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Trafficking and surface expression of hyperpolarization-activated cyclic nucleotide-gated channels in hippocampal neurons.
Noam Y, Zha Q, Phan L, Wu RL, Chetkovich DM, Wadman WJ, Baram TZ
(2010) J Biol Chem 285: 14724-36
MeSH Terms: Actins, Animals, Animals, Newborn, Biotinylation, Cells, Cultured, Cyclic Nucleotide-Gated Cation Channels, Dendrites, Electrophysiology, Green Fluorescent Proteins, Hippocampus, Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels, Immunoenzyme Techniques, Ion Channel Gating, Microtubules, Neurons, Potassium Channels, Rats, Rats, Sprague-Dawley, Recombinant Fusion Proteins
Show Abstract · Added April 2, 2019
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate the hyperpolarization-activated current I(h) and thus play important roles in the regulation of brain excitability. The subcellular distribution pattern of the HCN channels influences the effects that they exert on the properties and activity of neurons. However, little is known about the mechanisms that control HCN channel trafficking to subcellular compartments or that regulate their surface expression. Here we studied the dynamics of HCN channel trafficking in hippocampal neurons using dissociated cultures coupled with time lapse imaging of fluorophore-fused HCN channels. HCN1-green fluorescence protein (HCN1-GFP) channels resided in vesicle-like organelles that moved in distinct patterns along neuronal dendrites, and these properties were isoform-specific. HCN1 trafficking required intact actin and tubulin and was rapidly inhibited by activation of either NMDA or AMPA-type ionotropic glutamate receptors in a calcium-dependent manner. Glutamate-induced inhibition of the movement of HCN1-GFP-expressing puncta was associated with increased surface expression of both native and transfected HCN1 channels, and this surface expression was accompanied by augmented I(h). Taken together, the results reveal the highly dynamic nature of HCN1 channel trafficking in hippocampal neurons and provide a novel potential mechanism for rapid regulation of I(h), and hence of neuronal properties, via alterations of HCN1 trafficking and surface expression.
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MeSH Terms
An azido-biotin reagent for use in the isolation of protein adducts of lipid-derived electrophiles by streptavidin catch and photorelease.
Kim HY, Tallman KA, Liebler DC, Porter NA
(2009) Mol Cell Proteomics 8: 2080-9
MeSH Terms: Aldehydes, Amino Acid Sequence, Azides, Biotin, Biotinylation, Blood Proteins, Blotting, Western, Humans, Indicators and Reagents, Light, Lipids, Mass Spectrometry, Molecular Sequence Data, Peptides, Proteins, Proteomics, Streptavidin
Show Abstract · Added March 7, 2014
HNE (4-hydroxynonenal), a byproduct of lipid peroxidation, reacts with nucleophilic centers on proteins. A terminal alkynyl analog of HNE (alkynyl HNE, aHNE) serves as a surrogate for HNE itself, both compounds reacting with protein amine and thiol functional groups by similar chemistry. Proteins modified with aHNE undergo reaction with a click reagent that bears azido and biotin groups separated by a photocleavable linker. Peptides and proteins modified in this way are affinity purified on streptavidin beads. Photolysis of the beads with a low intensity UV light releases bound biotinylated proteins or peptides, i.e. proteins or peptides modified by aHNE. Two strategies, (a) protein catch and photorelease and (b) peptide catch and photorelease, are employed to enrich adducted proteins or peptide mixtures highly enriched in adducts. Proteomics analysis of the streptavidin-purified peptides by LC-MS/MS permits identification of the adduction site. Identification of 30 separate peptides from human serum albumin by peptide catch and photorelease reveals 18 different aHNE adduction sites on the protein. Protein catch and photorelease shows that both HSA and ApoA1 in human plasma undergo significant modification by aHNE.
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17 MeSH Terms
Global analysis of protein damage by the lipid electrophile 4-hydroxy-2-nonenal.
Codreanu SG, Zhang B, Sobecki SM, Billheimer DD, Liebler DC
(2009) Mol Cell Proteomics 8: 670-80
MeSH Terms: Aldehydes, Biotin, Biotinylation, Cell Extracts, Cell Line, Tumor, Chromatography, Liquid, Humans, Hydrazines, Immunoblotting, Lipid Peroxidation, Lipids, Mass Spectrometry, Neoplasm Proteins, Peptides, Protein Carbonylation, Reproducibility of Results
Show Abstract · Added August 2, 2013
Lipid peroxidation yields a variety of electrophiles, which are thought to contribute to the molecular pathogenesis of diseases involving oxidative stress, yet little is known of the scope of protein damage caused by lipid electrophiles. We identified protein targets of the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE) in RKO cells treated with 50 or 100 mum HNE. HNE Michael adducts were biotinylated by reaction with biotinamidohexanoic acid hydrazide, captured with streptavidin, and the captured proteins were resolved by one dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin, and identified by liquid chromatography-tandem mass spectrometry. Of the 1500+ proteins identified, 417 displayed a statistically significant increase in adduction with increasing HNE exposure concentration. We further identified 18 biotin hydrazide-modified, HNE-adducted peptides by specific capture using anti-biotin antibody and analysis by high resolution liquid chromatography-tandem mass spectrometry. A subset of the identified HNE targets were validated with a streptavidin capture and immunoblotting approach, which enabled detection of adducts at HNE exposures as low as 1 mum. Protein interaction network analysis indicated several subsystems impacted by endogenous electrophiles in oxidative stress, including the 26 S proteasomal and chaperonin containing TCP-1 (CCT) systems involved in protein-folding and degradation, as well as the COP9 signalosome, translation initiation complex, and a large network of ribonucleoproteins. Global analyses of protein lipid electrophile adducts provide a systems-level perspective on the mechanisms of diseases involving oxidative stress.
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16 MeSH Terms
A high-throughput and sensitive method to measure global DNA methylation: application in lung cancer.
Anisowicz A, Huang H, Braunschweiger KI, Liu Z, Giese H, Wang H, Mamaev S, Olejnik J, Massion PP, Del Mastro RG
(2008) BMC Cancer 8: 222
MeSH Terms: Aged, Aged, 80 and over, Avidin, Biotinylation, Carcinoma, Non-Small-Cell Lung, Cell Line, Tumor, CpG Islands, DNA Methylation, Epigenesis, Genetic, Female, Humans, Lung Neoplasms, Male, Middle Aged, Regression Analysis
Show Abstract · Added March 10, 2014
BACKGROUND - Genome-wide changes in DNA methylation are an epigenetic phenomenon that can lead to the development of disease. The study of global DNA methylation utilizes technology that requires both expensive equipment and highly specialized skill sets.
METHODS - We have designed and developed an assay, CpGlobal, which is easy-to-use, does not utilize PCR, radioactivity and expensive equipment. CpGlobal utilizes methyl-sensitive restriction enzymes, HRP Neutravidin to detect the biotinylated nucleotides incorporated in an end-fill reaction and a luminometer to measure the chemiluminescence. The assay shows high accuracy and reproducibility in measuring global DNA methylation. Furthermore, CpGlobal correlates significantly with High Performance Capillary Electrophoresis (HPCE), a gold standard technology. We have applied the technology to understand the role of global DNA methylation in the natural history of lung cancer. World-wide, it is the leading cause of death attributed to any cancer. The survival rate is 15% over 5 years due to the lack of any clinical symptoms until the disease has progressed to a stage where cure is limited.
RESULTS - Through the use of cell lines and paired normal/tumor samples from patients with non-small cell lung cancer (NSCLC) we show that global DNA hypomethylation is highly associated with the progression of the tumor. In addition, the results provide the first indication that the normal part of the lung from a cancer patient has already experienced a loss of methylation compared to a normal individual.
CONCLUSION - By detecting these changes in global DNA methylation, CpGlobal may have a role as a barometer for the onset and development of lung cancer.
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15 MeSH Terms
Anomalous dopamine release associated with a human dopamine transporter coding variant.
Mazei-Robison MS, Bowton E, Holy M, Schmudermaier M, Freissmuth M, Sitte HH, Galli A, Blakely RD
(2008) J Neurosci 28: 7040-6
MeSH Terms: Alanine, Amphetamine, Analysis of Variance, Biotinylation, Cell Line, Transformed, Dopamine, Dopamine Plasma Membrane Transport Proteins, Dopamine Uptake Inhibitors, Humans, Membrane Potentials, Methylphenidate, Mutation, Patch-Clamp Techniques, Protein Transport, Sodium, Transfection, Valine
Show Abstract · Added December 10, 2013
Dopamine (DA) signaling at synapses is tightly coordinated through opposing mechanisms of vesicular fusion-mediated DA release and transporter-mediated DA clearance. Altered brain DA signaling is suspected to underlie multiple brain disorders, including schizophrenia, Parkinson's disease, bipolar disorder, and attention-deficit hyperactivity disorder (ADHD). We identified a pedigree containing two male children diagnosed with ADHD who share a rare human DA transporter (DAT; SLC6A3) coding variant, Ala559Val. Among >1000 control and affected subjects, the Val559 variant has only been isolated once previously, in a female subject with bipolar disorder. Although hDAT Ala559Val supports normal DAT protein and cell surface expression, as well as normal DA uptake, the variant exhibits anomalous DA efflux from DA-loaded cells. We also demonstrate that hDAT Ala599Val exhibits increased sensitivity to intracellular Na(+), but not intracellular DA, and displays exaggerated DA efflux at depolarized potentials. Remarkably, the two most common ADHD medications, amphetamine and methylphenidate, both block hDAT Ala559Val-mediated DA efflux, whereas these drugs have opposite actions at wild-type hDAT. Our findings reveal that DA efflux, typically associated with amphetamine-like psychostimulants, can be produced through a heritable change in hDAT structure. Because multiple gene products are known to coordinate to support amphetamine-mediated DA efflux, the properties of hDAT Ala559Val may have broader significance in identifying a new mechanism through which DA signaling disorders arise. Additionally, they suggest that block of inappropriate neurotransmitter efflux may be an unsuspected mechanism supporting the therapeutic actions of existing transporter-directed medications.
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17 MeSH Terms
Identification of protein targets of 4-hydroxynonenal using click chemistry for ex vivo biotinylation of azido and alkynyl derivatives.
Vila A, Tallman KA, Jacobs AT, Liebler DC, Porter NA, Marnett LJ
(2008) Chem Res Toxicol 21: 432-44
MeSH Terms: Aldehydes, Alkynes, Azides, Bacterial Proteins, Biotinylation, Combinatorial Chemistry Techniques, Cross-Linking Reagents, HSP70 Heat-Shock Proteins, HSP90 Heat-Shock Proteins, Horseradish Peroxidase, Magnetic Resonance Spectroscopy, Proteins, Proteomics
Show Abstract · Added March 5, 2014
Polyunsaturated fatty acids (PUFA) are primary targets of free radical damage during oxidative stress. Diffusible electrophilic alpha,beta-unsaturated aldehydes, such as 4-hydroxynonenal (HNE), have been shown to modify proteins that mediate cell signaling (e.g., IKK and Keap1) and alter gene expression pathways responsible for inducing antioxidant genes, heat shock proteins, and the DNA damage response. To fully understand cellular responses to HNE, it is important to determine its protein targets in an unbiased fashion. This requires a strategy for detecting and isolating HNE-modified proteins regardless of the nature of the chemical linkage between HNE and its targets. Azido or alkynyl derivatives of HNE were synthesized and demonstrated to be equivalent to HNE in their ability to induce heme oxygenase induction and induce apoptosis in colon cancer (RKO) cells. Cells exposed to the tagged HNE derivatives were lysed and exposed to reagents to effect Staudinger ligation or copper-catalyzed Huisgen 1,3 dipolar cycloaddition reaction (click chemistry) to conjugate HNE-adducted proteins with biotin for subsequent affinity purification. Both strategies yielded efficient biotinylation of tagged HNE-protein conjugates, but click chemistry was found to be superior for the recovery of biotinylated proteins from streptavidin-coated beads. Biotinylated proteins were detected in lysates from RKO cell incubations with azido-HNE at concentrations as low as 1 microM. These proteins were affinity purified with streptavidin beads, and proteomic analysis was performed by linear ion trap mass spectrometry. Proteomic analysis revealed a dose-dependent increase in labeled proteins with increased sequence coverage at higher concentrations. Several proteins involved in stress signaling (heat shock proteins 70 and 90 and the 78-kDa glucose-regulated protein) were selectively adducted by azido- and alkynyl-HNE. The use of azido and alkynyl derivatives in conjunction with click chemistry appears to be a valuable approach for the identification of the protein targets of HNE.
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13 MeSH Terms
Phospholipid-protein adducts of lipid peroxidation: synthesis and study of new biotinylated phosphatidylcholines.
Tallman KA, Kim HY, Ji JX, Szapacs ME, Yin H, McIntosh TJ, Liebler DC, Porter NA
(2007) Chem Res Toxicol 20: 227-34
MeSH Terms: Azo Compounds, Biotin, Biotinylation, Humans, Imidazoles, Lipid Peroxidation, Molecular Structure, Phosphatidylcholines, Phospholipids, Proteins, Reference Values, Serum Albumin, Stereoisomerism, X-Ray Diffraction
Show Abstract · Added March 20, 2014
Oxidative stress gives rise to a number of electrophilic aldehydes from membrane phospholipids, and these compounds have been linked to pathophysiologic events associated with the progression of cardiovascular disease. A headgroup biotinylated phosphatidylcholine (PC) has been prepared, and its oxidation chemistry has been studied. Biotin or biotin-sulfoxide groups were attached to PC at the ammonium headgroup via a di-ethylene glycol link. The modified phospholipids have calorimetric and colloidal properties similar to those of the parent. The oxidation of PLPBSO (the biotin-sulfoxide analogue of 1-palmitoyl-2-linoleoylglycerylphosphatidylcholine, PLPC) was studied under a variety of conditions. PLPBSO, like PLPC, undergoes oxidation to give electrophiles that adduct to small model peptides as well as to isolated proteins such as human serum albumin. PLPBSO incorporates into human blood plasma, and treatment of the plasma with water soluble free radical initiators gives rise to a number of biotinylated plasma proteins that can be isolated via (strept)avidin affinity. Isolated peptide or protein-lipid adducts can be identified by proteomics analyses, and studies on model peptides show that phospholipid-protein adduction sites can be identified by known algorithms. Biotinylated lipids such as PLPBSO and modern proteomics tools would appear to provide a new approach to exploring the chemistry and biology of membrane peroxidation associated with oxidative stress.
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14 MeSH Terms
Amphetamine induces a calcium/calmodulin-dependent protein kinase II-dependent reduction in norepinephrine transporter surface expression linked to changes in syntaxin 1A/transporter complexes.
Dipace C, Sung U, Binda F, Blakely RD, Galli A
(2007) Mol Pharmacol 71: 230-9
MeSH Terms: Amphetamine, Biotinylation, Calcium, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Calcium-Calmodulin-Dependent Protein Kinases, Cell Line, Cell Membrane, Enzyme Activation, Gene Expression Regulation, Humans, Kinetics, Norepinephrine Plasma Membrane Transport Proteins, Syntaxin 1
Show Abstract · Added December 10, 2013
Norepinephrine (NE) transporters (NETs) are high-affinity transport proteins that mediate the synaptic clearance of NE after vesicular release. NETs represent a major therapeutic target for antidepressants and are targets of multiple psychostimulants including amphetamine (AMPH) and cocaine. Recently, we demonstrated that syntaxin 1A (SYN1A) regulates NET surface expression and, through binding to the transporter's NH(2) terminus, regulates transporter catalytic function. AMPH induces NE efflux and may also regulate transporter trafficking. We monitored NET distribution and function in catecholaminergic cell lines (CAD) stably transfected with either full-length human NET (CAD-hNET) or with an hNET N-terminal deletion (CAD-hNETDelta(28-47) cells). In hNET-CAD cells, AMPH causes a slow and small reduction of surface hNET with a modest increase in hNET/SYN1A associations at the plasma membrane. In contrast, in CAD-hNETDelta(28-47) cells, AMPH induces a rapid and substantial reduction in surface hNETDelta(28-47) accompanied by a large increase in plasma membrane hNETDelta(28-47)/SYN1A complexes. We also found that AMPH in CAD-hNETDelta(28-47) cells induces a robust increase in cytosolic Ca2+ and concomitant activation of calcium/calmodulin-dependent protein kinase II (CaMKII). Inhibition of either the increase in intracellular Ca2+ or CaMKII activity blocks AMPH-stimulated hNETDelta(28-47) trafficking and the formation of hNETDelta(28-47)/SYN1A complexes. Here, we demonstrate that AMPH stimulation of CAMKII stabilizes an hNET/SYN1A complex. This hNET/SYN1A complex rapidly redistributes, upon AMPH treatment, when mechanisms supported by the transporter's NH2 terminus are eliminated.
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13 MeSH Terms
High affinity inhibitors of the dopamine transporter (DAT): novel biotinylated ligands for conjugation to quantum dots.
Tomlinson ID, Mason JN, Blakely RD, Rosenthal SJ
(2006) Bioorg Med Chem Lett 16: 4664-7
MeSH Terms: Biotinylation, Dopamine, Dopamine Plasma Membrane Transport Proteins, Humans, Inhibitory Concentration 50, Ligands, Molecular Structure, Quantum Dots
Show Abstract · Added July 10, 2013
Compounds capable of inhibiting the dopamine transporter protein (DAT) that can be conjugated to cadmium selenide/zinc sulfide/core shell nanocrystals may be used to image the location and distribution of the DAT in neuronal cell membranes. This letter describes the synthesis of biotinylated analogs of the DAT antagonists GBR 12909 and GBR 12935 that can be attached to streptavidin coated cadmium selenide/zinc sulfide/core shell nanocrystals.
1 Communities
2 Members
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8 MeSH Terms
Expression studies of naturally occurring human dopamine transporter variants identifies a novel state of transporter inactivation associated with Val382Ala.
Mazei-Robison MS, Blakely RD
(2005) Neuropharmacology 49: 737-49
MeSH Terms: Alanine, Amphetamine, Animals, Biotinylation, COS Cells, Cattle, Cell Line, Chlorocebus aethiops, Cocaine, Dopamine, Dopamine Plasma Membrane Transport Proteins, Dopamine Uptake Inhibitors, Drug Interactions, Electrophoresis, Polyacrylamide Gel, Gene Expression, Gene Expression Regulation, Genetic Variation, Humans, Mice, Models, Biological, Mutagenesis, Neuroblastoma, Norepinephrine, Phorbol Esters, Protein Transport, Rats, Time Factors, Transfection, Valine
Show Abstract · Added July 10, 2013
Multiple, rare, human dopamine (DA) transporter (hDAT, SLC6A3) coding variants have been described, though to date they have been incompletely characterized. Here we present studies analyzing the function and regulation of five naturally occurring coding variants, V55A, R237Q, V382A, A559V and E602G, expressed in COS-7 and SH-SY5Y cells. All variants, except V382A, exhibited levels of surface protein expression and DA transport activity comparable to hDAT. V382A, divergent at the most highly conserved residue among reported hDAT variants, exhibited significantly diminished surface expression, likely derived from inefficient plasma membrane delivery. Moreover, a greater expression of V382A protein was required to achieve comparable levels of transport to hDAT, consistent with a loss of transport function. V382A displayed a decrease in sensitivity to phorbol ester (PMA)-induced internalization, as well as an altered substrate selectivity for DA versus norepinephrine (NE). Analysis of PMA-induced V382A internalization revealed a trafficking-independent action of PMA, consistent with the existence of a surface-localized, transport-inactive state.
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29 MeSH Terms