Research in the Lowe-Krentz lab is supported by research grant HL54269 from the National Institutes of Health to LLK.

<ol>
	<li>Pugh, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmembrane+Protein+184A+Is+a+Receptor+Required+for+Vascular+Smooth+Muscle+Cell+Responses+to+Heparin.">Transmembrane Protein 184A Is a Receptor Required for Vascular Smooth Muscle Cell Responses to Heparin.</a> J Biol Chem. 291, 5326-5341 (2016).</li><li>Daher, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Toxoplasma+TgPH1,+a+pleckstrin+homology+domain-containing+protein+that+binds+to+the+phosphoinositide+PI(3,5)P.">Identification of Toxoplasma TgPH1, a pleckstrin homology domain-containing protein that binds to the phosphoinositide PI(3,5)P.</a> Mol Biochem Parasitol. , (2016).</li><li>Vit, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+identification+of+membrane+proteins+based+on+analysis+of+trypsin-protected+transmembrane+segments.">Large-scale identification of membrane proteins based on analysis of trypsin-protected transmembrane segments.</a> J Proteomics. , (2016).</li><li>Attwood, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topology+based+identification+and+comprehensive+classification+of+four-transmembrane+helix+containing+proteins+(4TMs)+in+the+human+genome.">Topology based identification and comprehensive classification of four-transmembrane helix containing proteins (4TMs) in the human genome.</a> BMC genomics. 17, 268 (2016).</li><li>Zou, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Identification+of+Jatropha+curcas+Aquaporin+Genes+and+the+Comparative+Analysis+Provides+Insights+into+the+Gene+Family+Expansion+and+Evolution+in+Hevea+brasiliensis.">Genome-Wide Identification of Jatropha curcas Aquaporin Genes and the Comparative Analysis Provides Insights into the Gene Family Expansion and Evolution in Hevea brasiliensis.</a> Front Plant Sci. 7, 395 (2016).</li><li>Gilotti, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin+responses+in+vascular+smooth+muscle+cells+involve+cGMP-dependent+protein+kinase+(PKG).">Heparin responses in vascular smooth muscle cells involve cGMP-dependent protein kinase (PKG).</a> J Cell Physiol. 229, 2142-2152 (2014).</li><li>Farwell, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin+Decreases+in+Tumor+Necrosis+Factor+alpha+(TNFalpha)-induced+Endothelial+Stress+Responses+Require+Transmembrane+Protein+184A+and+Induction+of+Dual+Specificity+Phosphatase+1.">Heparin Decreases in Tumor Necrosis Factor alpha (TNFalpha)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1.</a> J Biol Chem. 291, 5342-5354 (2016).</li><li>Xu, D., Esko, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demystifying+heparan+sulfate-protein+interactions.">Demystifying heparan sulfate-protein interactions.</a> Annu Rev Biochem. 83, 129-157 (2014).</li><li>Chiodelli, P., Bugatti, A., Urbinati, C., Rusnati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin/Heparan+sulfate+proteoglycans+glycomic+interactome+in+angiogenesis:+biological+implications+and+therapeutical+use.">Heparin/Heparan sulfate proteoglycans glycomic interactome in angiogenesis: biological implications and therapeutical use.</a> Molecules. 20, 6342-6388 (2015).</li><li>Slee, J. B., Lowe-Krentz, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+realignment+and+cofilin+regulation+are+essential+for+barrier+integrity+during+shear+stress.">Actin realignment and cofilin regulation are essential for barrier integrity during shear stress.</a> J Cell Biochem. 114, 782-795 (2013).</li><li>Patton, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+heparin-binding+protein+using+monoclonal+antibodies+that+block+heparin+binding+to+porcine+aortic+endothelial+cells.">Identification of a heparin-binding protein using monoclonal antibodies that block heparin binding to porcine aortic endothelial cells.</a> The Biochemical journal. 311, 461-469 (1995).</li><li>Doggett, T. M., Breslin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+actin+cytoskeleton+in+live+endothelial+cells+expressing+GFP-actin.">Study of the actin cytoskeleton in live endothelial cells expressing GFP-actin.</a> J Vis Exp. , (2011).</li><li>Skalamera, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+genome+scale+lentiviral+vector+library+for+EF1alpha+promoter-driven+expression+of+human+ORFs+and+identification+of+human+genes+affecting+viral+titer.">Generation of a genome scale lentiviral vector library for EF1alpha promoter-driven expression of human ORFs and identification of human genes affecting viral titer.</a> PloS one. 7, 51733 (2012).</li><li>Castro, M., Nikolaev, V. O., Palm, D., Lohse, M. J., Vilardaga, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turn-on+switch+in+parathyroid+hormone+receptor+by+a+two-step+parathyroid+hormone+binding+mechanism.">Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism.</a> Proc Natl Acad Sci U S A. 102, 16084-16089 (2005).</li><li>Albertazzi, L., Arosio, D., Marchetti, L., Ricci, F., Beltram, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+FRET+analysis+with+the+EGFP-mCherry+fluorescent+protein+pair.">Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair.</a> Photochem Photobiol. 85, 287-297 (2009).</li><li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain+organization+of+the+ATP-sensitive+potassium+channel+complex+examined+by+fluorescence+resonance+energy+transfer.">Domain organization of the ATP-sensitive potassium channel complex examined by fluorescence resonance energy transfer.</a> J Biol Chem. 288, 4378-4388 (2013).</li><li>Christiansen, E., Hudson, B. D., Hansen, A. H., Milligan, G., Ulven, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Characterization+of+a+Potent+Free+Fatty+Acid+Receptor+1+(FFA1)+Fluorescent+Tracer.">Development and Characterization of a Potent Free Fatty Acid Receptor 1 (FFA1) Fluorescent Tracer.</a> J Med Chem. 59, 4849-4858 (2016).</li><li>Chiang, C. F., Okou, D. T., Griffin, T. B., Verret, C. R., Green Williams, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein+rendered+susceptible+to+proteolysis:+positions+for+protease-sensitive+insertions.">fluorescent protein rendered susceptible to proteolysis: positions for protease-sensitive insertions.</a> Arch Biochem Biophys. 394, 229-235 (2001).</li></ol>

Die Autoren haben nichts zu offenbaren.

Die Protokolle , die hier berichtet wurden entwickelt 1 bestätigenden Beweis für die Identifizierung von TMEM184A als Heparin - Rezeptor in vaskulären Zellen. Knockdown Techniken werden als ein Mechanismus zur Bestätigung der Identifizierung neuer Proteine ​​routinemäßig eingesetzt. Allerdings ist einige Funktionsverlust nach Zuschlags typischerweise nicht ausreichenden Nachweis, dass ein Kandidat Protein ist eigentlich die richtige Rezeptor (oder andere funktionelle Protein). Es ist au...

Identifizierung von Kandidatenproteine ​​für neuartige Funktionen hängt oft von Affinität basierende Isolation Protokolle durch partielle Sequenzbestimmung gefolgt. Jüngste Beispiele für neu identifizierten Proteine sind Transmembranprotein 184A (TMEM184A), eine Heparin - Rezeptor identifiziert nach Heparin - Affinität Wechselwirkungen 1 und TgPH1, eine pleckstrin Homologie - Domäne - Protein , das Phosphoinositid PI (3,5) P 2 2 bindet. Andere neuartige Proteinidentifikation beinhaltet direkte Sequenzanalyse von Peptiden , wie sie von Vit, et al. die verwendeten Trans Peptide Proteinprodukte von bisher nicht charakterisierte Gene 3 zu erkennen. In ähnlicher Weise kann die Identifizierung neuer Proteinsequenzen erreicht werden Bioinformatik unter Verwendung von zuvor charakterisierten Proteinfamilien suchen, wie Identifizierung neuer 4TM Proteine 4. Die Prüfung der Aquaporinfamilie Gensequenzen hat also ergab die Identifizierung neuer Mitglieder mit neuen Funktionen 5. Nach der Identifizierung, Analyse der Proteinfunktion ist typischerweise ein nächster Schritt, die manchmal einen spezifischen Assay von Protein-Funktion, wie beispielsweise in dem Aquaporin Fall untersucht werden können. Wenn möglich, die Funktion eines neu identifizierte Protein kann mit spezifischen enzymatischen oder ähnlichen in vitro - Assays Funktion untersucht werden. Da viele Funktionen der neuen Proteine in komplexen Wechselwirkungen abhängen , die nur in intakten Zellen oder Organismen auftreten, in - vitro - Tests sind nicht immer wirksam. Jedoch müssen die in - vivo - Tests in der Weise ausgebildet sein , dass sie auf der Gensequenz abhängen. In Zellkultur und / oder einfachen Modellorganismen, kann Knockdown Nachweise für sorgen für die Protein / Funktion Identifizierung 6. Mit neuartigen Proteine ​​identifiziert, wie oben erwähnt, ist es oft nicht einfach ein Protein abreißen zu Funktion bestätigen, eind die Gestaltung der in vivo funktionelle Assays , die auf Gensequenz abhängen wird zur Charakterisierung von neuen Proteinen wichtig. Die kürzliche Identifizierung von TMEM184A als Heparin - Rezeptor (die Proliferation in der glatten Gefäßmuskulatur und Entzündungsreaktionen in Endothelzellen moduliert) unter Verwendung von Affinitätschromatographie und MALDI - MS - 1, 7 versehen , die Möglichkeit , eine Sammlung von Assays zu entwickeln , nach dem Zuschlags Ergebnisse erzielt , die mit der Identifizierung . Eine aktuelle Überprüfung bestätigt , dass Heparin spezifisch mit vielen Wachstumsfaktoren in Wechselwirkung tritt, ihre Rezeptoren, extrazellulären Matrixkomponenten, Zelladhäsion Rezeptoren und anderen Proteinen 8. Im Gefäßsystem, Heparin und Heparansulfat - Proteoglykane (enthaltend Heparansulfatketten ähnlich in Struktur zu Heparin) interagieren mit mehreren hundert Proteine 9. Um funktionell bestätigen that TMEM184A wurde mit Heparin-Aufnahme beteiligt und Bindung, Techniken, die das Genkonstrukt für TMEM184A eingesetzt wurden entwickelt. Der vorliegende Bericht enthält eine Sammlung von Assays auf Basis eines GFP-TMEM184A zur Verwendung konstruieren in die Identität von TMEM184A als Heparin-Rezeptor bestätigt.

<table><tbody><tr><td>GFP-TMEM184A construct</td><td>OriGene</td><td>RG213192</td><td></td></tr><tr><td>Rhodamine-Heparin</td><td>Creative PEGWorks</td><td>HP-204</td><td>Light Sensitive</td></tr><tr><td>Fluorescein-Heparin</td><td>Creative PEGWorks</td><td>HP-201</td><td>Light Sensitive</td></tr><tr><td>Mowiol</td><td>EMD Millipore</td><td>475904-100GM</td><td></td></tr><tr><td>Paraformaldehyde (PFA, methanol free)</td><td>Thermo Sci Pierce Biotech, available through Fisher Scientific</td><td>PI28908 at Fisher</td><td>Use in Fume Hood</td></tr><tr><td>Reacti-bind neutravidin plates (Avidin coated black 96-well dishes)</td><td>Thermo Sci Pierce Biotech, through Fisher Scientific</td><td>PI15510 at Fisher</td><td>Pay attention to shelf-life</td></tr><tr><td>Black 96-well plates</td><td>Corning Life Sciences Plastic, purchased through Fisher Scientific</td><td>064432 at Fisher</td><td></td></tr><tr><td>A7r5 vascular smooth muscle cell line</td><td>ATCC</td><td>CRL 1444</td><td>Can be exchanged into MEM medium&lt;sup&gt;1&lt;/sup&gt;</td></tr><tr><td>BAOEC bovine aortic endothelial cells</td><td>Cell Applications, Inc.</td><td>B304-05</td><td>Culture as recommended initially, can be exchanged into MEM medium for continuing culture&lt;sup&gt;1,7&lt;/sup&gt;</td></tr><tr><td>BAOSMC bovine aortic smooth muscle cells</td><td>Cell Applications, Inc.</td><td>B354-05</td><td>Culture as recommended initially, can be exchanged into MEM medium for continuing culture&lt;sup&gt;1&lt;/sup&gt;</td></tr><tr><td>RAOEC rat aortic endothelial cells</td><td>Cell Applications, Inc.</td><td>R304-05a</td><td>Culture as recommended initially, can be exchanged into MEM medium for continuing culture&lt;sup&gt;7&lt;/sup&gt;</td></tr><tr><td>Biotinylated anti-GFP</td><td>Thermo Sci Pierce Biotech, through Fisher Scientific</td><td>MA5-15256-BTIN</td><td></td></tr><tr><td>Streptavidin-coated beads</td><td>Sigma</td><td>S1638</td><td></td></tr><tr><td>HeBS</td><td>Available from Bio-Rad</td><td></td><td>Can be prepared in the lab. The pH is 6.8</td></tr><tr><td>TMEM184A antibody to the N-terminus</td><td>Santa Cruz Biotechnology</td><td>sc292006</td><td>Only known TMEM184A antibody to N-terminal region.</td></tr><tr><td>TMEM184A antibody to the C-terminus</td><td>Obtained from ProSci Inc, Poway, CA&amp;nbsp;</td><td>Pro Sci 5681</td><td>ProSci used in &lt;strong&gt;Figure 1&lt;/strong&gt;</td></tr><tr><td>GFP antibodies</td><td>Santa Cruz Biotechnology</td><td>sc9996</td><td>Used in &lt;strong&gt;Figure 5&lt;/strong&gt;</td></tr><tr><td>Secondary antibodies, labeled with TRITC or Cy3</td><td>Jackson ImmunoResearch Laboratories, Inc, West Grove, PA</td><td>711 025 152 (donkey anti-rabbit, TRITC)&lt;br/&gt; 715 165 150 (donkey anti-mouse, Cy3)</td><td>Minimal cross-reactivity to minimize any nonspecific staining.</td></tr><tr><td>CHAPS</td><td>Purchased from Sigma</td><td>C5849</td><td>Note that this specific catalog number has been discontinued.&amp;nbsp; Supplier will provide information regarding replacement.</td></tr><tr><td>Live imaging 35-mm dishes</td><td>MatTek (Ashland MA)</td><td>P35G-1.0 &amp;ndash; 20 mm - C</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM 510 Meta with a 63X oil-immersion lens</td><td>Used for images and live-imaging in &lt;strong&gt;Figures 1, 2 and 3&lt;/strong&gt;</td></tr><tr><td>Confocal Microscope</td><td>Nikon</td><td>C2+ confocal with a 60X oil-immersion lens</td><td>Used for images in &lt;strong&gt;Figure 5&lt;/strong&gt;</td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>Zeiss LSM 880 with a 63X oil-immersion lens</td><td>Used for images in &lt;strong&gt;Figure 2C&lt;/strong&gt;</td></tr><tr><td>Electroporation equipment</td><td>Bio-Rad</td><td>Gene Pulser X-Cell System</td><td></td></tr><tr><td>Electroporation cuvettes</td><td>Available from MidSci</td><td>EC2L</td><td>Can also be obtained from equipment supplier</td></tr><tr><td>Plate reader</td><td>TECAN</td><td>TECAN Infinite&amp;reg; m200 Pro plate reader</td><td>Readings in the middle of the wells rather than at the surface.</td></tr><tr><td>Computer program for measuring staining intensity</td><td>Image J</td><td>https://imagej.nih.gov/ij/&lt;br/&gt; Program and information available on-line</td><td>Any appropriate program can be used. See https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html for additional detail&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Cell Culture trypsin solution</td><td>Sigma</td><td>T4174</td><td>purchased as a 10x solution</td></tr></tbody></table>

using a gfp-tagged tmem184a construct for confirmation of heparin receptor identity

Wenn neue Proteine ​​identifiziert werden durch Affinitäts-basierte Isolierung und Bioinformatik-Analyse, sind sie oft weitgehend uncharacterized. Antikörper, die gegen spezifische Peptide innerhalb der vorhergesagten Sequenz einige Lokalisierungs Experimente erlauben. Jedoch können auch andere mögliche Wechselwirkungen mit den Antikörpern oft nicht ausgeschlossen werden kann. Diese Situation bot die Möglichkeit, eine Reihe von Tests, abhängig von der Proteinsequenz zu entwickeln. Insbesondere ist ein Konstrukt, das das Gen-Sequenz am C-terminalen Ende des Proteins an das GFP-codierende Sequenz gekoppelt enthielt, wurde für diese Zwecke gewonnen und eingesetzt. Experimente Lokalisierung, Ligandenaffinität zu charakterisieren, und die Verstärkung der Funktion wurden ursprünglich entworfen und ausgeführt , um die Identifizierung von TMEM184A als Heparin - Rezeptor 1 bis bestätigen. Zusätzlich kann das Konstrukt für Studien Adressieren Membrantopologie Fragen und detaillierter Protein-Ligand-Wechselwirkungen eingesetzt werden. Der vorliegende Bericht stellt arange der experimentellen Protokolle auf der Basis der GFP-Konstrukt TMEM184A in vaskulären Zellen exprimiert, die leicht für andere neue Proteine ​​angepasst werden könnte.

Während in der Theorie, die Transfektion von DNA in Zellen jeder konstruieren könnte mit lipophilen Transfektionsreagenzien durchgeführt werden, zeigen früheren Berichten effektiver Transfektion von GFP - Konstrukten in endothelial Elektroporation 12 unter Verwendung von Zellen. Das Protokoll hier bereitgestellt typischerweise erreicht GFP-Konstrukt die Expression in mehr als 80% der primären abgeleitete Endothelzellen und glatte Muskelzellen verwendet. Entwu...

Watch this Scientific Journal Video about Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity at JoVE.com

Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity

Ein Konstrukt Codierung TMEM184A mit einem GFP-Tag am Carboxy-Terminus für eukaryotische Expressions entworfen wurde in Assays entwickelt, um zu bestätigen die Identifizierung von TMEM184A als Heparin-Rezeptor in vaskulären Zellen eingesetzt.

ein GFP-markierten TMEM184A Verwendung für die Bestätigung der Heparin-Rezeptor Identität Construct

When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies ...

using-gfp-tagged-tmem184a-construct-for-confirmation-heparin-receptor

Research

JoVE Journal

Genetics

7.9K Aufrufe.  Lehigh University.  Ein Konstrukt Codierung TMEM184A mit einem GFP-Tag am Carboxy-Terminus für eukaryotische Expressions entworfen wurde in Assays entwickelt, um zu bestätigen die Identifizierung von TMEM184A als Heparin-Rezeptor in vaskulären Zellen eingesetzt. 

Serie: Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Ausdruck und Reinigung des menschlichen Lipid-Sensitive kation Kanal TRPC3 für die Strukturaufklärung von Single-Teilchen Kryo-Elektronenmikroskopie

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Forschung

Biochemie

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

Optimierung der genetischen Einbeziehung der chemischen Sonden in GPCRs für Foto-Vernetzung-Mapping und Bioorthogonal Chemie in Live Säugerzellen

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Chemie

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

Mess TCR-pMHC Binding<em&gt; In-situ-</em&gt; Mit einem FRET-Mikroskopie Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Biotechnik

<meta charset="utf8"></head><body>Fluoreszierende Proteinmarkierung in Zebrafischlarven mittels CRISPR/Cas9-Knock-in

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

<meta charset="utf8"></head><body>Fluoreszenzmarkierung zur Visualisierung niedrig exprimierter Proteine im Zebrafisch

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

Fluoreszenzmarkierung zur Visualisierung niedrig exprimierter Proteine im Zebrafisch

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

Genetik

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

Mit Hilfe eines α-Bungarotoxin Binding Site Tag an GABA A-Rezeptor-Membranlokalisierung und Handel Studieren

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

Neurowissenschaften

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Imaging Protein-Protein-Wechselwirkungen In vivo</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Biologie

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

Genetisch kodierte Molecular Probes, um G-Protein-gekoppelte Rezeptoren Studieren

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Bestimmung der relativen Zelloberfläche und Total Expression rekombinanter Ionenkanäle Mit Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

ein GFP-markierten TMEM184A Verwendung für die Bestätigung der Heparin-Rezeptor Identität Construct

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Ausdruck und Reinigung des menschlichen Lipid-Sensitive kation Kanal TRPC3 für die Strukturaufklärung von Single-Teilchen Kryo-Elektronenmikroskopie

Optimierung der genetischen Einbeziehung der chemischen Sonden in GPCRs für Foto-Vernetzung-Mapping und Bioorthogonal Chemie in Live Säugerzellen

Mess TCR-pMHC Binding

Fluoreszenzmarkierung zur Visualisierung niedrig exprimierter Proteine im Zebrafisch

Fluoreszenzmarkierung zur Visualisierung niedrig exprimierter Proteine im Zebrafisch

Fluoreszenzmarkierung zur Visualisierung niedrig exprimierter Proteine im Zebrafisch

Mit Hilfe eines α-Bungarotoxin Binding Site Tag an GABA A-Rezeptor-Membranlokalisierung und Handel Studieren

Imaging Protein-Protein-Wechselwirkungen In vivo

Genetisch kodierte Molecular Probes, um G-Protein-gekoppelte Rezeptoren Studieren

Bestimmung der relativen Zelloberfläche und Total Expression rekombinanter Ionenkanäle Mit Flow Cytometry