Distinct roles of the extracellular surface residues of glucagon-like peptide-1 receptor in ß-arrestin 1/2 signaling.

Glucagon-like peptide-1 receptor (GLP-1R) is a prime drug target for type 2 diabetes and obesity. The ligand initiated GLP-1R interaction with G protein has been well studied, but not with ß-arrestin 1/2. Therefore, bioluminescence resonance energy transfer (BRET), mutagenesis and an operational model were used to evaluate the roles of 85 extracellular surface residues on GLP-1R in ß-arrestin 1/2 recruitment triggered by three representative GLP-1R agonists (GLP-1, exendin-4 and oxyntomodulin). Residues selectively regulated ß-arrestin 1/2 recruitment for diverse ligands, and ß-arrestin isoforms were identified. Mutation of residues K130-S136, L142 and Y145 on the transmembrane helix 1 (TM1)-extracellular domain (ECD) linker decreased ß-arrestin 1 recruitment but increased ß-arrestin 2 recruitment. Other extracellular loop (ECL) mutations, including P137A, Q211A, D222A and M303A selectively affected ß-arrestin 1 recruitment while D215A, L217A, Q221A, S223A, Y289A, S301A, F381A and I382A involved more in ß-arrestin 2 recruitment for the ligands. Oxyntomodulin engaged more broadly with GLP-1R extracellular surface to drive ß-arrestin 1/2 recruitment than GLP-1 and exendin-4; I147, W214 and L218 involved in ß-arrestin 1 recruitment, while L141, D215, L218, D293 and F381 in ß-arrestin 2 recruitment for oxyntomodulin particularly. Additionally, the non-conserved residues on ß-arrestin 1/2 C-domains contributed to interaction with GLP-1R. Further proteomic profiling of GLP-1R stably expressed cell line upon ligand stimulation with or without ß-arrestin 1/2 overexpression demonstrated both commonly and biasedly regulated proteins and pathways associated with cognate ligands and ß-arrestins. Our study offers valuable information about ligand induced ß-arrestin recruitment mediated by GLP-1R and consequent intracellular signaling events.

Highly Luminescent Chiral Double π-Helical Nanoribbons.

Unveiling the mechanism behind chirality propagation and dissymmetry amplification at the molecular level is of significance for the development of chiral systems with comprehensively outstanding chiroptical performances. Herein, we have presented a straightforward Cu-mediated Ullmann homocoupling approach to synthesize perylene diimide-entwined double π-helical nanoribbons encompassing dimer, trimer, and tetramer while producing homochiral or heterochiral linking of chiral centers. A significant dissymmetry amplification was achieved, with absorption dissymmetry factors (|gabs|) increasing from 0.009 to 0.017 and further to 0.019, and luminescence dissymmetry factors (|glum|) rising from 0.007 to 0.013 and eventually to 0.015 for homochiral double π-helical oligomers. The disparity of magnetic transition dipole moment (m) densities in homochiral and heterochiral tetramers by time-dependent density functional theory calculations confirmed that homochiral oligomerization can maximize the total m, which is favorable for achieving ever-increasing g factors. Notably, these double π-helices exhibited exceptional photoluminescence quantum yields (ΦPL) ranging from 83 to 95%. The circularly polarized luminescence brightness (BCPL) eventually reached a remarkable 575 M-1 cm-1 for the homochiral tetramer, which is among the highest values reported for chiral small molecules. This kind of linearly extended double π-helices offers a platform for a comprehensive understanding of the mechanism behind chirality propagation and dissymmetry amplification.

Single-cell transcriptomics reveals the role of antigen presentation in liver metastatic breast cancer.

Liver metastasis (LM) is the primary cause of cancer-related mortality in late-stage breast cancer (BC) patients. Here we report an in-depth analysis of the transcriptional landscape of LM of 11 patients with secondary hepatic carcinoma at single-cell resolution. Our study reveals that terminally exhausted CD4+ and dysfunctional CD8+ T cells were enriched in LM along with low antigen presentation. We also found that macrophages were associated with the tumor infiltrating CD4+ T cells, while FCN3+ macrophages, type 1 conventional dendritic cells (cDC1) and LAMP3+ DC regulated T cell functions, probably via antigen processing and presentation. Major histocompatibility complex expression in FCN3+ macrophage, cDC1 and LAMP3+ DC was reduced in LM compared to those in normal tissues and primary BC. Malfunctioned antigen presentation in these cells is linked to a worse prognosis in invasive BC and hepatocellular carcinoma. Our results provide valuable insights into the role of tumor infiltrating T cells in LM.

Molecular features of the ligand-free GLP-1R, GCGR and GIPR in complex with Gs proteins.

Class B1 G protein-coupled receptors (GPCRs) are important regulators of many physiological functions such as glucose homeostasis, which is mainly mediated by three peptide hormones, i.e., glucagon-like peptide-1 (GLP-1), glucagon (GCG), and glucose-dependent insulinotropic polypeptide (GIP). They trigger a cascade of signaling events leading to the formation of an active agonist-receptor-G protein complex. However, intracellular signal transducers can also activate the receptor independent of extracellular stimuli, suggesting an intrinsic role of G proteins in this process. Here, we report cryo-electron microscopy structures of the human GLP-1 receptor (GLP-1R), GCG receptor (GCGR), and GIP receptor (GIPR) in complex with Gs proteins without the presence of cognate ligands. These ligand-free complexes share a similar intracellular architecture to those bound by endogenous peptides, in which, the Gs protein alone directly opens the intracellular binding cavity and rewires the extracellular orthosteric pocket to stabilize the receptor in a state unseen before. While the peptide-binding site is partially occupied by the inward folded transmembrane helix 6 (TM6)-extracellular loop 3 (ECL3) juncture of GIPR or a segment of GCGR ECL2, the extracellular portion of GLP-1R adopts a conformation close to the active state. Our findings offer valuable insights into the distinct activation mechanisms of these three important receptors. It is possible that in the absence of a ligand, the intracellular half of transmembrane domain is mobilized with the help of Gs protein, which in turn rearranges the extracellular half to form a transitional conformation, facilitating the entry of the peptide N-terminus.

CCDC92 promotes podocyte injury by regulating PA28α/ABCA1/cholesterol efflux axis in type 2 diabetic mice.

Podocyte lipotoxicity mediated by impaired cellular cholesterol efflux plays a crucial role in the development of diabetic kidney disease (DKD), and the identification of potential therapeutic targets that regulate podocyte cholesterol homeostasis has clinical significance. Coiled-coil domain containing 92 (CCDC92) is a novel molecule related to metabolic disorders and insulin resistance. However, whether the expression level of CCDC92 is changed in kidney parenchymal cells and the role of CCDC92 in podocytes remain unclear. In this study, we found that Ccdc92 was significantly induced in glomeruli from type 2 diabetic mice, especially in podocytes. Importantly, upregulation of Ccdc92 in glomeruli was positively correlated with an increased urine albumin-to-creatinine ratio (UACR) and podocyte loss. Functionally, podocyte-specific deletion of Ccdc92 attenuated proteinuria, glomerular expansion and podocyte injury in mice with DKD. We further demonstrated that Ccdc92 contributed to lipid accumulation by inhibiting cholesterol efflux, finally promoting podocyte injury. Mechanistically, Ccdc92 promoted the degradation of ABCA1 by regulating PA28α-mediated proteasome activity and then reduced cholesterol efflux. Thus, our studies indicate that Ccdc92 contributes to podocyte injury by regulating the PA28α/ABCA1/cholesterol efflux axis in DKD.

Structure of GPR101-Gs enables identification of ligands with rejuvenating potential.

GPR101 is an orphan G protein-coupled receptor actively participating in energy homeostasis. Here we report the cryo-electron microscopy structure of GPR101 constitutively coupled to Gs heterotrimer, which reveals unique features of GPR101, including the interaction of extracellular loop 2 within the 7TM bundle, a hydrophobic chain packing-mediated activation mechanism and the structural basis of disease-related mutants. Importantly, a side pocket is identified in GPR101 that facilitates in silico screening to identify four small-molecule agonists, including AA-14. The structure of AA-14-GPR101-Gs provides direct evidence of the AA-14 binding at the side pocket. Functionally, AA-14 partially restores the functions of GH/IGF-1 axis and exhibits several rejuvenating effects in wild-type mice, which are abrogated in Gpr101-deficient mice. In summary, we provide a structural basis for the constitutive activity of GPR101. The structure-facilitated identification of GPR101 agonists and functional analysis suggest that targeting this orphan receptor has rejuvenating potential.

Molecular basis of signal transduction mediated by the human GIPR splice variants.

Glucose-dependent insulinotropic polypeptide receptor (GIPR) is a potential drug target for metabolic disorders. It works with glucagon-like peptide-1 receptor and glucagon receptor in humans to maintain glucose homeostasis. Unlike the other two receptors, GIPR has at least 13 reported splice variants (SVs), more than half of which have sequence variations at either C or N terminus. To explore their roles in endogenous peptide-mediated GIPR signaling, we determined the cryoelectron microscopy (cryo-EM) structures of the two N terminus-altered SVs (referred as GIPR-202 and GIPR-209 in the Ensembl database, SV1 and SV2 here, respectively) and investigated the outcome of coexpressing each of them in question with GIPR in HEK293T cells with respect to ligand binding, receptor expression, cAMP (adenosine 3,5-cyclic monophosphate) accumulation, ß-arrestin recruitment, and cell surface localization. It was found that while both N terminus-altered SVs of GIPR neither bound to the hormone nor elicited signal transduction per se, they suppressed ligand binding and cAMP accumulation of GIPR. Meanwhile, SV1 reduced GIPR-mediated ß-arrestin 2 responses. The cryo-EM structures of SV1 and SV2 showed that they reorganized the extracellular halves of transmembrane helices 1, 6, and 7 and extracellular loops 2 and 3 to adopt a ligand-binding pocket-occupied conformation, thereby losing binding ability to the peptide. The results suggest a form of signal bias that is constitutive and ligand-independent, thus expanding our knowledge of biased signaling beyond pharmacological manipulation (i.e., ligand specific) as well as constitutive and ligand-independent (e.g., SV1 of the growth hormone-releasing hormone receptor).

Molecular Carbons: How Far Can We Go?

The creation and development of carbon nanomaterials promoted material science significantly. Bottom-up synthesis has emerged as an efficient strategy to synthesize atomically precise carbon nanomaterials, namely, molecular carbons, with various sizes and topologies. Different from the properties of the feasibly obtained mixture of carbon nanomaterials, numerous properties of single-component molecular carbons have been discovered owing to their well-defined structures as well as potential applications in various fields. This Perspective introduces recent advances in molecular carbons derived from fullerene, graphene, carbon nanotube, carbyne, graphyne, and Schwarzite carbon acquired with different synthesis strategies. By selecting a variety of representative examples, we elaborate on the relationship between molecular carbons and carbon nanomaterials. We hope these multiple points of view presented may facilitate further advancement in this field.

Structure-Based Ligand Discovery Targeting the Transmembrane Domain of Frizzled Receptor FZD7.

Despite the essential roles of Frizzled receptors (FZDs) in mediating Wnt signaling in embryonic development and tissue homeostasis, ligands targeting FZDs are rare. A few antibodies and peptide modulators have been developed that mainly bind to the family-conserved extracellular cysteine-rich domain of FZDs, while the canonical binding sites in the transmembrane domain (TMD) are far from sufficiently addressed. Based on the recent structures of FZDs, we explored small-molecule ligand discovery by targeting TMD. From the ChemDiv library with ∼1.6 million compounds, we identified compound F7H as an antagonist of FZD7 with an IC50 at 1.25 ± 0.38 µM. Focusing on this hit, the structural dissection study, together with computing studies such as molecular docking, molecular dynamics simulation, and free energy perturbation calculations, defined the binding pocket with key residue recognition. Our results revealed the structural basis of ligand recognition and demonstrated the feasibility of structure-guided ligand discovery for FZD7-TMD.

Validation of the Efficacy of Ultrasound Speckle Tracking in Measuring Tendon Gliding After Finger Flexor Tendon Repair.

OBJECTIVE: Restricted tendon gliding is commonly observed in patients after finger flexor tendon (FFT) repair. The study described here was aimed at quantifying the amount of FFT gliding to evaluate the recovery of post-operative tendons using a 2-D radiofrequency (RF)-based ultrasound speckle tracking algorithm (UST). METHODS: Ex vivo uniaxial tensile testing of porcine flexor tendons and in vivo isometric testing of human FFT were implemented to verify the efficacy of UST beforehand. The verified UST was then applied to the patients after FFT repair to compare tendon gliding between affected and healthy sides and to investigate its correlation with the joint range of motion (ROM). RESULTS: Excellent validity was confirmed with the average R2 value of 0.98, mean absolute error of 0.15 ± 0.08 mm and mean absolute percentage error of 5.19 ± 2.43% between results from UST and ex vivo testing. The test-retest reliability was verified with good agreement of ICC (0.90). The affected side exhibited less gliding (p = 0.001) and smaller active ROM (p = 0.002) than the healthy side. Meanwhile, a significant correlation between tendon gliding and passive ROM was found only on the healthy side (ρ = 0.711, p = 0.009). CONCLUSION: The present study provides a promising protocol to evaluate post-operative tendon recovery by quantifying the amount of FFT gliding with a validated UST. FFT gliding in patients with different levels of ROM restriction should be further explored for categorizing the severity of tendon adhesion.

GPCR activation and GRK2 assembly by a biased intracellular agonist.

Phosphorylation of G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) desensitizes G-protein signalling and promotes arrestin signalling, which is also modulated by biased ligands1-6. The molecular assembly of GRKs on GPCRs and the basis of GRK-mediated biased signalling remain largely unknown owing to the weak GPCR-GRK interactions. Here we report the complex structure of neurotensin receptor 1 (NTSR1) bound to GRK2, Gαq and the arrestin-biased ligand SBI-5537. The density map reveals the arrangement of the intact GRK2 with the receptor, with the N-terminal helix of GRK2 docking into the open cytoplasmic pocket formed by the outward movement of the receptor transmembrane helix 6, analogous to the binding of the G protein to the receptor. SBI-553 binds at the interface between GRK2 and NTSR1 to enhance GRK2 binding. The binding mode of SBI-553 is compatible with arrestin binding but clashes with the binding of Gαq protein, thus providing a mechanism for its arrestin-biased signalling capability. In sum, our structure provides a rational model for understanding the details of GPCR-GRK interactions and GRK2-mediated biased signalling.

Structural analysis of the dual agonism at GLP-1R and GCGR.

Glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR), two members of class B1 G protein-coupled receptors, play important roles in glucose homeostasis and energy metabolism. They share a high degree of sequence homology but have different functionalities. Unimolecular dual agonists of both receptors developed recently displayed better clinical efficacies than that of monotherapy. To study the underlying molecular mechanisms, we determined high-resolution cryo-electron microscopy structures of GLP-1R or GCGR in complex with heterotrimeric Gs protein and three GLP-1R/GCGR dual agonists including peptide 15, MEDI0382 (cotadutide) and SAR425899 with variable activating profiles at GLP-1R versus GCGR. Compared with related structures reported previously and supported by our published pharmacological data, key residues responsible for ligand recognition and dual agonism were identified. Analyses of peptide conformational features revealed a difference in side chain orientations within the first three residues, indicating that distinct engagements in the deep binding pocket are required to achieve receptor selectivity. The middle region recognizes extracellular loop 1 (ECL1), ECL2, and the top of transmembrane helix 1 (TM1) resulting in specific conformational changes of both ligand and receptor, especially the dual agonists reshaped ECL1 conformation of GLP-1R relative to that of GCGR, suggesting an important role of ECL1 interaction in executing dual agonism. Structural investigation of lipid modification showed a better interaction between lipid moiety of MEDI0382 and TM1-TM2 cleft, in line with its increased potency at GCGR than SAR425899. Together, the results provide insightful information for the design and development of improved therapeutics targeting these two receptors simultaneously.

Structural insights into the interaction of three Y-shaped ligands with PI3Kα.

Class IA phosphoinositide 3-kinase alpha (PI3Kα) is an important drug target because it is one of the most frequently mutated proteins in human cancers. However, small molecule inhibitors currently on the market or under development have safety concerns due to a lack of selectivity. Therefore, other chemical scaffolds or unique mechanisms of catalytic kinase inhibition are needed. Here, we report the cryo-electron microscopy structures of wild-type PI3Kα, the dimer of p110α and p85α, in complex with three Y-shaped ligands [cpd16 (compound 16), cpd17 (compound 17), and cpd18 (compound 18)] of different affinities and no inhibitory effect on the kinase activity. Unlike ATP-competitive inhibitors, cpd17 adopts a Y-shaped conformation with one arm inserted into a binding pocket formed by R770 and W780 and the other arm lodged in the ATP-binding pocket at an angle that is different from that of the ATP phosphate tail. Such a special interaction induces a conformation of PI3Kα resembling that of the unliganded protein. These observations were confirmed with two isomers (cpd16 and cpd18). Further analysis of these Y-shaped ligands revealed the structural basis of differential binding affinities caused by stereo- or regiochemical modifications. Our results may offer a different direction toward the design of therapeutic agents against PI3Kα.

GPCRana: A web server for quantitative analysis of GPCR structures.

G protein-coupled receptors (GPCRs) attract tremendous attention from both industrial and academic researchers with currently over 900 released structures. Structural analysis is widely used to understand receptor functionality and pharmacology, but more user-friendly tools are needed. Residue-residue contact score (RRCS) is an atomic distance-based method that allows a quantitative description of GPCR structures. Here, we present GPCRana, a web server that provides a user-friendly interface to analyze GPCR structures. After uploading selected structures, GPCRana immediately generates a comprehensive report covering four aspects: (i) RRCS for all residue pairs incorporated with real-time 3D visualization; (ii) ligand-receptor interactions; (iii) activation pathway analysis; and (iv) RRCS_TMs that indicates the global movements of transmembrane helices. Moreover, conformational changes between two structures can be analyzed. Applying GPCRana on AlphaFold2-predicted models reveals differentiated inter-helical packing forms in a receptor-dependent manner. Our web server offers a fast and precise way to study GPCR structures and is freely available at http://gpcranalysis.com/#/.

Structural and mechanistic insights provided by single particle cryo-EM analysis of phosphoinositide 3-kinase (PI3Kα).

Recent cryo-electron microscopic (cryo-EM) investigations have succeeded in the analysis of various structural conformations and functional states of PI3Kα, a dimer consisting of the catalytic subunit p110α and the regulatory subunit p85α of class IA of phosphoinositide 3-kinase. High resolution structures have been obtained of the unliganded and of BYL-719-bound PI3Kα. The latter provides information on excessively flexible domains of p85α that are then further analyzed with nanobodies and CXMS (chemical cross-linking, digestion and mass spectrometry). Analysis of p110α helical and kinase domain mutations reveals mutant-specific features that can be linked to the gain of function in enzymatic and signaling activities.

Structural insights into ligand recognition and subtype selectivity of the human melanocortin-3 and melanocortin-5 receptors.

Members of the melanocortin receptor (MCR) family that recognize different melanocortin peptides mediate a broad spectrum of cellular processes including energy homeostasis, inflammation and skin pigmentation through five MCR subtypes (MC1R-MC5R). The structural basis of subtype selectivity of the endogenous agonist γ-MSH and non-selectivity of agonist α-MSH remains elusive, as the two agonists are highly similar with a conserved HFRW motif. Here, we report three cryo-electron microscopy structures of MC3R-Gs in complex with γ-MSH and MC5R-Gs in the presence of α-MSH or a potent synthetic agonist PG-901. The structures reveal that α-MSH and γ-MSH adopt a "U-shape" conformation, penetrate into the wide-open orthosteric pocket and form massive common contacts with MCRs via the HFRW motif. The C-terminus of γ-MSH occupies an MC3R-specific complementary binding groove likely conferring subtype selectivity, whereas that of α-MSH distances itself from the receptor with neglectable contacts. PG-901 achieves the same potency as α-MSH with a shorter length by rebalancing the recognition site and mimicking the intra-peptide salt bridge in α-MSH by cyclization. Solid density confirmed the calcium ion binding in MC3R and MC5R, and the distinct modulation effects of divalent ions were demonstrated. Our results provide insights into ligand recognition and subtype selectivity among MCRs, and expand the knowledge of signal transduction among MCR family members.

Single hormone or synthetic agonist induces Gs/Gi coupling selectivity of EP receptors via distinct binding modes and propagating paths.

To accomplish concerted physiological reactions, nature has diversified functions of a single hormone at at least two primary levels: 1) Different receptors recognize the same hormone, and 2) different cellular effectors couple to the same hormone-receptor pair [R.P. Xiao, Sci STKE 2001, re15 (2001); L. Hein, J. D. Altman, B.K. Kobilka, Nature 402, 181-184 (1999); Y. Daaka, L. M. Luttrell, R. J. Lefkowitz, Nature 390, 88-91 (1997)]. Not only these questions lie in the heart of hormone actions and receptor signaling but also dissecting mechanisms underlying these questions could offer therapeutic routes for refractory diseases, such as kidney injury (KI) or X-linked nephrogenic diabetes insipidus (NDI). Here, we identified that Gs-biased signaling, but not Gi activation downstream of EP4, showed beneficial effects for both KI and NDI treatments. Notably, by solving Cryo-electron microscope (cryo-EM) structures of EP3-Gi, EP4-Gs, and EP4-Gi in complex with endogenous prostaglandin E2 (PGE2)or two synthetic agonists and comparing with PGE2-EP2-Gs structures, we found that unique primary sequences of prostaglandin E2 receptor (EP) receptors and distinct conformational states of the EP4 ligand pocket govern the Gs/Gi transducer coupling selectivity through different structural propagation paths, especially via TM6 and TM7, to generate selective cytoplasmic structural features. In particular, the orientation of the PGE2 ω-chain and two distinct pockets encompassing agonist L902688 of EP4 were differentiated by their Gs/Gi coupling ability. Further, we identified common and distinct features of cytoplasmic side of EP receptors for Gs/Gi coupling and provide a structural basis for selective and biased agonist design of EP4 with therapeutic potential.

Ascorbate peroxidase plays an important role in photoacclimation in the extremophilic red alga Cyanidiococcus yangmingshanensis.

Introduction: Acidothermophilic cyanidiophytes in natural habitats can survive under a wide variety of light regimes, and the exploration and elucidation of their long-term photoacclimation mechanisms promises great potential for further biotechnological applications. Ascorbic acid was previously identified as an important protectant against high light stress in Galdieria partita under mixotrophic conditions, yet whether ascorbic acid and its related enzymatic reactive oxygen species (ROS) scavenging system was crucial in photoacclimation for photoautotrophic cyanidiophytes was unclear. Methods: The significance of ascorbic acid and related ROS scavenging and antioxidant regenerating enzymes in photoacclimation in the extremophilic red alga Cyanidiococcus yangmingshanensis was investigated by measuring the cellular content of ascorbic acid and the activities of ascorbate-related enzymes. Results and discussion: Accumulation of ascorbic acid and activation of the ascorbate-related enzymatic ROS scavenging system characterized the photoacclimation response after cells were transferred from a low light condition at 20 µmol photons m-2 s-1 to various light conditions in the range from 0 to 1000 µmol photons m-2 s-1. The activity of ascorbate peroxidase (APX) was most remarkably enhanced with increasing light intensities and illumination periods among the enzymatic activities being measured. Light-dependent regulation of the APX activity was associated with transcriptional regulation of the chloroplast-targeted APX gene. The important role of the APX activity in photoacclimation was evidenced by the effect of the APX inhibitors on the photosystem II activity and the chlorophyll a content under the high light condition at 1000 µmol photons m-2 s-1. Our findings provide a mechanistic explanation for the acclimation of C. yangmingshanensis to a wide range of light regimes in natural habitats.