Biology subjects

Lind, S.

Publications and source records attributed to Lind, S..

12 recordsLinked to original sources

bioRxiv2025

Free Fatty Acid Receptor 2 Allosterism is Defined By Cellular Context

BackgroundAllosteric modulators offer a way to fine-tune GPCR signaling in the presence of endogenous ligands. The short-chain fatty acid receptor FFA2R (GPR43) recognizes propionate and allosteric ligands such as Cmp58 and AZ1729. We characterized FFA2R signaling and allosteric modulation using multiple cell models including HEK293, HL60 cells and primary human neutrophils. MethodsFFA2R activation was assessed using complementary assays in HEK293 and HL60 cells as well as primary human neutrophils. G protein activation and {beta}-arrestin recruitment were profiled using ebBRET biosensors. Ca2+ mobilization was measured with Fura-2, and reactive oxygen species (ROS) generation was quantified by isoluminol chemiluminescence. Pharmacological tools included the FFA2R antagonist CATPB, the Gq inhibitor YM-254890, and pertussis toxin (PTX). ResultsPropionate activated all tested G proteins except G12 in HEK293 cells and recruited both {beta}-arrestin1 and {beta}-arrestin2. The allosteric ligands Cmp58 and AZ1729 behaved as pathway-selective ago-PAMs. Alone they engaged a limited subset of G proteins with minimal {beta}-arrestin recruitment, whereas in the presence of propionate they selectively potentiated Gi1 while attenuating Gq/11 and Gi2/3. Fura-2 measurements coupled to YM-254890 treatment established that FFA2R couples to Gq-dependent Ca2+ mobilization in HEK293 cells; Cmp58, but not AZ1729, enhanced Ca2+ responses at submaximal propionate concentrations. In primary neutrophils, propionate elicited Ca2+ transients but did not trigger NADPH oxidase-dependent ROS on its own. Either Cmp58 or AZ1729 enabled propionate-driven ROS, and their combination produced robust ROS. Transient FFA2R expression in HL60 cells reconstituted this neutrophil-like functional profile, including allosteric activation of ROS. ConclusionsAcross systems, Ca2+ mobilization emerged as a conserved, receptor-proximal output of FFA2R, while allosteric modulation by Cmp58 and AZ1729 promoted Gi/o-biased signaling that enabled ROS generation. These data define pathway-selective allosterism at FFA2R and highlight Ca2+ mobilization and ROS as informative readouts for therapeutic strategies that exploit allosteric control.

cell biology↗

bioRxiv2025

Profiling of HCAR1 signaling reveals Gαi/o and Gαs activation without β-arrestin recruitment and the discovery of an allosteric agonist

Lactate was long considered a byproduct of glycolysis and associated with various harmful effects. However, the role of lactate was expanded with the finding that it also can act as a signaling molecule through the G protein-coupled receptor Hydroxycarboxylic Acid Receptor 1 (HCAR1). The receptor was shown to be primarily expressed in adipocytes but is also expressed in many other tissues and cell types. Activation of HCAR1 can help regulate lipolysis and improve insulin sensitivity, making it a promising target for managing obesity and other metabolic disorders. While HCAR1 activation offers therapeutic benefits for metabolic diseases, it can also promote cancer cell survival and metastasis, necessitating a nuanced approach to avoid unintended tumor growth. However, only a few ligands have been reported for HCAR1, and their signaling pathways remain unexplored. Using enhanced bystander bioluminescence resonance energy transfer (ebBRET) to study G protein activation and {beta}-arrestin recruitment following ligand addition, we were able to identify compounds such as AZ7136, a potent HCAR1 agonist, AZ2114 a partial agonist, and establish GPR81 agonist 1 as an ago-positive allosteric modulator. We also show that HCAR1 preferentially activates the Gi/o and Gs pathways without recruiting {beta}-arrestins. These findings enhance our understanding of the signaling profile of HCAR1 and the newly characterized ligands could be used as molecular tools to understand more about HCAR1 in metabolic disease. One Sentence SummaryThis study used the ebBRET platform to identify and characterize several synthetic ligands for the lactate receptor HCAR1, significantly advancing our understanding of HCAR1 signaling.

pharmacology and toxicology↗

bioRxiv2022

The ketone body acetoacetate activates human neutrophils through FFA2R

Neutrophils express many surface receptors that sense environmental changes. One such sensor is FFA2R (free fatty acid receptor 2), a receptor that detects gut microbiota-derived short chain fatty acids. As such, FFA2R has been regarded as a molecular link between metabolism and inflammation. Our recent studies on FFA2R, using its endogenous agonist propionate in combination with allosteric modulators, have identified several novel aspects of FFA2R regulation. A recent study has also identified the ketone body acetoacetate as an endogenous ligand for mouse FFA2R. Whether human FFA2R also recognizes acetoacetate and how this recognition modulates human neutrophil functions has not been earlier investigated. In this study, we found that acetoacetate can induce a decrease of cAMP and translocation of {beta}-arrestin in cells overexpressing FFAR2. In addition, we show that similar to propionate, FFA2R specific allosteric modulators enhance acetoacetate-induced transient rise in cytosolic calcium, production of reactive oxygen species and cell migration in human neutrophils. In summary, we demonstrate that human neutrophils recognize the ketone body acetoacetate through FFA2R. Thus, our data further highlight the key role of FFA2R in inflammation and metabolism.

cell biology↗

bioRxiv2022

THE ALLOSTERICALLY MODULATED FREE FATTY ACID RECEPTOR 2 IS TRANSACTIVATED BY AN INCREASE IN THE CYTOSOLIC CONCENTRATION OF CALCIUM IONS

Allosterically modulated free fatty acid receptor 2 (FFA2R/GPR43) can be activated without the involvement of any orthosteric FFA2R agonist, by signals generated for example by P2Y2R, the G protein coupled receptor for ATP. An FFA2R specific positive allosteric modulator (PAM; Cmp58) was used to disclose the molecular mechanism by which signals generated by ATP/P2Y2R transactivates FFA2R. The P2Y2R induced signal that transactivates the allosterically modulated FFA2R was generated downstream of the Gq containing G protein that couple to P2Y2R. A receptor induced rise in the cytosolic concentration of ionized calcium ([Ca2+]i) was hypothesized to be the receptor transactivation signal. The Gq dependent transient rise in [Ca2+]i induced by the ATP activated P2Y2Rs was not affected by Cmp58. The hypothesis gained, however, support from the finding that the modulator transferred FFA2R to a Ca2+ sensitive state. The rise in [Ca2+]i induced by the Ca2+ specific ionophore ionomycin, activated the allosterically modulated FFA2R. The response induced by ionomycin was rapidly terminated and the FFA2Rs could then no longer be activated by the orthosteric FFA2R agonist propionate or be transactivated by the signal generated by the activated ATP receptor. The desensitized/non-responding state of FFA2R was, however, revoked by an earlier described cross-sensitizing/activating allosteric FFA2R modulator. The receptor transactivation of the allosterically modulated FFA2Rs, represent a unique regulatory receptor cross-talk mechanism by which the activity of a G protein coupled receptor is controlled by a signaling system operating from the cytosolic side of the plasma membrane. One-sentence summaryA P2Y2 receptor signal generated downstream of a Gq containing G protein transactivates the allosterically modulated FFA2 receptor

cell biology↗

bioRxiv2021

Structural determinants in the Staphylococcus aureus derived phenol-soluble modulin-α2 peptide required for neutrophil formyl peptide receptor activation

Highly pathogenic Staphylococcus aureus strains produce phenol-soluble modulins (PSMs), peptides which are formylated N-terminally. Nanomolar concentrations of PSM2 are recognized by formyl peptide receptor 2 (FPR2), but unlike the prototypic FPR2 agonist WKYMVM, PSM2 is a biased signaling agonist. A shortened N-terminal PSM2 variant, consisting of the five N- terminal residues, is selectively recognized by the closely related FPR1, showing that the C- terminal part of PSM2 confers FPR2 selectivity, while the N-terminal part may interact with the FPR1 binding site. In the present study, a combined pharmacological and genetic approach, involving primary neutrophils and engineered FPR "knock-in" and "knock-out" cells, was used to gain molecular insights into FPR1 and FPR2 recognition of formyl peptides and the receptor downstream signaling induced by these peptides. In comparison to the full-length PSM2, we show that the peptide in which the N-terminal part of PSM2 was replaced by fMIFL (an FPR1- selective peptide agonist) potently activates both FPRs for production of superoxide anions and {beta}- arrestin recruitment. A shortened analogue of PSM2 (PSM21-12), lacking the nine C-terminal residues activated both FPR1 and FPR2 to produce ROS, whereas {beta}-arrestin recruitment was only mediated through FPR1. However, a single amino acid replacement (Gly-2 to Ile-2) in PSM21-12 was sufficient to alter FPR2 signaling to include {beta}-arrestin recruitment, highlighting a key role of Gly-2 in conferring FPR2 biased signaling. In conclusion, we provide novel structural insights into FPR1 and FPR2 recognition as well as the signaling induced by interaction with formyl peptides derived from PSM2, originating from Staphylococcus aureus bacteria.

immunology↗

bioRxiv2021

Allosteric Receptor Modulation uncovers an FFAR2 antagonist as a positive orthosteric modulator/agonist in disguise

Two earlier described Free Fatty Acid Receptor 2 (FFAR2)-specific antagonists (CATPB and GLPG0974) have different receptor-interaction characteristics at the molecular/functional level. The inhibitory effect of the two antagonists, on the novel receptor-cross-talk activation signals generated by the ATP-receptor, show that both antagonists inhibit the effect of the positive allosteric FFAR2 modulators (PAMs) AZ1729 and Cmp58. No neutrophil activation was induced by AZ1729 or Cmp58 alone, but together they were co-agonistic PAMs and activated the superoxide generating NADPH-oxidase in neutrophils. This response was inhibited by CATPB but not by GLPG0974; in contrast, GLPG0974 acted as a positive modulator that increased the potency but not the efficacy of the response. At the signaling level, GLPG0974 changed the biased signaling induced by the co-agonistic PAMs, to include a rise in the cytosolic concentration of free calcium ions (Ca2+). This effect was reciprocal, i.e., GLPG0974 triggers a rise in intracellular Ca2+, demonstrating that GLPG0974 may act as an FFAR2 agonist. In summary, by studying the effects of the FFAR2 ligand GLPG0974 on neutrophils activation induced by the co-agonists AZ1729 and Cmp58, we reveal that GLPG0974 in addition to be an antagonist, displays also agonistic and positive FFAR2 modulating functions that affects the NADPH-oxidase activity and the receptor down-stream signaling induced by the two co-agonistic PAMs.

pharmacology and toxicology↗

bioRxiv2020

Multiple ligand recognition sites in free fatty acid receptor 2 (FFAR2) direct distinct neutrophil activation patterns

Non-activating positive allosteric modulators specific for free fatty acid receptor 2 (FFAR2) increased the activity induced by orthosteric agonists to trigger a rise in intracellular Ca2+ ([Ca2+]i) and activate the O2- producing neutrophil NADPH-oxidase. In addition, two allosteric modulators (Cmp58 and AZ1729) recognized by different receptor domains on FFAR2, cooperatively triggered activation without any rise in [Ca2+]i. To gain insights into FFAR2 modulation and signaling, we set out to identify structurally diverse allosteric FFAR2 modulators. Initially, we identified two molecules that directly activate neutrophils and these were classified as an allosteric FFAR2 agonists and an orthosteric agonist, respectively. Based on the sensitizing effect on the neutrophil response to propionate, ten non-direct-activating molecules were classified as allosteric FFAR2 modulators. One of these synergistically activated neutrophils when combined with AZ1729, but not when combined with Cmp58. The remaining nine compounds synergistically induced the same type of biased neutrophil signaling but only when combined with Cmp58. The activation signals down-stream of FFAR2 when stimulated by two allosteric modulators with different binding sites were in most cases biased in that two complementary modulators together triggered an activation of the NADPH-oxidase, but no increase in [Ca2+]i. The neutrophil activation pattern achieved when two functionally "AZ1729- or "Cmp58-like" allosteric FFAR2 modulators were combined, supporting a model for activation in which FFAR2 has two different sites that selectively bind allosteric modulators. The novel neutrophil activation patterns and receptor down-stream signaling mediated by two cross-sensitizing allosteric modulators represent a new regulatory mechanism that controls FFAR2 receptor function.

cell biology↗

bioRxiv2020

Functional selective FPR1 signaling in favor of an activation of the neutrophil superoxide generating NOX2-complex

Two formyl peptide receptors (FPR1 and FPR2), abundantly expressed by neutrophils, regulate both pro-inflammatory tissue recruitment of neutrophils and resolution of inflammatory reactions. This dual functionality of the FPRs, opens for a possibility to develop receptor selective therapeutics as mechanism for novel anti-inflammatory treatments. In line with this, high throughput screening studies have identified numerous FPR ligands belonging to different structural classes, but a potent FPR1 agonist with defined biased signaling and functional selectivity has not yet been reported. In this study, we used an FPR1 selective small compound agonist (RE) that represents a chemical entity developed from NOX2 activators identified from our earlier screening studies (WO2012127214). This FPR1 agonist potently activates neutrophils to produce reactive oxygen species (ROS, EC50 ~1 nM), whereas it is a weaker chemoattractant than the prototype FPR1 agonist fMLF. At the signaling level, RE has a strong bias towards the PLC-PIP2-Ca2+ pathway and ERK1/2 activation but away from {beta}-arrestin recruitment and the ability to recruit neutrophils chemotactically. In addition, FPR1 when activated by RE could cross-regulate other receptor-mediated neutrophil functions. In comparison to the peptide agonist fMLF, RE is more resistant to oxidization-induced inactivation by the MPO-H2O2-halide system. In summary, this study describes as a novel FPR1 agonist displaying a biased signaling and functional selectivity when activating FPR1 in human blood neutrophils. RE could possibly be a useful tool compound not only for further mechanistic studies of the regulatory role of FPR1 in inflammation in vitro and in vivo, but also for developing FPR1specific drug therapeutics.

cell biology↗

bioRxiv2019

Interdependent allosteric FFA2R modulators synergistically induce functional selective activation and desensitization in neutrophils

The non-activating allosteric modulator AZ1729, specific for free fatty acid receptor 2 (FFA2R), transfers the orthosteric FFA2R agonists propionate and the P2Y2R specific agonist ATP into activating ligands that trigger an assembly of the neutrophil superoxide generating NADPH-oxidase. The homologous priming effect on the propionate response and the heterologous receptor cross-talk sensitized ATP response mediated by AZ1729 are functional characteristics shared with Cmp58, another non-activating allosteric FFA2R modulator. In addition, AZ1729 also turned Cmp58 into a potent activator of the superoxide generating neutrophil NADPH-oxidase, and in agreement with the allosteric modulation concept, the effect was reciprocal in that Cmp58 turned AZ1729 into a potent activating allosteric agonist. The activation signals down-stream of FFA2R when stimulated by the two interdependent allosteric modulators were biased in that, unlike for orthosteric agonists, the two complementary modulators together triggered an activation of the NADPH-oxidase, but not any transient rise in the cytosolic concentration of free Ca2+. In addition, following AZ1729/Cmp58 activation, signaling by the desensitized FFA2Rs was functionally selective in that the orthosteric agonist propionate could still induce a transient rise in intracellular Ca2+. The novel neutrophil activation and receptor down-stream signaling pattern mediated by the two cross-sensitizing allosteric FFA2R modulators represents a new regulatory mechanism that controls receptor signaling. Significance StatementA novel activation mechanism of a G-protein coupled free fatty acid receptor (FFA2R), is synergistically triggered by two otherwise non-activating allosteric modulators in the absence of orthosteric agonists. The receptor down-stream signaling proved to be functionally selective (biased); a superoxide generating enzyme is assembled and activated without involvement of the Ca2+ signaling pathway. The novel activation mechanism and the receptor down-stream signaling pattern mediated by the two cross-sensitizing allosteric FFA2R modulators, represents a new regulatory mechanism for control of GPCR-signaling.

pharmacology and toxicology↗

bioRxiv2019

The putative Gαq-inhibiting pepducin P4Pal10 distinctly modulates function of the Gαi-coupled receptors FPR2 and FFA2R in neutrophils

A novel mechanism of action was described, when a protease-activated receptor 4 (P4Pal10) derived lipopeptide (pepducin) was shown to inhibit signaling downstream of several unrelated Gq-coupled receptors. We show that this putative Gq-inhibiting pepducin lacks inhibitory effects on signaling downstream of the Gq-coupled receptors for ATP (P2Y2R) and PAF (PAFR) expressed in human neutrophils. P4Pal10 inhibited however, signaling in neutrophils activated with agonists for the Gi-coupled formyl peptide receptor 2 (FPR2) but not the closely related FPR1. In addition, the P4Pal10 pepducin was turned into an activating agonist in the presence of an allosteric modulator selective for free fatty acid receptor 2 (FFA2R). The results presented thus reveal Gq-independent effects of P4Pal10 in modulating FPR2- and FFA2R-mediated neutrophil activation.

immunology↗

bioRxiv2019

Functional and signaling characterization of the neutrophil FPR2 selective agonist Act-389949

Despite the steadily increased numbers of formyl peptide receptor (FPR) ligands identified over the years, few have been characterized in studies using animal disease models and even less have entered clinical trials in human subjects. A small-molecule compound, Act-389949, was however recently tested in a phase I clinical trial and found to be safe and well tolerated in healthy human subjects. The desired anti-inflammatory property of Act-389949 was proposed to be mediated through FPR2, one of the FPRs expressed in neutrophils, but no basic characterization was included in the study. To gain more insights into FPR2 recognition of this first-in-class compound for future utility of the agonist, we have in this study determined the receptor preference and down-stream signaling characteristics induced by Act-389949 in human blood neutrophils isolated from healthy donors. Our data demonstrate that Act-389949 is an agonist for FPR2 that triggers functional/signaling repertoires comparable to what has been earlier described for other FPR2 agonists, including neutrophil chemotaxis, granule mobilization and activation of the NADPH-oxidase. In fact, Act-389949 was found to be as potent as the prototype FPR2 peptide agonist WKYMVM and had the advantage of being resistant to oxidation by the MPO-H2O2-halide derived oxidants, as compared to the sensitive WKYMVM. The down-stream signals generated by Act-389949 include an FPR2-dependent and Gq-independent transient rise in intracellular Ca2+ and recruitment of {beta}-arrestin. In summary, our data show that Act-389949 serves as an excellent tool-compound for further dissection of FPR2-regulated activities in vitro and in vivo. Potent and stable FPR ligands such as Act-389949 may therefore be used to develop the next generation of FPR signaling regulating anti-inflammatory therapeutics.

pharmacology and toxicology↗

bioRxiv2018

Discordant expression profile between RNA and protein for the genes involved in immune response network in adenovirus type 2 infected cells

Alternation of cellular genes expressions during Adenovirus type 2 (Ad2) infection in IMR-90 cells was studied using paired-end sequencing and stable isotope labeling of amino acids in cell culture mass spectrometric analysis (SILAC-MS). At transcriptional level, cellular genes involved in different pathways revealed distinct expression profiles. At early phase, the genes involved in regulation of cellular immune response, cellular signaling and cell growth control were among the most deregulated. Later follows, in an orderly fashion, genes involved in cell cycle control, DNA replication and further on genes engaged in RNA processing and protein translation. Comparison of cellular gene expression at transcriptional and posttranscriptional levels revealed low correlation. Here we highlight the genes which expose opposite expression profiles with an emphasis on key factors that play important roles in cellular immune pathways including NF{kappa}B, JAK/STAT, caspases and MAVS. Transcription of many of these genes was transiently induced early, but became down-regulated in the late phase. In contrast, their expressions at protein level were up-regulated early and so sustained until late phase of infection. Suppression at the transcriptional level and enhancement at the protein level of immune response genes most likely illustrate counteractions between Ad2 and its host cell.\n\nImportanceOur paper comprises a state of the art quality transcriptomics data set unravelling the alterations in gene expression that take place during different phases of an adenovirus infection. The information allows us to draw conclusion about the cellular pathways that are perturbed by the virus. The data set also provides an important resource for scientists in general for future studies on mechanisms behind host/virus interactions in efforts to design tools for combatting virus infections.\n\nMoreover, our paper includes novel proteomics information unravelling an unexpected role of post transcriptional events in cellular gene expression, demonstrating that the current picture of the adenovirus replication cycle is simplified.

microbiology↗