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The subcortical maternal complex (SCMC) is essential for safeguarding female fertility in mammals. Assembled in oocytes, the SCMC maintains the cleavage of early embryos, but the underlying mechanism remains unclear. Here, we report that 14-3-3, a multifunctional protein, is a component of the SCMC. By resolving the structure of the 14-3-3-containing SCMC, we discover that phosphorylation of TLE6 contributes to the recruitment of 14-3-3. Mechanistically, during maternal-to-embryo transition, the SCMC stabilizes 14-3-3 protein and contributes to the proper control of CDC25B, thus ensuring the activation of the maturation-promoting factor and mitotic entry in mouse zygotes. Notably, the SCMC establishes a conserved molecular link with 14-3-3 and CDC25B in human oocytes/embryos. This study discloses the molecular mechanism through which the SCMC regulates the cell cycle in early embryos and elucidates the function of the SCMC in mammalian early embryogenesis. The subcortical maternal complex (SCMC) maintains cleavage of early embryos. Here the authors identify 14-3-3 as a component of the SCMC and show that phosphorylation of TLE6 contributes to 14-3-3 recruitment and stabilization, ensuring mitotic entry in mouse zygotes. [ABSTRACT FROM AUTHOR]

In developing B cells, V(D)J recombination assembles exons encoding IgH and Igκ variable regions from hundreds of gene segments clustered across Igh and Igk loci. V, D and J gene segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease1. RAG orchestrates Igh V(D)J recombination upon capturing a JH-RSS within the JH-RSS-based recombination centre1–3 (RC). JH-RSS orientation programmes RAG to scan upstream D- and VH-containing chromatin that is presented in a linear manner by cohesin-mediated loop extrusion4–7. During Igh scanning, RAG robustly utilizes only D-RSSs or VH-RSSs in convergent (deletional) orientation with JH-RSSs4–7. However, for Vκ-to-Jκ joining, RAG utilizes Vκ-RSSs from deletional- and inversional-oriented clusters8, inconsistent with linear scanning2. Here we characterize the Vκ-to-Jκ joining mechanism. Igk undergoes robust primary and secondary rearrangements9,10, which confounds scanning assays. We therefore engineered cells to undergo only primary Vκ-to-Jκ rearrangements and found that RAG scanning from the primary Jκ-RC terminates just 8 kb upstream within the CTCF-site-based Sis element11. Whereas Sis and the Jκ-RC barely interacted with the Vκ locus, the CTCF-site-based Cer element12 4 kb upstream of Sis interacted with various loop extrusion impediments across the locus. Similar to VH locus inversion7, DJH inversion abrogated VH-to-DJH joining; yet Vκ locus or Jκ inversion allowed robust Vκ-to-Jκ joining. Together, these experiments implicated loop extrusion in bringing Vκ segments near Cer for short-range diffusion-mediated capture by RC-based RAG. To identify key mechanistic elements for diffusional V(D)J recombination in Igk versus Igh, we assayed Vκ-to-JH and D-to-Jκ rearrangements in hybrid Igh–Igk loci generated by targeted chromosomal translocations, and pinpointed remarkably strong Vκ and Jκ RSSs. Indeed, RSS replacements in hybrid or normal Igk and Igh loci confirmed the ability of Igk-RSSs to promote robust diffusional joining compared with Igh-RSSs. We propose that Igk evolved strong RSSs to mediate diffusional Vκ-to-Jκ joining, whereas Igh evolved weaker RSSs requisite for modulating VH joining by RAG-scanning impediments.Experiments in mouse models, and in cell lines that only allow primary Vκ-to-Jκ rearrangements, enable characterization of the mechanisms of V(D)J recombination. [ABSTRACT FROM AUTHOR]

This document, published in the journal Cell Discovery, provides a detailed analysis of the structure and binding mechanisms of the CXCR3 receptor and its ligands, CXCL10 and AMG487. The study utilized cryo-electron microscopy to determine the structures of the CXCR3-CXCL10 and CXCR3-AMG487 complexes. The findings suggest that both ligands bind to specific regions of the receptor and contribute to its activation. The study also identified the presence of lipids in the CXCR3-AMG487 complex, which may have implications for drug development. The authors acknowledge their sources of support and funding, and the data and coordinates for the complex structures are available in the Protein Data Bank/Electron Microscopy Data Bank. No competing interests were declared by the authors, and supplementary figures and tables can be accessed online. [Extracted from the article]

Coal-fired power plants (CPP) usually release massive numerous amounts of potentially toxic metal(loid)s (PTMs) into nearby ecosystems. There have been relatively few studies targeted on the ecological influences of PTMs related to the CPP in arid area. In this work, the distribution pattern, source apportionment and environmental risks of As, Cd, Cr, Hg, Pb and a couple of seldom monitored PTMs (Se, Zn, Co, Cu, Fe, Mn and Ni) in the soils near a coal electricity integration base were investigated in Hami, a city in northwestern China. Nemerow synthesis pollution index, geo-accumulation index and ecological risk index were used to assess pollution state of these PTMs in soils, and ordinary Kriging interpolation was used to analyze the spatial distribution for these elements. Methods of CA, PCA, CA and PAM were carried out for quantitative source analysis. The research outcome includes: (1) the contents of individual PTMs in most samples were greater than the background values, the pollution degrees of Se, Pb, Hg, Cd and As were significant, and some areas exceeded the warning threshold value; (2) the main sources of these PTMs were natural sources (35%), coal mine sewage (11%), atmospheric release during coal combustion (21%), dust generated from coal and combustion products (33%); (3) attention should be paid to the open-pit coal mines, shaft coal mines and ash dumps where the contents of metal elements were significantly polluted; and (4) wind is the main driving forces of PTMs migration in arid areas. [ABSTRACT FROM AUTHOR]

The formyl peptide receptor 1 (FPR1) is primarily responsible for detection of short peptides bearing N-formylated methionine (fMet) that are characteristic of protein synthesis in bacteria and mitochondria. As a result, FPR1 is critical to phagocyte migration and activation in bacterial infection, tissue injury and inflammation. How FPR1 distinguishes between formyl peptides and non-formyl peptides remains elusive. Here we report cryo-EM structures of human FPR1-Gi protein complex bound to S. aureus-derived peptide fMet-Ile-Phe-Leu (fMIFL) and E. coli-derived peptide fMet-Leu-Phe (fMLF). Both structures of FPR1 adopt an active conformation and exhibit a binding pocket containing the R2015.38XXXR2055.42 (RGIIR) motif for formyl group interaction and receptor activation. This motif works together with D1063.33 for hydrogen bond formation with the N-formyl group and with fMet, a model supported by MD simulation and functional assays of mutant receptors with key residues for recognition substituted by alanine. The cryo-EM model of agonist-bound FPR1 provides a structural basis for recognition of bacteria-derived chemotactic peptides with potential applications in developing FPR1-targeting agents. Detection of invading bacteria is key to immunity. Here, the authors report cryo-electron microscopy structures of agonist-bound formyl peptide receptor 1 (FPR1), that reveal structural basis for recognition of bacteria-derived formyl peptides. [ABSTRACT FROM AUTHOR]

Porous monolithic ceramic materials have been fabricated based on poly(vinyl alcohol) (PVA) and sol nanoparticles by a series of procedures, including thermally impacted and non-solvent induced phase separations, solvent exchange, ambient pressure drying, and calcination. The morphology and properties of the porous ceramic materials could be tailored by tuning cooling temperatures, PVA concentrations, ratios of sol nanoparticles to PVA, and sizes of sol nanoparticles. These parameters exert important influence on the phase separation process, and eventually affect the morphology and properties of porous ceramic materials. For monolithic SiO2 materials, a low density of 0.19 g/cm3, a high porosity of 92%, and a large specific surface area of 30 m2/g were achieved, exhibiting potential applications in adsorption, catalysis, and filtration applications fields. Moreover, as a general and facile method, it could also be extended to fabricate porous monolithic TiO2, ZrO2 and other oxide ceramic materials. Highlights: A facile and general approach for the fabrication of porous monolithic ceramic materials is developed via phase separations in solutions of PVA and sol nanoparticles. Monolithic SiO2, TiO2, and ZrO2 materials with hierarchical and three-dimensional interconnected pores have been successfully fabricated. The porous structures of monolithic ceramic materials are mainly determined by the phase separation process, whose influences factors are systematically elucidated. [ABSTRACT FROM AUTHOR]

A non-enzymatic glucose-sensing material composed of α-nickel hydroxide nanosheets grown on reduced graphene oxide sheets (α-Ni(OH)2-rGO) is fabricated by a straightforward co-precipitation method. The morphology and composition of the α-Ni(OH)2-rGO are analyzed by field emission scanning electron microscopy, transmission electron microscopy, X-ray diffraction, Raman spectroscopy, thermogravimetric analyzer, and X-ray photoelectron spectroscopy. The characterization results show that the content of Ni(OH)2 in the α-Ni(OH)2-rGO composites is about 82.77 wt%. The introduction of rGO can effectively prevent α-Ni(OH)2 from agglomeration. The thickness of the α-Ni(OH)2 sheets grown vertically on the rGO is less than 10 nm, and they are interwoven to form a porous network structure. Then, the α-Ni(OH)2-rGO/NiF (Nickel Foam)-sensing electrode is prepared with NiF as the substrate. The sensing mechanism and detection property of the α-Ni(OH)2-rGO/NiF-sensing electrode are researched through cyclic voltammetry (CV) and amperometry. The test results demonstrate that α-Ni(OH)2-rGO/NiF-sensing electrode has excellent glucose-sensing performance. Its linear detection range is as wide as 0.5–22.5 mM, and the sensitivity is as high as 95.5 μAmM−1 cm−2, which is obviously much better than previously studied glucose-sensing materials. Moreover, it is found that the prepared sensing electrode has excellent anti-interference and stability. After repeated measurements, the anodic peak current is only 6% lower than the initial current. More importantly, the sensing electrode also showed a good detection ability for actual samples, which fully confirms that α-Ni(OH)2-rGO/NiF-sensing electrode has good practical application potential. [ABSTRACT FROM AUTHOR]

RAG endonuclease initiates Igh V(D)J recombination in progenitor B cells by binding a JH-recombination signal sequence (RSS) within a recombination centre (RC) and then linearly scanning upstream chromatin, presented by loop extrusion mediated by cohesin, for convergent D-RSSs1,2. The utilization of convergently oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning3. Scanning of RAG from the DJH-RC-RSS to upstream convergent VH-RSSs is impeded by D-proximal CTCF-binding elements (CBEs)2–5. Primary progenitor B cells undergo a mechanistically undefined contraction of the VH locus that is proposed to provide distal VHs access to the DJH-RC6–9. Here we report that an inversion of the entire 2.4-Mb VH locus in mouse primary progenitor B cells abrogates rearrangement of both VH-RSSs and normally convergent cryptic RSSs, even though locus contraction still occurs. In addition, this inversion activated both the utilization of cryptic VH-RSSs that are normally in opposite orientation and RAG scanning beyond the VH locus through several convergent CBE domains to the telomere. Together, these findings imply that broad deregulation of CBE impediments in primary progenitor B cells promotes RAG scanning of the VH locus mediated by loop extrusion. We further found that the expression of wings apart-like protein homologue (WAPL)10, a cohesin-unloading factor, was low in primary progenitor B cells compared with v-Abl-transformed progenitor B cell lines that lacked contraction and RAG scanning of the VH locus. Correspondingly, depletion of WAPL in v-Abl-transformed lines activated both processes, further implicating loop extrusion in the locus contraction mechanism.Long-distance V(D)J recombination is facilitated by contraction of the Igh locus and linear RAG scanning along chromatin, both driven by cohesin-mediated loop extrusion, which allows recombination of widely separated gene segments to occur. [ABSTRACT FROM AUTHOR]

After activation by an agonist, G-protein-coupled receptors (GPCRs) recruit β-arrestin, which desensitizes heterotrimeric G-protein signalling and promotes receptor endocytosis1. Additionally, β-arrestin directly regulates many cell signalling pathways that can induce cellular responses distinct from that of G proteins2. In contrast to G proteins, for which there are many high-resolution structures in complex with GPCRs, the molecular mechanisms underlying the interaction of β-arrestin with GPCRs are much less understood. Here we present a cryo-electron microscopy structure of β-arrestin 1 (βarr1) in complex with M2 muscarinic receptor (M2R) reconstituted in lipid nanodiscs. The M2R–βarr1 complex displays a multimodal network of flexible interactions, including binding of the N domain of βarr1 to phosphorylated receptor residues and insertion of the finger loop of βarr1 into the M2R seven-transmembrane bundle, which adopts a conformation similar to that in the M2R–heterotrimeric Go protein complex3. Moreover, the cryo-electron microscopy map reveals that the C-edge of βarr1 engages the lipid bilayer. Through atomistic simulations and biophysical, biochemical and cellular assays, we show that the C-edge is critical for stable complex formation, βarr1 recruitment, receptor internalization, and desensitization of G-protein activation. Taken together, these data suggest that the cooperative interactions of β-arrestin with both the receptor and the phospholipid bilayer contribute to its functional versatility. A cryo-electron microscopy structure of β-arrestin 1 in complex with the M2 muscarinic receptor reconstituted in lipid nanodiscs is reported. [ABSTRACT FROM AUTHOR]

The RAG endonuclease initiates Igh V(D)J assembly in B cell progenitors by joining D segments to JH segments, before joining upstream VH segments to DJH intermediates1. In mouse progenitor B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain2 at the 3′ end of Igh contains an internal subdomain that spans the 5′ CBE anchor (IGbibr1)3, the DH segments, and a RAG-bound recombination centre (RC)4. The RC comprises the JH-proximal D segment (DQ52), four JH segments, and the intronic enhancer (iEμ)5. Robust RAG-mediated cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSS and 23RSS)6. D segments are flanked downstream and upstream by 12RSSs that mediate deletional joining with convergently oriented JH-23RSSs and VH-23RSSs, respectively6. Despite 12/23 compatibility, inversional D-to-JH joining via upstream D-12RSSs is rare7,8. Plasmid-based assays have attributed the lack of inversional D-to-JH joining to sequence-based preference for downstream D-12RSSs9, as opposed to putative linear scanning mechanisms10,11. As RAG linearly scans convergent CBE-anchored chromatin loops4,12–14, potentially formed by cohesin-mediated loop extrusion15–18, we revisited its scanning role. Here we show that the chromosomal orientation of JH-23RSS programs RC-bound RAG to linearly scan upstream chromatin in the 3′ Igh subdomain for convergently oriented D-12RSSs and, thereby, to mediate deletional joining of all D segments except RC-based DQ52, which joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of JH segments, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3′ Igh subdomain, in which scanning can be impeded by targeted binding of nuclease-dead Cas9, by transcription through repetitive Igh switch sequences, and by the 3′ Igh CBE-based loop anchor. Each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High-resolution mapping of chromatin interactions in the RC reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG. V(D)J recombination in B cells involves cohesin-mediated extrusion of chromatin loops to present DNA targets for cleavage and joining. [ABSTRACT FROM AUTHOR]

Neurotensin receptor 1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temperature, weight and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric Gi1 protein, at a resolution of 3 Å. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR–Gi/o complexes (in which the nucleotide-binding pocket adopts more flexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45 degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by molecular dynamics simulations and functional studies, provides insights into the complex process of G-protein activation. Cryo-electron microscopy structures of human neurotensin receptor 1 in complex with Gi1 protein and the agonist JMV449 reveal a non-canonical state that may represent an intermediate form in G-protein activation. [ABSTRACT FROM AUTHOR]

Fusarium graminearum is a prominent fungal pathogen that causes economically important losses by infesting a wide variety of cereal crops. F. graminearum produces both asexual and sexual spores which disseminate and inoculate hosts. Therefore, to better understand the disease cycle and to develop strategies to improve disease management, it is important to further clarify molecular mechanisms of F. graminearum conidiogenesis. In this study, we functionally characterized the FgMed1, a gene encoding an ortholog of a conserved MedA transcription factor known to be a key conidiogenesis regulator in Aspergillus nidulans. The gene deletion mutants ΔFgMed1 produced significantly less conidia, and these were generated from abnormal conidiophores devoid of phialides. Additionally, we observed defective sexual development along with reduced virulence and deoxynivalenol (DON) production in ΔFgMed1. The GFP-tagged FgMed1 protein localized to the nuclei of conidiophores and phialides during early conidiogenesis. Significantly, RNA-Seq analyses showed that a number of the conidiation- and toxin-related genes are differentially expressed in the ΔFgMed1 mutant in early conidiogenesis. These data strongly suggest that FgMed1 involved in regulation of genes associated with early conidiogenesis, DON production, and virulence in F. graminearum. [ABSTRACT FROM AUTHOR]

Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich domains to their 7-transmembrane domains. Spectroscopic studies show that signalling is a dynamic process, in which large-scale conformational changes underlie the transmission of signals from the extracellular Venus flytraps to the G protein-coupling domains—the 7-transmembrane domains—in the membrane. Here, using a combination of X-ray crystallography, cryo-electron microscopy and signalling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the Venus flytraps leads to a compaction of the intersubunit dimer interface, thereby bringing the cysteine-rich domains into close proximity. Interactions between the cysteine-rich domains and the second extracellular loops of the receptor enable the rigid-body repositioning of the 7-transmembrane domains, which come into contact with each other to initiate signalling. The activation mechanism of metabotropic glutamate receptor subtype 5, a member of the family C G-protein-coupled receptors, is characterized by a combination of cryo-electron microscopy, crystallography and signalling studies. [ABSTRACT FROM AUTHOR]

The μ-opioid receptor (μOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by μOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Here we present the 3.5 Å resolution cryo-electron microscopy structure of the μOR bound to the agonist peptide DAMGO and nucleotide-free Gi. DAMGO occupies the morphinan ligand pocket, with its N terminus interacting with conserved receptor residues and its C terminus engaging regions important for opioid-ligand selectivity. Comparison of the μOR-Gi complex to previously determined structures of other GPCRs bound to the stimulatory G protein Gs reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein α-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the Gi protein-coupling specificity of the µOR. A cryo-electron structure of the µ-opioid receptor in complex with the peptide agonist DAMGO and the inhibitory G protein Gi reveals structural determinants of its G protein-binding specificity. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide 1 (GLP-1) is a hormone with essential roles in regulating insulin secretion, carbohydrate metabolism and appetite. GLP-1 effects are mediated through binding to the GLP-1 receptor (GLP-1R), a class B G-protein-coupled receptor (GPCR) that signals primarily through the stimulatory G protein Gs. Class B GPCRs are important therapeutic targets; however, our understanding of their mechanism of action is limited by the lack of structural information on activated and full-length receptors. Here we report the cryo-electron microscopy structure of the peptide-activated GLP-1R-Gs complex at near atomic resolution. The peptide is clasped between the N-terminal domain and the transmembrane core of the receptor, and further stabilized by extracellular loops. Conformational changes in the transmembrane domain result in a sharp kink in the middle of transmembrane helix 6, which pivots its intracellular half outward to accommodate the α5-helix of the Ras-like domain of Gs. These results provide a structural framework for understanding class B GPCR activation through hormone binding. [ABSTRACT FROM AUTHOR]

F-box and WD repeat domain-containing 7 (FBW7) is the substrate recognition component of the Skp1-Cul1-F-box (SCF) ubiquitin ligase complex and functions as a major tumor suppressor by targeting various oncoproteins for degradation. Genomic deletion or mutation of FBW7 has frequently been identified in many human cancers but not in pancreatic ductal adenocarcinoma. Thus it is important to know how the tumor suppressive function of FBW7 is impaired in pancreatic cancer. In this study, we first observed that low FBW7 expression correlated significantly with ERK activation in pancreatic cancer clinical samples, primarily due to KRAS mutations in pancreatic cancer. We further showed that ERK directly interacted with FBW7 and phosphorylated FBW7 at Thr205, which sequentially promoted FBW7 ubiquitination and proteasomal degradation. Furthermore, the phospho-deficient T205A FBW7 mutant is resistant to ERK activation and could significantly suppress pancreatic cancer cell proliferation and tumorigenesis. These results collectively demonstrate how the oncogenic KRAS mutation inhibits the tumor suppressor FBW7, thus revealing an important function of KRAS mutations in promoting pancreatic cancer progression. [ABSTRACT FROM AUTHOR]

The surname of author Toon Laeremans was misspelled 'Laermans'. This error has been corrected online. [ABSTRACT FROM AUTHOR]

Single-particle cryo-electron microscopy (cryo-EM) has recently enabled high-resolution structure determination of numerous biological macromolecular complexes. Despite this progress, the application of high-resolution cryo-EM to G protein coupled receptors (GPCRs) in complex with heterotrimeric G proteins remains challenging, owning to both the relative small size and the limited stability of these assemblies. Here we describe the development of antibody fragments that bind and stabilize GPCR-G protein complexes for the application of high-resolution cryo-EM. One antibody in particular, mAb16, stabilizes GPCR/G-protein complexes by recognizing an interface between Gα and Gβγ subunits in the heterotrimer, and confers resistance to GTPγS-triggered dissociation. The unique recognition mode of this antibody makes it possible to transfer its binding and stabilizing effect to other G-protein subtypes through minimal protein engineering. This antibody fragment is thus a broadly applicable tool for structural studies of GPCR/G-protein complexes. [ABSTRACT FROM AUTHOR]

Small heat shock proteins (sHSPs) are molecular chaperones ubiquitously present in all forms of life, but their function mechanisms remain controversial. Here we show by cryo-electron microscopy and single particle 3D reconstruction that, at the low temperatures (4-25°C), CeHSP17 (a sHSP from Caenorhabditis elegans) exists as a 24-subunit spherical oligomer with tetrahedral symmetry. Our studies demonstrate that CeHSP17 forms large sheet-like super-molecular assemblies (SMAs) at the high temperatures (45-60°C), and such SMAs are apparently the form that exhibits chaperone-like activity. Our findings suggest a novel molecular mechanism for sHSPs to function as molecular chaperones. [ABSTRACT FROM AUTHOR]

Dihydropyridine receptor (DHPR), an L-type Ca2+ channel complex, plays an essential role in muscle contraction, secretion, integration of synaptic input in neurons and synaptic transmission. The molecular architecture of DHPR complex remains elusive. Here we present a 15-Å resolution cryo-electron microscopy structure of the skeletal DHPR/L-type Ca2+ channel complex. The DHPR has an asymmetrical main body joined by a hook-like extension. The main body is composed of a 'trapezoid' and a 'tetrahedroid'. Homologous crystal structure docking and site-specific antibody labelling revealed that the α1 and α2 subunits are located in the 'trapezoid' and the β subunit is located in the 'tetrahedroid'. This structure revealed the molecular architecture of a eukaryotic Ca2+ channel complex. Furthermore, this structure provides structural insights into the key elements of DHPR involved in physical coupling with the RyR/Ca2+ release channel and shed light onto the mechanism of excitation-contraction coupling. [ABSTRACT FROM AUTHOR]