Biology subjects

Cong, X.

Publications and source records attributed to Cong, X..

17 recordsLinked to original sources

bioRxiv2026

Divergent activation of the RXFP1 relaxin receptor by protein and small molecule agonists

RXFP1 is an unusual G protein-coupled receptor (GPCR) that mediates physiological adaptations to pregnancy and is a therapeutic target due to its benefits in treatment of fibrosis and heart failure. Protein and small-molecule agonists are currently in mid-stage clinical trials. Here, we present three cryo-electron microscopy structures of RXFP1: ligand-free, bound to the native agonist relaxin-2, and bound to the small-molecule drug candidate AZD5462. These structures show that relaxin-2 engages the receptors ectodomain while AZD5462 binds within transmembrane domain. Together with hydrogen-deuterium exchange mass spectrometry, we show that relaxin-2 induces conformational reorganization of the linker domain into a helical secondary structure. In contrast, AZD5462 stabilizes a unique active conformation capable of inducing {beta}-arrestin recruitment. Both mechanisms are distinctive from the "push-pull" mechanism of glycoprotein hormone receptors. Altogether, our findings define divergent activation mechanisms for protein and small-molecule agonists of RXFP1 and provide structural framework for next-generation drug discovery targeting relaxin receptors and their relatives.

biochemistry↗

bioRxiv2025

Unveiling the Hidden: Dissecting Liraglutide Oligomerization Dual Pathways via Direct Mass Technology, Electron-Capture Dissociation, and Molecular Dynamics

Peptide therapeutics have revolutionized drug design strategies, yet the inherent structural flexibility and conjugated moieties of drug molecules present challenges in discovery, rational design, and manufacturing. Liraglutide, a GLP-1 receptor agonist conjugated with palmitic acid at its lysine residue, exemplifies these challenges by forming oligomers, which may compromise efficacy through progressive formation of aggregates. Here, we incorporate native mass spectrometry platforms including electron-capture dissociation (ECD), direct mass technology (DMT), and molecular dynamics (MD) to capture the early oligomerization process of liraglutide. Our findings reveal a restricted C-terminal region upon oligomer formation, as indicated by the reduced release of z-ions in ECD analysis. Additionally, we identified the formation of higher-order oligomers (n=25-62) by DMT, primarily stabilized by hydrophilic interactions involving preformed stable oligomers (n=14). Together, these integrative mass spectrometry results delineate a dual-pathway oligomerization process for liraglutide, demonstrating the power of mass spectrometry in uncovering hidden pathways of self-association. This approach underscores the potential of mass spectrometry as a key tool in the rational design and optimization of peptide-based therapeutics.

biophysics↗

bioRxiv2024

A photoswitchable positive allosteric modulator to control the activation of the metabotropic glutamate receptor 5 by light

1.The metabotropic glutamate receptor 5 (mGlu5) is widely expressed in the brain, where it plays an important role in synaptic plasticity, learning and memory, making it a therapeutic target of interest in various neurological disorders. In this study, we developed a photoswitchable positive allosteric modulator (PAM) of the mGlu5, as a novel tool for this clinically relevant drug target. To that aim, we used an azologisation strategy of the mGlu5 PAM agonist VU0424465 leading to the molecule azoglurax. We observed a reversible photoisomerization of azoglurax in solution with optimal wavelengths of 365 nm and 435 nm for trans to cis and cis to trans isomerization, respectively. In cell-based assays, azoglurax potentiates the agonist-induced activity of mGlu5 with a sub-micromolar potency in the dark. This potency is reduced under UV illumination. Similar to its parent molecule, azoglurax acts as an allosteric agonist of mGlu5, activating the receptor in absence of glutamate, as demonstrated on a glutamate-insensitive mutant receptor. Docking and site-directed mutagenesis experiments also suggest that azoglurax and VU0424465 bind the same pocket. In addition, molecular dynamics on cis-azoglurax-bound mGlu5 suggests that it azoglurax cis isomer does not bind stably in the receptor, in contrast to the trans-isomer, explaining the difference of activity between the two isomers. In conclusion, azoglurax is the first mGlu5 photoswitchable PAM agonist reported to date, retaining the properties and the binding mode of its parent in the dark, while the insertion of an azobenzene confers light-regulated activity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/629646v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1e0c1f4org.highwire.dtl.DTLVardef@dd7e36org.highwire.dtl.DTLVardef@1892657org.highwire.dtl.DTLVardef@1343743_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

pharmacology and toxicology↗

bioRxiv2024

Functional characterisation of neuropeptides that act as ligands for both calcitonin-type and pigment-dispersing factor-type receptors in a deuterostome

The calcitonin (CT) family of related peptides exerts diverse physiological effects in mammals via two G-protein coupled receptors: CTR and the CTR-like receptor CLR. Phylogenetic analysis of CT-type signaling has revealed the presence of CT-type peptides and CTR/CLR-type proteins in both deuterostome and protostome invertebrates. Furthermore, experimental studies have demonstrated that in the protostome Drosophila melanogaster the CT-like peptide DH31 can act as a ligand for a CTR/CLR-type receptor and a pigment-dispersing factor (PDF) receptor. Here we investigated the signaling mechanisms and functions of CT-type neuropeptides in a deuterostome invertebrate, the sea cucumber Apostichopus japonicus (phylum Echinodermata). In A. japonicus, a single gene encodes two CT-type peptides (AjCT1 and AjCT2) and both peptides act as ligands for a CTR/CLR-type receptor (AjCTR) and two PDF-type receptors (AjPDFR1, AjPDFR2), but with differential activation of downstream cAMP/PKA, Gq/Ca2+/PKC and ERK1/2 signaling pathways. AjCT1/AjCT2-encoding transcripts were detected in the central nervous system and a variety of organ systems and neuropeptide expression was visualized immunohistochemically using an antiserum to a starfish CT-type peptide (ArCT). In vitro pharmacological experiments demonstrated that AjCT1 and/or AjCT2 cause dose-dependent relaxation of longitudinal muscle and intestine preparations. Furthermore, in vivo pharmacological experiments, combined with gain-and loss-of-function experiments, revealed a potential physiological role for AjCT2/AjPDFR2 signaling in promoting feeding and growth in A. japonicus. To our knowledge, this is the first study to obtain evidence that CT-type peptides can act as ligands for both CTR/CLR-type and PDF-type receptors in a deuterostome. Moreover, it provides the first evidence for appetite-stimulating and growth-promoting effects of CT-type neuropeptides in bilaterians. Given the economic importance of A. japonicus as a foodstuff, the discovery of CT-type peptides as potential regulators of feeding and growth in this species may offer novel strategies for aquaculture applications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/610391v3_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@663885org.highwire.dtl.DTLVardef@108bea3org.highwire.dtl.DTLVardef@11bd14dorg.highwire.dtl.DTLVardef@14a2008_HPS_FORMAT_FIGEXP M_FIG C_FIG A schematic showing proposed molecular mechanisms by which CT-type signaling regulates feeding and growth in A. japonicus. AjCT1 and AjCT2 are represented by purple and yellow circles, respectively. For cell signaling, black arrows indicate that this process is activated, whilst the pink arrow indicates that ERK1/2 is activated in the AjCT1/AjPDFR1, AjCT2/AjPDFR1 and AjCT2/AjPDFR2 signaling pathways.

neuroscience↗

bioRxiv2023

Conformational dynamics underlying Atypical Chemokine Receptor 3 activation

Atypical Chemokine Receptor 3 (ACKR3) is a G protein-coupled receptor that does not signal through G proteins. It is known as a chemokine scavenger involved in various pathologies, making it an appealing yet intriguing therapeutic target. Indeed, the structural properties that govern ACKR3 functional selectivity and the overall conformational dynamics of ACKR3 activation are poorly understood. Here we combined Hydrogen/Deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics simulations to examine the binding mode and mechanism of action of various small-molecule ACKR3 ligands of different efficacy for {beta}-arrestin recruitment. Our results show that activation or inhibition of ACKR3 is largely governed by intracellular conformational changes of helix 6, intracellular loop 2 and helix 7, while the DRY motif becomes protected during both processes. Moreover, HDX-MS identifies the binding sites and the allosteric modulation of ACKR3 upon {beta}-arrestin 1 binding. In summary, this study highlights the structure-function relationship of small-molecule ligands, the overall activation dynamics of ACKR3, the binding mode of {beta}-arrestin 1 and the atypical dynamic features in ACKR3 that may contribute to its inability to activate G proteins.

biochemistry↗

bioRxiv2023

Biased activation of the vasopressin V2 receptor probed by NMR, paramagnetic ligands, and molecular dynamics simulations

G protein-coupled receptors (GPCRs) control critical intercellular communications by responding to extracellular stimuli and undertaking conformational changes to convey signals to intracellular effectors. We combined NMR, molecular pharmacology, and molecular dynamics (MD) simulations to study the conformational diversity of the vasopressin V2 GPCR subtype (V2R) bound to different types of ligands: the antagonist tolvaptan, the endogenous unbiased agonist arginine-vasopressin, and MCF14, a Gs-protein biased agonist. We developed a double-labeling NMR scheme to study the conformational dynamics: V2R was subjected to lysine 13CH3 methylation, whereas the agonists were tagged with a paramagnetic probe. Paramagnetic relaxation enhancements were used to validate the ligand binding poses in the MD simulations. We found that the bias for the Gs protein over the {beta}-arrestin pathway involves interactions between the conserved NPxxY motif in the transmembrane helix (TM) 7 and a central hydrophobic patch in TM3, which constrains TM7 and likely inhibits {beta}-arrestin signaling. A similar mechanism was observed for the pathogenic mutation, I1303.43N, which constitutively activates the Gs protein without concomitant {beta}-arrestin recruitment. This mechanism resembles to opioid receptors findings indicating common patterns in class A GPCRs.

biophysics↗

bioRxiv2022

Structure of the vasopressin hormone-V2 receptor-β-arrestin1 ternary complex

Arrestins interact with G protein-coupled receptors (GPCRs) to stop G protein activation and to initiate key signaling pathways. Recent structural studies shed light on the molecular mechanisms involved in GPCR-arrestin coupling, but whether this process is conserved among GPCRs is poorly understood. Here, we report the cryo-electron microscopy active structure of the wild-type arginine-vasopressin V2 receptor (V2R) in complex with {beta}-arrestin1. It reveals an atypical position of {beta}-arrestin1 compared to previously described GPCR-arrestin assemblies, associated with an original V2R/{beta}-arrestin1 interface involving all receptor intracellular loops. Phosphorylated sites of the V2R C-terminus are clearly identified and interact extensively with the {beta}-arrestin1 N-lobe, in agreement with structural data obtained with chimeric or synthetic systems. Overall, these findings highlight a striking structural variability among GPCR-arrestin signaling complexes.

biochemistry↗

bioRxiv2022

The relaxin receptor RXFP1 signals through a mechanism of autoinhibition

The relaxin family peptide receptor 1 (RXFP1) is the receptor for relaxin-2, an important regulator of reproductive and cardiovascular physiology. RXFP1 is a multi-domain G protein-coupled receptor (GPCR) with an ectodomain consisting of an LDLa module and leucine-rich repeats. The mechanism of RXFP1 signal transduction is clearly distinct from that of other GPCRs, but remains very poorly understood. Here, we present the cryo-electron microscopy structure of active-state human RXFP1, bound to a single-chain version of the endogenous agonist relaxin-2 and to the heterotrimeric Gs protein. Evolutionary coupling analysis and structure-guided functional experiments reveal that RXFP1 signals through a mechanism of autoinhibition, wherein the receptors extracellular loop 2 occupies the orthosteric site in the active state but is inhibited by the ectodomain in the absence of relaxin-2. Our results explain how an unusual GPCR family functions, providing a path to rational drug development targeting the relaxin receptors.

pharmacology and toxicology↗

bioRxiv2021

Extracellular loop 2 of G protein-coupled olfactory receptors is critical for odorant recognition

G protein-coupled olfactory receptors (ORs) enable us to detect innumerous odorants. They are also ectopically expressed, emerging as attractive drug targets. ORs can be promiscuous or highly specific, which is part of Natures strategy for odor discrimination. This work demonstrates that the extracellular loop 2 (ECL2) plays critical roles in OR promiscuity and specificity. Using site-directed mutagenesis and molecular modeling, we constructed 3D OR models in which ECL2 forms a lid of the orthosteric pocket. ECL2 controls the shape and the volume of the odorant-binding pocket, maintains the pocket hydrophobicity and acts as a gatekeeper of odorant binding. The interplay between the specific orthosteric pocket and the variable, less specific ECL2 controls OR specificity and promiscuity. The 3D models enabled virtual screening of new OR agonists and antagonists, exhibiting 78% hit rate in cell assays. This approach can be generalized to structure-based ligand screening for other GPCRs that lack high-resolution 3D structures.

molecular biology↗

bioRxiv2021

Structural insights into recognition of chemokine receptors by Staphylococcus aureus leukotoxins

Staphylococcus aureus (SA) leukocidin LukED belongs to a family of bicomponent pore forming toxins that play important roles in SA immune evasion and nutrient acquisition. LukED targets specific G protein-coupled chemokine receptors to lyse human erythrocytes and leukocytes. The first recognition step of receptors is critical for specific cell targeting and lysis. The structural and molecular bases for this mechanism are not well understood but could constitute essential information to guide antibiotic development. Here, we characterized the interaction of LukE with chemokine receptors ACKR1, CCR2 and CCR5 using a combination of structural, pharmacological and computational approaches. First, crystal structures of LukE in complex with a small molecule mimicking sulfotyrosine side chain (p-cresyl sulfate) and with peptides containing sulfotyrosines issued from receptor sequences revealed the location of receptor sulfotyrosine binding sites in the toxins. Then, by combining the available experimental information with protein docking, classical and accelerated weight histogram (AWH) molecular dynamics we propose models of the ACKR1-LukE and CCR5-LukE complexes. This work provides novel insights into chemokine receptor recognition by leukotoxins and suggests that the conserved sulfotyrosine binding pocket could be a target of choice for future drug development.

biochemistry↗

bioRxiv2021

Discovery and mechanism of action of small molecule inhibitors of ceramidases

Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes such as energy utilisation and cell proliferation. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to ultimately produce pro-proliferative sphingosine-1-phosphate. Human ceramidases can be soluble proteins (acid and neutral ceramidase) or integral membrane proteins (alkaline ceramidases). Increasing ceramide levels to increase apoptosis has shown efficacy as a cancer treatment using small molecules inhibiting a soluble ceramidase. Due to the transmembrane nature of alkaline ceramidases, no specific small molecule inhibitors have been reported. Here, we report novel fluorescent substrates (FRETceramides) of ceramidases that can be used to monitor enzyme activity in real-time. We use FRETceramides to discover the first drug-like inhibitors of alkaline ceramidase 3 (ACER3) which are active in cell-based assays. Biophysical characterization of enzyme:inhibitor interactions reveal a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules. Table of contents summaryUse of synthetic fluorescent ceramide molecules allows the discovery of the first selective drug-like small molecule inhibitors for alkaline ceramidase 3, an intra-membrane enzyme involved in sphingolipid metabolism in health and disease.

biochemistry↗

bioRxiv2021

An automated platform for structural analysis of membrane proteins through serial crystallography

Membrane proteins are central to many pathophysiological processes yet remain very difficult to analyze at a structural level. Moreover, high-throughput structure-based drug discovery has not yet been exploited for membrane proteins due to lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperature and in a single support. We apply this approach to two human integral membrane proteins, which allowed us to capture different conformational states of intramembrane enzyme-product complexes and analyze the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting and serial crystallography enabling screening of small molecule libraries with membrane protein crystals grown in meso. This approach brings badly needed automation for this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins. HighlightsO_LIA fully automated, online workflow enables rapid determination of membrane protein structures by serial X-ray crystallography (SSX). C_LIO_LIHigh resolution room temperature and cryogenic structures of ADIPOR2 provide insights into the dynamic nature of receptor:ligand interactions. C_LIO_LIA web-based application allows remote user-guided experimental design and execution. C_LIO_LIAn automated SSX-based ligand discovery pipeline for integral membrane proteins is introduced. C_LI

biochemistry↗

bioRxiv2021

Molecular insights into the μ-opioid receptorselective signaling

GPCR functional selectivity has opened new opportunities for the design of safer drugs. Ligands orchestrate GPCR signaling cascades by modulating the receptor conformational landscape. Our study provides insights into the dynamic mechanism enabling opioid ligands to preferentially activate the G protein over the {beta}-arrestin pathways through the -opioid receptor (OR). We combined functional assays in living cells, solution NMR spectroscopy and enhanced-sampling molecular dynamic simulations to identify the specific OR conformations induced by G protein-biased agonists. In particular, we describe the dynamic and allosteric communications between the ligand-binding pocket and the receptor intracellular domains, through conserved motifs in class A GPCRs. Most strikingly, the biased agonists triggered OR conformational changes in the intracellular loop 1 and helix 8 domains, which may impair {beta}-arrestin binding or signaling. The findings may apply to other GPCR families and provide key molecular information that could facilitate the design of biased ligands.

bioinformatics↗

bioRxiv2020

Phosphoproteomics Reveals AMPK Substrate Network in Response to DNA Damage and Histone Acetylation

AMPK is a conservative energy sensor that plays roles in diverse biologic processes via direct phosphorylation on various substrates. Emerging studies have demonstrated the regulatory roles of AMPK in DNA repair, but the underlying mechanisms remain to be fully understood. Herein, using mass spectrometry-based proteomic technologies, we systematically investigate the regulatory network of AMPK in DNA damage response. Our system-wide phosphoproteome study uncovers a variety of newly-identified potential substrates involved in diverse biologic processes, whereas our system-wide histone modification analysis reveals a linkage between AMPK and histone acetylation. Together with these findings, we discover that AMPK promotes apoptosis by phosphorylating ASPP2 in irradiation-dependent way and regulates histone acetylation by phosphorylating HDAC9 in irradiation-independent way. Besides, we reveal that disturbing the histone acetylation by the bromodomain BRD4 inhibitor JQ-1 enhanced the sensitivity of AMPK-deficient cells to irradiation. Therefore, our studies provided a source to study the phosphorylation and histone acetylation underlying the regulatory network of AMPK, which could be beneficial to understand the exact role of AMPK in DNA damage response.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2020

Functions of olfactory receptors are decoded from their sequence

G protein-coupled receptors (GPCRs) conserve common structural folds and activation mechanisms, yet their ligand spectra and functions are highly diversified. This work investigated how the functional variations in olfactory GPCRs (ORs)-the largest GPCR family-are encoded in the primary sequence. With the aid of site-directed mutagenesis and molecular simulations, we built machine learning models to predict OR-ligand pairs as well as basal activity of ORs. In vitro functional assay confirmed 20 new OR-odorant pairs, including 9 orphan ORs. Residues around the odorant-binding pocket dictate the odorant selectivity/specificity of the ORs. Residues that encode the varied basal activities of the ORs were found to mostly surround the conserved motifs as well as the binding pocket. The machine learning approach, which is readily applicable to mammalian OR families, will accelerate OR-odorant mapping and the decoding of combinatorial OR codes for odors.

bioinformatics↗

bioRxiv2019

Effect of recombinant phycocyanin on photosynthetic system in Chlamydomonas reinhardtii

The phycobilisome is an important photosynthetic antenna in the photosynthetic cyanobacteria, and phycocyanin is one of the main components of phycobilisomes. It helps cells absorb green light that green-lineage photo-synthetic organisms cannot. In this work, phycocyanin, heme oxidase and ferredoxin oxidoreductase from Arthrospira platensis FACHB 314 were successfully expressed in the green algae Chlamydomonas reinhardtii. Then the effects of this expression on the photosynthesis and growth of C. reinhardtii were detected. Transcriptional level analysis showed that the phycocyanin gene was successfully expressed stably in the transgenic strains. The results of low-temperature fluorescence emission spectra and chlorophyll fluorescence showed that recombinant phycocyanin has considerable optical activity. The expression of phycocyanin, heme oxidase and ferredoxin oxidoreductase in low-light conditions is particularly evident in the promotion of photosynthesis in C. reinhardtii. The growth of transgenic strains was significantly promoted in the early growth phase under low-light conditions. However, the final growth and biomass accumulation of transgenic C. reinhardti were inhibited by this expression. In this paper, the possibility of photoenergy transfer between phycocyanin and heterologous host thylakoid membrane was researched, which provided a useful attempt for the construction of a new photosynthetic system using phycobiliprotein from cyanobacteria.\n\nOne-sentence summaryPhycocyanin from Arthrospira platensis FACHB 314 expressed in Chlamydomonas reinhardtii can effect the photosynthetic system of C. reinhardtii.

molecular biology↗

bioRxiv2018

Regulatory B cells contribute to the alleviation of colitis induced by Dextran Sulphate Sodium after H.pylori infection

BackgroundEpidemiological studies showed that there was an inverse relationship between Helicobacter pylori infection and the incidence of inflammatory bowel disease. Our previous research indicated that the regulatory immune responses induced by H.pylori infection were not limited to gastric mucosa, and the balance of intestinal mucosal immunity was influenced. In this study, we investigated the role of the regulatory B cells in the effects of the H.pylori infection on acute and chronic colitis induced by dextran sulphate sodium.\n\nMethodsMice were infected with H.pylori and then colitis was induced by 3% DSS, the CD19+IL-10+Breg cells and CD4+CD25+Foxp3+Treg cells in blood spleen mesenteric lymph nodes (MLN) Peyers Patches (PP) and gastrointestinal mucosa were measured and examined. The anti- and pro-inflammatory cytokines were also detected at mRNA level.\n\nResultsIn the acute and chronic colitis groups, DAI and colonic histological scores reduced significantly and colon length shorted less; the anti-inflammatory cytokines mRNA expression upregulated while the pro-inflammatory cytokines mRNA expression downregulated in colonic mucosa; and the percentages of CD19+IL-10+Breg cells in blood spleen MLN PP were higher in the H.pylori/DSS co-treated group compared with the DSS treated group.\n\nConclusionH.pylori infection can alleviate the acute and chronic colitis induced by DSS. CD19+IL-10+Breg cells may play a critical role in the alleviation of acute and chronic colitis following H.pylori infection.

immunology↗