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Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from the emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy (cryoEM) to decipher the mechanism of action of a potent HA head-directed monoclonal antibody (mAb) bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, prefusion conformation that typifies all HA structures to date. Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the prefusion conformation. The "breathing" of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously.
Transforming growth factors (TGFs) were discovered as activities that were secreted by cancer cells, and later by normal cells, and had the ability to phenotypically and reversibly transform immortalized fibroblasts. TGF-β distinguished itself from TGF-α because it did not bind to the same epidermal growth factor (EGF) receptor as TGF-α and, therefore, acted through different cell-surface receptors and signaling mediators. This review summarizes the discovery of TGF-β, the early developments in its molecular and biological characterization with its many biological activities in different cell and tissue contexts and its roles in disease, the realization that there is a family of secreted TGF-β-related proteins with many differentiation functions in development and activities in normal cell and tissue physiology, and the subsequent identification and characterization of the receptors and effectors that mediate TGF-β family signaling responses.
Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.
BACKGROUND - The CRISPR/Cas9 system is increasingly used for gene inactivation in mouse zygotes, but homology-directed mutagenesis and use of inbred embryos are less established. In particular, Rosa26 knock-in alleles for the insertion of transgenes in a genomic 'safe harbor' site, have not been produced. Here we applied CRISPR/Cas9 for the knock-in of 8-11 kb inserts into Rosa26 of C57BL/6 zygotes.
RESULTS - We found that 10-20 % of live pups derived from microinjected zygotes were founder mutants, without apparent off-target effects, and up to 50 % knock-in embryos were recovered upon coinjection of Cas9 mRNA and protein. Using this approach, we established a new mouse line for the Cre/loxP-dependent expression of Cas9.
CONCLUSIONS - Altogether, our protocols and resources support the fast and direct generation of new Rosa26 knock-in alleles and of Cas9-mediated in vivo gene editing in the widely used C57BL/6 inbred strain.
Glucagon antagonism is a potential treatment for diabetes. One potential side effect is α-cell hyperplasia, which has been noted in several approaches to antagonize glucagon action. To investigate the molecular mechanism of the α-cell hyperplasia and to identify the responsible factor, we created a zebrafish model in which glucagon receptor (gcgr) signaling has been interrupted. The genetically and chemically tractable zebrafish, which provides a robust discovery platform, has two gcgr genes (gcgra and gcgrb) in its genome. Sequence, phylogenetic, and synteny analyses suggest that these are co-orthologs of the human GCGR. Similar to its mammalian counterparts, gcgra and gcgrb are mainly expressed in the liver. We inactivated the zebrafish gcgra and gcgrb using transcription activator-like effector nuclease (TALEN) first individually and then both genes, and assessed the number of α-cells using an α-cell reporter line, Tg(gcga:GFP). Compared to WT fish at 7 days postfertilization, there were more α-cells in gcgra-/-, gcgrb-/-, and gcgra-/-;gcgrb-/- fish and there was an increased rate of α-cell proliferation in the gcgra-/-;gcgrb-/- fish. Glucagon levels were higher but free glucose levels were lower in gcgra-/-, gcgrb-/-, and gcgra-/-;gcgrb-/- fish, similar to Gcgr-/- mice. These results indicate that the compensatory α-cell hyperplasia in response to interruption of glucagon signaling is conserved in zebrafish. The robust α-cell hyperplasia in gcgra-/-;gcgrb-/- larvae provides a platform to screen for chemical and genetic suppressors, and ultimately to identify the stimulus of α-cell hyperplasia and its signaling mechanism.
© 2015 Society for Endocrinology.
Precise regulation of Notch signaling is essential for normal vertebrate development. Mind bomb (Mib) is a ubiquitin ligase that is required for activation of Notch by Notch׳s ligand, Delta. Sorting Nexin 5 (SNX5) co-localizes with Mib and Delta complexes and has been shown to directly bind to Mib. We show that microRNA-216a (miR-216a) is expressed in the retina during early development and regulates snx5 to precisely regulate Notch signaling. miR-216a and snx5 have complementary expression patterns. Knocking down miR-216a and/or overexpression of snx5 resulted in increased Notch activation. Conversely, knocking down snx5 and/or miR-216a overexpression caused a decrease in Notch activation. We propose a model in which SNX5, precisely controlled by miR-216a, is a vital partner of Mib in promoting endocytosis of Delta and subsequent activation of Notch signaling.
Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
OBJECTIVE - To construct, express and purify human fusion proteins composed of a single-chain antibody fragment scFv that recognizes the prostate specific membrane antigen (PSMA) protein, Fdt, HA2 and tp, and to analyze the binding activity of the expressed fusion proteins.
METHODS - The fusion protein genes scFv, scFv-tp, and scFv-Fdt-HA2-tp were amplified by PCR, and the genes obtained were then cloned into the expression vector pET28 and expressed in E. coli BL21. The expressed products were identified by SDS-PAGE and Western blot and purified with Ni(2+)-NTA chelating agarose. The antigen-binding activity of the fusion proteins was determined by ELISA.
RESULTS - The human anti-PSMA fusion gene was successfully constructed and expressed in M15 as the inclusion body after induced with IPTG. All the target proteins expressed could bind the PSMA antigen.
CONCLUSION - Fusion proteins can specifically bind the PSMA antigen. This finding contributes to the study of the targeted delivery of siRNA.
Adipocytes have key endocrine roles, mediated in large part by secreted protein hormones termed adipokines. The adipokine adiponectin is well known for its role in sensitizing peripheral tissues to insulin, and several lines of evidence suggest that adiponectin might also modulate stem cells/precursors. It remains unclear, however, how adiponectin signaling controls stem cells and whether this role is secondary to its insulin-sensitizing effects or distinct. Drosophila adipocytes also function as an endocrine organ and, although no obvious adiponectin homolog has been identified, Drosophila AdipoR encodes a well-conserved homolog of mammalian adiponectin receptors. Here, we generate a null AdipoR allele and use clonal analysis to demonstrate an intrinsic requirement for AdipoR in germline stem cell (GSC) maintenance in the Drosophila ovary. AdipoR null GSCs are not fully responsive to bone morphogenetic protein ligands from the niche and have a slight reduction in E-cadherin levels at the GSC-niche junction. Conversely, germline-specific overexpression of AdipoR inhibits natural GSC loss, suggesting that reduction in adiponectin signaling might contribute to the normal decline in GSC numbers observed over time in wild-type females. Surprisingly, AdipoR is not required for insulin sensitization of the germline, leading us to speculate that insulin sensitization is a more recently acquired function than stem cell regulation in the evolutionary history of adiponectin signaling. Our findings establish Drosophila female GSCs as a new system for future studies addressing the molecular mechanisms whereby adiponectin receptor signaling modulates stem cell fate.
Copyright © 2015 Elsevier Inc. All rights reserved.
The Ste20-related kinase SPAK regulates sodium, potassium, and chloride transport in a variety of tissues. Recently, SPAK fragments, which lack the catalytic domain and are inhibitory to Na(+) transporters, have been detected in kidney. It has been hypothesized that the fragments originate from alternative translation start sites, but their precise origin is unknown. Here, we demonstrate that kidney lysate possesses proteolytic cleavage activity toward SPAK. Ion exchange and size exclusion chromatography combined with mass spectrometry identified the protease as aspartyl aminopeptidase. The presence of the protease was verified in the active fractions, and recombinant aspartyl aminopeptidase recapitulated the cleavage pattern observed with kidney lysate. Identification of the sites of cleavage by mass spectrometry allowed us to test the function of the smaller fragments and demonstrate their inhibitory action toward the Na(+)-K(+)-2Cl(-) cotransporter, NKCC2.
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Knowledge of an individual protein's modifications, binding partners, and localization is essential for understanding complex biological networks. We recently described a fluorescent protein-based (mVenus) multifunctional affinity purification (MAP) tag that can be used both to purify a given protein and determine its localization (Ma et al., Mol Cell Proteomics 11:501-511, 2012). MAP purified protein complexes can be further analyzed to identify binding partners and posttranslational modifications by LC-MS/MS. The MAP approach offers rapid FACS-selection of stable clonal cell lines based on the expression level/fluorescence of the MAP-protein fusion. The MAP tag is highly efficient and shows little variability between proteins. Here we describe the general MAP purification method in detail, and show how it can be applied to a specific protein using the human Cdc14B phosphatase as an example.
Directional export of messenger RNA (mRNA) protein particles (mRNPs) through nuclear pore complexes (NPCs) requires multiple factors. In Saccharomyces cerevisiae, the NPC proteins Nup159 and Nup42 are asymmetrically localized to the cytoplasmic face and have distinct functional domains: a phenylalanine-glycine (FG) repeat domain that docks mRNP transport receptors and domains that bind the DEAD-box ATPase Dbp5 and its activating cofactor Gle1, respectively. We speculated that the Nup42 and Nup159 FG domains play a role in positioning mRNPs for the terminal mRNP-remodeling steps carried out by Dbp5. Here we find that deletion (Δ) of both the Nup42 and Nup159 FG domains results in a cold-sensitive poly(A)+ mRNA export defect. The nup42ΔFG nup159ΔFG mutant also has synthetic lethal genetic interactions with dbp5 and gle1 mutants. RNA cross-linking experiments further indicate that the nup42ΔFG nup159ΔFG mutant has a reduced capacity for mRNP remodeling during export. To further analyze the role of these FG domains, we replaced the Nup159 or Nup42 FG domains with FG domains from other Nups. These FG "swaps" demonstrate that only certain FG domains are functional at the NPC cytoplasmic face. Strikingly, fusing the Nup42 FG domain to the carboxy-terminus of Gle1 bypasses the need for the endogenous Nup42 FG domain, highlighting the importance of proximal positioning for these factors. We conclude that the Nup42 and Nup159 FG domains target the mRNP to Gle1 and Dbp5 for mRNP remodeling at the NPC. Moreover, these results provide key evidence that character and context play a direct role in FG domain function and mRNA export.
Copyright © 2014 by the Genetics Society of America.