

Keywords:  NHE3; clathrin; epididymis; epithelial cells; luminal acidification

DOI:  https://doi.org/10.1111/andr.13820
J Mol Biol. 2024 Dec 09. pii: S0022-2836(24)00522-9. [Epub ahead of print] 168892

Regulating IL-2 immune signaling function via a core allosteric structural network.

Claire H Woodward, Shahlo O Solieva, Daniel Hwang, Viviane S De Paula, Charina S Fabilane, Michael C Young, Tony Trent, Ella C Teeley, Ananya Majumdar, Jamie B Spangler, Gregory R Bowman, Nikolaos G Sgourakis.

  Human interleukin-2 (IL-2) is a crucial cytokine for T cell regulation, with therapeutic potential in cancer and autoimmune diseases. However, IL-2's pleiotropic effects across different immune cell types often lead to toxicity and limited efficacy. Previous efforts to enhance IL-2's therapeutic profile have focused on modifying its receptor binding sites. Yet, the underlying dynamics and intramolecular networks contributing to IL-2 receptor recognition remain unexplored. This study presents a detailed characterization of IL-2 dynamics compared to two engineered IL-2 mutants, "superkines" S15 and S1, which exhibit biased signaling towards effector T cells. Using NMR spectroscopy and molecular dynamics simulations, we demonstrate significant variations in core dynamic pathways and conformational exchange rates across these three IL-2 variants. We identify distinct allosteric networks and minor state conformations in the superkines, despite their structural similarity to wild-type IL-2. Furthermore, we rationally design a mutation (L56A) in the S1 superkine's core network, which partially reverts its dynamics, receptor binding affinity, and T cell signaling behavior towards that of wild-type IL-2. Our results reveal that IL-2 superkine core dynamics play a critical role in their enhanced receptor binding and function, suggesting that modulating IL-2 dynamics and core allostery represents an untapped approach for designing immunotherapies with improved immune cell selectivity profiles.

Keywords:  Interleukin-2; T cells; allostery; conformational exchange; molecular dynamics simulations; nuclear magnetic resonance spectroscopy; receptor binding

DOI:  https://doi.org/10.1016/j.jmb.2024.168892
Elife. 2024 Dec 09. pii: RP96841. [Epub ahead of print]13

Endogenous oligomer formation underlies DVL2 condensates and promotes Wnt/β-catenin signaling.

Senem Ntourmas, Martin Sachs, Petra Paclíková, Martina Brückner, Vítězslav Bryja, Jürgen Behrens, Dominic B Bernkopf.

  Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.

Keywords:  DVL2; Wnt signaling; biochemistry; biomolecular condensates; cell biology; chemical biology; dishevelled; human; paralogs

DOI:  https://doi.org/10.7554/eLife.96841
Fish Physiol Biochem. 2025 Feb;51(1): 1-12

Membrane progesterone receptor e (paqr9) is necessary for chorion elevation in zebrafish.

Md Razain Tanvir, Takumi Mouri, Eisei Tsutsumi, Umme Habiba Mustary, Md Almamun Farid, Md Forhad Hossain, Yuki Omori, Chihiro Yamamoto, Akiteru Maeno, Toshinobu Tokumoto.

  Paqr9 is a gene encoding membrane progestin receptor e (mPRe), the fifth subtype of the five mPR subtypes, and is currently classified a member of the progestin and adipoQ receptor (PAQR) family, which consists of 11 genes. To elucidate the physiological functions of the mPR subtypes, we established gene knockout (KO) fish via genome editing of seven paqr genes in zebrafish and analyzed their phenotypes. The null-mutant strain of paqr9 (paqr9-/-) that we established in this study presented reduced chorion elevation and a high percentage of abnormal embryos. Embryos exhibit various kinds of abnormal morphology, which are thought to be caused by insufficient elevation of the chorion. Immunohistochemical staining of ovaries with an anti-Paqr9 antibody revealed that Paqr9 was expressed in the periplasm of oocytes and the surface of chorion in the wild type, whereas signals were absent in paqr9-/- zebrafish. In histological sections, the periplasmic connection between the oocyte plasma membrane and chorion was absent in paqr9-/- oocytes. The number of cortical alveoli (CA) that are responsible for chorion elevation was significantly reduced in paqr9-/- zebrafish. SEM revealed that fiber-supported knob-like structures (KSs) on the chorion were absent in paqr9-/- zebrafish. These results indicate that Paqr9 is required for the preparation of CA during oogenesis. Insufficient formation of the chorion resulted in the abnormal development of embryos.

Keywords:   paqr9 ; Progesterone receptor; Zebrafish

DOI:  https://doi.org/10.1007/s10695-024-01435-1
bioRxiv. 2024 Dec 01. pii: 2024.11.26.625421. [Epub ahead of print]

LNS8801: An enantiomerically pure agonist of the G protein-coupled estrogen receptor suitable for clinical development.

Christopher A Natale, Sophia Mercado, Richard Zhuang, Cristina Aguirre-Portolés, Israel Olayide, Christopher K Arnatt, John T Seykora, Tina K Garyantes, Wayne Luke, Todd W Ridky.

Estrogen effects in tissue are mediated in part through activation of the surface estrogen receptor GPER, a broadly expressed G protein-coupled receptor that impacts a wide range of normal and pathologic processes, including metabolism, vascular health, inflammation, and cancer. A commonly used synthetic and specific GPER agonist, named G-1, antagonizes tumors by promoting cellular differentiation and enhancing tumor immunogenicity. G-1 is a racemic compound, and since its discovery, the question of whether both enantiomers display agonist activity or the agonist activity resides primarily in a single enantiomer has never been fully resolved. Herein, we disclose the isolation of the pure enantiomers of G-1 and determine that the desirable activity resides exclusively in 1 enantiomer, named LNS8801, whose configuration we have unambiguously determined by single crystal x-ray structure analysis. Using preclinical models, we show that LNS8801 suppresses cancer in a GPER-dependent manner and that LNS8801 is efficacious when administered orally. Further, we show that GPER is widely, but not ubiquitously, expressed in both normal and malignant human tissues. In addition, an attenuated response to LNS8801 is observed in a common germline coding variant in human GPER. These findings support ongoing human cancer trials with LNS8801 and suggest that the germline GPER genotype may serve as a predictive biomarker of therapeutic response.

DOI: https://doi.org/10.1101/2024.11.26.625421
Heliyon. 2024 Dec 15. 10(23): e40416

Characteristics of Gracilariopsis lemaneiformis hydrocolloids and their effects on intestine PPAR signaling and liver lipid metabolism in Oreochromis niloticus: A multiomics analysis.

Jia-Wei Shen, Po-Kai Pan, Yin-Yu Chen, Fan-Hua Nan, Yu-Sheng Wu.

  This study evaluated the effects of Gracilariopsis lemaneiformis hydrocolloids on Nile tilapia (Oreochromis niloticus) using an advanced multiomics approach (transcriptome and proteome) linked with genomic isoform structure to elucidate the biofunctions of G. lemaneiformis hydrocolloids. The results showed that G. lemaneiformis hydrocolloids did not affect growth, as indicated by the nonsignificant differences in growth and blood biochemical indicators. Regarding the response, both intestine and liver tissues were assessed. These findings indicate that 20 % G. lemaneiformis hydrocolloids enhanced cytokine expression, which may contribute to a biological function in the intestine and liver of O. niloticus. Genome and proteome profiles indicated that G. lemaneiformis hydrocolloids upregulated the intestine and liver peroxisome proliferator-activated receptor (PPAR) signaling pathway, nucleocytoplasmic transport, steroid biosynthesis, and histidine metabolism. In contrast, co-factor biosynthesis, nucleocytoplasmic transport, tryptophan metabolism, arginine and proline metabolism, arginine biosynthesis, and ribosome activity were downregulated. These findings indicate that G. lemaneiformis hydrocolloids significantly affect liver lipid and carbohydrate metabolism. Proteomics analysis revealed that G. lemaneiformis hydrocolloids upregulated the PPAR signaling pathway, playing a crucial role in lipid metabolism. In summary, 20 % G. lemaneiformis hydrocolloids are primarily involved in modulating the intestine and liver PPAR signaling pathway to regulate lipid metabolism.

Keywords:  Genomic isoform structure; Hydrocolloid; Lipid metabolism; Marine algae; Multiomics

DOI:  https://doi.org/10.1016/j.heliyon.2024.e40416
Insect Sci. 2024 Dec 11.

Juvenile hormone induces phosphorylation of insulin/insulin-like growth factor signaling proteins in previtellogenic Aedes aegypti mosquitoes.

Wenhao Zhao, Pengcheng Liu, Thomas R Saunders, Jinsong Zhu.

  Juvenile hormone (JH) plays a pivotal role in regulating post-emergence development and metabolism in previtellogenic female Aedes aegypti mosquitoes. In contrast, yolk protein precursor production and egg maturation after a blood meal are regulated by the steroid hormone 20-hydroxyecdysone, the insulin-like growth factor (IGF)/insulin signaling (IIS) pathway, and the mammalian target of rapamycin (mTOR) pathway. The role of IIS/mTOR signaling in female adults prior to blood feeding has not been thoroughly investigated. In this study, we identified a significant increase in the phosphorylation of key effector proteins in the IIS/mTOR signaling pathway, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), ribosomal protein S6 kinase (S6K) and forkhead box protein O1 (FoxO1), in previtellogenic females. In vitro fat body culture experiments suggest that JH induces these phosphorylations through rapid nongenomic signaling mediated by the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mTOR network. RNA interference experiments demonstrated that activation of IIS/mTOR signaling in previtellogenic females modulate metabolic gene expression, promoting the accumulation of energy reserves (glycogen and triglycerides), which influence mosquito fecundity. Additionally, depletion of either the insulin receptor (InR) or the JH receptor Methoprene-tolerant (Met) in adult mosquitoes abolished the phosphorylation of these proteins, indicating that both receptors are involved in JH-induced membrane-initiated signal transduction. Although the precise mechanisms remain unclear, this study uncovers a novel function of the IIS/mTOR pathway in adult mosquitoes before blood feeding, as well as a new mode of JH action through its crosstalk with the IIS pathway.

Keywords:  RNAi; insulin signaling; juvenile hormone; mosquito; phosphorylation; reproduction

DOI:  https://doi.org/10.1111/1744-7917.13482
J Hazard Mater. 2024 Dec 07. pii: S0304-3894(24)03399-5. [Epub ahead of print]485 136818

Multiomics unraveled that gibberellin signaling underlies adaptation of rice to ciprofloxacin stress: Calling for concerns on the adverse effects of pharmaceutical residues in water during agricultural irrigations.

Xin Qi, Rui Zhao, Xiaona Zhang, Shaoguo Ru, Jiu-Qiang Xiong.

  Residual concentrations of antibiotics in water can reach ng mL-1 - µg mL-1 levels, which pose high risks to crops during irrigation; however, the interactions between rice and antibiotics, as well as the defense mechanisms of rice at their early growth phase remain unclear. In this study, we investigated the uptake dynamics of a ubiquitously found antibiotic, ciprofloxacin (CIP) at 0.1, 1, 6.5, and 20 µg mL-1 in rice seedlings. We found gradually bioaccumulated CIP induced significant physiological changes including inhibited growth of roots and leaves of rice seedlings, and decreased pigment contents, which can be caused by disrupted homeostasis of reactive oxygen species. Integrating roots transcriptomics, metabolomics, and validation experiments, we found that rice seedlings synthesized more gibberellins to trigger the expression of transcription factors such as group VII ethylene response factors, which induced metabolic reprogramming to yield more fatty acids derivates. These compounds including eicosanoids, isoprenoids, and fatty acids and conjugates can act as signaling molecules, as well as antioxidants and energy sources to achieve rice recovery. This conclusion is supported by the evidence showing that adding gibberellins in rice seedlings culture decreased the accumulated CIP and improved rice growth; whilst, disrupting gibberellin signaling pathway using paclobutrazol as an inhibitor increased uptaken CIP in both roots and leaves with augmenting the antibiotic stress on rice. This study has demonstrated a gibberellin-based defense mechanism in rice for defense of CIP stress, which might have significant environmental applications since we can add minor gibberellins to reduce bioaccumulated CIP with simultaneously promoting rice growth at their early phases.

Keywords:  Antibiotics; Ciprofloxacin; Gibberellins; Integrated transcriptomics and metabolomics; Metabolic reprogramming; Oryza sativa

DOI:  https://doi.org/10.1016/j.jhazmat.2024.136818
J Virol. 2024 Dec 10. e0180124

Third intracellular loop of HCMV US28 is necessary for signaling and viral reactivation.

Samuel Medica, Michael Denton, Nicole L Diggins, Olivia Kramer-Hansen, Lindsey B Crawford, Adam T Mayo, Wilma D Perez, Michael A Daily, Christopher J Parkins, Luke E Slind, Lydia J Pung, Whitney C Weber, Hannah K Jaeger, Zachary J Streblow, Gauthami Sulgey, Craig N Kreklywich, Timothy Alexander, Mette M Rosenkilde, Patrizia Caposio, Meaghan H Hancock, Daniel N Streblow.

  The human cytomegalovirus (HCMV) encoded chemokine receptor US28 plays a critical role in viral pathogenesis, mediating several processes such as cellular migration, differentiation, transformation, and viral latency and reactivation. Despite significant research examining the signal transduction pathways utilized by US28, the precise mechanism by which US28 activates these pathways remains unclear. We performed a mutational analysis of US28 to identify signaling domains that are critical for functional activities. Our results indicate that specific residues within the third intracellular loop (ICL3) of US28 are major determinants of G-protein coupling and downstream signaling activity. Alanine substitutions at positions S218, K223, and R225 attenuated US28-mediated activation of MAPK and RhoA signal transduction pathways. Furthermore, we show that mutations at positions S218, K223, or R225 result in impaired coupling to multiple Gα isoforms. However, these substitutions did not affect US28 plasma membrane localization or the receptor internalization rate. Utilizing CD34+ HPC models, we demonstrate that attenuation of US28 signaling via mutation of residues within the ICL3 region results in an inability of the virus to efficiently reactivate from latency. These results were recapitulated in vivo, utilizing a humanized mouse model of HCMV infection. Together, our results provide new insights into the mechanism by which US28 manipulates host signaling networks to mediate viral latency and reactivation. The results reported here will guide the development of targeted therapies to prevent HCMV-associated disease.IMPORTANCEHuman cytomegalovirus (HCMV) is a β-herpesvirus that infects between 44% and 100% of the world population. Primary infection is typically asymptomatic and results in the establishment of latent infection within CD34+hematopoietic progenitor cells (HPCs). However, reactivation from latent infection remains a significant cause of morbidity and mortality in immunocompromised individuals. The viral chemokine receptor US28 influences various cellular processes crucial for viral latency and reactivation, yet the precise mechanism by which US28 functions remains unclear. Through mutational analysis, we identified key residues within the third intracellular loop (ICL3) of US28 that govern G-protein coupling, downstream signaling, and viral reactivation in vitro and in vivo. These findings offer novel insights into how US28 manipulates host signaling networks to regulate HCMV latency and reactivation and expand our understanding of HCMV pathogenesis.

Keywords:  G-protein-coupled receptor; cytomegalovirus; latency; reactivation; signal transduction

DOI:  https://doi.org/10.1128/jvi.01801-24
Anal Chim Acta. 2025 Jan 15. pii: S0003-2670(24)01246-7. [Epub ahead of print]1335 343445

Comprehensive host cell proteins profiling in biopharmaceuticals by a sensitivity enhanced mass spectrometry strategy using TMT-labeling and signal boosting.

Andong Xue, Linlin Kong, Jialin Li, Yuxin Jiao, Zhishang Hu, Bin Fu, Guibin Wang, Wanjun Zhang, Jianheng Li, Weijie Qin.

   BACKGROUND: Host Cell Proteins (HCPs) are impurities expressed in host cells during the biopharmaceutical production process, whichmay compromise product quality and potentially leading to immunogenic reactions or other adverse effects. Mass spectrometry (MS)-based strategy is more and more considered as a promising method for HCPs analysis, since it is capable of simultaneously quantifying thousands of proteins in a single test. However, considering the large excess biopharmaceutical product protein present in the system and the extremely low abundance of HCPs, sensitive MS methods are urgently needed in HCPs analysis.
RESULTS: In this work, we introduced a novel approach that leveraged host cell lysate as a boosting channel to enhance the MS signal of the residue HCPs in biopharmaceutical products using isobaric TMT labeling, thereby elevating the low-abundant HCPs to detectable and quantifiable levels of current MS without using enrichment or depletion method to avoid disturbance of the original concentration of the HCPs. Our method surpassed previous benchmarks by identifying a significantly higher number (23844 unique peptides for 3475 proteins) compared to existing records (4541 unique peptides for 848 proteins) for HCPs analysis in RM8671 NIST monoclonal antibody (mAb), demonstrating unparalleled sensitivity and robustness. Furthermore, our workflow successfully identified 44 of 48 UPS1 proteins across a concentration range of 0.32-4.15 ppm in monoclonal antibodies (mAbs), proving its effectiveness for in-depth HCPs analysis in biopharmaceuticals.
SIGNIFICANCE: Present even at sub-ppm levels, HCPs may compromise the stability and safety of product proteins and alter pharmacokinetics or neutralization of therapeutic effects. Our MS signal enhancing method presented an advancement in HCP analysis, combining improved sensitivity and increased scale of HCPs with a streamlined and robust workflow. This method allowed HCPs quantification at <1 ppm level without disturbance of the original HCPs concentration, which is still rare in the field.

Keywords:  Biopharmaceuticals; Enrichment; Host cell proteins; Mass spectrometry; Proteomics

DOI:  https://doi.org/10.1016/j.aca.2024.343445
J Virol. 2024 Dec 12. e0169124

Equine lentivirus Gag protein degrades mitochondrial antiviral signaling protein via the E3 ubiquitin ligase Smurf1.

Kewei Chen, Bingqian Zhou, Xinhui Wang, Guangpu Yang, Yuezhi Lin, Xuefeng Wang, Cheng Du, Xiaojun Wang.

  Equine infectious anemia virus (EIAV) and HIV-1 are both members of the Lentivirus genus and are similar in virological characters. EIAV is of great concern in the equine industry. Lentiviruses establish a complex interaction with the host cell to counteract the antiviral responses. There are various pattern recognition receptors in the host, for instance, the cytosolic RNA helicases interact with viral RNA to activate the mitochondrial antiviral signaling protein (MAVS) and subsequent interferon (IFN) response. However, viruses also exploit multiple strategies to resist host immunity by targeting MAVS, but the mechanism by which lentiviruses are able to target MAVS has remained unclear. In this study, we found that EIAV infection induced MAVS degradation, and that EIAV Gag protein recruited the E3 ubiquitin ligase Smurf1 to polyubiquitinate and degrade MAVS. The CARD domain of MAVS and the WW domain of Smurf1 are responsible for the interaction with Gag. EIAV Gag is a precursor polyprotein of the membrane-interacting matrix p15, the capsid p26, and the RNA-binding nucleocapsid proteins p11 and p9. Therefore, we analyzed which protein domain of Gag could interact with MAVS and Smurf1. We found that p15 and p26, but not p11 or p9, target MAVS for degradation. Moreover, we identified the key amino acid residues that support the interactions between p15 or p26 and MAVS or Smurf1. The present study describes a novel role of the EIAV structural protein Gag in targeting MAVS to counteract innate immunity, and reveals the mechanism by which the equine lentivirus can antagonize against MAVS.IMPORTANCEHost anti-RNA virus innate immunity relies mainly on the recognition by retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), and subsequently initiates downstream signaling through interaction with mitochondrial antiviral signaling protein (MAVS). However, viruses have developed various strategies to counteract MAVS-mediated signaling, although the method of antagonism of MAVS by lentiviruses is still unknown. In this article, we demonstrate that the precursor (Pr55gag) polyprotein of EIAV and its protein domains p15 and p26 target MAVS for ubiquitin-mediated degradation through E3 ubiquitin ligase Smurf1. MAVS degradation leads to the inhibition of the downstream IFN-β pathway. This is the first time that lentiviral structural protein has been found to have antagonistic effects on MAVS pathway. Overall, our study reveals a novel mechanism by which equine lentiviruses can evade host innate immunity, and provides insight into potential therapeutic strategies for the control of lentivirus infection.

Keywords:  EIAV; HIV; MAVS; RLR; lentivirus

DOI:  https://doi.org/10.1128/jvi.01691-24