Recognition of EGF-like domains by the Notch-modifying O-fucosyltransferase POFUT1.

Protein O-fucosyltransferase 1 (POFUT1) fucosylates the epidermal growth factor (EGF)-like domains found in cell-surface and secreted glycoproteins including Notch and its ligands. Although Notch fucosylation is critical for development, and POFUT1 deficiency leads to human disease, how this enzyme binds and catalyzes the fucosylation of its diverse EGF-like domain substrates has not been determined. Reported here is the X-ray crystal structure of mouse POFUT1 in complex with several EGF-like domains, including EGF12 and EGF26 of Notch. Overall shape complementarity, interactions with invariant atoms of the fucosylation motif and flexible segments on POFUT1 all define its EGF-like-domain binding properties. Using large-scale structural and sequence analysis, we also show that POFUT1 binds EGF-like domains of the hEGF type and that the highly correlated presence of POFUT1 and fucosylatable hEGFs has accompanied animal evolution.

Simple piggyBac transposon-based mammalian cell expression system for inducible protein production.

Reported here is a piggyBac transposon-based expression system for the generation of doxycycline-inducible, stably transfected mammalian cell cultures for large-scale protein production. The system works with commonly used adherent and suspension-adapted mammalian cell lines and requires only a single transfection step. Moreover, the high uniform expression levels observed among clones allow for the use of stable bulk cell cultures, thereby eliminating time-consuming cloning steps. Under continuous doxycycline induction, protein expression levels have been shown to be stable for at least 2 mo in the absence of drug selection. The high efficiency of the system also allows for the generation of stable bulk cell cultures in 96-well format, a capability leading to the possibility of generating stable cell cultures for entire families of membrane or secreted proteins. Finally, we demonstrate the utility of the system through the large-scale production (140-750 mg scale) of an endoplasmic reticulum-resident fucosyltransferase and two potential anticancer protein therapeutic agents.

The X-ray crystal structure of human aminopeptidase N reveals a novel dimer and the basis for peptide processing.

Human aminopeptidase N (hAPN/hCD13) is a dimeric membrane protein and a member of the M1 family of zinc metallopeptidases. Within the rennin-angiotensin system, its enzymatic activity is responsible for processing peptide hormones angiotensin III and IV. In addition, hAPN is also involved in cell adhesion, endocytosis, and signal transduction and it is an important target for cancer therapy. Reported here are the high resolution x-ray crystal structures of the dimeric ectodomain of hAPN and its complexes with angiotensin IV and the peptidomimetic inhibitors, amastatin and bestatin. Each monomer of the dimer is found in what has been termed the closed form in other M1 enzymes and each monomer is characterized by an internal cavity surrounding the catalytic site as well as a unique substrate/inhibitor-dependent loop ordering, which in the case of the bestatin complex suggests a new route to inhibitor design. The hAPN structure provides the first example of a dimeric M1 family member and the observed structural features, in conjunction with a model for the open form, provide novel insights into the mechanism of peptide processing and signal transduction.

miR-130b-3p is high-expressed in polycystic ovarian syndrome and promotes granulosa cell proliferation by targeting SMAD4.

BACKGROUND: Being one of the most prevalent metabolic and endocrine disorders, Polycystic Ovary Syndrome (PCOS) has been proven to be associated with microRNA-130b-3p (miR-130b-3p). However, the exact role played by miR-130b-3p in the pathogenesis and progression of PCOS remains unknown. Thus, this article is focused on elucidating the function of miR-130b-3p in the pathogenesis of PCOS. METHODS: The expression levels of miR-130b-3p and SMAD4 in tissues and cells responsible for the development of PCOS were determined by RT-qPCR and western blot. A miR-130b-3p mimic/inhibitor or si-SMAD4 were transfected into KGN cells. The cell viability was detected by CCK-8 and EDU methods. The activity of caspase-3 was measured by caspase-3 analysis. Subsequently, apoptosis and the cell cycle were measured via flow cytometry. The correlation between SMAD4 and miR-130b-3p was confirmed using an RNA pull-down assay and a dual luciferase reporter system assay. RESULTS: MiR-130b-3p was upregulated in the KGN cells and ovarian granulosa cells (GCs) of PCOS patients. It was found that miR-130b-3p overexpression or SMAD4 silencing can promote KGN cell proliferation and positive EDU rates, induce S phase arrest, inhibit apoptosis and caspase-3 activity. On the other hand, miR-130b-3p inhibitors reduce KGN cell proliferation, inhibit apoptosis and reverse the effect of si-SMAD4. CONCLUSION: MiR-130b-3p directly interacts with SMAD4 to induce KGN cell proliferation, inhibit apoptosis, suggesting that miR-130b-3p expression is positively correlated with the development of PCOS. This may serve as new evidence for the abnormal proliferation of GCs in PCOS.

Downregulation of lncRNA FGF12-AS2 suppresses the tumorigenesis of NSCLC via sponging miR-188-3p.

BACKGROUND: Non-small-cell lung carcinoma (NSCLC) seriously threatens the health of human beings. Aberrant expression of lncRNAs has been confirmed to be related with the progression of multiple malignant tumors, including NSCLC. LncRNA FGF12-AS2 has been considered to be upregulated in NSCLC. However, the mechanism by which FGF12-AS2 promotes the tumorigenesis of NSCLC remains elusive. METHODS: Gene and protein expressions in NSCLC cells were measured by q-PCR and western blot, respectively. CCK-8 and immunofluorescence staining were performed to detect the cell proliferation. Cell apoptosis was tested by flow cytometry. Transwell assay was used to detect the cell migration and invasion. Finally, the dual luciferase report assay was used to verify the relation among FGF12-AS2, miR-188-3p, and NCAPG2. RESULTS: Downregulation of FGF12-AS2 significantly inhibited the proliferation of NSCLC cells via inducing apoptosis. In addition, FGF12-AS2 silencing notably suppressed the migration and invasion of A549 cells. Meanwhile, FGF12-AS2 modulated the progression of NSCLC via regulation of miR-188-3p/NCAPG2 axis. Finally, knockdown of FGF12-AS2 inhibited the tumorigenesis of NSCLC via suppressing the EMT process of NSCLC. CONCLUSION: Downregulation of lncRNA FGF12-AS2 suppressed the tumorigenesis of NSCLC via sponging miR-188-3p. Thus, FGF12-AS2 may serve as a potential target for the treatment of NSCLC.

Exploiting the diphtheria toxin internalization receptor enhances delivery of proteins to lysosomes for enzyme replacement therapy.

Enzyme replacement therapy, in which a functional copy of an enzyme is injected either systemically or directly into the brain of affected individuals, has proven to be an effective strategy for treating certain lysosomal storage diseases. The inefficient uptake of recombinant enzymes via the mannose-6-phosphate receptor, however, prohibits the broad utility of replacement therapy. Here, to improve the efficiency and efficacy of lysosomal enzyme uptake, we exploited the strategy used by diphtheria toxin to enter into the endolysosomal network of cells by creating a chimera between the receptor-binding fragment of diphtheria toxin and the lysosomal hydrolase TPP1. We show that chimeric TPP1 binds with high affinity to target cells and is efficiently delivered into lysosomes. Further, we show superior uptake of chimeric TPP1 over TPP1 alone in brain tissue following intracerebroventricular injection in mice lacking TPP1, demonstrating the potential of this strategy for enhancing lysosomal storage disease therapy.

The human coronavirus HCoV-229E S-protein structure and receptor binding.

The coronavirus S-protein mediates receptor binding and fusion of the viral and host cell membranes. In HCoV-229E, its receptor binding domain (RBD) shows extensive sequence variation but how S-protein function is maintained is not understood. Reported are the X-ray crystal structures of Class III-V RBDs in complex with human aminopeptidase N (hAPN), as well as the electron cryomicroscopy structure of the 229E S-protein. The structures show that common core interactions define the specificity for hAPN and that the peripheral RBD sequence variation is accommodated by loop plasticity. The results provide insight into immune evasion and the cross-species transmission of 229E and related coronaviruses. We also find that the 229E S-protein can expose a portion of its helical core to solvent. This is undoubtedly facilitated by hydrophilic subunit interfaces that we show are conserved among coronaviruses. These interfaces likely play a role in the S-protein conformational changes associated with membrane fusion.

Structural basis of Notch O-glucosylation and O-xylosylation by mammalian protein-O-glucosyltransferase 1 (POGLUT1).

Protein O-glucosyltransferase 1/Rumi-mediated glucosylation of Notch epidermal growth factor-like (EGF-like) domains plays an important role in Notch signaling. Protein O-glucosyltransferase 1 shows specificity for folded EGF-like domains, it can only glycosylate serine residues in the C1XSXPC2 motif, and it possesses an uncommon dual donor substrate specificity. Using several EGF-like domains and donor substrate analogs, we have determined the structures of human Protein O-glucosyltransferase 1 substrate/product complexes that provide mechanistic insight into the basis for these properties. Notably, we show that Protein O-glucosyltransferase 1's requirement for folded EGF-like domains also leads to its serine specificity and that two distinct local conformational states are likely responsible for its ability to transfer both glucose and xylose. We also show that Protein O-glucosyltransferase 1 possesses the potential to xylosylate a much broader range of EGF-like domain substrates than was previously thought. Finally, we show that Protein O-glucosyltransferase 1 has co-evolved with EGF-like domains of the type found in Notch.POGLUT1 is a protein-O-glucosyltransferase that transfers glucose and xylose to the EGF-like domains of Notch and other signaling receptors. Here the authors report the structure of human POGLUT1 in complexes with 3 different EGF-like domains and donor substrates and shed light on the enzyme's substrate specificity and catalytic mechanism.

Receptor-binding loops in alphacoronavirus adaptation and evolution.

RNA viruses are characterized by a high mutation rate, a buffer against environmental change. Nevertheless, the means by which random mutation improves viral fitness is not well characterized. Here we report the X-ray crystal structure of the receptor-binding domain (RBD) of the human coronavirus, HCoV-229E, in complex with the ectodomain of its receptor, aminopeptidase N (APN). Three extended loops are solely responsible for receptor binding and the evolution of HCoV-229E and its close relatives is accompanied by changing loop-receptor interactions. Phylogenetic analysis shows that the natural HCoV-229E receptor-binding loop variation observed defines six RBD classes whose viruses have successively replaced each other in the human population over the past 50 years. These RBD classes differ in their affinity for APN and their ability to bind an HCoV-229E neutralizing antibody. Together, our results provide a model for alphacoronavirus adaptation and evolution based on the use of extended loops for receptor binding.