ABSTRACT
Pathogenic variants in the JAG1 gene are a primary cause of the multi-system disorder Alagille syndrome. Although variant detection rates are high for this disease, there is uncertainty associated with the classification of missense variants that leads to reduced diagnostic yield. Consequently, up to 85% of reported JAG1 missense variants have uncertain or conflicting classifications. We generated a library of 2,832 JAG1 nucleotide variants within exons 1-7, a region with a high number of reported missense variants, and designed a high-throughput assay to measure JAG1 membrane expression, a requirement for normal function. After calibration using a set of 175 known or predicted pathogenic and benign variants included within the variant library, 486 variants were characterized as functionally abnormal (n = 277 abnormal and n = 209 likely abnormal), of which 439 (90.3%) were missense. We identified divergent membrane expression occurring at specific residues, indicating that loss of the wild-type residue itself does not drive pathogenicity, a finding supported by structural modeling data and with broad implications for clinical variant classification both for Alagille syndrome and globally across other disease genes. Of 144 uncertain variants reported in patients undergoing clinical or research testing, 27 had functionally abnormal membrane expression, and inclusion of our data resulted in the reclassification of 26 to likely pathogenic. Functional evidence augments the classification of genomic variants, reducing uncertainty and improving diagnostics. Inclusion of this repository of functional evidence during JAG1 variant reclassification will significantly affect resolution of variant pathogenicity, making a critical impact on the molecular diagnosis of Alagille syndrome.
Subject(s)
Alagille Syndrome , Jagged-1 Protein , Mutation, Missense , Alagille Syndrome/genetics , Jagged-1 Protein/genetics , Humans , Exons/geneticsABSTRACT
Immunotherapy efficacy is limited in melanoma, and combinations of immunotherapies with other modalities have yielded limited improvements but also adverse events requiring cessation of treatment. In addition to ineffective patient stratification, efficacy is impaired by paucity of intratumoral immune cells (itICs); thus, effective strategies to safely increase itICs are needed. We report that dietary administration of L-fucose induces fucosylation and cell surface enrichment of the major histocompatibility complex (MHC)-II protein HLA-DRB1 in melanoma cells, triggering CD4+ T cell-mediated increases in itICs and anti-tumor immunity, enhancing immune checkpoint blockade responses. Melanoma fucosylation and fucosylated HLA-DRB1 associate with intratumoral T cell abundance and anti-programmed cell death protein 1 (PD1) responder status in patient melanoma specimens, suggesting the potential use of melanoma fucosylation as a strategy for stratifying patients for immunotherapies. Our findings demonstrate that fucosylation is a key mediator of anti-tumor immunity and, importantly, suggest that L-fucose is a powerful agent for safely increasing itICs and immunotherapy efficacy in melanoma.
Subject(s)
Fucose , Melanoma , Humans , HLA-DRB1 Chains/genetics , HLA-DRB1 Chains/metabolism , Fucose/metabolism , Melanoma/drug therapy , Immunotherapy , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/pathologyABSTRACT
The immune checkpoint receptor lymphocyte activation gene 3 protein (LAG3) inhibits T cell function upon binding to major histocompatibility complex class II (MHC class II) or fibrinogen-like protein 1 (FGL1). Despite the emergence of LAG3 as a target for next-generation immunotherapies, we have little information describing the molecular structure of the LAG3 protein or how it engages cellular ligands. Here we determined the structures of human and murine LAG3 ectodomains, revealing a dimeric assembly mediated by Ig domain 2. Epitope mapping indicates that a potent LAG3 antagonist antibody blocks interactions with MHC class II and FGL1 by binding to a flexible 'loop 2' region in LAG3 domain 1. We also defined the LAG3-FGL1 interface by mapping mutations onto structures of LAG3 and FGL1 and established that FGL1 cross-linking induces the formation of higher-order LAG3 oligomers. These insights can guide LAG3-based drug development and implicate ligand-mediated LAG3 clustering as a mechanism for disrupting T cell activation.
Subject(s)
Antigens, CD/metabolism , Lymphocyte Activation , Animals , Antibodies , Fibrinogen , Histocompatibility Antigens Class II/metabolism , Humans , Immunotherapy , Ligands , Mice , Receptors, Immunologic , Lymphocyte Activation Gene 3 ProteinABSTRACT
Glucose homeostasis, regulated by glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and glucagon (GCG) is critical to human health. Several multi-targeting agonists at GIPR, GLP-1R or GCGR, developed to maximize metabolic benefits with reduced side-effects, are in clinical trials to treat type 2 diabetes and obesity. To elucidate the molecular mechanisms by which tirzepatide, a GIPR/GLP-1R dual agonist, and peptide 20, a GIPR/GLP-1R/GCGR triagonist, manifest their multiplexed pharmacological actions over monoagonists such as semaglutide, we determine cryo-electron microscopy structures of tirzepatide-bound GIPR and GLP-1R as well as peptide 20-bound GIPR, GLP-1R and GCGR. The structures reveal both common and unique features for the dual and triple agonism by illustrating key interactions of clinical relevance at the near-atomic level. Retention of glucagon function is required to achieve such an advantage over GLP-1 monotherapy. Our findings provide valuable insights into the structural basis of functional versatility of tirzepatide and peptide 20.
Subject(s)
Diabetes Mellitus, Type 2 , Receptors, Glucagon , Cryoelectron Microscopy , Diabetes Mellitus, Type 2/drug therapy , Gastric Inhibitory Polypeptide , Glucagon/metabolism , Glucagon-Like Peptide 1/therapeutic use , Glucagon-Like Peptide-1 Receptor/metabolism , Glucose/therapeutic use , Humans , Peptides/chemistry , Receptors, G-Protein-CoupledABSTRACT
Much effort has been invested in the investigation of the structural basis of G protein-coupled receptors (GPCRs) activation. Inverse agonists, which can inhibit GPCRs with constitutive activity, are considered useful therapeutic agents, but the molecular mechanism of such ligands remains insufficiently understood. Here, we report a crystal structure of the ghrelin receptor bound to the inverse agonist PF-05190457 and a cryo-electron microscopy structure of the active ghrelin receptor-Go complex bound to the endogenous agonist ghrelin. Our structures reveal a distinct binding mode of the inverse agonist PF-05190457 in the ghrelin receptor, different from the binding mode of agonists and neutral antagonists. Combining the structural comparisons and cellular function assays, we find that a polar network and a notable hydrophobic cluster are required for receptor activation and constitutive activity. Together, our study provides insights into the detailed mechanism of ghrelin receptor binding to agonists and inverse agonists, and paves the way to design specific ligands targeting ghrelin receptors.
Subject(s)
Receptors, Ghrelin/agonists , Receptors, Ghrelin/antagonists & inhibitors , Cryoelectron Microscopy , Crystallography, X-Ray , Ghrelin/agonists , Ghrelin/genetics , Humans , Ligands , Mutation , Protein Binding , Receptors, Ghrelin/geneticsABSTRACT
Aberrant activation of Wnt/ß-catenin signaling is strongly associated with many diseases including cancer invasion and metastasis. Small-molecule targeting of the central signaling node of this pathway, ß-catenin, is a biologically rational approach to abolish hyperactivation of ß-catenin signaling but has been demonstrated to be a difficult task. Herein, we report a drug-like small molecule, ZW4864, that binds with ß-catenin and selectively disrupts the protein-protein interaction (PPI) between B-cell lymphoma 9 (BCL9) and ß-catenin while sparing the ß-catenin/E-cadherin PPI. ZW4864 dose-dependently suppresses ß-catenin signaling activation, downregulates oncogenic ß-catenin target genes, and abrogates invasiveness of ß-catenin-dependent cancer cells. More importantly, ZW4864 shows good pharmacokinetic properties and effectively suppresses ß-catenin target gene expression in the patient-derived xenograft mouse model. This study offers a selective chemical probe to explore ß-catenin-related biology and a drug-like small-molecule ß-catenin/BCL9 disruptor for future drug development.
Subject(s)
Antineoplastic Agents/therapeutic use , Neoplasms/drug therapy , Piperidines/therapeutic use , Protein Binding/drug effects , Transcription Factors/antagonists & inhibitors , beta Catenin/antagonists & inhibitors , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Cell Movement/drug effects , Drug Design , Female , Humans , Male , Mice, Inbred C57BL , Mice, SCID , Molecular Structure , Piperidines/chemical synthesis , Piperidines/pharmacokinetics , Structure-Activity Relationship , Transcription Factors/metabolism , Xenograft Model Antitumor Assays , beta Catenin/metabolismABSTRACT
Gamma delta (γδ) T cells infiltrate most human tumors, but current immunotherapies fail to exploit their in situ major histocompatibility complex-independent tumoricidal potential. Activation of γδ T cells can be elicited by butyrophilin and butyrophilin-like molecules that are structurally similar to the immunosuppressive B7 family members, yet how they regulate and coordinate αß and γδ T cell responses remains unknown. Here, we report that the butyrophilin BTN3A1 inhibits tumor-reactive αß T cell receptor activation by preventing segregation of N-glycosylated CD45 from the immune synapse. Notably, CD277-specific antibodies elicit coordinated restoration of αß T cell effector activity and BTN2A1-dependent γδ lymphocyte cytotoxicity against BTN3A1+ cancer cells, abrogating malignant progression. Targeting BTN3A1 therefore orchestrates cooperative killing of established tumors by αß and γδ T cells and may present a treatment strategy for tumors resistant to existing immunotherapies.
Subject(s)
Antigens, CD/immunology , Butyrophilins/antagonists & inhibitors , Butyrophilins/immunology , Intraepithelial Lymphocytes/immunology , Ovarian Neoplasms/immunology , Ovarian Neoplasms/therapy , Animals , Antibodies, Monoclonal/therapeutic use , Antigens, CD/genetics , Butyrophilins/genetics , Female , Humans , Immunotherapy/methods , Mice , Mice, Transgenic , Receptors, Antigen, T-Cell, alpha-beta/immunology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
The G-protein-coupled bile acid receptor (GPBAR) conveys the cross-membrane signalling of a vast variety of bile acids and is a signalling hub in the liver-bile acid-microbiota-metabolism axis1-3. Here we report the cryo-electron microscopy structures of GPBAR-Gs complexes stabilized by either the high-affinity P3954 or the semisynthesized bile acid derivative INT-7771,3 at 3 Å resolution. These structures revealed a large oval pocket that contains several polar groups positioned to accommodate the amphipathic cholic core of bile acids, a fingerprint of key residues to recognize diverse bile acids in the orthosteric site, a putative second bile acid-binding site with allosteric properties and structural features that contribute to bias properties. Moreover, GPBAR undertakes an atypical mode of activation and G protein coupling that features a different set of key residues connecting the ligand-binding pocket to the Gs-coupling site, and a specific interaction motif that is localized in intracellular loop 3. Overall, our study not only reveals unique structural features of GPBAR that are involved in bile acid recognition and allosteric effects, but also suggests the presence of distinct connecting mechanisms between the ligand-binding pocket and the G-protein-binding site in the G-protein-coupled receptor superfamily.
Subject(s)
Bile Acids and Salts/metabolism , Cryoelectron Microscopy , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/ultrastructure , Allosteric Regulation/drug effects , Bile Acids and Salts/chemistry , Binding Sites/drug effects , Cholic Acids/chemistry , Cholic Acids/pharmacology , GTP-Binding Protein alpha Subunits, Gs/chemistry , GTP-Binding Protein alpha Subunits, Gs/metabolism , GTP-Binding Protein alpha Subunits, Gs/ultrastructure , Humans , Ligands , Models, Molecular , Protein Binding , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/chemistry , Substrate SpecificityABSTRACT
IMPACT STATEMENT: CKLF1, a recently identified chemokine, has been reported by a number of studies to play important roles in quite many diseases. However, the potential pathways that CKLF1 may be involved are not manifested well yet. In our review, we showed the basic molecular structure and major functions of this novel chemokine, and implication in human diseases, such as tumors. To attract more attention, we summarized its signaling pathways and clearly present them in a set of figures. With the overview of the experimental trial of CKLF1-targeting medicines in animal models, we hope to provide a few important insights about CKLF1 to both medical researchers and pharmacy.
Subject(s)
Chemokines/metabolism , Disease , Molecular Targeted Therapy , Animals , Chemokines/chemistry , Chemokines/genetics , Chemotaxis , Humans , Models, Biological , Signal TransductionABSTRACT
Grainyhead (Grh)/CP2 transcription factors are highly conserved in multicellular organisms as key regulators of epithelial differentiation, organ development and skin barrier formation. In addition, they have been implicated as being tumor suppressors in a variety of human cancers. Despite their physiological importance, little is known about their structure and DNA binding mode. Here, we report the first structural study of mammalian Grh/CP2 factors. Crystal structures of the DNA-binding domains of grainyhead-like (Grhl) 1 and Grhl2 reveal a closely similar conformation with immunoglobulin-like core. Both share a common fold with the tumor suppressor p53, but differ in important structural features. The Grhl1 DNA-binding domain binds duplex DNA containing the consensus recognition element in a dimeric arrangement, supporting parsimonious target-sequence selection through two conserved arginine residues. We elucidate the molecular basis of a cancer-related mutation in Grhl1 involving one of these arginines, which completely abrogates DNA binding in biochemical assays and transcriptional activation of a reporter gene in a human cell line. Thus, our studies establish the structural basis of DNA target-site recognition by Grh transcription factors and reveal how tumor-associated mutations inactivate Grhl proteins. They may serve as points of departure for the structure-based development of Grh/CP2 inhibitors for therapeutic applications.
Subject(s)
DNA/chemistry , Repressor Proteins/chemistry , Transcriptional Activation , Animals , Arginine/chemistry , Cell Line , Claudin-4/genetics , DNA/metabolism , DNA-Binding Proteins/chemistry , Humans , Mice , Models, Molecular , Mutation , Promoter Regions, Genetic , Protein Binding , Protein Domains , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transcription Factors/chemistryABSTRACT
The RecQ helicase from Deinococcus radiodurans (DrRecQ) distinguishes from other helicases in that it utilizes its three 'helicase and RNaseD C-terminal' domains (HRDC1, HRDC2 and HRDC3) to regulate its activity. These HRDC domains have different influence on the biochemical functions of DrRecQ. Currently, only the structure of HRDC3 was reported. Here, we determined the NMR structure of the N-terminal-most HRDC1, revealing a potential DNA binding domain. Fluorescence anisotropy assay indicates that HRDC1 has binding affinity weaker than 70 µM to all DNA substrates without any specificity. Biochemical assays suggested that HRDC1 cooperates with other domains to enhance full-length DrRecQ interactions with DNA.
Subject(s)
Bacterial Proteins/chemistry , Deinococcus/enzymology , RecQ Helicases/chemistry , Amino Acid Sequence , DNA/chemistry , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Sequence Homology, Amino AcidABSTRACT
A series of substituted 5-benzyl-2-phenylpyrazolo[1,5-a]pyrazin-4,6(5H,7H)-dione derivatives was synthesized by one-step reaction of ethyl 3-phenyl-1H-pyrazole-5-carboxylate derivatives and N-arylalkyl-2-chloroacetamide. Structures of the compounds were determined by IR, (1)H NMR and mass spectroscopy. In addition, a representative single-crystal structure was characterized by using X-ray diffraction analysis. The compound 5j could selectively inhibit the growth of H322 lung cancer cells which contain a mutated p53 gene in a dose-dependent manner through inducing apoptosis of cells.