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Proximity ligation assay

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https://www.readbyqxmd.com/read/28342889/heteromeric-complexes-of-aldo-keto-reductase-auxiliary-kv%C3%AE-subunits-akr6a-regulate-sarcolemmal-localization-of-kv1-5-in-coronary-arterial-myocytes
#1
Matthew A Nystoriak, Deqing Zhang, Ganapathy Jagatheesan, Aruni Bhatnagar
Redox-sensitive potassium channels consisting of the voltage-gated K(+) (KV) channel pore subunit KV1.5 regulate resting membrane potential and thereby contractility of vascular smooth muscle cells. Members of the KV1 family associate with cytosolic auxiliary β subunits, which are members of the aldo-keto reductase (AKR) superfamily (AKR6A subfamily). The Kvβ subunits have been proposed to regulate Kv1 gating via pyridine nucleotide cofactor binding. However, the molecular identity of KVβ subunits that associate with native KV1...
March 22, 2017: Chemico-biological Interactions
https://www.readbyqxmd.com/read/28336810/role-of-transient-receptor-potential-vanilloid-1-trpv1-in-the-modulation-of-airway-smooth-muscle-tone-and-calcium-handling
#2
Gene T Yocum, Jun Chen, Christine H Choi, Elizabeth A Townsend, Yi Zhang, Dingbang Xu, Xiao Wen Fu, Michael J Sanderson, Charles W Emala
Asthma is a common disorder characterized, in part, by airway smooth muscle (ASM) hyperresponsiveness. Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel expressed on airway nerve fibers that modulates afferent signals resulting in cough, and potentially bronchoconstriction. In the present study, the TRPV1 transcript was detected by RT-PCR in primary cultured human ASM cells, and the TRPV1 protein was detected in ASM of human trachea by immunohistochemistry. Proximity ligation assays suggest that TRPV1 is expressed in the sarcoplasmic reticulum membrane of human ASM cells in close association with sarco/endoplasmic reticulum Ca2+ ATPase 2...
March 23, 2017: American Journal of Physiology. Lung Cellular and Molecular Physiology
https://www.readbyqxmd.com/read/28303576/nonmuscle-nm-myosin-heavy-chain-phosphorylation-regulates-the-formation-of-nm-myosin-filaments-adhesome-assembly-and-smooth-muscle-contraction
#3
Wenwu Zhang, Susan J Gunst
The molecular function of nonmuscle (NM) isoforms of myosin II in smooth muscle (SM) tissues, and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain (HC) on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro...
March 17, 2017: Journal of Physiology
https://www.readbyqxmd.com/read/28300546/cocaine-self-administration-specifically-increases-a2ar-d2r-and-d2r-sigma1r-heteroreceptor-complexes-in-the-rat-nucleus-accumbens-shell-relevance-for-cocaine-use-disorder
#4
Dasiel O Borroto-Escuela, Manuel Narváez, Karolina Wydra, Julia Pintsuk, Luca Pinton, Antonio Jimenez-Beristain, Michael Di Palma, Joanna Jastrzębska, Malgorzata Filip, Kjell Fuxe
Adenosine 2A receptor (A2AR) agonists were indicated to reduce cocaine reward and cocaine seeking mainly through activation of antagonistic allosteric A2AR-dopamine D2R (D2R) interactions in A2AR-D2R heteroreceptor complexes. Furthermore, it was shown that modulation of cocaine reward involves antagonistic A2AR-D2R interactions in the ventral but not the dorsal striatum in rats. In the current work the proximity ligation assay (PLA) was used to further study the A2AR-D2R heteroreceptor complexes in the nucleus accumbens shell and core as well as the dorsal striatum under the influence of cocaine self-administration in rats...
March 12, 2017: Pharmacology, Biochemistry, and Behavior
https://www.readbyqxmd.com/read/28298474/tumor-protein-d52-expression-is-post-transcriptionally-regulated-by-t-cell-intercellular-antigen-tia-1-and-tia-related-protein-via-mrna-stability
#5
Hiromi Motohashi, Yoshiki Mukudai, Chihiro Ito, Kosuke Kato, Toshikazu Shimane, Seiji Kondo, Tatsuo Shirota
Although tumor protein D52 (TPD) family proteins were first identified nearly 20 years ago, their molecular regulatory mechanisms remain unclear. Therefore, we investigated the post-transcriptional regulation of TPD52 family genes. An RNA immunoprecipitation assay showed the potential binding ability of TPD52 family mRNAs to several RNA-binding proteins, and an RNA degradation assay revealed that TPD52 is subject to more prominent post-transcriptional regulation than TPD53 and 54. We subsequently focused on the 3'-untranslated region (3'-UTR) of TPD52 as a cis -acting element in post-transcriptional gene regulation...
March 15, 2017: Biochemical Journal
https://www.readbyqxmd.com/read/28288880/dual-agonist-occupancy-of-orexin-receptor-1-and-cholecystokinin-a-receptor-heterodimers-decreases-g-protein-dependent-signaling-and-migration-in-the-human-colon-cancer-cell-line-ht-29
#6
Bo Bai, Xiaoyu Chen, Rumin Zhang, Xin Wang, Yunlu Jiang, Dandan Li, Zhengwen Wang, Jing Chen
The orexin (OX1R) and cholecystokinin A (CCK1R) receptors play opposing roles in the migration of the human colon cancer cell line HT-29, and may be involved in the pathogenesis and pathophysiology of cancer cell invasion and metastasis. OX1R and CCK1R belong to family A of the G-protein-coupled receptors (GPCRs), but the detailed mechanisms underlying their functions in solid tumor development remain unclear. In this study, we investigated whether these two receptors heterodimerize, and the results revealed novel signal transduction mechanisms...
March 10, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28266547/the-molecular-motor-myosin-va-interacts-with-the-cilia-centrosomal-protein-rpgrip1l
#7
L H P Assis, R M P Silva-Junior, L G Dolce, M R Alborghetti, R V Honorato, A F Z Nascimento, T D Melo-Hanchuk, D M Trindade, C C C Tonoli, C T Santos, P S L Oliveira, R E Larson, J Kobarg, E M Espreafico, P O Giuseppe, M T Murakami
Myosin Va (MyoVa) is an actin-based molecular motor abundantly found at the centrosome. However, the role of MyoVa at this organelle has been elusive due to the lack of evidence on interacting partners or functional data. Herein, we combined yeast two-hybrid screen, biochemical studies and cellular assays to demonstrate that MyoVa interacts with RPGRIP1L, a cilia-centrosomal protein that controls ciliary signaling and positioning. MyoVa binds to the C2 domains of RPGRIP1L via residues located near or in the Rab11a-binding site, a conserved site in the globular tail domain (GTD) from class V myosins...
March 7, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28258189/selective-expression-of-tspan2-in-vascular-smooth-muscle-is-independently-regulated-by-tgf-%C3%AE-1-smad-and-myocardin-serum-response-factor
#8
Jinjing Zhao, Wen Wu, Wei Zhang, Yao Wei Lu, Emiley Tou, Jiemei Ye, Ping Gao, David Jourd'heuil, Harold A Singer, Mingfu Wu, Xiaochun Long
Tetraspanins (TSPANs) comprise a large family of 4-transmembrane domain proteins. The importance of TSPANs in vascular smooth muscle cells (VSMCs) is unexplored. Given that TGF-β1 and myocardin (MYOCD) are potent activators for VSMC differentiation, we screened for TGF-β1 and MYOCD/serum response factor (SRF)-regulated TSPANs in VSMC by using RNA-seq analyses and RNA-arrays. TSPAN2 was found to be the only TSPAN family gene induced by TGF-β1 and MYOCD, and reduced by SRF deficiency in VSMCs. We also found that TSPAN2 is highly expressed in smooth muscle-enriched tissues and down-regulated in in vitro models of VSMC phenotypic modulation...
March 3, 2017: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
https://www.readbyqxmd.com/read/28257821/tight-junction-protein-zo-1-controls-organic-cation-carnitine-transporter-octn2-slc22a5-in-a-protein-kinase-c-dependent-way
#9
Dominika Jurkiewicz, Katarzyna Michalec, Krzysztof Skowronek, Katarzyna A Nałęcz
OCTN2 (SLC22A5) is an organic cation/carnitine transporter belonging to the solute carrier transporters (SLC) family. OCTN2 is ubiquitously expressed and its presence was shown in various brain cells, including the endothelial cells forming blood-brain barrier, where it was mainly detected at abluminal membrane and in proximity of tight junctions (TJ). Since OCTN2 contains a PDZ-binding domain, the present study was focused on a possible role of transporter interaction with a TJ-associated protein ZO-1, containing PDZ domains and detected in rat Octn2 proteome...
May 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28256719/rna-whole-mount-in-situ-hybridization-proximity-ligation-assay-rish-pla-an-assay-for-detecting-rna-protein-complexes-in-intact-cells
#10
Ioannis M Roussis, Fiona A Myers, Garry P Scarlett
Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein interactions as a consequence of the complexities associated with working with RNA. This unit describes a method for the adaptation of the In Situ Hybridization-Proximity Ligation Assay (ISH-PLA) to the study of RNA regulation (rISH-PLA). The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single-cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogeneous cell populations from which conventional RNA-protein interaction detection techniques suffer...
March 3, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28249776/lack-of-interaction-between-between-nemo-and-sharpin-impairs-linear-ubiquitination-and-nf-%C3%AE%C2%BAb-activation-and-leads-to-incontinentia-pigmenti
#11
E Bal, E Laplantine, Y Hamel, V Dubosclard, B Boisson, A Pesacatore, C Picard, S Hadj-Rabia, G Royer, J Steffann, J P Bonnefont, V M Ursini, P Vabres, A Munnich, J L Casanova, C Bodemer, R Weil, F Agou, A Smahi
BACKGROUND: Incontinentia pigmenti (IP; MIM308300) is a severe, male-lethal, X-linked, dominant genodermatosis resulting from loss-of-function mutations in the IKBKG gene encoding NF-κB essential modulator (NEMO, the regulatory subunit of the IKK complex). In 80% of cases of IP, the deletion of exons 4 to 10 leads to the absence of NEMO and total inhibition of NF-κB signaling. Here, we described a new IKBKG mutation responsible for IP resulting in an inactive truncated form of NEMO...
February 26, 2017: Journal of Allergy and Clinical Immunology
https://www.readbyqxmd.com/read/28248246/let-there-be-light
#12
REVIEW
Doroteya Raykova, Björn Koos, Anna Asplund, Márton Gelléri, Ylva Ivarsson, U Helena Danielson, Ola Söderberg
The invention of the microscope has been fundamental for the understanding of tissue architecture and subcellular structures. With the advancement of higher magnification microscopes came the development of various molecular biology tools such as Förster resonance energy transfer (FRET) and in situ proximity ligation assay (in situ PLA) to monitor protein interactions. Microscopy has become a commonly used method for the investigation of molecular events within the cell, for the identification of key players in signaling networks, and the activation of these pathways...
November 29, 2016: Proteomes
https://www.readbyqxmd.com/read/28239340/dopamine-induced-changes-in-g%C3%AE-olf-protein-levels-in-striatonigral-and-striatopallidal-medium-spiny-neurons-underlie-the-genesis-of-l-dopa-induced-dyskinesia-in-parkinsonian-mice
#13
Ryoma Morigaki, Shinya Okita, Satoshi Goto
The dopamine precursor, l-3,4-dihydroxyphenylalanine (l-DOPA), exerts powerful therapeutic effects but eventually generates l-DOPA-induced dyskinesia (LID) in patients with Parkinson's disease (PD). LID has a close link with deregulation of striatal dopamine/cAMP signaling, which is integrated by medium spiny neurons (MSNs). Olfactory type G-protein α subunit (Gαolf), a stimulatory GTP-binding protein encoded by the GNAL gene, is highly concentrated in the striatum, where it positively couples with dopamine D1 (D1R) receptor and adenosine A2A receptor (A2AR) to increase intracellular cAMP levels in MSNs...
2017: Frontiers in Cellular Neuroscience
https://www.readbyqxmd.com/read/28197073/association-of-trka-and-app-is-promoted-by-ngf-and-reduced-by-cell-death-promoting-agents
#14
Nadia Canu, Ilaria Pagano, Luca Rosario La Rosa, Marsha Pellegrino, Maria Teresa Ciotti, Delio Mercanti, Fabiola Moretti, Valentina Sposato, Viviana Triaca, Carla Petrella, Ichiro N Maruyama, Andrea Levi, Pietro Calissano
The amyloid precursor protein (APP) interacts with the tropomyosin receptor kinase A (TrkA) in normal rat, mouse, and human brain tissue but not in Alzheimer's disease (AD) brain tissue. However, it has not been reported whether the two proteins interact directly, and if so, which domains are involved. Clarifying these points will increase our understanding of the role and regulation of the TrkA/APP interaction in normal brain functioning as well as in AD. Here we addressed these questions using bimolecular fluorescence complementation (BiFC) and the proximity ligation assay (PLA)...
2017: Frontiers in Molecular Neuroscience
https://www.readbyqxmd.com/read/28187919/dopamine-d3-and-acetylcholine-nicotinic-receptor-heteromerization-in-midbrain-dopamine-neurons-relevance-for-neuroplasticity
#15
Leonardo Bontempi, Paola Savoia, Federica Bono, Chiara Fiorentini, Cristina Missale
Activation of nicotinic acetylcholine receptors (nAChR) promotes the morphological remodeling of cultured dopamine (DA) neurons, an effect requiring functional DA D3 receptors (D3R). The aim of this study was to investigate the mechanisms mediating D3R-nAChR cross-talk in the modulation of DA neuron structural plasticity. By using bioluminescence resonance energy transfer(2) (BRET(2)) and proximity ligation assay (PLA), evidence for the existence of D3R-nAChR heteromers has been obtained. In particular, BRET(2) showed that the D3R directly and specifically interacts with the β2 subunit of the nAChR...
February 7, 2017: European Neuropsychopharmacology: the Journal of the European College of Neuropsychopharmacology
https://www.readbyqxmd.com/read/28182009/hetero-oligomerization-between-the-tnf-receptor-superfamily-members-cd40-fas-and-trailr2-modulate-cd40-signalling
#16
Cristian R Smulski, Marion Decossas, Neila Chekkat, Julien Beyrath, Laure Willen, Gilles Guichard, Raquel Lorenzetti, Marta Rizzi, Hermann Eibel, Pascal Schneider, Sylvie Fournel
TNF receptor superfamily members (TNFRSF) such as CD40, Fas and TRAIL receptor 2 (TRAILR2) participate to the adaptive immune response by eliciting survival, proliferation, differentiation and/or cell death signals. The balance between these signals determines the fate of the immune response. It was previously reported that these receptors are able to self-assemble in the absence of ligand through their extracellular regions. However, the role of this oligomerization is not well understood, and none of the proposed hypotheses take into account potential hetero-association of receptors...
February 9, 2017: Cell Death & Disease
https://www.readbyqxmd.com/read/28174191/augmentation-of-muscle-blood-flow-by-ultrasound-cavitation-is-mediated-by-atp-and-purinergic-signaling
#17
J Todd Belcik, Brian P Davidson, Aris Xie, Melinda D Wu, Mrinal Yadava, Yue Qi, Sherry Liang, Chae Ryung Chon, Azzdine Y Ammi, Joshua Field, Leanne Harmann, William M Chilian, Joel Linden, Jonathan R Lindner
BACKGROUND: - Augmentation of tissue blood flow by therapeutic ultrasound is thought to rely on convective shear. Microbubble contrast agents that undergo ultrasound-mediated cavitation markedly amplify these effects. We hypothesized that purinergic signalling is responsible for shear-dependent increases in muscle perfusion during therapeutic cavitation. METHODS: -Unilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ultrasound (1...
February 7, 2017: Circulation
https://www.readbyqxmd.com/read/28171705/endothelin-converting-enzymes-degrade-%C3%AE-synuclein-and-are-reduced-in-dementia-with-lewy-bodies
#18
J Scott Miners, Seth Love
We have examined the roles of the endothelin-converting enzyme-1 and -2 (ECE-1 and ECE-2) in the homeostasis of α-synuclein (α-syn) and pathogenesis of Lewy body disease. The ECEs are named for their ability to convert inactive big endothelin to the vasoactive peptide endothelin-1 (EDN1). We have found that ECE-1 and ECE-2 cleave and degrade α-syn in vitro and siRNA-mediated knockdown of ECE-1 and ECE-2 in SH-SY5Y neuroblastoma cells significantly increased α-syn both intracellularly (within the cell lysate) (P < 0...
February 7, 2017: Journal of Neurochemistry
https://www.readbyqxmd.com/read/28155571/the-augmentation-of-bk-channel-activity-by-nitric-oxide-signaling-in-rat-cerebral-arteries-involves-co-localized-regulatory-elements
#19
Barry D Kyle, Ramesh C Mishra, Andrew P Braun
Large conductance, Ca(2+)-activated K(+) (BK) channels control cerebrovascular tone; however, the regulatory processes influencing these channels remain poorly understood. Here, we investigate the cellular mechanisms underlying the enhancement of BK current in rat cerebral arteries by nitric oxide (NO) signaling. In isolated cerebral myocytes, BK current magnitude was reversibly increased by sodium nitroprusside (SNP, 100 μM) and sensitive to the BK channel inhibitor, penitrem-A (100 nM). Fostriecin (30 nM), a protein phosphatase type 2A (PP2A) inhibitor, significantly prolonged the SNP-induced augmentation of BK current and a similar effect was produced by sildenafil (30 nM), a phosphodiesterase 5 (PDE5) inhibitor...
January 1, 2017: Journal of Cerebral Blood Flow and Metabolism
https://www.readbyqxmd.com/read/28148497/shear-stress-induces-g%C3%AE-q-11-activation-independent-of-g-protein-coupled-receptor-activation-in-endothelial-cells
#20
Nathaniel G Dela Paz, Benoît Melchior, John A Frangos
Mechanochemical signal transduction occurs when mechanical forces, such as fluid shear stress, are converted into biochemical responses within the cell. The molecular mechanisms by which endothelial cells (ECs) sense/transduce shear stress into biological signals, including the nature of the mechanosensor, are still unclear. G proteins and G protein-coupled receptors (GPCRs) have been postulated independently to mediate mechanotransduction. In this study, we used in situ proximity ligation assay (PLA) to investigate the role of a specific GPCR/Gαq/11 pair in EC shear stress-induced mechanotransduction...
February 1, 2017: American Journal of Physiology. Cell Physiology
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