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Mutational analysis of cysteine residues of the insect odorant co-receptor (Orco) from Drosophila melanogaster reveals differential effects on agonist- and odorant-tuning receptor-dependent activation.
Turner RM, Derryberry SL, Kumar BN, Brittain T, Zwiebel LJ, Newcomb RD, Christie DL
(2014) J Biol Chem 289: 31837-45
MeSH Terms: Allosteric Site, Animals, Biotinylation, Calcium, Cysteine, DNA Mutational Analysis, Drosophila Proteins, Drosophila melanogaster, Epitopes, HEK293 Cells, Humans, Ion Channels, Kinetics, Mutagenesis, Site-Directed, Mutation, Odorants, Patch-Clamp Techniques, Protein Binding, Protein Structure, Tertiary, Receptors, Odorant, Thioglycolates, Triazoles
Show Abstract · Added February 19, 2015
Insect odorant receptors are heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor (ORx) and a highly conserved co-receptor known as Orco. Orco is found only in insects, and very little is known about its structure and the mechanism leading to channel activation. In the absence of an ORx, Orco forms homomeric channels that can be activated by a synthetic agonist, VUAA1. Drosophila melanogaster Orco (DmelOrco) contains eight cysteine amino acid residues, six of which are highly conserved. In this study, we replaced individual cysteine residues with serine or alanine and expressed Orco mutants in Flp-In 293 T-Rex cells. Changes in intracellular Ca(2+) levels were used to determine responses to VUAA1. Replacement of two cysteines (Cys-429 and Cys-449) in a predicted intracellular loop (ICL3), individually or together, gave variants that all showed similar increases in the rate of response and sensitivity to VUAA1 compared with wild-type DmelOrco. Kinetic modeling indicated that the response of the Orco mutants to VUAA1 was faster than wild-type Orco. The enhanced sensitivity and faster response of the Cys mutants was confirmed by whole-cell voltage clamp electrophysiology. In contrast to the results from direct agonist activation of Orco, the two cysteine replacement mutants when co-expressed with a tuning receptor (DmelOR22a) showed an ∼10-fold decrease in potency for activation by 2-methyl hexanoate. Our work has shown that intracellular loop 3 is important for Orco channel activation. Importantly, this study also suggests differences in the structural requirements for the activation of homomeric and heteromeric Orco channel complexes.
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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22 MeSH Terms
Validation of DTI tractography-based measures of primary motor area connectivity in the squirrel monkey brain.
Gao Y, Choe AS, Stepniewska I, Li X, Avison MJ, Anderson AW
(2013) PLoS One 8: e75065
MeSH Terms: Animals, Biotinylation, Brain Mapping, Dextrans, Diffusion Tensor Imaging, Motor Cortex, Nerve Net, Saimiri, Staining and Labeling
Show Abstract · Added July 21, 2014
Diffusion tensor imaging (DTI) tractography provides noninvasive measures of structural cortico-cortical connectivity of the brain. However, the agreement between DTI-tractography-based measures and histological 'ground truth' has not been quantified. In this study, we reconstructed the 3D density distribution maps (DDM) of fibers labeled with an anatomical tracer, biotinylated dextran amine (BDA), as well as DTI tractography-derived streamlines connecting the primary motor (M1) cortex to other cortical regions in the squirrel monkey brain. We evaluated the agreement in M1-cortical connectivity between the fibers labeled in the brain tissue and DTI streamlines on a regional and voxel-by-voxel basis. We found that DTI tractography is capable of providing inter-regional connectivity comparable to the neuroanatomical connectivity, but is less reliable measuring voxel-to-voxel variations within regions.
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9 MeSH Terms
The sodium channel accessory subunit Navβ1 regulates neuronal excitability through modulation of repolarizing voltage-gated K⁺ channels.
Marionneau C, Carrasquillo Y, Norris AJ, Townsend RR, Isom LL, Link AJ, Nerbonne JM
(2012) J Neurosci 32: 5716-27
MeSH Terms: Analysis of Variance, Animals, Bacterial Proteins, Biophysics, Biotinylation, Cell Line, Transformed, Cerebral Cortex, Cycloheximide, Electric Stimulation, Endocytosis, Gene Expression Regulation, Green Fluorescent Proteins, Humans, Immunoprecipitation, Luminescent Proteins, Mass Spectrometry, Mice, Mice, Inbred C57BL, Mice, Knockout, Neurons, Patch-Clamp Techniques, Protein Synthesis Inhibitors, Proteomics, RNA, Small Interfering, Receptors, Transferrin, Shal Potassium Channels, Sodium Channels, Transfection, Voltage-Gated Sodium Channel beta-1 Subunit
Show Abstract · Added February 20, 2015
The channel pore-forming α subunit Kv4.2 is a major constituent of A-type (I(A)) potassium currents and a key regulator of neuronal membrane excitability. Multiple mechanisms regulate the properties, subcellular targeting, and cell-surface expression of Kv4.2-encoded channels. In the present study, shotgun proteomic analyses of immunoprecipitated mouse brain Kv4.2 channel complexes unexpectedly identified the voltage-gated Na⁺ channel accessory subunit Navβ1. Voltage-clamp and current-clamp recordings revealed that knockdown of Navβ1 decreases I(A) densities in isolated cortical neurons and that action potential waveforms are prolonged and repetitive firing is increased in Scn1b-null cortical pyramidal neurons lacking Navβ1. Biochemical and voltage-clamp experiments further demonstrated that Navβ1 interacts with and increases the stability of the heterologously expressed Kv4.2 protein, resulting in greater total and cell-surface Kv4.2 protein expression and in larger Kv4.2-encoded current densities. Together, the results presented here identify Navβ1 as a component of native neuronal Kv4.2-encoded I(A) channel complexes and a novel regulator of I(A) channel densities and neuronal excitability.
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29 MeSH Terms
Attention deficit/hyperactivity disorder-derived coding variation in the dopamine transporter disrupts microdomain targeting and trafficking regulation.
Sakrikar D, Mazei-Robison MS, Mergy MA, Richtand NW, Han Q, Hamilton PJ, Bowton E, Galli A, Veenstra-Vanderweele J, Gill M, Blakely RD
(2012) J Neurosci 32: 5385-97
MeSH Terms: Adolescent, Amphetamine, Analysis of Variance, Attention Deficit Disorder with Hyperactivity, Bacterial Proteins, Benzylamines, Biotinylation, Calcium, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Cell Line, Transformed, Child, Child, Preschool, Cholera Toxin, Cohort Studies, Dopamine, Dopamine Plasma Membrane Transport Proteins, Dopamine Uptake Inhibitors, Dose-Response Relationship, Drug, Electrochemistry, Female, Humans, Immunoprecipitation, Luminescent Proteins, Male, Membrane Microdomains, Membrane Proteins, Piperazines, Polymorphism, Single Nucleotide, Protein Kinase Inhibitors, Protein Transport, Sulfonamides, Transfection, Tritium
Show Abstract · Added December 5, 2013
Attention deficit/hyperactivity disorder (ADHD) is the most commonly diagnosed disorder of school-age children. Although genetic and brain-imaging studies suggest a contribution of altered dopamine (DA) signaling in ADHD, evidence of signaling perturbations contributing to risk is largely circumstantial. The presynaptic, cocaine- and amphetamine (AMPH)-sensitive DA transporter (DAT) constrains DA availability at presynaptic and postsynaptic receptors following vesicular release and is targeted by the most commonly prescribed ADHD therapeutics. Using polymorphism discovery approaches with an ADHD cohort, we identified a hDAT (human DAT) coding variant, R615C, located in the distal C terminus of the transporter, a region previously implicated in constitutive and regulated transporter trafficking. Here, we demonstrate that, whereas wild-type DAT proteins traffic in a highly regulated manner, DAT 615C proteins recycle constitutively and demonstrate insensitivity to the endocytic effects of AMPH and PKC (protein kinase C) activation. The disrupted regulation of DAT 615C parallels a redistribution of the transporter variant away from GM1 ganglioside- and flotillin1-enriched membranes, and is accompanied by altered CaMKII (calcium/calmodulin-dependent protein kinase II) and flotillin-1 interactions. Using C-terminal peptides derived from wild-type DAT and the R615C variant, we establish that the DAT 615C C terminus can act dominantly to preclude AMPH regulation of wild-type DAT. Mutagenesis of DAT C-terminal sequences suggests that phosphorylation of T613 may be important in sorting DAT between constitutive and regulated pathways. Together, our studies support a coupling of DAT microdomain localization with transporter regulation and provide evidence of perturbed DAT activity and DA signaling as a risk determinant for ADHD.
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33 MeSH Terms
Biotinylated probes for the analysis of protein modification by electrophiles.
Codreanu SG, Kim HY, Porter NA, Liebler DC
(2012) Methods Mol Biol 803: 77-95
MeSH Terms: Aldehydes, Amino Acid Sequence, Biochemistry, Biotin, Biotinylation, Blood Proteins, Blotting, Western, Cell Extracts, Cell Line, Tumor, Databases, Protein, Humans, Immunoblotting, Indicators and Reagents, Mass Spectrometry, Molecular Probes, Molecular Sequence Data, Peptides, Protein Processing, Post-Translational, Proteins, Streptavidin, Trypsin
Show Abstract · Added March 7, 2014
Formation of covalent protein adducts by lipid electrophiles contributes to diseases and toxicities linked to oxidative stress, but analysis of the adducts presents a challenging analytical problem. We describe selective adduct capture using biotin affinity probes to enrich protein and peptide adducts for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). One approach employs biotinamidohexanoic acid hydrazide to covalently label residual carbonyl groups on adducts. The other employs alkynyl analogs of lipid electrophiles, which form adducts that can be postlabeled with azidobiotin tags by Cu(+)-catalyzed cycloaddition (Click chemistry). To enhance the selectivity of adduct capture, we use an azidobiotin reagent with a photocleavable linker, which allows recovery of adducted proteins and peptides under mild conditions. This approach allows both the identification of protein targets of lipid electrophiles and sequence mapping of the adducts.
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21 MeSH Terms
Impaired striatal Akt signaling disrupts dopamine homeostasis and increases feeding.
Speed N, Saunders C, Davis AR, Owens WA, Matthies HJ, Saadat S, Kennedy JP, Vaughan RA, Neve RL, Lindsley CW, Russo SJ, Daws LC, Niswender KD, Galli A
(2011) PLoS One 6: e25169
MeSH Terms: Animals, Biological Transport, Biotinylation, Brain, Cell Membrane, Corpus Striatum, Diet, High-Fat, Dopamine, Homeostasis, Insulin, Locomotion, Male, Obesity, Proto-Oncogene Proteins c-akt, Rats, Rats, Sprague-Dawley, Signal Transduction, Substantia Nigra
Show Abstract · Added September 17, 2013
BACKGROUND - The prevalence of obesity has increased dramatically worldwide. The obesity epidemic begs for novel concepts and therapeutic targets that cohesively address "food-abuse" disorders. We demonstrate a molecular link between impairment of a central kinase (Akt) involved in insulin signaling induced by exposure to a high-fat (HF) diet and dysregulation of higher order circuitry involved in feeding. Dopamine (DA) rich brain structures, such as striatum, provide motivation stimuli for feeding. In these central circuitries, DA dysfunction is posited to contribute to obesity pathogenesis. We identified a mechanistic link between metabolic dysregulation and the maladaptive behaviors that potentiate weight gain. Insulin, a hormone in the periphery, also acts centrally to regulate both homeostatic and reward-based HF feeding. It regulates DA homeostasis, in part, by controlling a key element in DA clearance, the DA transporter (DAT). Upon HF feeding, nigro-striatal neurons rapidly develop insulin signaling deficiencies, causing increased HF calorie intake.
METHODOLOGY/PRINCIPAL FINDINGS - We show that consumption of fat-rich food impairs striatal activation of the insulin-activated signaling kinase, Akt. HF-induced Akt impairment, in turn, reduces DAT cell surface expression and function, thereby decreasing DA homeostasis and amphetamine (AMPH)-induced DA efflux. In addition, HF-mediated dysregulation of Akt signaling impairs DA-related behaviors such as (AMPH)-induced locomotion and increased caloric intake. We restored nigro-striatal Akt phosphorylation using recombinant viral vector expression technology. We observed a rescue of DAT expression in HF fed rats, which was associated with a return of locomotor responses to AMPH and normalization of HF diet-induced hyperphagia.
CONCLUSIONS/SIGNIFICANCE - Acquired disruption of brain insulin action may confer risk for and/or underlie "food-abuse" disorders and the recalcitrance of obesity. This molecular model, thus, explains how even short-term exposure to "the fast food lifestyle" creates a cycle of disordered eating that cements pathological changes in DA signaling leading to weight gain and obesity.
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18 MeSH Terms
ERp29 regulates DeltaF508 and wild-type cystic fibrosis transmembrane conductance regulator (CFTR) trafficking to the plasma membrane in cystic fibrosis (CF) and non-CF epithelial cells.
Suaud L, Miller K, Alvey L, Yan W, Robay A, Kebler C, Kreindler JL, Guttentag S, Hubbard MJ, Rubenstein RC
(2011) J Biol Chem 286: 21239-53
MeSH Terms: Animals, Biotinylation, Cell Membrane, Cystic Fibrosis, Cystic Fibrosis Transmembrane Conductance Regulator, Electrophysiology, Endoplasmic Reticulum, Epithelial Cells, Heat-Shock Proteins, Humans, Ions, Oocytes, Phenylbutyrates, Protein Transport, Xenopus
Show Abstract · Added January 20, 2015
Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ΔF508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ΔF508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ΔF508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (∼1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ΔF508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o- WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ΔF508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ΔF508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ΔF508-CFTR trafficking in CF epithelial cells.
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15 MeSH Terms
Cysteinyl peptide capture for shotgun proteomics: global assessment of chemoselective fractionation.
Lin D, Li J, Slebos RJ, Liebler DC
(2010) J Proteome Res 9: 5461-72
MeSH Terms: Analysis of Variance, Biotinylation, Cell Line, Tumor, Chemical Fractionation, Chromatography, Liquid, Colonic Neoplasms, Cysteine, Humans, Isoelectric Focusing, Peptides, Proteomics, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Tandem Mass Spectrometry
Show Abstract · Added March 5, 2014
The complexity of cell and tissue proteomes presents one of the most significant technical challenges in proteomic biomarker discovery. Multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based shotgun proteomics can be coupled with selective enrichment of cysteinyl peptides (Cys-peptides) to reduce sample complexity and increase proteome coverage. Here we evaluated the impact of Cys-peptide enrichment on global proteomic inventories. We employed a new cleavable thiol-reactive biotinylating probe, N-(2-(2-(2-(2-(3-(1-hydroxy-2-oxo-2-phenylethyl)phenoxy)acetamido)ethoxy)-ethoxy)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (IBB), to capture Cys-peptides after digestion. Treatment of tryptic digests with the IBB reagent followed by streptavidin capture and mild alkaline hydrolysis releases a highly purified population of Cys-peptides with a residual S-carboxymethyl tag. Isoelectric focusing (IEF) followed by LC-MS/MS of Cys-peptides significantly expanded proteome coverage in Saccharomyces cerevisiae (yeast) and in human colon carcinoma RKO cells. IBB-based fractionation enhanced detection of Cys-proteins in direct proportion to their cysteine content. The degree of enrichment typically was 2-8-fold but ranged up to almost 20-fold for a few proteins. Published copy number annotation for the yeast proteome enabled benchmarking of MS/MS spectral count data to yeast protein abundance and revealed selective enrichment of cysteine-rich, lower abundance proteins. Spectral count data further established this relationship in RKO cells. Enhanced detection of low abundance proteins was due to the chemoselectivity of Cys-peptide capture, rather than simplification of the peptide mixture through fractionation.
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14 MeSH Terms
Rab11 supports amphetamine-stimulated norepinephrine transporter trafficking.
Matthies HJ, Moore JL, Saunders C, Matthies DS, Lapierre LA, Goldenring JR, Blakely RD, Galli A
(2010) J Neurosci 30: 7863-77
MeSH Terms: Adrenergic Agents, Amphetamine, Animals, Biotinylation, Carrier Proteins, Cells, Cultured, Fluorescent Antibody Technique, Ganglia, Sympathetic, Membrane Proteins, Mice, Mice, Inbred C57BL, Microscopy, Confocal, Mutation, Neurons, Norepinephrine Plasma Membrane Transport Proteins, Protein Transport, Rats, Signal Transduction, Synaptic Vesicles, rab GTP-Binding Proteins, rab4 GTP-Binding Proteins
Show Abstract · Added December 10, 2013
The norepinephrine transporter (NET) is a presynaptic plasma membrane protein that mediates reuptake of synaptically released norepinephrine. NET is also a major target for medications used for the treatment of depression, attention deficit/hyperactivity disorder, narcolepsy, and obesity. NET is regulated by numerous mechanisms, including catalytic activation and membrane trafficking. Amphetamine (AMPH), a psychostimulant and NET substrate, has also been shown to induce NET trafficking. However, neither the molecular basis nor the nature of the relevant membrane compartments of AMPH-modulated NET trafficking has been defined. Indeed, direct visualization of drug-modulated NET trafficking in neurons has yet to be demonstrated. In this study, we used a recently developed NET antibody and the presence of large presynaptic boutons in sympathetic neurons to examine basal and AMPH-modulated NET trafficking. Specifically, we establish a role for Rab11 in AMPH-induced NET trafficking. First, we found that, in cortical slices, AMPH induces a reduction in surface NET. Next, we observed AMPH-induced accumulation and colocalization of NET with Rab11a and Rab4 in presynaptic boutons of cultured neurons. Using tagged proteins, we demonstrated that NET and a truncated Rab11 effector (FIP2DeltaC2) do not redistribute in synchrony, whereas NET and wild-type Rab11a do. Analysis of various Rab11a/b mutants further demonstrates that Rab11 regulates NET trafficking. Expression of the truncated Rab11a effector (FIP2DeltaC2) attenuates endogenous Rab11 function and prevented AMPH-induced NET internalization as does GDP-locked Rab4 S22N. Our data demonstrate that AMPH leads to an increase of NET in endosomes of single boutons and varicosities in a Rab11-dependent manner.
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21 MeSH Terms
Use of dimedone-based chemical probes for sulfenic acid detection methods to visualize and identify labeled proteins.
Nelson KJ, Klomsiri C, Codreanu SG, Soito L, Liebler DC, Rogers LC, Daniel LW, Poole LB
(2010) Methods Enzymol 473: 95-115
MeSH Terms: Animals, Biotinylation, Cyclohexanones, Humans, Mass Spectrometry, Protein Processing, Post-Translational, Proteins, Staining and Labeling, Sulfenic Acids
Show Abstract · Added March 20, 2014
Reversible thiol modification is a major component of the modulation of cell-signaling pathways by reactive oxygen species. Hydrogen peroxide, peroxynitrite, or lipid hydroperoxides are all able to oxidize cysteines to form cysteine sulfenic acids; this reactive intermediate can be directly reduced to thiol by cellular reductants such as thioredoxin or further participate in disulfide bond formation with glutathione or cysteine residues in the same or another protein. To identify the direct protein targets of cysteine modification and the conditions under which they are oxidized, a series of dimedone-based reagents linked to affinity or fluorescent tags have been developed that specifically alkylate and trap cysteine sulfenic acids. In this chapter, we provide detailed methods using one of our biotin-tagged reagents, DCP-Bio1, to identify and monitor proteins that are oxidized in vitro and in vivo. Using streptavidin-linked agarose beads, this biotin-linked reagent can be used to affinity capture labeled proteins. Stringent washing of the beads prior to elution minimizes the contamination of the enriched material with unlabeled proteins through coimmunoprecipitation or nonspecific binding. In particular, we suggest including DTT in one of the washes to remove proteins covalently linked to biotinylated proteins through a disulfide bond, except in cases where these linked proteins are of interest. We also provide methods for targeted approaches monitoring cysteine oxidation in individual proteins, global approaches to follow total cysteine oxidation in the cell, and guidelines for proteomic analyses to identify novel proteins with redox sensitive cysteines.
Copyright (c) 2010 Elsevier Inc. All rights reserved.
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9 MeSH Terms