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Proximity ligation assay

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https://www.readbyqxmd.com/read/28644885/organic-anion-transporting-polypeptide-1b3-can-form-homo-and-hetero-oligomers
#1
Yuchen Zhang, Kelli H Boxberger, Bruno Hagenbuch
OATP1B3 is a 12 transmembrane domain protein expressed at the basolateral membrane of human hepatocytes where it mediates the uptake of numerous drugs and endogenous compounds. Previous western blot results suggest the formation of OATP1B3 multimers. In order to better understand the function of OATP1B3 under normal physiological conditions, we investigated its oligomerization status. We transiently transfected OATP1B3 with a C-terminal His-, FLAG- or HA-tag in HEK293 cells and used co-immunoprecipitation and a Proximity Ligation Assay to detect interactions between the different constructs...
2017: PloS One
https://www.readbyqxmd.com/read/28644055/regulation-of-transient-receptor-potential-melastatin-4-channel-by-sarcoplasmic-reticulum-inositol-triphosphate-receptors-role-in-human-detrusor-smooth-muscle-function
#2
Aaron Provence, Eric S Rovner, Georgi V Petkov
We recently reported key physiological roles for Ca(2+)-activated transient receptor potential melastatin 4 (TRPM4) channels in detrusor smooth muscle (DSM). However, the Ca(2+)-signaling mechanisms governing TRPM4 channel activity in human DSM cells are unexplored. As the TRPM4 channels are activated by Ca(2+), inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca(2+) release from the sarcoplasmic reticulum represents a potential Ca(2+) source for TRPM4 channel activation. We used clinically-characterized human DSM tissues to investigate the molecular and functional interactions of the IP3Rs and TRPM4 channels...
June 23, 2017: Channels
https://www.readbyqxmd.com/read/28642261/transforming-growth-factor-beta-stimulates-smad1-5-signaling-in-pulmonary-artery-smooth-muscle-cells-and-fibroblasts-of-the-newborn-mouse-through-alk1
#3
Huili Zhang, Lili Du, Ying Zhong, Kathleen C Flanders, Jesse D Roberts
The intracellular signaling mechanisms through which TGFβ regulates pulmonary development are incompletely understood. Canonical TGFβ signaling involves Smad2/3 phosphorylation, Smad2/3·Smad4 complex formation and nuclear localization, and gene regulation. Here we show that physiologically relevant TGFβ1 levels also stimulate Smad1/5 phosphorylation, typically a mediator of bone morphogenetic protein (BMP) signaling, in mouse pup pulmonary artery smooth muscle cells (mPASMC) and lung fibroblasts and other interstitial lung cell lines...
June 22, 2017: American Journal of Physiology. Lung Cellular and Molecular Physiology
https://www.readbyqxmd.com/read/28640668/absence-of-the-fragile-x-mental-retardation-protein-results-in-defects-of-rna-editing-of-neuronal-mrnas-in-mouse
#4
Alice Filippini, Daniela Bonini, Caroline Lacoux, Laura Pacini, Maria Zingariello, Laura Sancillo, Daniela Bosisio, Valentina Salvi, Jessica Mingardi, Luca La Via, Francesca Zalfa, Claudia Bagni, Alessandro Barbon
The fragile X syndrome (FXS), the most common form of inherited intellectual disability, is due to the absence of FMRP, a protein regulating RNA metabolism. Recently, an unexpected function of FMRP in modulating the activity of Adenosine Deaminase Acting on RNA (ADAR) enzymes has been reported both in Drosophila and Zebrafish. ADARs are RNA-binding proteins that increase transcriptional complexity through a post-transcriptional mechanism called RNA editing. In order to evaluate the ADAR2-FMRP interaction in mammals we analysed several RNA editing re-coding sites in the fmr1 knockout (KO) mice...
June 22, 2017: RNA Biology
https://www.readbyqxmd.com/read/28617990/cell-specific-diversity-in-the-expression-and-organization-of-cytoplasmic-plaque-proteins-of-apical-junctions
#5
Ekaterina Vasileva, Sophie Sluysmans, Marie-Luce Bochaton-Piallat, Sandra Citi
Tight and adherens junctions play critical roles in the barrier, adhesion, and signaling functions of epithelial and endothelial cells. How the molecular organization of these junctions is tuned to the widely diverse physiological requirements of each tissue type is not well understood. Here, we address this question by examining the expression, localization, and interactions of major cytoplasmic plaque proteins of tight and adherens junctions in different cultured epithelial and endothelial cell lines. Immunoblotting and immunofluorescence analyses show that the expression profiles of cingulin, paracingulin, ZO-1, ZO-2, ZO-3, PLEKHA7, afadin, PDZD11, p120-catenin, and α-catenin, as well as the transmembrane junctional proteins occludin, E-cadherin, and VE-cadherin, are significantly diverse when comparing kidney cells (MDCK, mCCD), keratinocytes (HaCaT), lung carcinoma (A427, A549), and endothelium-derived cells (bEnd...
June 15, 2017: Annals of the New York Academy of Sciences
https://www.readbyqxmd.com/read/28615203/phosphorylation-of-serine-225-in-hepatitis-c-virus-ns5a-regulates-protein-protein-interactions
#6
Niluka Goonawardane, Anna Gebhardt, Christopher Bartlett, Andreas Pichlmair, Mark Harris
Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A, however the mechanistic details of the role of this post-translational modification in the virus life cycle remains obscure. We previously reported (Ross-Thriepland et al, 2015) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication...
June 14, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28611590/cryptochrome-is-a-regulator-of-synaptic-plasticity-in-the-visual-system-of-drosophila-melanogaster
#7
Milena Damulewicz, Gabriella M Mazzotta, Elena Sartori, Ezio Rosato, Rodolfo Costa, Elzbieta M Pyza
Drosophila CRYPTOCHROME (CRY) is a blue light sensitive protein with a key role in circadian photoreception. A main feature of CRY is that light promotes an interaction with the circadian protein TIMELESS (TIM) resulting in their ubiquitination and degradation, a mechanism that contributes to the synchronization of the circadian clock to the environment. Moreover, CRY participates in non-circadian functions such as magnetoreception, modulation of neuronal firing, phototransduction and regulation of synaptic plasticity...
2017: Frontiers in Molecular Neuroscience
https://www.readbyqxmd.com/read/28604965/-phenotypic-and-genetic-analysis-of-an-inv-dup-15-case-with-a-bp3-bp3-rearrangement
#8
Fuchun Zhong, Fenghua Lan, Xiao Zhang, Yuxiang Lin, Yanhong Lin, Aizhen Yan, Xiangdong Tu
OBJECTIVE: To analyze a case of supernumerary marker chromosome (SMC) with combined genetic techniques and explore its correlation with the clinical phenotype. METHODS: The SMC was analyzed with G-banded karyotyping, multiplex ligation dependent probe amplification (MLPA), fluorescence in situ hybridization (FISH), and single nucleotide polymorphism array (SNP-array). RESULTS: G-banding analysis indicated that the patient has a karyotype of 47,XX,+mar...
June 10, 2017: Zhonghua Yi Xue Yi Chuan Xue za Zhi, Zhonghua Yixue Yichuanxue Zazhi, Chinese Journal of Medical Genetics
https://www.readbyqxmd.com/read/28600321/importin-beta-and-crm1-control-a-ranbp2-spatiotemporal-switch-essential-for-mitotic-kinetochore-function
#9
Eugenia Gilistro, Valeria de Turris, Michela Damizia, Annalisa Verrico, Sara Moroni, Riccardo De Santis, Alessandro Rosa, Patrizia Lavia
Protein conjugation with SUMO is a post-translational modification that modulates protein interactions and localisation. RANBP2 is a large nucleoporin endowed with SUMO E3 ligase and SUMO-stabilising activity and is implicated in some cancer types. RANBP2 is part of a larger complex, comprising SUMO-modified RANGAP1, the GTP-hydrolysis activating factor for the GTPase RAN. During mitosis, the RANBP2/SUMO-RANGAP1 complex localises to the mitotic spindle and to kinetochores after microtubule attachment. Here we have addressed the mechanisms that regulate this localisation and how they affect kinetochore functions...
June 9, 2017: Journal of Cell Science
https://www.readbyqxmd.com/read/28572629/novel-approach-for-accurate-tissue-based-protein-colocalization-and-proximity-microscopy
#10
Mirjam I Lutz, Carmen Schwaiger, Bernhard Hochreiter, Gabor G Kovacs, Johannes A Schmid
Fluorescence colocalization microscopy is frequently used to assess potential links between distinct molecules; however, this method can lead to striking false-positive results and erroneous conclusions. Here we developed a novel approach with more sophisticated mathematical colocalization analyses together with visualization of physical proximity using fluorescence resonance energy transfer (FRET). To verify our results we used the proximity ligation assay (PLA). With these methods we could demonstrate that distinct neurodegeneration-related proteins either not or only rarely interact in human brain tissue...
June 1, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28547925/blockade-of-the-dissociation-of-nmmhc-iia-from-tnfr2-by-d39-a-saponin-from-liriope-muscari-suppresses-tissue-factor-expression-and-venous-thrombosis
#11
Ke-Feng Zhai, Jin-Rong Zheng, You-Mei Tang, Fang Li, Yan-Ni Lv, Yuan-Yuan Zhang, Zhen Gao, Jin Qi, Bo-Yang Yu, Jun-Ping Kou
BACKGROUND AND PURPOSE: Non-muscular myosin heavy chain IIA (NMMHC IIA) has been demonstrated to play a key role in tissue factor (TF) expression and venous thrombosis. However, whether natural products could inhibit thrombosis via NMMHC IIA remains unclear. In this study, we identified that a representative natural saponin D39 from Liriope muscari showed anti-thrombotic activity by targeting NMMHC IIA. EXPERIMENTAL APPROACH: TF expression and activity in endothelial cells were analysed in vitro by western blot and simplified chromogenic assays...
May 26, 2017: British Journal of Pharmacology
https://www.readbyqxmd.com/read/28522883/in-situ-evaluation-of-estrogen-receptor-dimers-in-breast-carcinoma-cells-visualization-of-protein-protein-interactions
#12
Erina Iwabuchi, Yasuhiro Miki, Katsuhiko Ono, Yoshiaki Onodera, Hironobu Sasano
The estrogen receptor (ER) functions as a dimer and is involved in several different biological functions. However ER dimeric proteins have not been identified by in situ methodologies. Structured illumination microscopy (SIM) has been recently developed, which enabled the localization of protein and protein interaction. Therefore, in this study, we firstly demonstrated that ERs formed both homodimers and heterodimers in breast carcinoma cell lines using Nikon's SIM (N-SIM). ERα/α homodimers were detected in the nuclei of both ERα-positive MCF-7 and T-47D cells; 23...
April 27, 2017: Acta Histochemica et Cytochemica
https://www.readbyqxmd.com/read/28500295/angiotensin-ii-type-1-adenosine-a-2a-receptor-oligomers-a-novel-target-for-tardive-dyskinesia
#13
Paulo A de Oliveira, James A R Dalton, Marc López-Cano, Adrià Ricarte, Xavier Morató, Filipe C Matheus, Andréia S Cunha, Christa E Müller, Reinaldo N Takahashi, Víctor Fernández-Dueñas, Jesús Giraldo, Rui D Prediger, Francisco Ciruela
Tardive dyskinesia (TD) is a serious motor side effect that may appear after long-term treatment with neuroleptics and mostly mediated by dopamine D2 receptors (D2Rs). Striatal D2R functioning may be finely regulated by either adenosine A2A receptor (A2AR) or angiotensin receptor type 1 (AT1R) through putative receptor heteromers. Here, we examined whether A2AR and AT1R may oligomerize in the striatum to synergistically modulate dopaminergic transmission. First, by using bioluminescence resonance energy transfer, we demonstrated a physical AT1R-A2AR interaction in cultured cells...
May 12, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28485577/site-selective-conjugation-of-native-proteins-with-dna
#14
Julie B Trads, Thomas Tørring, Kurt V Gothelf
Conjugation of DNA to proteins is increasingly used in academia and industry to provide proteins with tags for identification or handles for hybridization to other DNA strands. Assay technologies such as immuno-PCR and proximity ligation and the imaging technology DNA-PAINT require DNA-protein conjugates. In DNA nanotechnology, the DNA handle is exploited to precisely position proteins by self-assembly. For these applications, site-selective conjugation is almost always desired because fully functional proteins are required to maintain the specificity of antibodies and the activity of enzymes...
May 9, 2017: Accounts of Chemical Research
https://www.readbyqxmd.com/read/28475172/keratins-regulate-colonic-epithelial-cell-differentiation-through-the-notch1-signalling-pathway
#15
Iris A K Lähdeniemi, Julia O Misiorek, Christian J M Antila, Sebastian K-J Landor, Carl-Gustaf A Stenvall, Lina E Fortelius, Linda K Bergström, Cecilia Sahlgren, Diana M Toivola
Keratins (K) are intermediate filament proteins important in stress protection and mechanical support of epithelial tissues. K8, K18 and K19 are the main colonic keratins, and K8-knockout (K8(-/-)) mice display a keratin dose-dependent hyperproliferation of colonic crypts and a colitis-phenotype. However, the impact of the loss of K8 on intestinal cell differentiation has so far been unknown. Here we show that K8 regulates Notch1 signalling activity and differentiation in the epithelium of the large intestine...
June 2017: Cell Death and Differentiation
https://www.readbyqxmd.com/read/28473697/highly-sensitive-and-specific-protein-detection-via-combined-capillary-isoelectric-focusing-and-proximity-ligation
#16
Narendra Padhan, Junhong Yan, Annegret Boge, Elaine Scrivener, Helgi Birgisson, Agata Zieba, Mats Gullberg, Masood Kamali-Moghaddam, Lena Claesson-Welsh, Ulf Landegren
Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms...
May 4, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28471195/-application-of-pla-method-for-detection-of-p53-p63-p73-complexes-in-situ-in-tumour-cells-and-tumour-tissue
#17
V Hrabal, M Nekulová, R Nenutil, J Holčaková, P J Coates, B Vojtěšek
BACKGROUND: PLA (proximity ligation assay) can be used for detection of protein-protein interactions in situ directly in cells and tissues. Due to its high sensitivity and specificity it is useful for detection, localization and quantification of protein complexes with single molecule resolution. One of the mechanisms of mutated p53 gain of function is formation of proten-protein complexes with other members of p53 family - p63 and p73. These interactions influences chemosensitivity and invasivity of cancer cells and this is why these complexes are potential targets of anti-cancer therapy...
2017: Klinická Onkologie: Casopis Ceské a Slovenské Onkologické Spolecnosti
https://www.readbyqxmd.com/read/28456688/native-metabotropic-glutamate-receptor-4-depresses-synaptic-transmission-through-an-unusual-g%C3%AE-q-transduction-pathway
#18
Solenne Chardonnet, Thomas Bessiron, Cathy Isaura Ramos, Raoudha Dammak, Marie-Ange Richard, Céline Boursier, Christelle Cadilhac, Frédéric M Coquelle, Simon Bossi, Fabrice Ango, Pierre Le Maréchal, Paulette Decottignies, Catherine Berrier, Heather McLean, Hervé Daniel
In cerebellar cortex, mGlu4 receptors located on parallel fibers play an essential role in normal motor function, but the molecular mechanisms involved are not yet completely understood. Using a strategy combining biochemical and electrophysiological approaches in the rodent cerebellum, we demonstrate that presynaptic mGlu4 receptors control synaptic transmission through an atypical activation of Gαq proteins. First, the Gαq subunit, PLC and PKC signaling proteins present in cerebellar extracts are retained on affinity chromatography columns grafted with different sequences of the cytoplasmic domain of mGlu4 receptor...
April 26, 2017: Neuropharmacology
https://www.readbyqxmd.com/read/28451223/imaging-specific-newly-synthesized-proteins-within-cells-by-fluorescence-resonance-energy-transfer
#19
Linfeng Sheng, Lesi Cai, Jie Liu, Sichun Zhang, Jing-Juan Xu, Xinrong Zhang, Hong-Yuan Chen
Metabolic azide amino acid labelling followed by the use of bioorthogonal chemistry is an efficient technique for imaging newly synthesized proteins. Recently, AHA-labelling together with the proximity-ligation assay was used to identify newly synthesized proteins of interest (POI) (Tom Dieck et al., Nat. Meth. 2015, 12, 411). Here we build on this study replacing the proximity-ligation assay with FRET to improve the spatial resolution. Herein, we develop a FRET-based strategy for imaging the newly synthesized endogenous POI within cells: a FRET acceptor is installed onto the newly synthesized proteins via click chemistry, and a FRET donor onto the POI via immunocytochemistry...
January 1, 2017: Chemical Science
https://www.readbyqxmd.com/read/28430879/role-of-phosphatase-and-tensin-homolog-pten-in-hypoxic-pulmonary-vasoconstriction
#20
Adrienn Krauszman, Tak Mak, Katalin Szaszi, Wolfgang M Kuebler
AIMS: Hypoxic pulmonary vasoconstriction (HPV) redistributes blood flow from poorly ventilated to better aerated areas in the lung, thereby optimizing ventilation-perfusion ratio (V/Q). Pulmonary artery smooth muscle cell (PASMC) contraction in response to hypoxia is triggered by Ca2+ influx via transient receptor potential canonical 6 (TRPC6) cation channels that have translocated to caveolae in the plasma membrane. Since phosphatase and tensin homolog (PTEN) was suggested to regulate TRPC6 in endothelial cells, we aimed to define its role in the hypoxic response of PASMCs and as a putative mediator of HPV...
April 18, 2017: Cardiovascular Research
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