Wir wünschen Herrn Dr. Scott Henderson für die Hilfe bei der konfokalen Mikroskopie zu bestätigen. Diese Forschung wurde durch Bewilligungen von den National Institutes of Health 1RO1CA127501 zu WAB sowie Pilotprojekt Mittel aus dem Massey Cancer Center and School of Medicine (VCU), um WAB-Mikroskopie wurde an der VCU-Abteilung durchgeführt unterstützt. für Neurobiologie und Anatomie Microscopy Facility, unterstützt, zum Teil mit Mitteln aus NIHNINDS Zentrum Kern gewähren 5P30NS047463.

Das Protokoll besteht aus drei Schritten. Der erste Schritt für das Klonen von Ihrem Gen von Interesse in einen Säugerexpressionsvektor Amino-terminal an die monomeren Versionen von GFP und / oder YFP wird nur kurz besprochen werden. Die zweite und dritte Schritt, die Transfektion von Vektor-DNA in EA.hy926 Endothelzellen und konfokale Bildgebung mit FRET, sind in mehr Tiefe im Folgenden erläutert. 1. Bau von CFP und YFP chimäre Rezeptoren Rezeptoren von Interesse sollte Amino-terminal an monomeren erweiterte Versionen von CFP und YFP geklont werden. Bestimmung von FRET ist empirisch und hängt entscheidend von der Länge des Linkers zwischen den Transmembran Spanning Region und zu Beginn des Fluorophors 1. Deshalb müssen verschiedene Längen von Linker für jeden neuen Rezeptor-Paar zu untersuchenden erkundet werden. <ol><li> In den pcDNA3.1 Hygro R oder neo R-Vektor, der monomeren C-oder YFP (dieses Vektors können aus unserem Labor angefordert werden), Klon Ihrer Rezeptor von Interesse in die NheI / EcoRI. * Hinweis: Wenn nötig, wird NheI kompatibel mehrere andere Enzyme, einschließlich SpeI und XbaI. </li><li> Verwandeln Sie Ihren Ligationsreaktion in eine geeignete endA1 recA zuständige Zelllinie (verwenden wir routinemäßig Mach1). Screen-Kolonien auf die Anwesenheit des Einsatzes gefolgt von DNA-Sequenzierung, um die Abwesenheit von Mutationen zu überprüfen. </li><li> Nach Erhalt Konformation des erfolgreichen molekularen Klonen vorbereiten Transfektion-grade Vektor-DNA in sterilem Wasser oder TE mit Qiagen, oder ähnliche, DNA-Kits. Bewerten Konzentration und Reinheit mittels UV-Spektroskopie. Typische Ausbeuten von DNA sollte im Bereich von 100-200 pg, die routinemäßig an eine funktionierende Bestand von 1 ug / ul verdünnt werden. </li></ol> 2. Die Transfektion von EA.hy 926 Cells <ol><li> Split EA.hy 926 Zellen 4, in Komplett-DMEM (DMEM +10% Fötales Rinderserum + Pen. / Strep.) In MatTek Deckglas (Nr. 0) 35 mm Gerichte mit 2 mL Medium. Sie können konservativ zu erhalten ~ 15 Gerichte (~ 90% konfluent am folgenden Tag) aus einer 15 cm konfluenten Schale. Bereiten Sie ein Gericht um die Expression von einzelnen Rezeptor, sowie ein Gericht für jeden Rezeptor-Paar zu bewerten. </li><li> Lassen Sie die Zellen zu inkubieren in 5% CO 2-Atmosphäre über Nacht bei 37 ° C. Am nächsten Tag sollte der Kulturen ~ 70-90% konfluent sein. </li><li> Am Tag der Transfektion, bereiten die Nukleolipid Komplexe durch Mischen von 2 ug des chimären Rezeptors Vektor-DNA (2 mL) mit 8 ul Fugene6 in 200 ul vorgewärmtes OptiMEM. Für Rezeptor-Paare, transfizieren mit 1 ug jeder Rezeptor, für eine Gesamtmenge von 2 pg (Siehe auch die Diskussion unten). </li><li> Vorsichtig mischen durch Umdrehen. </li><li> Weiter bei Raumtemperatur für 30 Minuten inkubieren. </li><li> Während der Inkubationszeit, bereiten die Zellen für die Transfektion. Absaugen frischen Medien und waschen jede Platte vorsichtig und kurz mit warmem PBS vor der Zugabe von 2 mL vorgewärmten complete-DMEM ohne Antibiotika (DMEM + FBS). Es ist wichtig, um die Anwesenheit von Penicillin und Streptomycin zu vermeiden, da sie wesentlich mehr Zellen toxisch sind während der Behandlung mit Fugene6. </li><li> Fügen Sie die komplexen tropfenweise zu den Zellen und inkubieren für eine weitere 20-24 Stunden bei 37 ° C in einer 5% CO 2-Atmosphäre. </li><li> Nach 24 Stunden sorgfältig absaugen die Medien aus den Zellen und Pipette frisches vollständiges-DMEM mit Antibiotika. Nach der Transfektion die Lebensfähigkeit der Zellen sollte relativ hoch bleiben, also wenig bis gar keine schwimmenden Zellen beobachtet werden sollte. </li><li> Die Genexpression wird in der Regel maximal nach einer weiteren 48-60 Stunden nach der Transfektion. Allerdings ist Bildgebung in der Regel möglich, in so kurz wie 24 Stunden. Zeitlicher Verlauf Experimente für eine optimale Genexpression durchgeführt werden soll. </li></ol> 3. Konfokale Bildgebung und FRET-Analyse Live Cell Imaging ist auf einem Leica TCS-SP2 AOBS konfokalen Laser-Scanning-Mikroskop mit blauen Diode (405 nm), Argon (458, 476, 488, 514 nm) ausgestattet war. grünen HeNe (543 nm), orange HeNe (594 nm) und roten HeNe (633 nm)-Laser, ein HCX PI Apo 63x/1.3 na Glycerin-Immersions-Objektiv, einem motorisierten XY-Tisch (Märzhäuser) und einem kontrollierten Umweltbedingungen ( Temperatur, Luftfeuchtigkeit und CO 2) Bühne Inkubator (Pecon). Experimentelle Kontrollen sind wichtig, um Fluorophor cross-talk zwischen der Emission Kanäle sowie Ausdruck auswerten Fragen und unspezifische Fluorophor Multimerisierung beseitigen. Als solche sind einzelne Fluorophor Transfektionen für die Einrichtung jedes Bild Session genutzt. Negative FRET Kontrollen (unter Verwendung von bekannten wechselwirkenden Rezeptoren) müssen durchgeführt werden, um dadurch den Hintergrund herauszurechnen FRET, das auftritt, durch Überexpression. <ol><li> Zur Vermeidung von Übersprechen zwischen den Emissions-Kanäle für CFP und YFP, Ort der GFP Expression Kontrolle in die Stufe Inkubator. Mit weißem Licht, stellen Sie die Z-Platte zu konzentrierenzu den Zellen. </li><li> Set SP-Fenster Einstellungen für Emissions-Detektion von GFP 465-505 nm und YFP zu 525-600 nm. </li><li> Während die Überwachung sowohl Emissions-Kanäle, erregen die GFP bei 458 nm. Mit dem AOTF, setzen Sie die Laserleistung auf nennenswerte Blutung über der GFP zu beseitigen in der YFP-Emission-Kanal. Speichern Sie diese Einstellung. </li><li> Führen Sie die gleiche Steuerung für YFP Übersprechen (mit dem YFP Ausdruck control) in der GFP-Kanal durch die Anpassung der Erregung Leistung bei 514 nm für die Zellen, die nur YFP. Speichern Sie diese Einstellung. </li><li> Nächster Ort Zellen co-exprimierenden CFP und YFP in das Stadium Inkubator. Anhand der Sequenzdaten Einstellung für die Leica konfokalen Software, suchen Sie exprimieren ähnliches Niveau von CFP und YFP mit der richtigen Membran Lokalisierung. </li><li> Mit der Leica Software Akzeptor Photobleaching Programm, stellen Sie den Donor-und Akzeptor-Einstellungen, um Ihre gespeicherten CFP und YFP-Einstellungen, anständig. </li><li> Zoomen auf die Zelle, markieren Sie den ROI (Region of Interest), in dem das Foto-Zerstörung von YFP ist auf der Zellmembran auftreten und beginnen das Programm. </li><li> Für Foto-Zerstörung des Akzeptors, passen Sie die AOTF zu 100% bei 514 nm und damit der Laser-Bleaching, weiterhin bis zu 70% der YFP-Emission erschöpft ist. Zur Beschleunigung Bleichen, sind ROIs in der Regel im Raster-Achse des Lasers (X-Achse) gewählt. </li><li> Während Foto-Bleichen, überwachen die zelluläre Bewegung und lehnen Messungen mit nennenswerten Bewegung in der XY-Ebene erhalten, da diese Messungen können Bericht falsch FRET Effizienz. </li><li> Pre-und Post-Bleiche Bilder sind für beide Donor (CFP) und Akzeptor (YFP) erfasst und FRET-Effizienz wie folgt berechnet: FRET Eff = (D post-D pre) / D Beitrag für alle D Beitrag&gt; D vor dem D vor und D Beitrag ist der Spender Intensität vor und nach dem Ausbleichen bzw.. </li><li> JMP Software (SAS) wird verwendet, um das Ausmaß der experimentellen Variabilität zu bestimmen. </li></ol> 4. Repräsentative Ergebnisse Transfektionseffektivitäten sind in der Regel zwischen 30 bis 40%. Trotz früherer Befunde von anderen, haben wir nicht gesehen, dass die Transfektion und Expression von einem Vektor, der anderen begünstigt. In der Tat, wir häufig beobachten, exklusive Expression von einem Fluorophor Chimäre oder das andere. Typische FRET Effizienzen variieren zwischen Rezeptor-Systeme. Für den Tie-Rezeptor-System, sind typische Werte 20-28% für Epithelzellen und 19-23% in Endothelzellen. Für negative Kontrollen sind typische Wirkungsgrade unter 2-3%. Das Ausmaß der Variabilität wird deutlich sinken mit Erfahrung. <img src="/files/ftp_upload/2149/2149fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Repräsentative Bilder von transfizierten EA.hy 926 Zellen. A) DIC Bild EA.hy 926 Zellmonolayer. B) Fluoreszenzbild von Zellen in (A) angezeigt. C) Überlagerung von (A) und (B) demonstriert ~ 20-30% Transfektionseffizienz. <img src="/files/ftp_upload/2149/2149fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Vertreter Akzeptor Photobleaching Analyse von FRET zwischen CFP und YFP vorkommenden in einer EA.hy926 Zelle. Die GFP-Emission-Kanal (obere Bilder) und YFP-Emission-Kanal (unten zwei Tafeln) wurden separat vor überwacht und post-Akzeptor Bleichen. Photobleaching Experimente wurden eingeschränkt, und FRET-Werte aus, die Region in der grünen Box berechnet. FRET-Effizienz wird als absolute reichen von hoch (rot-1.0) zu niedrig ist (lila-0.0) auf einem vergrößerten Überlagerung eines CFP / YFP angezeigt zusammengefügte Bild nur zur Orientierung.

<ol>
	<li>Kim, M., Carman, C. V., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+transmembrane+signaling+by+cytoplasmic+domain+separation+in+integrins.">Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins.</a> Science. 301, 1720-1725 (2003).</li><li>Zacharias, D. A., Violin, J. D., Newton, A. C., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+of+lipidmodified+monomeric+GFPs+into+membrane+microdomains+of+live+cells.">Partitioning of lipidmodified monomeric GFPs into membrane microdomains of live cells.</a> Science. 296, 913-916 (2002).</li><li>Seegar, T. C. M., Eller, B., Tzvetkova-Robev, D., Kolev, M., Henderson, S. C., Nikolov, D. B., Barton, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tie1-Tie2+interactions+mediate+functional+differences+between+angiopoietin+ligands.">Tie1-Tie2 interactions mediate functional differences between angiopoietin ligands.</a> Molecular Cell. 37 (5), 643-655 (2010).</li><li>Edgell, C. J., McDonald, C. C., Graham, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permanent+cell+line+expressing+human+factor+VIII-related+antigen+established+by+hybridization.">Permanent cell line expressing human factor VIII-related antigen established by hybridization.</a> Proc Natl Acad Sci U S A. 80 (12), 3734-3737 (1983).</li></ol>

Es gibt mehrere Schritte, die für den Erfolg entscheidend sind. Der prominenteste unter ihnen ist die relative Bedeutung der Ausdruck zwischen den beiden chimären Rezeptoren. Zur Umgehung dieses Problems, kann man machen, stabilen Zelllinien, sowohl Proteine ​​von Interesse, oder ermitteln die optimale Verhältnis von Vektor-DNA zu gleichwertigen Ausdruck zu ermöglichen. Auch aufgrund der uneinheitlichen Transfektion in eine Schüssel, werden Protein-Ebene nur selten gleichwertig zwischen den Zellen. Deshalb ist darauf zu achten, um &quot;high&quot; Expressoren von «niedrig» zu unterscheiden. Diejenigen, die Anzeige &quot;durchschnittlichen&quot; Mengen an Protein in der Regel liefern zuverlässige und reproduzierbare FRET Effizienz. Eine Methode, unser Labor verfolgt, um dieses Problem zu lindern, ist die Verwendung von Adeno-und Lenti-Viren &quot;, auch&quot; Gen-Expression. Darüber hinaus FRET eine alternative Methode zur Akzeptor Photobleaching zur Bestimmung Effizienz ist sensibilisierte Emission. Obwohl trotz des Potenzials der sensibilisierte Emission auf einzelne Zellen in Echtzeit zu überwachen, haben wir Akzeptor Photobleaching empfindlicher und zuverlässiger gefunden. Schließlich kann mittels FRET zu Protein-Wechselwirkungen Monitor eine Herausforderung sein, und erfordert eine sorgfältige Auswahl der Linker Längen für membrangebundene Rezeptoren. Darüber hinaus Zugabe von C / YFP oft einen großen Einfluss auf die Proteinexpression Ebenen und die Ergebnisse in die Aggregation im endoplasmatischen Retikulum und Golgi-Apparat. Doch für transiente oder instabil, Interaktionen, ist FRET die ideale Methode zu nutzen.

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<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11960-069</td>
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<td>Penicillin- Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15070-063</td>
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<td>Hyclone</td>
<td>N/A</td>
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<td>Opti-MEM</td>
<td>&nbsp;</td>
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<td>Coverslip Dishes</td>
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imaging protein-protein interactions in vivo

Protein-Protein-Interaktionen sind ein Markenzeichen aller wesentlichen zellulären Prozessen. Allerdings sind viele dieser Wechselwirkungen transiente oder energetisch schwach, die Verhinderung ihrer Identifizierung und Analyse durch traditionelle biochemische Methoden wie Co-Immunopräzipitation. In dieser Hinsicht haben die genetisch kodierbaren fluoreszierende Proteine ​​(GFP, RFP, etc.) und die damit verbundenen überlappenden Fluoreszenzspektrum revolutioniert unsere Fähigkeit, schwache Wechselwirkungen in vivo mit Förster resonance energy transfer (FRET) 1-3 überwachen. Hier Detail haben wir den Einsatz eines FRET-basierten Proximity Assay zur Überwachung Rezeptor-Rezeptor-Wechselwirkungen auf der endothelialen Zelloberfläche.

Watch this Scientific Journal Video about Imaging Protein-protein Interactions in vivo at JoVE.com

Imaging Protein-protein Interactions in vivo

Dieses Protokoll beschreibt, wie Bild-Protein-Protein-Interaktionen mit Hilfe eines FRET-basierten 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, ...

imaging-protein-protein-interactions-in-vivo

Research

JoVE Journal

Biology

Imaging Protein-protein Interactions in vivo

21.2K Aufrufe.  Virginia Commonwealth University. Dieses Protokoll beschreibt, wie Bild-Protein-Protein-Interaktionen mit Hilfe eines FRET-basierten Proximity Assay.

Serie: Imaging Protein-protein Interactions in vivo

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-Ubiquitin Basierend Membrane Yeast Two-Hybrid (Mythos) System: Ein wirksames Instrument zur Ermittlung von Protein-Protein-Wechselwirkungen

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Forschung

Biologie

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

In-vivo-Detektion von Protein-Protein-Interaktionen auf Micro-gemusterten Oberflächen

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Protein-Protein-Wechselwirkungen durch Bimolekulare Fluoreszenz-Komplementation in Tabak-Protoplasten-und Blätter-Visualisierte

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Untersuchung von Protein-Protein-Interaktionen in lebenden Zellen Mit Biolumineszenz-Resonanz-Energie-Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

Probing hoher Dichte Funktionale Protein-Microarrays, um Protein-Protein Wechselwirkungen erkennen

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

Entwicklung von Inhibitoren von Protein-Protein-Wechselwirkungen durch REPLACE: Anwendung auf die Konzeption und Entwicklung Nicht ATP Competitive CDK-Inhibitoren

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Chromatinimmunpräzipitation Assay zur Identifizierung von Arabidopsis-Protein-DNA-Wechselwirkungen<em&gt; In-vivo-</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Untersuchung von Protein-Protein-Interaktionen in der Autophagie-Forschung

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Fortgeschrittene Mikroskopiertechniken der konfokalen zur Untersuchung von Protein-Protein-Wechselwirkungen und die Kinetik bei DNA-Läsionen

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Luciferase Komplementierung Imaging Assay in Nicotiana Benthamiana Blätter für Transient Bestimmung Proteinprotein Interaktion Dynamik

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...