Biology subjects

Holdfeldt, A.

Publications and source records attributed to Holdfeldt, A..

6 recordsLinked to original sources

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↗

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

Barbadin selectively modulates FPR2-mediated neutrophil functions independent of receptor endocytosis

Formyl peptide receptor 2 (FPR2), a member of the family of G protein-coupled receptors (GPCRs), mediates neutrophil migration, a response that has been linked to {beta}-arrestin recruitment. {beta}-Arrestin regulates GPCR endocytosis and can also elicit non-canonical receptor signaling. To determine the poorly understood role of {beta}-arrestin in FPR2 endocytosis and in NADPH-oxidase activation in neutrophils, Barbadin was used as a research tool in this study. Barbadin has been shown to bind the clathrin adaptor protein (AP2) and thereby prevent {beta}- arrestin/AP2 interaction and {beta}-arrestin-mediated GPCR endocytosis. In agreement with this, AP2/{beta}-arrestin interaction induced by an FPR2-specific agonist was inhibited by Barbadin. Unexpectedly, however, Barbadin did not inhibit FPR2 endocytosis, indicating that a mechanism independent of {beta}-arrestin/AP2 interaction may sustain FPR2 endocytosis. This was confirmed by the fact, that FPR2 also underwent agonist-promoted endocytosis in {beta}-arrestin deficient cells, albeit at a diminished level as compared to wild type cells. Dissection of the Barbadin effects on FPR2-mediated neutrophil functions including NADPH-oxidase activation mediated release of reactive oxygen species (ROS) and chemotaxis reveled that Barbadin had no effect on chemotactic migration whereas the release of ROS was potentiated/primed. The effect of Barbadin on ROS production was reversible, independent of {beta}-arrestin recruitment, and similar to that induced by latrunculin A. Taken together, our data demonstrate that endocytic uptake of FPR2 occurs independently of {beta}-arrestin, while Barbadin selectively augments FPR2-mediated neutrophil ROS production independently of receptor endocytosis. Given that Barbadin binds to AP2 and prevents the AP2/{beta}-arrestin interaction, our results indicate a role for AP2 in FPR2-mediated ROS release from human neutrophils.

pharmacology and toxicology↗

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↗

bioRxiv2018

Allosteric receptor modulation of FFA2R turns natural agonists into potent activators of the superoxide generating neutrophil NADPH-oxidase

Acetate, agonist for the free fatty acid receptor 2 (FFA2R/GPR43), triggers an increase in the cytosolic concentration of free Ca2+ in neutrophils without any assembly of the superoxide generating NADPH-oxidase. We show that the phenylacetamide compound 58 (Cmp58; (S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide, lacking a direct activating effect on neutrophils, acts as a positive allosteric FFA2R modulator that turns acetate into a potent activating agonist that triggers an assembly of the NADPH-oxidase. The NADPH-oxidase activity could be further increased in neutrophils treated with the pro-inflammatory cytokine TNF. Many neutrophil chemoattractant receptors are stored in secretory organelles but no FFA2R mobilization was induced in neutrophils treated with TNF. The receptor selectivity was demonstrated through the inhibition of the neutrophil response induced by the combined action of acetate and Cmp58 by the FFA2R antagonist CATPB. Allosteric modulators that positively co-operate with natural FFA2R agonists and prime neutrophils in their response to such agonists, may serve as good tools for further unraveling the physiological functions of the FFA2R and its involvement in various diseases. In this study, allosteric modulation of FFA2R is introduced as a novel receptor selective mechanism to prime neutrophils to produce increased amounts of reactive oxygen species.

cell biology↗