Oncostatin M receptor beta (OSMRβ) is an essential signal-transducing subunit in the IL-31/IL-31 receptor alpha (IL-31RA)/OSMRβ complex and is implicated in inflammatory and pruritic diseases. We previously developed a high-affinity monoclonal antibody (2O2) that targets the proximal extracellular domain (amino acids 619-734) of canine OSMRβ (cOSMRβ) and shows no cross-reactivity with human OSMRβ (hOSMRβ). Here, we elucidate its functional mechanism of action and determine the co-crystal structure of the 2O2 Fab fragment in complex with this domain at a resolution of 2.76 Å. Consistent with the epitope location far away from the cytokine-binding domain (CBD) of cOSMRβ, 2O2 does not compete with canine IL-31 (cIL-31) for cOSMRβ binding. However, the in vitro functional assay demonstrated that 2O2 inhibits cIL-31-induced phosphorylation of STAT5 in DH-82 cells. Mechanistically, this inhibition is achieved through antibody-induced internalization of cOSMRβ from the cell surface. Structural analysis reveals that 2O2 binds at an oblique angle relative to the membrane, creating steric hindrance that induces localized lipid bilayer invagination. The bivalent IgG exhibits ∼34-fold higher potency than its monovalent Fab, reflecting not only avidity but also mechanical consequences of dual receptor engagement: crosslinking two receptor complexes exerts a stretching force that amplifies membrane invagination and promotes efficient endocytic uptake. This mechanism enables clearance of the entire signaling complex, including the cytokine, from the cell surface-a functional advantage over conventional cytokine-blocking antibodies. These findings establish 2O2 as a novel canine therapeutic candidate with a distinct internalization-driven mechanism of action.

Cholesterol (CHL) serves as a building block for membrane biogenesis and a precursor to oxysterols, steroid hormones, bile acids, and vitamin D. The lysosome serves as a major sorting station for low-density lipoproteins (LDLs), which carry dietary CHL, and it is also the cellular site where the master growth regulator, the protein kinase mechanistic Target of Rapamycin Complex 1 (mTORC1), is activated. Recently, the lysosomal transmembrane protein GPR155 was reported to signals CHL sufficiency to mTORC1 through sequestration of the GTPase-activating protein towards the Rags 1 (GATOR1). Although the recently reported structures of GPR155 have revealed the CHL binding site, how the signal is transduced from the CHL binding site to the soluble parts of GPR155 and GATOR1 remains unknown. Here, with our three cryo-EM structures of GPR155 captured in different conformations in complex with CHL, complemented by long-time scale molecular dynamics simulations, the dynamic rearrangement of different domains was observed. CHL binding induces a widening of the crevice between the transporter and GPCR domains. The extending helix preceding transmembrane helix (TM) 16, which was unresolved in other structures, acts as a linkage lever that transmits the rotation of the GPCR domain to the soluble parts of GPR155 in response to CHL binding. This work not only answers the question of how CHL is sensed by GPR155, but also addresses a more profound question: how the signal perceived by the TMs regions is transduced to the LED and DEP domains.

In 2018, two novel influenza-like virus genomes were first identified in basal vertebrates: the Asiatic toads (Bufo gargarizans) and spiny eels (Mastacembelus aculeatus). Their hemagglutinin (HA) proteins exhibit remarkably low amino acid sequences homology (23.0% and 42.8%, respectively) compared to influenza B virus (IBV), their closest canonical influenza virus relative. This study revealed that the Asiatic toad influenza-like virus HA (tHA) demonstrates dual receptor specificity, bound both α2-3 (avian-type) and α2-6 (human-type) sialic acid (SA) receptors, whereas the spiny eel influenza-like virus HA (eHA) lacks this capability. Biophysical characterization showed reduced thermal stability (lower Tm values) for both tHA and eHA compared to canonical influenza HA. Furthermore, we determined the cryo-EM structures of apo-tHA, tHA in complex with either α2-3 SA receptor or α2-6 SA receptor, as well as apo-eHA and eHA bound to GM2 complex. Our analysis revealed that tHA has a shorter length and looser HA trimer packing compared to canonical HA. These findings collectively indicate that influenza-like viruses in basal vertebrates have evolutionarily acquired dual SA receptor-binding capacity, a trait critical for cross-species transmission in influenza viruses. However, the observed thermolability of these HA proteins suggests that host physiological temperatures may impose a barrier to zoonotic spillover.

Pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenases (MDHs), the periplasmic metalloenzymes in Gram-negative methylotrophic bacteria, play a pivotal role in methane and methanol bio-utilization. Although the structures of many PQQ-dependent MDHs have been resolved, including the canonical heterotetrameric enzymes composed of two MxaF and two MxaI subunits with a molecule of PQQ and a calcium ion in the active site in MxaF, the biogenesis of these enzymes remains elusive. Here, we characterize a chaperone, MxaJ, responsible for PQQ incorporation by reconstructing a PQQ-dependent MDH assembly system in Escherichia coli. Using cryo-electron microscopy, we capture the structures of the intermediate complexes formed by the chaperone MxaJ and catalytic subunit MxaF during PQQ-dependent MDH maturation, revealing a chaperone-mediated molecular mechanism of cofactor incorporation. These findings not only advance our understanding on the biogenesis of PQQ-dependent MDH, but also provide an alternative engineering way for methane and methanol bioconversion.

Interleukin-31 (IL-31) signals through the IL-31 receptor alpha (IL-31RA) and oncostatin M receptor beta (OSMRβ) heterodimer, mediating pruritus, dermatitis, inflammatory responses, neuroimmune interactions, and certain cancers. Here, we present the crystal structure of canine IL-31 (cIL-31) in complex with a neutralizing caninized monoclonal antibody (2D10-2). This antibody competitively inhibited cIL-31 binding to canine OSMRβ (cOSMRβ) but not to canine IL-31RA (cIL-31RA). Moreover, it effectively blocked cIL-31-induced STAT5 phosphorylation in vitro and alleviated cIL-31-induced pruritus in beagle dogs. Structural analysis identified key antibody-binding residues in α-helical A, α-helical D, and the AB loop of cIL-31. Systematic mutagenesis based on the complex structure further defined the conformational epitopes of cIL-31 recognized by cOSMRβ. In summary, this study reports the IL-31 structure, revealing a four-α-helical bundle cytokine, and elucidates 2D10-2's neutralizing mechanism by targeting the cIL-31-cOSMRβ interaction. These findings advance our understanding of IL-31 and offer insights for developing IL-31-targeted therapeutics.

Various precise gene editing techniques at the DNA/RNA level,driven by clustered regularly interspaced short palin-drome repeats(CRISPR)/CRISPR-associated protein 9(Cas9)technology,have gained significant prominence.Yet,research on targeted protein editing techniques remains limited.Only a few attempts have been made,including the use of specific proteases and de-O-glycosylating enzymes as editing enzymes.Here,we propose direct editing of N-glycosylated proteins using de-N-glycosylating enzymes to modify N-glycosylation and simultaneously alter the rele-vant asparagine residue to aspartate in living cells.Selective protein deglycosylation editors were developed by fusing high-affinity protein-targeting peptides with active peptide:N-glycanases(PNGases).Three crucial cell membrane pro-teins,programmed cell death protein-1(PD-1),programmed cell death-1 ligand 1(PD-L1),and severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)spike protein,were chosen to be tested as a proof of concept.N-linked glycans were removed,and the relevant sites were converted from Asn to Asp in living mammalian cells,destabilizing target proteins and accelerating their degradation.Further investigation focused on SARS-CoV-2 spike protein deglycosyla-tion editing.The collaboration of LCB1-PNGase F(PNGF)effectively reduced syncytia formation,inhibited pseudovirus packaging,and significantly hindered virus entry into host cells,which provides insights for coronavirus disease 2019(COVID-19)treatment.This tool enables editing protein sequences post-de-N-glycosylation in living human cells,shed-ding light on protein N-glycosylation functions,and Asn to Asp editing in organisms.It also offers the potential for devel-oping protein degradation technologies.

Various precise gene editing techniques at the DNA/RNA level, driven by clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology, have gained significant prominence. Yet, research on targeted protein editing techniques remains limited. Only a few attempts have been made, including the use of specific proteases and de-O-glycosylating enzymes as editing enzymes. Here, we propose direct editing of N-glycosylated proteins using de-N-glycosylating enzymes to modify N-glycosylation and simultaneously alter the relevant asparagine residue to aspartate in living cells. Selective protein deglycosylation editors were developed by fusing high-affinity protein-targeting peptides with active peptide:N-glycanases (PNGases). Three crucial cell membrane proteins, PD-1, PD-L1, and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein, were chosen to be tested as a proof of concept. N-linked glycans were removed, and the relevant sites were converted from Asn to Asp in living mammalian cells, destabilizing target proteins and accelerating their degradation. Further investigation focused on SARS-CoV-2 spike protein deglycosylation editing. The collaboration of LCB1-PNGase F (PNGF) effectively reduced syncytia formation, inhibited pseudovirus packaging, and significantly hindered virus entry into host cells, which provides insights for coronavirus disease 2019 (COVID-19) treatment. This tool enables editing protein sequences post-de-N-glycosylation in living human cells, shedding light on protein N-glycosylation functions and Asn to Asp editing in organisms. It also offers the potential for developing protein degradation technologies.

EfpA, the first major facilitator superfamily (MFS) protein identified inMycobacterium tuberculosis(Mtb), is an essential efflux pump implicated in resistance to multiple drugs. EfpA-inhibitors have been developed to kill drug-tolerant Mtb. However, the biological function of EfpA has not yet been elucidated. Here, we present the cryo-EM structures of EfpA complexed with lipids or the inhibitor BRD-8000.3 at resolutions of 2.9 Å and 3.4 Å, respectively. Unexpectedly, EfpA forms an antiparallel dimer. Functional studies reveal that EfpA is a lipid transporter and BRD-8000.3 inhibits its lipid transport activity. Intriguingly, the mutation V319F, known to confer resistance to BRD-8000.3, alters the expression level and oligomeric state of EfpA. Based on our results and the observation of other antiparallel dimers in the MFS family, we propose an antiparallel-function model of EfpA. Collectively, our work provides structural and functional insights into EfpA’s role in lipid transport and drug resistance, which would accelerate the development of antibiotics against this promising drug target.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high fatality rate of up to 30% caused by SFTS virus (SFTSV). However, no specific vaccine or antiviral therapy has been approved for clinical use. To develop an effective treatment, we isolated a panel of human monoclonal antibodies (mAbs). SF5 and SF83 are two neutralizing mAbs that recognize two viral glycoproteins (Gn and Gc), respectively. We found that their epitopes are closely located, and we then engineered them as several bispecific antibodies (bsAbs). Neutralization and animal experiments indicated that bsAbs display more potent protective effects than the parental mAbs, and the cryoelectron microscopy structure of a bsAb3 Fab-Gn-Gc complex elucidated the mechanism of protection. In vivo virus passage in the presence of antibodies indicated that two bsAbs resulted in less selective pressure and could efficiently bind to all single parental mAb-escape mutants. Furthermore, epitope analysis of the protective mAbs against SFTSV and RVFV indicated that they are all located on the Gn subdomain I, where may be the hot spots in the phleboviruses. Collectively, these data provide potential therapeutic agents and molecular basis for the rational design of vaccines against SFTSV infection.

Interleukin-31 (IL-31), belonging to the IL-6 cytokine family, is involved in skin inflammation and pruritus, as well as some tumors' progression. Here, we reported the expression and purification of recombinant human IL-31 (rhIL-31) using a prokaryotic system. This recombinant protein was expressed in the form of inclusion bodies, refolded and purified by size-exclusion chromatography. Circular dichroism analysis revealed that the secondary structure of rhIL-31 was mainly composed of alpha-helix, which is in consistence with the 3D model structure built by AlphaFold server. In vitro studies showed that rhIL-31 exhibited a good binding ability to the recombinant hIL-31 receptor alpha fused with human Fc fragment (rhIL-31RA-hFc) with EC50 value of 16.36 µg/mL in ELISA assay. Meanwhile, flow cytometry demonstrated that rhIL-31 was able to bind to hIL-31RA or hOSMRβ expressed on the cell surface, independently. Furthermore, rhIL-31 could induce the phosphorylation of STAT3 in A549 cells. In conclusion, the prepared rhIL-31 in this study possesses the binding ability to its receptors, and can activate the signal pathway of JAK/STAT. Thus, it can be applied in further studies, including investigation of hIL-31-related diseases, structural analysis, and development of therapeutic drugs, and monoclonal antibodies targeting hIL-31.

Interleukin-31 (IL-31) is a pro-inflammatory cytokine involved in skin inflammation and tumor progression. The IL-31 signaling cascade is initiated by its binding to two receptors, IL-31 receptor alpha (IL-31RA) and oncostatin M receptor subunit beta (OSMRβ). The previous study suggested that human IL-31 (hIL-31) directly interacts with IL-31RA and OSMRβ, independently, but the binding ability of hIL-31 to IL-31RA is stronger than to OSMRβ. In different to its human ortholog, feline IL-31 (fIL-31) has a higher binding affinity for feline OSMRβ. However, the binding pattern of canine IL-31 to its receptors remains to be elucidated. In this study, we purified the recombinant canine IL-31 (rcIL-31) protein and revealed its secondary structure to be mainly composed of alpha-helices. Moreover, in vitro studies show that rcIL-31 has the ability to induce the phosphorylation of signal transducer activator of transcription 3 (STAT3) and STAT5 in DH-82 cells. In the following, the binding efficacies of bioactive rcIL-31 for its individual receptor components have been measured using a flow cytometry assay. The result demonstrates that correctly refolded rcIL-31 binds independently with cIL-31RA and cOSMRβ which were expressed on the cell surface. Of note, rcIL-31 has a greater than tenfold higher affinity to OSMRβ than to IL-31RA. Additionally, we demonstrated that D1–D4, especially D4 of cOSMRβ, is crucial for its binding to cIL-31. Furthermore, this study proved that rcIL-31 has a high binding affinity to the soluble cOSMRβ with a KD value of 3.59 × 10–8 M. The results presented in the current study will have a significant implication in the development of drugs or antibodies against diseases induced by cIL-31 signaling.

Influenza B virus is one of the causes for seasonal influenza, which can account for serious illness or even death in some cases. We tested the expression of extracellular domain of hemagglutinin (HA-ecto) of influenza B viruses in mammalian cells, and then determined the immunogenicity of HA-ecto in mice. The gene sequence encoding influenza B virus HA-ecto, foldon sequence, and HIS tag was optimized and inserted into pCAGGS vector. The opening reading frame (ORF) of neuraminidase was also cloned into pCAGGS. The pCAGGS-HA-ecto and pCAGGS-NA were co-transfected into 293T cells using linear polyethylenimine. Cell supernatant after transfection was collected after 96 h, and the secreted trimmeric HA-ecto protein was purified by nickel ion affinity chromatography and size exclusion chromatography. Subsequently, the mice were immunized with HA-ecto protein, and the corresponding antibody titers were detected by ELISA and hemagglutination inhibition (HAI) assays. The results showed that soluble trimeric HA-ecto protein could be obtained using mammalian cell expression system. Moreover, trimeric HA-ecto protein, in combination with the adjuvant, induced high levels of ELISA and HAI antibodies against homogenous and heterologous antigens in mice. Thus, the soluble HA-ecto protein expressed in mammalian cells could be used as a recombinant subunit vaccine candidate for influenza B virus.

Oncostatin M receptor beta (OSMRβ) mediates signaling of Oncostatin M (OSM) and interleukine-31 (IL-31), two key cytokines involved in many important biological processes including inflammation and cancer progression. More importantly, OSMRβ might be a potential biomarker and therapeutic target for some diseases, such as inflammatory bowel disease, pruritus and ovarian cancer. In this study, soluble recombinant canine OSMRβ (cOSMRβ) was experimentally expressed as a native antigen to develop an effective cOSMRβ-specific monoclonal antibody (mAb), 2O2, using hybridoma technology. It was demonstrated that 2O2 is able to detect OSMRβ expressed on cell surface using immunofluorescence assay (IFA) and flow cytometry (FACS). This mAb exhibits very high binding affinity to cOSMRβ with the KD and half-maximal effective concentration (EC50) values of 2.49 nM and 96.96 ng/ml, respectively. Meanwhile, it didn't show any cross-relativities with feline OSMRβ (fOSMRβ) and human OSMRβ (hOSMRβ). Moreover, we determined the binding epitope of 2O2, which localizes in the domain VI (DVI, amino acids 623-734) of cOSMRβ. In conclusion, this novel mAb, 2O2, can be used in immunoassays, including IFA, FACS and enzyme-linked immunosorbent assay (ELISA) to facilitate studies in dogs.

Influenza virus neuraminidase (NA) is an important target for antiviral development because it plays a crucial role in releasing newly assembled viruses. Two unique influenza-like virus genomes were recently reported in the Wuhan Asiatic toad and Wuhan spiny eel. Their NA genes appear to be highly divergent from all known influenza NAs, raising key questions as to whether the Asiatic toad influenza-like virus NA (tNA) and spiny eel NA (eNA) have canonical NA activities and structures and whether they show sensitivity to NA inhibitors (NAIs). Here, we found that both tNA and eNA have neuraminidase activities. A detailed structural analysis revealed that tNA and eNA present similar overall structures to currently known NAs, with a conserved calcium binding site. Inhibition assays indicated that tNA is resistant to NAIs, while eNA is still sensitive to NAIs. E119 is conserved in canonical NAs. The P119E substitution in tNA can restore sensitivity to NAIs, and, in contrast, the E119P substitution in eNA decreased its sensitivity to NAIs. The structures of NA-inhibitor complexes further provide a detailed insight into NA-inhibitor interactions at the atomic level. Moreover, tNA and eNA have unique N-glycosylation sites compared with canonical NAs. Collectively, the structural features, NA activities, and sensitivities to NAIs suggest that fish-and amphibian-derived influenza-like viruses may circulate in these vertebrates. More attention should be paid to these influenza-like viruses because their NA molecules may play roles in the emergence of NAI resistance.

Significance Neuraminidase (NA) is a good target for antiviral drugs due to its function of releasing progeny viruses. Recently, two influenza-like virus genomes derived from Asiatic toad and spiny eel were discovered. They appear to be highly divergent from all known influenza NAs, raising key questions about whether tNA and eNA have canonical activities and can be inhibited by NA inhibitors (NAIs). Here, we demonstrated that these two NAs display canonical NA activities and overall structures. However, eNA is sensitive to NAIs, while tNA is highly resistant to multiple NAIs. We found that residue 119 is the key amino acid that determines sensitivity to NAIs and further illustrated the drug-resistance mechanism by solving the high-resolution crystal structures of NA–NAI complexes.

The avian influenza A (H7N9) virus is a zoonotic virus that is closely associated with live poultry markets. It has caused infections in humans in China since 2013. Five waves of the H7N9 influenza epidemic occurred in China between March 2013 and September 2017. H7N9 with low-pathogenicity dominated in the first four waves, whereas highly pathogenic H7N9 influenza emerged in poultry and spread to humans during the fifth wave, causing wide concern. Specialists and officials from China and other countries responded quickly, controlled the epidemic well thus far, and characterized the virus by using new technologies and surveillance tools that were made possible by their preparedness efforts. Here, we review the characteristics of the H7N9 viruses that were identified while controlling the spread of the disease. It was summarized and discussed from the perspectives of molecular epidemiology, clinical features, virulence and pathogenesis, receptor binding, T-cell responses, monoclonal antibody development, vaccine development, and disease burden. These data provide tools for minimizing the future threat of H7N9 and other emerging and re-emerging viruses, such as SARS-CoV-2.

SARS-CoV-2 can infect many domestic animals, including dogs. Herein, we show that dog angiotensin-converting enzyme 2 (dACE2) can bind to the SARS-CoV-2 spike (S) protein receptor binding domain (RBD), and that both pseudotyped and authentic SARS-CoV-2 can infect dACE2-expressing cells. We solved the crystal structure of RBD in complex with dACE2 and found that the total number of contact residues, contact atoms, hydrogen bonds and salt bridges at the binding interface in this complex are slightly fewer than those in the complex of the RBD and human ACE2 (hACE2). This result is consistent with the fact that the binding affinity of RBD to dACE2 is lower than that of hACE2. We further show that a few important mutations in the RBD binding interface play a pivotal role in the binding affinity of RBD to both dACE2 and hACE2. Our work reveals a molecular basis for cross-species transmission and potential animal spread of SARS-CoV-2, and provides new clues to block the potential transmission chains of this virus.

SARS-CoV-2 can infect many domestic animals, including dogs. Herein, we show that dog angiotensin converting enzyme 2 (dACE2) can bind to SARS-CoV-2 spike (S) protein receptor binding region (RBD), and that both pseudotyped and authentic SARS-CoV-2 can infect dACE2-expressing cells. we solved the crystal structure of RBD in complex with dACE2 and found that the total numbers of contact residues, contact atoms, hydrogen bonds and salt bridges at the binding interface in this complex are slightly fewer than those in the complex of the RBD and human ACE2 (hACE2). This result is consistent with the fact that the binding affinity of RBD to dACE2 is lower than that to hACE2. We further show that a few important mutations in the RBD binding interface play a pivotal role in the binding affinity of RBD to both dACE2 and hACE2, and need intense monitoring and controlling.