ABSTRACT
Arthritogenic alphaviruses, such as Chikungunya virus (CHIKV), cause severe and debilitating rheumatic diseases worldwide, resulting in severe morbidity and economic costs. Recently, MXRA8 was reported as an entry receptor. Here, we present the crystal structures of the mouse MXRA8, human MXRA8 in complex with the CHIKV E protein, and the cryo-electron microscopy structure of human MXRA8 and CHIKV virus-like particle. MXRA8 has two Ig-like domains with unique structural topologies. This receptor binds in the "canyon" between two protomers of the E spike on the surface of the virion. The atomic details at the interface between the two binding entities reveal that both the two domains and the hinge region of MXRA8 are involved in interaction with CHIKV E1-E2 residues from two protomers. Notably, the stalk region of MXRA8 is critical for CHIKV virus entry. This finding provides important information regarding the development of therapeutic countermeasures against those arthritogenic alphaviruses.
Subject(s)
Chikungunya virus/chemistry , Membrane Proteins/chemistry , Viral Envelope Proteins/chemistry , Virus Internalization , Animals , Chikungunya virus/metabolism , Chlorocebus aethiops , HEK293 Cells , Humans , Membrane Proteins/metabolism , Protein Domains , Vero Cells , Viral Envelope Proteins/metabolismABSTRACT
Enterovirus B (EV-B), a major proportion of the genus Enterovirus in the family Picornaviridae, is the causative agent of severe human infectious diseases. Although cellular receptors for coxsackievirus B in EV-B have been identified, receptors mediating virus entry, especially the uncoating process of echovirus and other EV-B remain obscure. Here, we found that human neonatal Fc receptor (FcRn) is the uncoating receptor for major EV-B. FcRn binds to the virus particles in the "canyon" through its FCGRT subunit. By obtaining multiple cryo-electron microscopy structures at different stages of virus entry at atomic or near-atomic resolution, we deciphered the underlying mechanisms of enterovirus attachment and uncoating. These structures revealed that different from the attachment receptor CD55, binding of FcRn to the virions induces efficient release of "pocket factor" under acidic conditions and initiates the conformational changes in viral particle, providing a structural basis for understanding the mechanisms of enterovirus entry.
Subject(s)
Enterovirus B, Human/metabolism , Histocompatibility Antigens Class I/metabolism , Histocompatibility Antigens Class I/ultrastructure , Receptors, Fc/metabolism , Receptors, Fc/ultrastructure , Capsid/metabolism , Cryoelectron Microscopy , Enterovirus , Enterovirus B, Human/pathogenicity , Enterovirus Infections/metabolism , Histocompatibility Antigens Class I/physiology , Humans , Models, Molecular , Phylogeny , Receptors, Fc/physiology , Virion , Virus InternalizationABSTRACT
Somatostatin receptor 5 (SSTR5) is an important G protein-coupled receptor and drug target for neuroendocrine tumors and pituitary disorders. This study presents two high-resolution cryogenicelectron microscope structures of the SSTR5-Gi complexes bound to the cyclic neuropeptide agonists, cortistatin-17 (CST17) and octreotide, with resolutions of 2.7 Å and 2.9 Å, respectively. The structures reveal that binding of these peptides causes rearrangement of a "hydrophobic lock", consisting of residues from transmembrane helices TM3 and TM6. This rearrangement triggers outward movement of TM6, enabling Gαi protein engagement and receptor activation. In addition to hydrophobic interactions, CST17 forms conserved polar contacts similar to somatostatin-14 binding to SSTR2, while further structural and functional analysis shows that extracellular loops differently recognize CST17 and octreotide. These insights elucidate agonist selectivity and activation mechanisms of SSTR5, providing valuable guidance for structure-based drug development targeting this therapeutically relevant receptor.
Subject(s)
Octreotide , Receptors, Somatostatin , Receptors, Somatostatin/metabolism , Receptors, Somatostatin/agonists , Receptors, Somatostatin/chemistry , Humans , Octreotide/chemistry , Octreotide/pharmacology , Octreotide/metabolism , Neuropeptides/metabolism , Neuropeptides/chemistry , Cryoelectron Microscopy , Protein Binding , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Peptides, Cyclic/metabolism , Somatostatin/metabolism , Somatostatin/chemistry , Somatostatin/analogs & derivatives , Models, Molecular , HEK293 CellsABSTRACT
African swine fever (ASF) is a highly contagious viral disease that affects domestic and wild pigs. The causative agent of ASF is African swine fever virus (ASFV), a large double-stranded DNA virus with a complex virion structure. Among the various proteins encoded by ASFV, A137R is a crucial structural protein associated with its virulence. However, the structure and molecular mechanisms underlying the functions of A137R remain largely unknown. In this study, we present the structure of A137R determined by cryogenic electron microscopy single-particle reconstruction, which reveals that A137R self-oligomerizes to form a dodecahedron-shaped cage composed of 60 polymers. The dodecahedron is literally equivalent to a T = 1 icosahedron where the icosahedral vertexes are located in the center of each dodecahedral facet. Within each facet, five A137R protomers are arranged in a head-to-tail orientation with a long N-terminal helix forming the edge through which adjacent facets stitch together to form the dodecahedral cage. Combining structural analysis and biochemical evidence, we demonstrate that the N-terminal domain of A137R is crucial and sufficient for mediating the assembly of the dodecahedron. These findings imply the role of A137R cage as a core component in the icosahedral ASFV virion and suggest a promising molecular scaffold for nanotechnology applications. IMPORTANCE: African swine fever (ASF) is a lethal viral disease of pigs caused by African swine fever virus (ASFV). No commercial vaccines and antiviral treatments are available for the prevention and control of the disease. A137R is a structural protein of ASFV that is associated with its virulence. The discovery of the dodecahedron-shaped cage structure of A137R in this study is of great importance in understanding ASFV pathogenicity. This finding sheds light on the molecular mechanisms underlying the functions of A137R. Furthermore, the dodecahedral cage formed by A137R shows promise as a molecular scaffold for nanoparticle vectors. Overall, this study provides valuable insights into the structure and function of A137R, contributing to our understanding of ASFV and potentially opening up new avenues for the development of vaccines or treatments for ASF.
Subject(s)
African Swine Fever Virus , Swine , Viral Structural Proteins , Animals , African Swine Fever/virology , African Swine Fever Virus/chemistry , African Swine Fever Virus/growth & development , African Swine Fever Virus/pathogenicity , African Swine Fever Virus/ultrastructure , Cryoelectron Microscopy , Structure-Activity Relationship , Swine/virology , Viral Structural Proteins/chemistry , Viral Structural Proteins/metabolism , Viral Structural Proteins/ultrastructure , Virion/chemistry , Virion/metabolism , Virion/ultrastructure , VirulenceABSTRACT
African swine fever (ASF) is a viral hemorrhagic disease that affects domestic pigs and wild boar and is caused by the African swine fever virus (ASFV). The ASFV virion contains a long double-stranded DNA genome, which encodes more than 150 proteins. However, the immune escape mechanism and pathogenesis of ASFV remain poorly understood. Here, we report that the pyroptosis execution protein gasdermin D (GSDMD) is a new binding partner of ASFV-encoded protein S273R (pS273R), which belongs to the SUMO-1 cysteine protease family. Further experiments demonstrated that ASFV pS273R-cleaved swine GSDMD in a manner dependent on its protease activity. ASFV pS273R specifically cleaved GSDMD at G107-A108 to produce a shorter N-terminal fragment of GSDMD consisting of residues 1 to 107 (GSDMD-N1-107). Interestingly, unlike the effect of GSDMD-N1-279 fragment produced by caspase-1-mediated cleavage, the assay of LDH release, cell viability, and virus replication showed that GSDMD-N1-107 did not trigger pyroptosis or inhibit ASFV replication. Our findings reveal a previously unrecognized mechanism involved in the inhibition of ASFV infection-induced pyroptosis, which highlights an important function of pS273R in inflammatory responses and ASFV replication.
Subject(s)
African Swine Fever Virus , African Swine Fever , Cysteine Proteases , Phosphate-Binding Proteins , Pore Forming Cytotoxic Proteins , Viral Proteins , African Swine Fever/virology , African Swine Fever Virus/enzymology , African Swine Fever Virus/metabolism , Animals , Cysteine Proteases/metabolism , Phosphate-Binding Proteins/metabolism , Pore Forming Cytotoxic Proteins/metabolism , Pyroptosis , Sus scrofa , Swine , Viral Proteins/metabolismABSTRACT
Classical swine fever virus (CSFV) is an important pathogen in the swine industry. Virion attachment is mediated by envelope proteins Erns and E2, and E2 is indispensable. Using a pull-down assay with soluble E2 as the bait, we demonstrated that ADAM17, a disintegrin and metalloproteinase 17, is essential for CSFV entry. Loss of ADAM17 in a permissive cell line eliminated E2 binding and viral entry, but compensation with pig ADAM17 cDNA completely rescued these phenotypes. Similarly, ADAM17 silencing in primary porcine fibroblasts significantly impaired virus infection. In addition, human and mouse ADAM17, which is highly homologous to pig ADAM17, also mediated CSFV entry. The metalloproteinase domain of ADAM17 bound directly to E2 protein in a zinc-dependent manner. A surface exposed region within this domain was mapped and shown to be critical for CSFV entry. These findings clearly demonstrate that ADAM17 serves as an essential attachment factor for CSFV.
Subject(s)
ADAM17 Protein/metabolism , Classical Swine Fever Virus/metabolism , Receptors, Virus/metabolism , Viral Envelope Proteins/metabolism , Virus Internalization , Animals , Classical Swine Fever , Classical Swine Fever Virus/pathogenicity , Humans , SwineABSTRACT
Inflammatory factors and type I interferons (IFNs) are key components of host antiviral innate immune responses, which can be released from the pathogen-infected macrophages. African swine fever virus (ASFV) has developed various strategies to evade host antiviral innate immune responses, including alteration of inflammatory responses and IFNs production. However, the molecular mechanism underlying inhibition of inflammatory responses and IFNs production by ASFV-encoded proteins has not been fully understood. Here we report that ASFV infection only induced low levels of IL-1ß and type I IFNs in porcine alveolar macrophages (PAMs), even in the presence of strong inducers such as LPS and poly(dA:dT). Through further exploration, we found that several members of the multigene family 360 (MGF360) and MGF505 strongly inhibited IL-1ß maturation and IFN-ß promoter activation. Among them, pMGF505-7R had the strongest inhibitory effect. To verify the function of pMGF505-7R in vivo, a recombinant ASFV with deletion of the MGF505-7R gene (ASFV-Δ7R) was constructed and assessed. As we expected, ASFV-Δ7R infection induced higher levels of IL-1ß and IFN-ß compared with its parental ASFV HLJ/18 strain. ASFV infection-induced IL-1ß production was then found to be dependent on TLRs/NF-κB signaling pathway and NLRP3 inflammasome. Furthermore, we demonstrated that pMGF505-7R interacted with IKKα in the IKK complex to inhibit NF-κB activation and bound to NLRP3 to inhibit inflammasome formation, leading to decreased IL-1ß production. Moreover, we found that pMGF505-7R interacted with and inhibited the nuclear translocation of IRF3 to block type I IFN production. Importantly, the virulence of ASFV-Δ7R is reduced in piglets compared with its parental ASFV HLJ/18 strain, which may due to induction of higher IL-1ß and type I IFN production in vivo. Our findings provide a new clue to understand the functions of ASFV-encoded pMGF505-7R and its role in viral infection-induced pathogenesis, which might help design antiviral agents or live attenuated vaccines to control ASF.
Subject(s)
African Swine Fever Virus/pathogenicity , African Swine Fever/immunology , Immune Evasion/immunology , Macrophages, Alveolar/immunology , Viral Proteins/immunology , African Swine Fever Virus/immunology , Animals , Immunity, Innate , Interferon Type I/biosynthesis , Interleukin-1beta/biosynthesis , Multigene Family , Swine , Virulence/immunologyABSTRACT
African swine fever is a severe animal infectious disease caused by African swine fever virus (ASFV), and the morbidity and mortality associated with virulent ASFV isolates are as high as 100%. Previous studies showed that the ability of ASFV to antagonize IFN production is closely related to its pathogenicity. Here, we report that ASFV HLJ/18 infection induced low levels of type I IFN and inhibited cGMP-AMP-induced type I IFN production in porcine alveolar macrophages that were isolated from specific pathogen-free Landrace piglets. Subsequently, an unbiased screen was performed to screen the ASFV genes with inhibitory effects on the type I IFN production. ASFV pI215L, a viral E2 ubiquitin-conjugating enzyme, was identified as one of the strongest inhibitory effectors on the production of type I IFN. Knockdown of pI215L expression inhibited ASFV replication and enhanced IFN-ß production. However, inhibition of type I IFN production by pI215L was independent of its E2 enzyme activity. Furthermore, we found that pI215L inhibited type I IFN production and K63-linked polyubiquitination of TANK-binding kinase 1 through pI215L-binding RING finger protein 138 (RNF138). ASFV pI215L enhanced the interaction between RNF138 and RNF128 and promoted RNF138 to degrade RNF128, which resulted in reduced K63-linked polyubiquitination of TANK-binding kinase 1 and type Ð IFN production. Taken together, our findings reveal a novel immune escape mechanism of ASFV, which provides a clue to the design and development of an immune-sensitive attenuated live vaccine.
Subject(s)
African Swine Fever Virus/immunology , Nucleotidyltransferases/immunology , Protein Serine-Threonine Kinases/immunology , Ubiquitin-Protein Ligases/immunology , Cells, Cultured , HEK293 Cells , Humans , Signal Transduction/immunology , UbiquitinationABSTRACT
Although hepatocellular carcinoma (HCC) is a type of carcinoma with high malignancy, high morbidity and low survival rate, its underlying mechanism has not been entirely elucidated. Abnormal lncRNAs involve many diseases such as neurological diseases, tumors, cardiovascular diseases, immune-mediated disorders, and genetic disorders. The abnormal expression of several lncRNAs is involved in HCC. This paper describes the roles of lncRNAs in promoting and maintaining tumor initiation and progression, interactions between lncRNAs and miRNAs, and exosomes associated with lncRNAs in HCC. We introduce their prospective clinical applications as tumor markers for the diagnosis, treatment, prognosis, and recurrence of HCC. This paper will help us better understand how lncRNAs produce their effects on HCC and provide general directions and strategies to conduct further research.
Subject(s)
Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/therapy , Liver Neoplasms/genetics , Liver Neoplasms/therapy , Metabolic Networks and Pathways/genetics , RNA, Long Noncoding/genetics , Animals , Carcinoma, Hepatocellular/diagnosis , Exosomes/genetics , Humans , Liver Neoplasms/diagnosisABSTRACT
Activation of quiescent hepatic stellate cells (HSCs) is the major event in liver fibrosis, along with enhancement of cell proliferation and overproduction of extracellular matrix. Recent findings suggest that senescence of activated HSCs might limit the development of liver fibrosis. The p53, a guardian of the genome is associated with liver fibrosis, has been shown to regulate HSCs senescence. In this study, we report that microRNA-145 (miR-145) and p53 were downregulated in vivo and in vitro, concomitant with the enhanced expression of zinc finger E-box binding homeobox 2 (ZEB2). In addition, overexpression of miR-145 and p53 led to upregulation of the number of senescence-associated ß-galactosidase-positive HSCs and the expression of senescence markers p16 and p21, along with the reduced abundance of HSC activation markers α-smooth muscle actin and type I collagen in activated HSCs. Furthermore, silencing of ZEB2 promoted senescence of activated HSCs. Moreover, we also demonstrated that miR-145 specifically targeted the 3'-untranslated regions of ZEB2. In vitro promoter regulation studies show that ZEB2 could bind to the E-box of the p53 promoter as well as inhibit its promoter activity and thus suppress the expression of p53, which in turn repressed activated HSCs senescence. Taken together, our results describe a novel miR-145-ZEB2-p53 regulatory line might participate in the senescence of activated HSCs and might carry potential therapeutic targets for restraining liver fibrosis.
Subject(s)
Cellular Senescence/genetics , Hepatic Stellate Cells/pathology , MicroRNAs/genetics , Tumor Suppressor Protein p53/genetics , Zinc Finger E-box Binding Homeobox 2/genetics , 3' Untranslated Regions/genetics , Animals , Cell Line , Cell Proliferation/genetics , Down-Regulation/genetics , Liver/pathology , Liver Cirrhosis/genetics , Male , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic/genetics , Signal Transduction/genetics , Up-Regulation/geneticsABSTRACT
TRAF family member-associated NF-κB activator (TANK) is a scaffold protein that assembles into the interferon (IFN) regulator factor 3 (IRF3)-phosphorylating TANK-binding kinase 1 (TBK1)-(IκB) kinase ε (IKKε) complex, where it is involved in regulating phosphorylation of the IRF3 and IFN production. However, the functions of TANK in encephalomyocarditis virus (EMCV) infection-induced type I IFN production are not fully understood. Here, we demonstrated that, instead of stimulating type I IFN production, the EMCV-HB10 strain infection potently inhibited Sendai virus- and polyI:C-induced IRF3 phosphorylation and type I IFN production in HEK293T cells. Mechanistically, EMCV 3C protease (EMCV 3C) cleaved TANK and disrupted the TANK-TBK1-IKKε-IRF3 complex, which resulted in the reduction in IRF3 phosphorylation and type I IFN production. Taken together, our findings demonstrate that EMCV adopts a novel strategy to evade host innate immune responses through cleavage of TANK.
Subject(s)
Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Cysteine Endopeptidases/metabolism , Encephalomyocarditis virus/enzymology , I-kappa B Kinase/antagonists & inhibitors , Interferon Regulatory Factor-3/antagonists & inhibitors , Interferon Type I/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Viral Proteins/metabolism , 3C Viral Proteases , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Substitution , Animals , Cell Line , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , Dogs , Gene Deletion , Humans , I-kappa B Kinase/chemistry , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Interferon Regulatory Factor-3/chemistry , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon Type I/biosynthesis , Mesocricetus , Mutagenesis, Site-Directed , Mutation , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphorylation , Protein Multimerization , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proteolysis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/geneticsABSTRACT
UNLABELLED: We isolated two H5N1 viruses, A/duck/Hunan/S4020/2008 (DK/08) and A/chicken/Guangxi/S2039/2009 (CK/09), from live-bird markets during routine surveillance and found that these two viruses are genetically similar but differ in their replication and virulence in mice. The CK/09 virus is lethal for mice with a 50% mouse lethal dose (MLD50) of 1.6 log10 50% egg infectious doses (EID50), whereas the DK/08 virus is nonpathogenic for mice with an MLD50 value of 6.2 log10 EID50. We explored the genetic basis of the virulence difference of these two viruses by generating a series of reassortant viruses and mutants in the lethal virus CK/09 background and evaluating their virulence in mice. We found that the PB1 gene of the DK/08 virus dramatically attenuated the virulence of the CK/09 virus and that the amino acid at position 622 in PB1 made an important contribution. We further demonstrated that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impaired the binding of PB1 to viral RNA, thereby dramatically decreasing the polymerase activity and attenuating H5N1 virus virulence in mice. Our results identify a novel virulence-related marker of H5N1 influenza viruses and provide a new target for live attenuated vaccine development. IMPORTANCE: H5N1 avian influenza viruses have caused the deaths of nearly 60% of the humans that they have infected since 1997 and clearly represent a threat to public health. A thorough understanding of the genetic basis of virulence determinants will provide important insights for antiviral drug and live attenuated vaccine development. Several virulence-related markers in the PB2, PA, M1, and NS1 proteins of H5N1 viruses have been identified. In this study, we isolated two H5N1 avian influenza viruses that are genetically similar but differ in their virulence in mice, and we identified a new virulence-related marker in the PB1 gene. We found that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impairs the binding of PB1 to viral RNA, thereby attenuating H5N1 virus virulence in mice. This newly identified virulence-related marker could be applied to the development of live attenuated vaccines against H5N1 influenza.
Subject(s)
Glycine/metabolism , Influenza A Virus, H5N1 Subtype/pathogenicity , Orthomyxoviridae Infections/pathology , Viral Proteins/metabolism , Virulence Factors/metabolism , Animals , Female , Glycine/genetics , Humans , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/physiology , Lethal Dose 50 , Mice, Inbred BALB C , Orthomyxoviridae Infections/virology , Reassortant Viruses/genetics , Reassortant Viruses/pathogenicity , Reassortant Viruses/physiology , Reverse Genetics , Survival Analysis , Viral Proteins/genetics , Virulence , Virulence Factors/genetics , Virus ReplicationABSTRACT
Manipulating the posttranslational modulator of p53 is central in the regulation of its activity and function. ISGylated p53 can be degraded by the 20S proteasome. During this process, HERC5/Ceb1, an IFN-induced HECT-type E3 ligase, mediated p53 ISGylation. In this study, we indicated that HERC5 was over-expressed in both HCC tissue samples and cell lines. Knockdown of HERC5 significantly induced the expression of p53, p21 and Bax/Bcl-2 in HCC cells, resulting in apoptosis augment. Whereas, opposite results were obtained by using HERC5 over-expression. On this basis, we screened a 7, 11-disubstituted quinazoline derivative HZ-6d that could bind to the HERC5 G-rich sequence in vitro. Interestingly, HZ-6d injection effectively delayed the growth of xenografts in nude mice. In vitro, HZ-6d significantly inhibited cell growth, suppressed cell migration, induced apoptosis in HCC cells. Further studies demonstrated the anti-cancer effect of HZ-6d was associated with down-regulation of HERC5 and accumulation of p53. Collectively, we demonstrated that HZ6d is a HERC5 G-quadruplex ligand with anti-tumor properties, an action that may offer an attractive idea for restoration of p53 function in cancers.
Subject(s)
Antineoplastic Agents/pharmacology , Benzofurans/pharmacology , Carcinoma, Hepatocellular/metabolism , Intracellular Signaling Peptides and Proteins/pharmacology , Liver Neoplasms/metabolism , Quinazolines/pharmacology , Tumor Suppressor Protein p53/metabolism , Antineoplastic Agents/administration & dosage , Benzofurans/administration & dosage , Cell Line, Tumor , Drug Delivery Systems/methods , Gene Expression Regulation, Neoplastic/drug effects , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Quinazolines/administration & dosage , Tumor Suppressor Protein p53/geneticsABSTRACT
TRAF family member-associated NF-κB activator (TANK) is a negative regulator of canonical NF-κB signaling in the Toll-like receptor- and B-cell receptor-mediated signaling pathways. However, functions of TANK in viral infection-mediated NF-κB activation remain unclear. Here, we reported that TANK was cleaved by encephalomyocarditis virus 3C at the 197 and 291 glutamine residues, which depends on its cysteine protease activity. In addition, encephalomyocarditis virus 3C impaired the ability of TANK to inhibit TRAF6-mediated NF-κB signaling. Interestingly, we found that several viral proteases encoded by the foot and mouth disease virus, porcine reproductive and respiratory syndrome virus, and equine arteritis virus also cleaved TANK. Our results suggest that TANK is a novel target of some viral proteases, indicating that some positive RNA viruses have evolved to utilize their major proteases to regulate NF-κB activation.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cysteine Endopeptidases/metabolism , Encephalomyocarditis virus/enzymology , NF-kappa B/metabolism , Proteolysis , TNF Receptor-Associated Factor 6/metabolism , Viral Proteins/metabolism , 3C Viral Proteases , Amino Acid Sequence , Cysteine Endopeptidases/genetics , Equartevirus/enzymology , Foot-and-Mouth Disease Virus/enzymology , HEK293 Cells , Humans , Molecular Sequence Data , Porcine respiratory and reproductive syndrome virus/enzymology , TNF Receptor-Associated Factor 6/antagonists & inhibitors , Viral Proteins/geneticsABSTRACT
Bombesin receptor subtype-3 (BRS3) is an important orphan G protein-coupled receptor that regulates energy homeostasis and insulin secretion. As a member of the bombesin receptor (BnR) family, the lack of known endogenous ligands and high-resolution structure has hindered the understanding of BRS3 signaling and function. We present two cryogenic electron microscopy (cryo-EM) structures of BRS3 in complex with the heterotrimeric Gq protein in its active states: one bound to the pan-BnR agonist BA1 and the other bound to the synthetic BRS3-specific agonist MK-5046. These structures reveal the architecture of the orthosteric ligand pocket underpinning molecular recognition and provide insights into the structural basis for BRS3's selectivity and low affinity for bombesin peptides. Examination of conserved micro-switches suggests a shared activation mechanism among BnRs. Our findings shed light on BRS3's ligand selectivity and signaling mechanisms, paving the way for exploring its therapeutic potential for diabetes, obesity, and related metabolic disorders.
ABSTRACT
Glycogen synthase kinase-3ß (GSK3ß) not only plays a crucial role in regulating sperm maturation but also is pivotal in orchestrating the acrosome reaction. Here, we integrated single-molecule long-read and short-read sequencing to comprehensively examine GSK3ß expression patterns in adult Diannan small-ear pig (DSE) testes. We identified the most important transcript ENSSSCT00000039364 of GSK3ß, obtaining its full-length coding sequence (CDS) spanning 1263 bp. Gene structure analysis located GSK3ß on pig chromosome 13 with 12 exons. Protein structure analysis reflected that GSK3ß consisted of 420 amino acids containing PKc-like conserved domains. Phylogenetic analysis underscored the evolutionary conservation and homology of GSK3ß across different mammalian species. The evaluation of the protein interaction network, KEGG, and GO pathways implied that GSK3ß interacted with 50 proteins, predominantly involved in the Wnt signaling pathway, papillomavirus infection, hippo signaling pathway, hepatocellular carcinoma, gastric cancer, colorectal cancer, breast cancer, endometrial cancer, basal cell carcinoma, and Alzheimer's disease. Functional annotation identified that GSK3ß was involved in thirteen GOs, including six molecular functions and seven biological processes. ceRNA network analysis suggested that DSE GSK3ß was regulated by 11 miRNA targets. Furthermore, qPCR expression analysis across 15 tissues highlighted that GSK3ß was highly expressed in the testis. Subcellular localization analysis indicated that the majority of the GSK3ß protein was located in the cytoplasm of ST (swine testis) cells, with a small amount detected in the nucleus. Overall, our findings shed new light on GSK3ß's role in DSE reproduction, providing a foundation for further functional studies of GSK3ß function.
Subject(s)
Glycogen Synthase Kinase 3 beta , Spermatogenesis , Animals , Glycogen Synthase Kinase 3 beta/genetics , Glycogen Synthase Kinase 3 beta/metabolism , Male , Swine/genetics , Spermatogenesis/genetics , Testis/metabolism , Phylogeny , Gene Expression RegulationABSTRACT
Prostaglandin F2α (PGF2α), an endogenous arachidonic acid metabolite, regulates diverse physiological functions in many tissues and cell types through binding and activation of a G-protein-coupled receptor (GPCR), the PGF2α receptor (FP), which also is the primary therapeutic target for glaucoma and several other diseases. Here, we report cryo-electron microscopy (cryo-EM) structures of the human FP bound to endogenous ligand PGF2α and anti-glaucoma drugs LTPA and TFPA at global resolutions of 2.67 Å, 2.78 Å, and 3.14 Å. These structures reveal distinct features of FP within the lipid receptor family in terms of ligand binding selectivity, its receptor activation, and G protein coupling mechanisms, including activation in the absence of canonical PIF and ERY motifs and Gq coupling through direct interactions with receptor transmembrane helix 1 and intracellular loop 1. Together with mutagenesis and functional studies, our structures reveal mechanisms of ligand recognition, receptor activation, and G protein coupling by FP, which could facilitate rational design of FP-targeting drugs.
Subject(s)
GTP-Binding Proteins , Prostaglandins , Humans , Cryoelectron Microscopy , Ligands , Arachidonic AcidABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious porcine pathogen that causes serious economic losses to the world swine industry. The inhibitor kappa B kinase ß (IKKß), a catalytic subunit of the IKK complex, plays multiple roles in regulating the nuclear transcription factor kappa B (NF-κB) activity and a variety of cytokines transcription involved in immune responses. Here, we reported that the nonstructural protein 4 (Nsp4) of PRRSV cleaved IKKß at the E378 site to inhibit the activation of NF-κB signaling pathway. Additionally, we clearly showed that cleavage of IKKß by PRRSV Nsp4 depends on the 3 C-like serine protease activity of Nsp4 because the catalytically inactivate mutants of Nsp4 lost the function to cleave IKKß. Furthermore, we found that hydrophobic patch at the KD-ULD junction of IKKß could be disrupted by PRRSV Nsp4 via the cleavage of the E378 site, resulting in disruption of NF-κB activity. Of note, the two cleavage fragments of IKKß lose their function to phosphorylate IκBα and activate NF-κB signaling pathway. Our findings provide a clue to better understand the pathogenic mechanism of PRRSV involved in PRRSV evasion of host antiviral innate immune responses.
Subject(s)
Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Swine Diseases , Swine , Animals , Porcine respiratory and reproductive syndrome virus/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Cell Line , Signal TransductionABSTRACT
African swine fever (ASF) is a severe hemorrhagic disease in swine characterized by massive lymphocyte depletion and cell death, with apoptosis and necrosis in infected lymphoid tissues. However, the molecular mechanism regarding ASFV-induced cell death remains largely unknown. In this study, 94 ASFV-encoded proteins were screened to determine the viral proteins involved in cell death in vitro, and pE199L showed the most significant effect. Ectopic expression of pE199L in porcine cells (CRL-2843) and human cells (HEK293T and HeLa cells) induced cell death remarkably, showing obvious shrinking, blistering, apoptotic bodies, and nuclear DNA fragments. Meanwhile, cell death was markedly alleviated when the expression of pE199L was knocked down during ASFV infection. Additionally, the expression of pE199L caused a loss of mitochondrial membrane potential, release of cytochrome C, and caspase-9 and -3/7 activation, indicating that the mitochondrial apoptotic pathway was involved in pE199L-induced apoptosis. Further investigations showed that pE199L interacted with several anti-apoptotic BCL-2 subfamily members (such as BCL-XL, MCL-1, BCL-W, and BCL-2A1) and competed with BAK for BCL-XL, which promoted BAK and BAX activation. Taken together, ASFV pE199L induces the mitochondrial-dependent apoptosis, which may provide clues for a comprehensive understanding of ASFV pathogenesis.