1.
Biased allosteric modulation of formyl peptide receptor 2 leads to distinct receptor conformational states for pro- and anti-inflammatory signaling.
Zhang, S, Gong, H, Ge, Y, Ye, RD
Pharmacological research. 2020;:105117
Abstract
BACKGROUND AND PURPOSE Formyl peptide receptor 2 (FPR2) is a Class A G protein-coupled receptor (GPCR) that interacts with multiple ligands and transduces both proinflammatory and anti-inflammatory signals. These ligands include weak agonists and modulators that are produced during inflammation. The present study investigates how prolonged exposure to FPR2 modulators influence receptor signaling. EXPERIMENTAL APPROACH Fluorescent biosensors of FPR2 were constructed based on single-molecule fluorescent resonance energy transfer (FRET) and used for measurement of ligand-induced receptor conformational changes. These changes were combined with FPR2-mediated signaling events and used as parameters for the conformational states of FPR2. Ternary complex models were developed to interpret ligand concentration-dependent changes in FPR2 conformational states. KEY RESULTS Incubation with Ac2-26, an anti-inflammatory ligand of FPR2, decreased FRET intensity at picomolar concentrations. In comparison, WKYMVm (W-pep) and Aβ42, both proinflammatory agonists of FPR2, increased FRET intensity. Preincubation with Ac2-26 at 10 pM diminished W-pep-induced Ca2+ flux but potentiated W-pep-stimulated β-arrestin2 membrane translocation and p38 MAPK phosphorylation. The opposite effects were observed with 10 pM of Aβ42. Neither Ac2-26 nor Aβ42 competed for W-pep binding at the picomolar concentrations. CONCLUSIONS AND IMPLICATIONS The results support the presence of two allosteric binding sites on FPR2, each for Ac2-26 and Aβ42, with high and low affinities. Sequential binding of the two allosteric ligands at increasing concentrations induce different conformational changes in FPR2, providing a novel mechanism by which biased allosteric modulators alter receptor conformations and generate pro- and anti-inflammatory signals.
2.
Elucidating the Role of Hydroxylated Phenylalanine in the Formation and Structure of Cross-Linked Aβ Oligomers.
Zhang, S, Fox, DM, Urbanc, B
The journal of physical chemistry. B. 2019;(5):1068-1084
Abstract
Amyloid β-protein (Aβ) oligomers play a seminal role in Alzheimer's disease (AD). Cross-linking (X-linking), which can be used to determine Aβ oligomer size distributions experimentally, was reported to stabilize Aβ oligomers. Aβ oligomers X-linked in the presence of copper and hydrogen peroxide may represent the proximate neurotoxic species in AD. Our previous computational study demonstrated that X-linking of Aβ40 and Aβ42 oligomers via tyrosines alone cannot explain experimental findings. Here, we explore three plausible X-linking mechanisms, which involve, in addition to tyrosine, also lysine (mechanism 1), histidine (mechanism 2), and hydroxylated phenylalanine (mechanism 3). By examining the effect of X-linking on oligomer size distributions, we show that only mechanism 3 is consistent with experimental data. Our findings provide important insights into the two-step X-linking via mechanism 3, which consists of a simple covalent bonding via tyrosines in the presence of hydroxylated phenylalanines, followed by covalent bonding among tyrosines and hydroxylated phenylalanines. Structural analysis of X-linked Aβ oligomers revealed increased solvent exposure at the N-terminal region, which was previously associated with increased oligomer toxicity. Our results elucidate a potentially important role of phenylalanine hydroxylation and increased toxicity of Aβ oligomers induced by X-linking.
3.
Insights into Formation and Structure of Aβ Oligomers Cross-Linked via Tyrosines.
Zhang, S, Fox, DM, Urbanc, B
The journal of physical chemistry. B. 2017;(22):5523-5535
Abstract
Alzheimer's disease (AD) pathology is hypothesized to be triggered by amyloid β-protein (Aβ) assembly into oligomers. Oligomer size distributions of both predominant Aβ alloforms, Aβ40 and Aβ42, can be determined in vitro using cross-linking followed by gel electrophoresis. Cross-linking, which can occur in vivo in the presence of copper and hydrogen peroxide, was recently shown to stabilize Aβ oligomers by inhibiting their conversion into fibrils. Whereas several studies showed that cross-linking is facilitated by dityrosine bond formation, the molecular-level mechanism of cross-linking remains unclear. Here, we use efficient discrete molecular dynamics with DMD4B-HYDRA force field to examine the effect of cross-linking via tyrosines on Aβ oligomer formation. Our results show that cross-linking via tyrosines promotes Aβ self-assembly, in particular that of Aβ40, but does not account for cross-linked oligomers larger than Aβ40 trimers and Aβ42 tetramers. Cross-linking via tyrosines profoundly alters Aβ40 and Aβ42 oligomer conformations by increasing the solvent exposure of hydrophobic residues, resulting in elongated oligomeric morphologies that differ from globular structures of noncross-linked oligomers. When compared to available experimental data, our findings imply that amino acids other than tyrosines are involved in Aβ cross-linking, a proposition that is currently under investigation.