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Results: 1 to 10 of 202

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Metabolic Labeling of Inositol Phosphates and Phosphatidylinositols in Yeast and Mammalian Cells.
Hale AT, Clarke BP, York JD
(2020) Methods Mol Biol 2091: 83-92
MeSH Terms: Animals, Biochemical Phenomena, COS Cells, Cell Line, Chlorocebus aethiops, HT29 Cells, HeLa Cells, Humans, Inositol Phosphates, Phosphatidylinositols, Saccharomyces cerevisiae, Signal Transduction
Show Abstract · Added March 30, 2020
Inositol phosphate (IP) and phosphatidylinositol (PI) signaling are critical signal transduction pathways responsible for generating numerous receptor-mediated cellular responses. Biochemical and genetic studies have revealed diverse roles of IP and PI signaling in eukaryotic signaling, but detailed characterization of unique IP and PI signaling profiles in response to different agonists and among cell types remains largely unexplored. Here, we outline steady-state inositol metabolic-labeling techniques that can be leveraged to assess the IP and PI signaling state in eukaryotic cells. This flexible technique can be amended and optimized to your cell line of interest, perturbed with biochemical, genetic, or pharmacological alteration, and used to provide comprehensive inositol profiling in various cellular systems.
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12 MeSH Terms
Mechanism of differential Zika and dengue virus neutralization by a public antibody lineage targeting the DIII lateral ridge.
Zhao H, Xu L, Bombardi R, Nargi R, Deng Z, Errico JM, Nelson CA, Dowd KA, Pierson TC, Crowe JE, Diamond MS, Fremont DH
(2020) J Exp Med 217:
MeSH Terms: Aedes, Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, Cell Line, Tumor, Chlorocebus aethiops, Cross Reactions, Crystallography, X-Ray, Dengue, Dengue Virus, Epitopes, HEK293 Cells, Humans, Hydrogen Bonding, Immunoglobulin Fab Fragments, Protein Binding, Protein Conformation, Protein Domains, Vero Cells, Viral Envelope Proteins, Zika Virus, Zika Virus Infection
Show Abstract · Added March 31, 2020
We previously generated a panel of human monoclonal antibodies (mAbs) against Zika virus (ZIKV) and identified one, ZIKV-116, that shares germline usage with mAbs identified in multiple donors. Here we show that ZIKV-116 interferes with ZIKV infection at a post-cellular attachment step by blocking viral fusion with host membranes. ZIKV-116 recognizes the lateral ridge of envelope protein domain III, with one critical residue varying between the Asian and African strains responsible for differential binding affinity and neutralization potency (E393D). ZIKV-116 also binds to and cross-neutralizes some dengue virus serotype 1 (DENV1) strains, with genotype-dependent inhibition explained by variation in a domain II residue (R204K) that potentially modulates exposure of the distally located, partially cryptic epitope. The V-J reverted germline configuration of ZIKV-116 preferentially binds to and neutralizes an Asian ZIKV strain, suggesting that this epitope may optimally induce related B cell clonotypes. Overall, these studies provide a structural and molecular mechanism for a cross-reactive mAb that uniquely neutralizes ZIKV and DENV1.
© 2019 Zhao et al.
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23 MeSH Terms
Using In Vitro Pull-Down and In-Cell Overexpression Assays to Study Protein Interactions with Arrestin.
Perry NA, Zhan X, Gurevich EV, Iverson TM, Gurevich VV
(2019) Methods Mol Biol 1957: 107-120
MeSH Terms: Animals, Arrestin, Biological Assay, COS Cells, Chlorocebus aethiops, HEK293 Cells, Humans, Immobilized Proteins, Mice, Protein Binding, Protein Interaction Mapping, Recombinant Fusion Proteins
Show Abstract · Added April 1, 2019
Nonvisual arrestins (arrestin-2/arrestin-3) interact with hundreds of G protein-coupled receptor (GPCR) subtypes and dozens of non-receptor signaling proteins. Here we describe the methods used to identify the interaction sites of arrestin-binding partners on arrestin-3 and the use of monofunctional individual arrestin-3 elements in cells. Our in vitro pull-down assay with purified proteins demonstrates that relatively few elements in arrestin engage each partner, whereas cell-based functional assays indicate that certain arrestin elements devoid of other functionalities can perform individual functions in living cells.
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12 MeSH Terms
The F-BAR Domain of Rga7 Relies on a Cooperative Mechanism of Membrane Binding with a Partner Protein during Fission Yeast Cytokinesis.
Liu Y, McDonald NA, Naegele SM, Gould KL, Wu JQ
(2019) Cell Rep 26: 2540-2548.e4
MeSH Terms: Animals, COS Cells, Cell Cycle Proteins, Cell Membrane, Chlorocebus aethiops, Cytokinesis, GTPase-Activating Proteins, Microscopy, Confocal, Protein Domains, Schizosaccharomyces, Schizosaccharomyces pombe Proteins, Transfection
Show Abstract · Added April 10, 2019
F-BAR proteins bind the plasma membrane (PM) to scaffold and organize the actin cytoskeleton. To understand how F-BAR proteins achieve their PM association, we studied the localization of a Schizosaccharomyces pombe F-BAR protein Rga7, which requires the coiled-coil protein Rng10 for targeting to the division site during cytokinesis. We find that the Rga7 F-BAR domain directly binds a motif in Rng10 simultaneously with the PM, and that an adjacent Rng10 motif independently binds the PM. Together, these multivalent interactions significantly enhance Rga7 F-BAR avidity for membranes at physiological protein concentrations, ensuring the division site localization of Rga7. Moreover, the requirement for the F-BAR domain in Rga7 localization and function in cytokinesis is bypassed by tethering an Rga7 construct lacking its F-BAR to Rng10, indicating that at least some F-BAR domains are necessary but not sufficient for PM targeting and are stably localized to specific cortical positions through adaptor proteins.
Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.
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12 MeSH Terms
Myosin IIA drives membrane bleb retraction.
Taneja N, Burnette DT
(2019) Mol Biol Cell 30: 1051-1059
MeSH Terms: Actins, Animals, Blister, COS Cells, Cell Membrane, Cell Membrane Structures, Cell Movement, Cell Surface Extensions, Chlorocebus aethiops, Cytokinesis, Cytoplasm, Cytoskeletal Proteins, HeLa Cells, Humans, Myosin Type II, Nerve Tissue Proteins, Nonmuscle Myosin Type IIA, Nonmuscle Myosin Type IIB
Show Abstract · Added March 27, 2019
Membrane blebs are specialized cellular protrusions that play diverse roles in processes such as cell division and cell migration. Blebbing can be divided into three distinct phases: bleb nucleation, bleb growth, and bleb retraction. Following nucleation and bleb growth, the actin cortex, comprising actin, cross-linking proteins, and nonmuscle myosin II (MII), begins to reassemble on the membrane. MII then drives the final phase, bleb retraction, which results in reintegration of the bleb into the cellular cortex. There are three MII paralogues with distinct biophysical properties expressed in mammalian cells: MIIA, MIIB, and MIIC. Here we show that MIIA specifically drives bleb retraction during cytokinesis. The motor domain and regulation of the nonhelical tailpiece of MIIA both contribute to its ability to drive bleb retraction. These experiments have also revealed a relationship between faster turnover of MIIA at the cortex and its ability to drive bleb retraction.
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18 MeSH Terms
Structural Model of Ghrelin Bound to its G Protein-Coupled Receptor.
Bender BJ, Vortmeier G, Ernicke S, Bosse M, Kaiser A, Els-Heindl S, Krug U, Beck-Sickinger A, Meiler J, Huster D
(2019) Structure 27: 537-544.e4
MeSH Terms: Animals, Binding Sites, COS Cells, Chlorocebus aethiops, Ghrelin, HEK293 Cells, Humans, Magnetic Resonance Spectroscopy, Models, Molecular, Mutagenesis, Site-Directed, Protein Binding, Protein Conformation, Receptors, Ghrelin
Show Abstract · Added March 21, 2020
The peptide ghrelin targets the growth hormone secretagogue receptor 1a (GHSR) to signal changes in cell metabolism and is a sought-after therapeutic target, although no structure is known to date. To investigate the structural basis of ghrelin binding to GHSR, we used solid-state nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, and Rosetta modeling. The use of saturation transfer difference NMR identified key residues in the peptide for receptor binding beyond the known motif. This information combined with assignment of the secondary structure of ghrelin in its receptor-bound state was incorporated into Rosetta using an approach that accounts for flexible binding partners. The NMR data and models revealed an extended binding surface that was confirmed via mutagenesis. Our results agree with a growing evidence of peptides interacting via two sites at G protein-coupled receptors.
Copyright © 2018 Elsevier Ltd. All rights reserved.
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Cleavage of arrestin-3 by caspases attenuates cell death by precluding arrestin-dependent JNK activation.
Kook S, Vishnivetskiy SA, Gurevich VV, Gurevich EV
(2019) Cell Signal 54: 161-169
MeSH Terms: Animals, Apoptosis, Arrestins, COS Cells, Caspases, Chlorocebus aethiops, Etoposide, MAP Kinase Kinase 4, MAP Kinase Kinase Kinase 5
Show Abstract · Added March 18, 2020
The two non-visual subtypes, arrestin-2 and arrestin-3, are ubiquitously expressed and bind hundreds of G protein-coupled receptors. In addition, these arrestins also interact with dozens of non-receptor signaling proteins, including c-Src, ERK and JNK, that regulate cell death and survival. Arrestin-3 facilitates the activation of JNK family kinases, which are important players in the regulation of apoptosis. Here we show that arrestin-3 is specifically cleaved at Asp366, Asp405 and Asp406 by caspases during the apoptotic cell death. This results in the generation of one main cleavage product, arrestin-3-(1-366). The formation of this fragment occurs in a dose-dependent manner with the increase of fraction of apoptotic cells upon etoposide treatment. In contrast to a caspase-resistant mutant (D366/405/406E) the arrestin-3-(1-366) fragment reduces the apoptosis of etoposide-treated cells. We found that caspase cleavage did not affect the binding of the arrestin-3 to JNK3, but prevented facilitation of its activation, in contrast to the caspase-resistant mutant, which facilitated JNK activation similar to WT arrestin-3, likely due to decreased binding of the upstream kinases ASK1 and MKK4/7. The data suggest that caspase-generated arrestin-3-(1-366) prevents the signaling in the ASK1-MKK4/7-JNK1/2/3 cascade and protects cells, thereby suppressing apoptosis.
Copyright © 2018 Elsevier Inc. All rights reserved.
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9 MeSH Terms
A protective human monoclonal antibody targeting the West Nile virus E protein preferentially recognizes mature virions.
Goo L, Debbink K, Kose N, Sapparapu G, Doyle MP, Wessel AW, Richner JM, Burgomaster KE, Larman BC, Dowd KA, Diamond MS, Crowe JE, Pierson TC
(2019) Nat Microbiol 4: 71-77
MeSH Terms: Aedes, Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, Cell Line, Chlorocebus aethiops, HEK293 Cells, Humans, Male, Mice, Mice, Inbred C57BL, Protein Domains, Vero Cells, Viral Envelope Proteins, West Nile Fever, West Nile Virus Vaccines, West Nile virus
Show Abstract · Added March 31, 2019
West Nile virus (WNV), a member of the Flavivirus genus, is a leading cause of viral encephalitis in the United States. The development of neutralizing antibodies against the flavivirus envelope (E) protein is critical for immunity and vaccine protection. Previously identified candidate therapeutic mouse and human neutralizing monoclonal antibodies (mAbs) target epitopes within the E domain III lateral ridge and the domain I-II hinge region, respectively. To explore the neutralizing antibody repertoire elicited by WNV infection for potential therapeutic application, we isolated ten mAbs from WNV-infected individuals. mAb WNV-86 neutralized WNV with a 50% inhibitory concentration of 2 ng ml, one of the most potently neutralizing flavivirus-specific antibodies ever isolated. WNV-86 targets an epitope in E domain II, and preferentially recognizes mature virions lacking an uncleaved form of the chaperone protein prM, unlike most flavivirus-specific antibodies. In vitro selection experiments revealed a neutralization escape mechanism involving a glycan addition to E domain II. Finally, a single dose of WNV-86 administered two days post-infection protected mice from lethal WNV challenge. This study identifies a highly potent human neutralizing mAb with therapeutic potential that targets an epitope preferentially displayed on mature virions.
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18 MeSH Terms
Protective antibodies against Eastern equine encephalitis virus bind to epitopes in domains A and B of the E2 glycoprotein.
Kim AS, Austin SK, Gardner CL, Zuiani A, Reed DS, Trobaugh DW, Sun C, Basore K, Williamson LE, Crowe JE, Slifka MK, Fremont DH, Klimstra WB, Diamond MS
(2019) Nat Microbiol 4: 187-197
MeSH Terms: Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, Chlorocebus aethiops, Cricetinae, Encephalitis Virus, Eastern Equine, Encephalomyelitis, Equine, Epitope Mapping, Epitopes, Female, HEK293 Cells, Humans, Mice, Protein Domains, Vero Cells, Viral Envelope Proteins
Show Abstract · Added March 31, 2019
Eastern equine encephalitis virus (EEEV) is a mosquito-transmitted alphavirus with a high case mortality rate in humans. EEEV is a biodefence concern because of its potential for aerosol spread and the lack of existing countermeasures. Here, we identify a panel of 18 neutralizing murine monoclonal antibodies (mAbs) against the EEEV E2 glycoprotein, several of which have 'elite' activity with 50 and 99% effective inhibitory concentrations (EC and EC) of less than 10 and 100 ng ml, respectively. Alanine-scanning mutagenesis and neutralization escape mapping analysis revealed epitopes for these mAbs in domains A or B of the E2 glycoprotein. A majority of the neutralizing mAbs blocked infection at a post-attachment stage, with several inhibiting viral membrane fusion. Administration of one dose of anti-EEEV mAb protected mice from lethal subcutaneous or aerosol challenge. These experiments define the mechanistic basis for neutralization by protective anti-EEEV mAbs and suggest a path forward for treatment and vaccine design.
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17 MeSH Terms
Multifunctional Pan-ebolavirus Antibody Recognizes a Site of Broad Vulnerability on the Ebolavirus Glycoprotein.
Gilchuk P, Kuzmina N, Ilinykh PA, Huang K, Gunn BM, Bryan A, Davidson E, Doranz BJ, Turner HL, Fusco ML, Bramble MS, Hoff NA, Binshtein E, Kose N, Flyak AI, Flinko R, Orlandi C, Carnahan R, Parrish EH, Sevy AM, Bombardi RG, Singh PK, Mukadi P, Muyembe-Tamfum JJ, Ohi MD, Saphire EO, Lewis GK, Alter G, Ward AB, Rimoin AW, Bukreyev A, Crowe JE
(2018) Immunity 49: 363-374.e10
MeSH Terms: 3T3 Cells, Adult, Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, CHO Cells, Cell Line, Chlorocebus aethiops, Cricetulus, Disease Models, Animal, Drosophila, Ebolavirus, Female, Ferrets, Glycoproteins, Guinea Pigs, Hemorrhagic Fever, Ebola, Humans, Immunoglobulin G, Jurkat Cells, Male, Mice, Mice, Inbred BALB C, Mice, Knockout, THP-1 Cells, Vero Cells
Show Abstract · Added March 3, 2020
Ebolaviruses cause severe disease in humans, and identification of monoclonal antibodies (mAbs) that are effective against multiple ebolaviruses are important for therapeutics development. Here we describe a distinct class of broadly neutralizing human mAbs with protective capacity against three ebolaviruses infectious for humans: Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) viruses. We isolated mAbs from human survivors of ebolavirus disease and identified a potent mAb, EBOV-520, which bound to an epitope in the glycoprotein (GP) base region. EBOV-520 efficiently neutralized EBOV, BDBV, and SUDV and also showed protective capacity in relevant animal models of these infections. EBOV-520 mediated protection principally by direct virus neutralization and exhibited multifunctional properties. This study identified a potent naturally occurring mAb and defined key features of the human antibody response that may contribute to broad protection. This multifunctional mAb and related clones are promising candidates for development as broadly protective pan-ebolavirus therapeutic molecules.
Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
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