Wir möchten Quentina Beatty, Universität Linz, Österreich, für ihre freundliche Hilfe, Katharina Strub, Universität Genf, Schweiz danken, für die hCD71-GFP-Konstrukt, und Daniel Legler, Universität Konstanz, der Schweiz, für die GPI-GFP -DAF zu konstruieren. Und die GEN-AU Projekt des österreichischen Bundesministeriums für Wissenschaft und Forschung; Diese Arbeit wurde vom FWF (Projekt Y250-B03 FWF) unterstützt.

Mikrokontaktdrucken: <ol><li> Cut out Bereich der Mikrostruktur des PDMS-Stempels mit einem Skalpell </li><li> Wash das Feld mit der Mikrostruktur durch Spülen mit Ethanol (100%) und destilliertem Wasser. </li><li> Trocknen Sie die PDMS-Stempel mit Stickstoff oder Argon. </li><li> Je 50 ul BSA-Cy5 Arbeit-Lösung (0,67 mg / ml) auf die PDMS-Stempel, so dass die gesamte Mikrostruktur Feld mit Lösung bedeckt ist. Inkubieren für 30 min bei Raumtemperatur. </li><li> Waschen Sie die Mikrostruktur Bereich durch Spülen mit Phosphat-gepufferter Saline (PBS) und destilliertem Wasser. </li><li> Trocknen Sie die PDMS mit Stickstoff oder Argon. </li><li> Legen Sie die PDMS-Stempel unter seinem eigenen Gewicht auf die Mitte einer Epoxy-beschichtete Deckglas und Inkubation für 30 min bei Raumtemperatur in einer Petrischale. </li><li> Beschriften Sie die Position der PDMS-Stempel auf der Rückseite des Deckglases und Streifen der Stempel aus der Folie mit einer Pinzette. </li><li> Stick eine sichere Abdichtung Hybridisierung Kammer über die Mikrokontaktstruktur bedruckte Bereich auf dem Deckglas. Das Label hilft zu lokalisieren der Mitte des Feldes. </li><li> Pipette 60 ul Streptavidin Arbeit-Lösung (50 pg / ml) in die Reaktionskammer und inkubieren Sie die Probe für 60 min bei Raumtemperatur. </li><li> Waschen Sie die Probe mit 1 ml PBS, indem Sie die Puffer in einem Hafen der Kammer zu entfernen und gleichzeitig an den zweiten Port mit einer Pumpe. </li><li> Pipette 60 ul biotinylierten Antikörper Arbeit-Lösung (10 pg / ml in PBS mit 0,1% Tween 20 ergänzt, &quot;PBST&quot;) in die Reaktionskammer und Inkubation für 60 min bei Raumtemperatur. </li><li> Waschen Sie die Probe mit 1 ml PBST und anschließend 1 ml PBS, indem Sie die Puffer in einem Hafen der Kammer und Entfernen wieder auf den zweiten Port mit einer Pumpe. </li><li> Bewahren Sie die mikrostrukturierten Oberflächen in PBS im Dunkeln bei Raumtemperatur, bis die Zellen bereit für die Aussaat sind. </li></ol> Die Inkubation von Zellen auf der mikrostrukturierten Oberfläche: <ol><li> Wachsen adhärente Zellen zu 50% Konfluenz in einer 3 cm Gewebekulturplatte. </li><li> Lösen Sie exprimieren Köder und Beute Proteine ​​von Interesse mit Ethylendiamintetraessigsäure (EDTA)-Lösung und Zentrifuge 5 min bei 1.000 Umdrehungen pro Minute. </li><li> Überstand verwerfen und lösen das Zellpellet in dem entsprechenden Wachstumsmedium. </li><li> Zentrifuge 5 min bei 1.000 Umdrehungen pro Minute. </li><li> Überstand verwerfen und lösen das Zellpellet in geeigneten Nährmedium. </li><li> Tauschen Sie die PBS aus der Reaktionskammer auf den mikrostrukturierten Deckglas von 60 ul der Zellsuspension. </li><li> Erstellen einer feuchten Kammer durch Einweichen eines sterilen Tupfer in destilliertem Wasser und legt es in die Petrischale, um die Probe vor Trockenlauf zu verhindern. </li><li> Inkubieren Sie die Zellen über Nacht bei 37 ° C in einer 5% CO 2-Atmosphäre auf den mikrostrukturierten Oberflächen. </li><li> Vor einer Analyse der Zellen unter dem Mikroskop, den Austausch des Mediums durch gepufferte Salz Hank s Lösung mit Ca 2 + und Mg 2 +. </li></ol> Mikroskopie: <ol><li> Das Deckglas wird auf einer geeigneten Halterung gelegt und die Zellmorphologie unter Weißlicht-geprüft. </li><li> Die Qualität der BSA-Cy5 Muster wird durch Anregung bei 647 nm überprüft. </li><li> Mikrostrukturen von fluoreszierenden Beute-Protein (GFP oder YFP) sind bei 488 oder 514 nm unter Total Internal Reflection (TIR) ​​Anregung aufgenommen. </li></ol> Repräsentative Ergebnisse: Wenn es Wechselwirkungen zwischen den Target-Proteine ​​in den Zellen auf einer mikrostrukturierten Oberfläche gewachsen ist, wird die Beute folgen den Köder Umverteilung. Die resultierende Mikrostruktur können visualisiert werden, indem die Fluoreszenz-Markierung der Beute-Protein. Wichtig ist der Kontrast erhalten ein direktes Maß für die Stärke der Wechselwirkung (siehe Abbildung 1). Daher ist eine einfache Auswertung der Interaktion von zwei Proteinen möglich wird - ohne die Notwendigkeit einer weiteren Prozess der gemessenen Primärdaten. <img src="/files/ftp_upload/1969/1969fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Umlagerung des Köders in der lebenden Zelle Plasmamembran an unterschiedlichen Stärke der Wechselwirkung. TIR Bilder von T24-Zellen mit (a) CD71-GFP auf CD71-Antikörper, transfiziert, (b) CD4 und cytosolische YFP auf CD4-Antikörper und (c) GPI-GFP-DAF auf CD59-Antikörper microbiochips gezeigt. Die Daten sind charakteristisch für stark (a), no (b) oder schwache Wechselwirkung (c). (A) aus (Weghuber et al., In press), (b) aus (Schwarzenbacher et al., 2008) wiedergegeben.

<ol>
	<li>Brameshuber, M., Schwarzenbacher, M., Kaltenbrunner, M., Hesch, C., Paster, W., Weghuber, J., Heise, B., Sonnleitner, A., Stockinger, H., Schuetz, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reply+to+"Uncoupling+diffusion+and+binding+in+FRAP+experiments.">Reply to "Uncoupling diffusion and binding in FRAP experiments.</a> Nat. Methods. 6, 183-184 (2009).</li><li>Kim, P. W., Sun, Z. J., Blacklow, S. C., Wagner, G., Eck, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Zinc+Clasp+Structure+Tethers+Lck+to+T+Cell+Coreceptors+CD4+and+CD8.">A Zinc Clasp Structure Tethers Lck to T Cell Coreceptors CD4 and CD8.</a> Science. 301, 1725-1728 (2003).</li><li>Ramm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+screening:+a+technology+in+transition.">Image-based screening: a technology in transition.</a> Curr. Opin. Biotechnol. 16, 41-48 (2005).</li><li>Schwarzenbacher, M., Kaltenbrunner, M., Brameshuber, M., Hesch, C., Paster, W., Weghuber, J., Heise, B., Sonnleitner, A., Stockinger, H., Schutz, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+for+quantitative+analysis+of+protein-protein+interactions+in+living+cells.">Micropatterning for quantitative analysis of protein-protein interactions in living cells.</a> Nat. Methods. 5, 1053-1060 (2008).</li><li>Weghuber, J., Brameshuber, M., Sunzenauer, S., Lehner, M., Paar, C., Haselbruebler, T., Schwarzenbacher, M., Kaltenbrunner, M., Paster, W., Heise, B., Sonnleitner, A., Stockinger, H., Schutz, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+protein-protein+interactions+in+the+live+cell+plasma+membrane+by+quantifying+prey+redistribution+upon+bait+micropatterning.">Detection of protein-protein interactions in the live cell plasma membrane by quantifying prey redistribution upon bait micropatterning.</a> Methods in Enzymology. , .</li></ol>

Das Video enthält Bilder, die von Olympus, von der Homepage des Visualization Research Lab entnommen, Brown University zur Verfügung gestellt wurden oder reproduziert aus (Kim et al., 2003).

Das dazugehörige Video zeigt ein Verfahren zum Nachweis von Protein-Protein-Wechselwirkungen in der Plasmamembran von lebenden Zellen (Schwarzenbacher et al, 2008;. Brameshuber et al, 2009;.. Weghuber et al, in press). Im Prinzip kann jeder TIRF-basierte Mikroskopie-Plattform als Anzeige verwendet werden. Nur wenn eine hohe Empfindlichkeit gewünscht wird (zB für die Detektion einzelner Moleküle), werden fortgeschrittene Mikroskope verlangt werden. Um dies zu erreichen beste Ergebnisse die folgenden kritischen Punkte während des Herstellungsprozesses erfordern besondere Aufmerksamkeit: <ol class="lroman"><li> Shop Cy5-Stammlösung bei schlechten Lichtverhältnissen zu vermeiden Bleichen des Fluorophors und bereiten die BSA-Cy5 Arbeit Lösung frisch vor dem Mikrokontaktdrucken. </li><li> Achten Sie darauf, nicht auf die Mikrostruktur auf dem PDMS Kratzer mit der Pipettenspitze und inkubieren Sie die Probe bei schlechten Lichtverhältnissen zu vermeiden Bleichen des Fluorophors. </li><li> Legen Sie das Deckglas auf einem sterilen Tupfer zu verhindern Verkleben der Glasscheibe auf die Petrischale. Sobald die PDMS-Stempel auf den Objektträger geklebt hat, bewegen Sie es nicht. </li><li> Vermeiden Sie Luftblasen in der Reaktionskammer und inkubieren Sie die Probe bei schlechten Lichtverhältnissen zu vermeiden Bleichen des Fluorophors. </li><li> Lassen Sie sich nicht die Probe trocken für mehr als 2 Minuten, um die Bildung von Salzkristallen zu vermeiden. </li><li> Für die Ablösung der Zellen nicht verwenden Trypsin als es Abspaltung der extrazellulären Domäne des bisher geäußert Membranproteinen. Vermeiden Trypsin wird hilfreich sein, die für Proteine ​​mit niedrigen Fluktuationsrate zu Mikrostrukturen unmittelbar nach Anheftung der Zellen auf der Oberfläche bilden. </li><li> Kontrollieren Sie die Anzahl der inkubierten Zellen im Mikroskop und stellen Sie sicher, dass die Zellen nicht erreichen mehr als 30-50% Konfluenz. Stellen Sie sicher, dass die übermäßige Zellen sofort sind auf die schnelle Befestigung von Zellen auf der Antikörper-beschichteten Oberfläche entfernt. </li><li> Nur Folien mit hoher Qualität BSA-Cy5 Muster können zur weiteren Analyse verwendet werden. </li><li> Stabile Expression des fluoreszierenden Proteins Beute ist vorzuziehen aufgrund der größeren Anzahl von Zellen, die pro Scan analysiert werden kann. </li><li> Angemessene TIRF Einstellung ist unerlässlich, um Muster durch cytosolische Hintergrund sichtbar zu machen. </li></ol> Die Mikro-Strukturierung Technik bietet mehrere Möglichkeiten für die Analyse von Protein-Protein-Interaktionen. Erstens, zusammen mit der Quantifizierung der lokalen, räumlich aufgelöster Protein-Protein-Interaktionen auch die Detektion von schwachen oder indirekten Wechselwirkungen ist, ohne den Nachteil, dass eine hohe Anzahl von falschen Positiven oder Negativen möglich. Zweitens ermöglicht die Forscher, um Protein-Protein-Wechselwirkungen in der Plasmamembran von lebenden Zellen, die nur schwer durch biochemische Ansätze wie die 2-Hybrid-Screen zu erreichen, ist zu analysieren. Drittens der Ansatz ermöglicht die Erkennung von Köder-Beute-Interaktionen, moduliert durch Veränderungen der Umwelt wie die Temperatur, die Anwesenheit verschiedener Proteine ​​oder andere Moleküle oder post-translationale Modifikationen sind. So ist die Assay ermöglicht die Screening-Modulatoren einer bestimmten Interaktion Paar im Kontext der lebenden Zellen. Darüber hinaus, indem die Flächendichte der Erfassung Liganden oder die Verwendung von monovalenten Liganden, wird die Analyse der Ruhezustand möglich. Schließlich, wenn ausreichende Scan-Plattformen verwendet werden, ist die Zahl der analysierten Zellen hoch genug, um einen hohen Durchsatz Anforderungen der Pharma-Unternehmen für Wirkstoff-Screening (Ramm, 2005) überein.

in-vivo detection of protein-protein interactions on micro-patterned surfaces

Die Aufklärung der Interaktion Netzwerk von Molekülen<em&gt; In-vivo</em&gt; Ist der Schlüssel zum Verständnis der Mechanismen, die die Zellfunktion und den Stoffwechsel regulieren. Eine Vielzahl von methodischen Möglichkeiten für die Behandlung molekularer Wechselwirkungen in Zellen entwickelt worden, aber die meisten dieser Methoden leiden darunter, eher indirekt und daher kaum quantitativ. Im Gegenteil, ein paar High-End-quantitative Ansätze wurden eingeführt, die jedoch schwer zu hohen Durchsatz zu verlängern. So kombinieren hohe Durchsatz mit der Möglichkeit, quantitative Informationen zu extrahieren, haben wir kürzlich ein neues Konzept entwickelt für die Identifizierung von Protein-Protein-Interaktionen (Schwarzenbacher<em&gt; Et al</em&gt;., 2008). Hier beschreiben wir ein detailliertes Protokoll für die Konzeption und den Bau dieses Systems, die für die Analyse von Interaktionen zwischen einem Fluorophor-markierten Protein (&quot;Beute&quot;) und ein Membranprotein ermöglicht (&quot;Köder&quot;)<em&gt; In-vivo</em&gt;. Die Zellen werden auf mikrostrukturierten Oberflächen mit Antikörpern gegen den Köder exoplasmic Domain-funktionalisierte überzogen. Bait-Beute-Interaktionen sind über die Neuverteilung der fluoreszierenden Beute untersucht. Das Verfahren ist durch eine hohe Empfindlichkeit bis auf die Ebene einzelner Moleküle, die Fähigkeit, schwache Wechselwirkungen zu erkennen und mit hohem Durchsatz Fähigkeit charakterisiert, so dass es für die Screening-Instrument.

Watch this Scientific Journal Video about In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces at JoVE.com

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

Dieses Video zeigt Experimente mit anschließender Analyse von Protein-Protein-Wechselwirkungen durch den Einsatz von Mikro-gemusterten Oberflächen. Der Ansatz bietet die Möglichkeit, Protein-Wechselwirkungen in lebenden Zellen zu erkennen und verbindet hohe Durchsatz mit der Möglichkeit, quantitative Informationen zu extrahieren.

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 ...

in-vivo-detection-protein-protein-interactions-on-micro-patterned

Research

JoVE Journal

Biology

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

10.7K Aufrufe.  Johannes Kepler Universitat Linz. Dieses Video zeigt Experimente mit anschließender Analyse von Protein-Protein-Wechselwirkungen durch den Einsatz von Mikro-gemusterten Oberflächen. Der Ansatz bietet die Möglichkeit, Protein-Wechselwirkungen in lebenden Zellen zu erkennen und verbindet hohe Durchsatz mit der Möglichkeit, quantitative Informationen zu extrahieren.

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

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

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, ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

In vivo Quantifizierung der G Protein-gekoppelten Rezeptor-Wechselwirkungen mit spektral aufgelöste Zwei-Photonen-Mikroskopie

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Ein Protokoll für die Identifizierung von Protein-Protein-Wechselwirkungen Basierend auf<sup&gt; 15</sup&gt; N Metabolische Markierung, Immunpräzipitation, Quantitative Massenspektrometrie und Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

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. ...