Structural basis for activation of somatostatin receptor 5 by cyclic neuropeptide agonists.

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.

Transcriptional Regulation Analysis Provides Insight into the Function of GSK3ß Gene in Diannan Small-Ear Pig Spermatogenesis.

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.

African swine fever virus A137R assembles into a dodecahedron cage.

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.

Ligand-induced activation and G protein coupling of prostaglandin F2α receptor.

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.

Porcine reproductive and respiratory syndrome virus infection inhibits NF-κB signaling pathway through cleavage of IKKß by Nsp4.

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.

African swine fever virus cysteine protease pS273R inhibits pyroptosis by noncanonically cleaving gasdermin D.

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.

African Swine Fever Virus pI215L Negatively Regulates cGAS-STING Signaling Pathway through Recruiting RNF138 to Inhibit K63-Linked Ubiquitination of TBK1.

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 І 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.

African Swine Fever Virus pE199L Induces Mitochondrial-Dependent Apoptosis.

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.

pMGF505-7R determines pathogenicity of African swine fever virus infection by inhibiting IL-1ß and type I IFN production.

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.

ADAM17 is an essential attachment factor for classical swine fever virus.

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.

Long non-coding RNAs in hepatocellular carcinoma: Ordering of the complicated lncRNA regulatory network and novel strategies for HCC clinical diagnosis and treatment.

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.

Crystal Structure of African Swine Fever Virus dUTPase Reveals a Potential Drug Target.

E165R, a highly specific dUTP nucleotidohydrolase (dUTPase) encoded by the African swine fever virus (ASFV) genome, is required for productive replication of ASFV in swine macrophages. Here, we solved the high-resolution crystal structures of E165R in its apo state and in complex with its product dUMP. Structural analysis explicitly defined the architecture of the active site of the enzyme as well as the interaction between the active site and the dUMP ligand. By comparing the ASFV E165R structure with dUTPase structures from other species, we found that the active site of E165R is highly similar to those of dUTPases from Mycobacterium tuberculosis and Plasmodium falciparum, against which small-molecule chemicals have been developed, which could be the potential drug or lead compound candidates for ASFV. Our results provide important basis for anti-ASFV drug design by targeting E165R.IMPORTANCE African swine fever virus (ASFV), an Asfivirus affecting pigs and wild boars with up to 100% case fatality rate, is currently rampaging throughout China and some other countries in Asia. There is an urgent need to develop therapeutic and preventive reagents against the virus. Our crystallographic and biochemical studies reveal that ASFV E165R is a member of trimeric dUTP nucleotidohydrolase (dUTPase) family that catalyzes the hydrolysis of dUTP into dUMP. Our apo-E165R and E165R-dUMP structures reveal the constitutive residues and the configuration of the active center of this enzyme in rich detail and give evidence that the active center of E165R is very similar to that of dUTPases from Plasmodium falciparum and Mycobacterium tuberculosis, which have already been used as targets for designing drugs. Therefore, our high-resolution structures of E165R provide useful structural information for chemotherapeutic drug design.

Molecular Basis of Arthritogenic Alphavirus Receptor MXRA8 Binding to Chikungunya Virus Envelope Protein.

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.

Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B.

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.

DDX19 Inhibits Type I Interferon Production by Disrupting TBK1-IKKε-IRF3 Interactions and Promoting TBK1 and IKKε Degradation.

DExD/H-box helicase members are key receptors for recognizing viral nucleic acids, and they regulate retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-mediated type I interferon (IFN) production. Here, we report that the DExD/H-box helicase family member DExD/H-box RNA helicase 19 (DDX19) is a negative regulator of type I IFN production. Ectopic expression of DDX19 suppressed poly(I:C) (polyinosinic-polycytidylic acid)- and Sendai-virus-induced type I IFN production, whereas knockdown of DDX19 expression enhanced type I IFN production. Mechanistically, DDX19 inhibited TANK-binds kinase 1 (TBK1)- and inhibitor-κb kinase ε (IKKε)-mediated phosphorylation of interferon regulatory factor 3 (IRF3) by disrupting the interaction between TBK1 or IKKε and IRF3. Additionally, DDX19 recruited Lamtor2 and then formed the TBK1-IKKε-Lamtor2-DDX19-IRF3 complex to suppress IFN production by promoting TBK1 and IKKε degradation. We generated Ddx19 knockout mice using transcription activator-like effector nucleases (TALENs) and found that Ddx19 deficiency in vivo augmented type I IFN production, resulting in suppression of encephalomyocarditis virus replication. These data show that DDX19 is an important negative regulator of RLR-mediated type I IFN production.

MicroRNA-145 induces the senescence of activated hepatic stellate cells through the activation of p53 pathway by ZEB2.

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.