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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, Cercopithecus 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
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, Cercopithecus 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, Cercopithecus 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
Broadly Neutralizing Antibody Mediated Clearance of Human Hepatitis C Virus Infection.
Kinchen VJ, Zahid MN, Flyak AI, Soliman MG, Learn GH, Wang S, Davidson E, Doranz BJ, Ray SC, Cox AL, Crowe JE, Bjorkman PJ, Shaw GM, Bailey JR
(2018) Cell Host Microbe 24: 717-730.e5
MeSH Terms: Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibody Specificity, Base Sequence, Binding Sites, Cell Line, Cricetulus, Epitopes, Female, HEK293 Cells, HIV-1, Hepacivirus, Hepatitis C, Hepatitis C Antibodies, Humans, Immunologic Memory, Male, Models, Molecular, Mutagenesis, Site-Directed, Viral Envelope Proteins, Viral Load
Show Abstract · Added March 31, 2019
The role that broadly neutralizing antibodies (bNAbs) play in natural clearance of human hepatitis C virus (HCV) infection and the underlying mechanisms remain unknown. Here, we investigate the mechanism by which bNAbs, isolated from two humans who spontaneously cleared HCV infection, contribute to HCV control. Using viral gene sequences amplified from longitudinal plasma of the two subjects, we found that these bNAbs, which target the front layer of the HCV envelope protein E2, neutralized most autologous HCV strains. Acquisition of resistance to bNAbs by some autologous strains was accompanied by progressive loss of E2 protein function, and temporally associated with HCV clearance. These data demonstrate that bNAbs can mediate clearance of human HCV infection by neutralizing infecting strains and driving escaped viruses to an unfit state. These immunopathologic events distinguish HCV from HIV-1 and suggest that development of an HCV vaccine may be achievable.
Copyright © 2018 Elsevier Inc. All rights reserved.
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22 MeSH Terms
Activated CaMKII Binds to the mGlu Metabotropic Glutamate Receptor and Modulates Calcium Mobilization.
Marks CR, Shonesy BC, Wang X, Stephenson JR, Niswender CM, Colbran RJ
(2018) Mol Pharmacol 94: 1352-1362
MeSH Terms: Animals, Calcium, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Calmodulin, Cell Line, Cell Membrane, Female, HEK293 Cells, Humans, Immunoprecipitation, Male, Mice, Mice, Knockout, Protein Binding, Receptor, Metabotropic Glutamate 5, Signal Transduction
Show Abstract · Added November 8, 2018
Ca/calmodulin-dependent protein kinase II (CaMKII) and metabotropic glutamate receptor 5 (mGlu) are critical signaling molecules in synaptic plasticity and learning/memory. Here, we demonstrate that mGlu is present in CaMKII complexes isolated from mouse forebrain. Further in vitro characterization showed that the membrane-proximal region of the C-terminal domain (CTD) of mGlu directly interacts with purified Thr286-autophosphorylated (activated) CaMKII However, the binding of CaMKII to this CTD fragment is reduced by the addition of excess Ca/calmodulin or by additional CaMKII autophosphorylation at non-Thr286 sites. Furthermore, in vitro binding of CaMKII is dependent on a tribasic residue motif Lys-Arg-Arg (KRR) at residues 866-868 of the mGlu-CTD, and mutation of this motif decreases the coimmunoprecipitation of CaMKII with full-length mGlu expressed in heterologous cells by about 50%. The KRR motif is required for two novel functional effects of coexpressing constitutively active CaMKII with mGlu in heterologous cells. First, cell-surface biotinylation studies showed that CaMKII increases the surface expression of mGlu Second, using Ca fluorimetry and single-cell Ca imaging, we found that CaMKII reduces the initial peak of mGlu-mediated Ca mobilization by about 25% while doubling the relative duration of the Ca signal. These findings provide new insights into the physical and functional coupling of these key regulators of postsynaptic signaling.
Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.
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16 MeSH Terms
Methylglyoxal-derived posttranslational arginine modifications are abundant histone marks.
Galligan JJ, Wepy JA, Streeter MD, Kingsley PJ, Mitchener MM, Wauchope OR, Beavers WN, Rose KL, Wang T, Spiegel DA, Marnett LJ
(2018) Proc Natl Acad Sci U S A 115: 9228-9233
MeSH Terms: Arginine, HEK293 Cells, Histones, Humans, Lactoylglutathione Lyase, Protein Processing, Post-Translational, Pyruvaldehyde, Transcription, Genetic
Show Abstract · Added April 12, 2019
Histone posttranslational modifications (PTMs) regulate chromatin dynamics, DNA accessibility, and transcription to expand the genetic code. Many of these PTMs are produced through cellular metabolism to offer both feedback and feedforward regulation. Herein we describe the existence of Lys and Arg modifications on histones by a glycolytic by-product, methylglyoxal (MGO). Our data demonstrate that adduction of histones by MGO is an abundant modification, present at the same order of magnitude as Arg methylation. These modifications were detected on all four core histones at critical residues involved in both nucleosome stability and reader domain binding. In addition, MGO treatment of cells lacking the major detoxifying enzyme, glyoxalase 1, results in marked disruption of H2B acetylation and ubiquitylation without affecting H2A, H3, and H4 modifications. Using RNA sequencing, we show that MGO is capable of altering gene transcription, most notably in cells lacking GLO1. Finally, we show that the deglycase DJ-1 protects histones from adduction by MGO. Collectively, our findings demonstrate the existence of a previously undetected histone modification derived from glycolysis, which may have far-reaching implications for the control of gene expression and protein transcription linked to metabolism.
Copyright © 2018 the Author(s). Published by PNAS.
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Structure-function analyses of the ion channel TRPC3 reveal that its cytoplasmic domain allosterically modulates channel gating.
Sierra-Valdez F, Azumaya CM, Romero LO, Nakagawa T, Cordero-Morales JF
(2018) J Biol Chem 293: 16102-16114
MeSH Terms: Allosteric Regulation, Ankyrin Repeat, HEK293 Cells, Humans, Ion Channel Gating, Mutation, Protein Conformation, alpha-Helical, Protein Domains, TRPC Cation Channels
Show Abstract · Added April 10, 2019
The transient receptor potential ion channels support Ca permeation in many organs, including the heart, brain, and kidney. Genetic mutations in transient receptor potential cation channel subfamily C member 3 (TRPC3) are associated with neurodegenerative diseases, memory loss, and hypertension. To better understand the conformational changes that regulate TRPC3 function, we solved the cryo-EM structures for the full-length human TRPC3 and its cytoplasmic domain (CPD) in the apo state at 5.8- and 4.0-Å resolution, respectively. These structures revealed that the TRPC3 transmembrane domain resembles those of other TRP channels and that the CPD is a stable module involved in channel assembly and gating. We observed the presence of a C-terminal domain swap at the center of the CPD where horizontal helices (HHs) transition into a coiled-coil bundle. Comparison of TRPC3 structures revealed that the HHs can reside in two distinct positions. Electrophysiological analyses disclosed that shortening the length of the C-terminal loop connecting the HH with the TRP helices increases TRPC3 activity and that elongating the length of the loop has the opposite effect. Our findings indicate that the C-terminal loop affects channel gating by altering the allosteric coupling between the cytoplasmic and transmembrane domains. We propose that molecules that target the HH may represent a promising strategy for controlling TRPC3-associated neurological disorders and hypertension.
© 2018 Sierra-Valdez et al.
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9 MeSH Terms
Rhodol-based thallium sensors for cellular imaging of potassium channel activity.
Dutter BF, Ender A, Sulikowski GA, Weaver CD
(2018) Org Biomol Chem 16: 5575-5579
MeSH Terms: Fluorescent Dyes, HEK293 Cells, Humans, Methylation, Microscopy, Confocal, Optical Imaging, Potassium Channels, Spectrometry, Fluorescence, Thallium, Xanthones
Show Abstract · Added April 10, 2019
Thallium (Tl+) flux assays enable imaging of potassium (K+) channel activity in cells and tissues by exploiting the permeability of K+ channels to Tl+ coupled with a fluorescent Tl+ sensitive dye. Common Tl+ sensing dyes utilize fluorescein as the fluorophore though fluorescein exhibits certain undesirable properties in these assays including short excitation wavelengths and pH sensitivity. To overcome these drawbacks, the replacement of fluorescein with rhodols was investigated. A library of 13 rhodol-based Tl+ sensors was synthesized and their properties and performance in Tl+ flux assays evaluated. The dimethyl rhodol Tl+ sensor emerged as the best of the series and performed comparably to fluorescein-based sensors while demonstrating greater pH tolerance in the physiological range and excitation and emission spectra 30 nm red-shifted from fluorescein.
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BVES is required for maintenance of colonic epithelial integrity in experimental colitis by modifying intestinal permeability.
Choksi YA, Reddy VK, Singh K, Barrett CW, Short SP, Parang B, Keating CE, Thompson JJ, Verriere TG, Brown RE, Piazuelo MB, Bader DM, Washington MK, Mittal MK, Brand T, Gobert AP, Coburn LA, Wilson KT, Williams CS
(2018) Mucosal Immunol 11: 1363-1374
MeSH Terms: Adult, Animals, Caco-2 Cells, Cell Line, Cell Line, Tumor, Citrobacter rodentium, Coculture Techniques, Colitis, Ulcerative, Colon, Dextran Sulfate, Epithelial Cells, Escherichia coli, Female, HEK293 Cells, Humans, Intestinal Absorption, Intestinal Mucosa, Male, Membrane Proteins, Mice, Mice, Inbred C57BL, Middle Aged, Permeability, RNA, Messenger, Signal Transduction, Tight Junctions
Show Abstract · Added June 23, 2018
Blood vessel epicardial substance (BVES), or POPDC1, is a tight junction-associated transmembrane protein that modulates epithelial-to-mesenchymal transition (EMT) via junctional signaling pathways. There have been no in vivo studies investigating the role of BVES in colitis. We hypothesized that BVES is critical for maintaining colonic epithelial integrity. At baseline, Bves mouse colons demonstrate increased crypt height, elevated proliferation, decreased apoptosis, altered intestinal lineage allocation, and dysregulation of tight junctions with functional deficits in permeability and altered intestinal immunity. Bves mice inoculated with Citrobacter rodentium had greater colonic injury, increased colonic and mesenteric lymph node bacterial colonization, and altered immune responses after infection. We propose that increased bacterial colonization and translocation result in amplified immune responses and worsened injury. Similarly, dextran sodium sulfate (DSS) treatment resulted in greater histologic injury in Bves mice. Two different human cell lines (Caco2 and HEK293Ts) co-cultured with enteropathogenic E. coli showed increased attaching/effacing lesions in the absence of BVES. Finally, BVES mRNA levels were reduced in human ulcerative colitis (UC) biopsy specimens. Collectively, these studies suggest that BVES plays a protective role both in ulcerative and infectious colitis and identify BVES as a critical protector of colonic mucosal integrity.
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26 MeSH Terms
Discovery, Characterization, and Effects on Renal Fluid and Electrolyte Excretion of the Kir4.1 Potassium Channel Pore Blocker, VU0134992.
Kharade SV, Kurata H, Bender AM, Blobaum AL, Figueroa EE, Duran A, Kramer M, Days E, Vinson P, Flores D, Satlin LM, Meiler J, Weaver CD, Lindsley CW, Hopkins CR, Denton JS
(2018) Mol Pharmacol 94: 926-937
MeSH Terms: Animals, Binding Sites, Diuretics, Electrolytes, HEK293 Cells, Humans, Male, Models, Molecular, Molecular Docking Simulation, Molecular Structure, Mutagenesis, Site-Directed, Potassium Channels, Inwardly Rectifying, Rats, Small Molecule Libraries, Substrate Specificity
Show Abstract · Added April 10, 2019
The inward rectifier potassium (Kir) channel Kir4.1 () carries out important physiologic roles in epithelial cells of the kidney, astrocytes in the central nervous system, and stria vascularis of the inner ear. Loss-of-function mutations in lead to EAST/SeSAME syndrome, which is characterized by epilepsy, ataxia, renal salt wasting, and sensorineural deafness. Although genetic approaches have been indispensable for establishing the importance of Kir4.1 in the normal function of these tissues, the availability of pharmacological tools for acutely manipulating the activity of Kir4.1 in genetically normal animals has been lacking. We therefore carried out a high-throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library for small-molecule modulators of Kir4.1. The most potent inhibitor identified was 2-(2-bromo-4-isopropylphenoxy)--(2,2,6,6-tetramethylpiperidin-4-yl)acetamide (VU0134992). In whole-cell patch-clamp electrophysiology experiments, VU0134992 inhibits Kir4.1 with an IC value of 0.97 M and is 9-fold selective for homomeric Kir4.1 over Kir4.1/5.1 concatemeric channels (IC = 9 M) at -120 mV. In thallium (Tl) flux assays, VU0134992 is greater than 30-fold selective for Kir4.1 over Kir1.1, Kir2.1, and Kir2.2; is weakly active toward Kir2.3, Kir6.2/SUR1, and Kir7.1; and is equally active toward Kir3.1/3.2, Kir3.1/3.4, and Kir4.2. This potency and selectivity profile is superior to Kir4.1 inhibitors amitriptyline, nortriptyline, and fluoxetine. Medicinal chemistry identified components of VU0134992 that are critical for inhibiting Kir4.1. Patch-clamp electrophysiology, molecular modeling, and site-directed mutagenesis identified pore-lining glutamate 158 and isoleucine 159 as critical residues for block of the channel. VU0134992 displayed a large free unbound fraction () in rat plasma ( = 0.213). Consistent with the known role of Kir4.1 in renal function, oral dosing of VU0134992 led to a dose-dependent diuresis, natriuresis, and kaliuresis in rats. Thus, VU0134992 represents the first in vivo active tool compound for probing the therapeutic potential of Kir4.1 as a novel diuretic target for the treatment of hypertension.
Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.
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