Nos gustaría dar las gracias a Quentina Beatty, de la Universidad de Linz, Austria, por su amable ayuda, Katharina Strub, de la Universidad de Ginebra, Suiza, para la construcción de hCD71-GFP, y Daniel Legler, Universidad de Constanza, Suiza, por el GPI-GFP -DAF construir. Este trabajo fue apoyado por el Fondo Científico de Austria (FMF; proyecto Y250-B03) y el proyecto GEN-AU del Ministerio Federal de Ciencia e Investigación.

Microcontacto de impresión: <ol><li> Recorta el campo de micropattern del sello PDMS con un bisturí </li><li> Lave el campo que contiene el micropattern por lavado con etanol (100%) y agua destilada. </li><li> Seque el sello PDMS con nitrógeno o argón. </li><li> Añadir 50 l de BSA-Cy5 solución de trabajo (0,67 mg / ml) en el sello PDMS, para que todo el campo micropattern está cubierto con la solución. Incubar durante 30 min a temperatura ambiente. </li><li> Lavar el campo micropattern lavándolo con tampón fosfato salino (PBS) y agua destilada. </li><li> Seque el PDMS con nitrógeno o argón. </li><li> Coloque el sello PDMS por su propio peso en medio de un revestimiento epóxico cubreobjetos y se incuba durante 30 min a temperatura ambiente en una placa de Petri. </li><li> Etiqueta de la posición de la marca de PDMS en la parte trasera del cubreobjetos y tira el sello de la diapositiva con una pinza. </li><li> Palo de una cámara de sello de hibridación seguro sobre el campo microcontacto impresos en el cubreobjetos. La etiqueta ayuda a localizar el centro del campo. </li><li> Pipeta de 60 estreptavidina l solución de trabajo (50 mg / ml) en la cámara de reacción e incubar la muestra durante 60 minutos a temperatura ambiente. </li><li> Lavar la muestra con 1 ml de PBS mediante la adición de la memoria intermedia en un puerto de la cámara y eliminarlo de forma simultánea en el segundo puerto con una bomba. </li><li> Pipeta de 60 l de anticuerpos con biotina solución de trabajo (10 mg / ml en PBS suplementado con 0,1% de Tween 20; &quot;PBST&quot;) en la cámara de reacción e incubar durante 60 min a temperatura ambiente. </li><li> Lavar la muestra con 1 ml de PBST y luego 1 ml de PBS mediante la adición de la memoria intermedia en un puerto de la cámara y la eliminación de nuevo en el segundo puerto con una bomba. </li><li> Tienda de las superficies micropatterned en PBS en la oscuridad a temperatura ambiente hasta que las células están listas para la siembra. </li></ol> La incubación de las células en la superficie micropatterned: <ol><li> Crecen las células adherentes al 50% de confluencia en una placa de cultivo de 3 cm de tejido. </li><li> Separar las células que expresan proteínas cebo y la presa de intereses con ácido etilendiaminotetraacético (EDTA) y se centrifuga 5 min a 1.000 rpm. </li><li> Eliminar el sobrenadante y disolver el pellet de células en el medio de cultivo de acuerdo. </li><li> Centrifugar 5 min a 1.000 rpm. </li><li> Eliminar el sobrenadante y disolver el pellet de células en medio de cultivo apropiado. </li><li> Intercambio de la PBS de la cámara de reacción en el cubreobjetos micropatterned en un 60 l de la suspensión celular. </li><li> Crear una cámara húmeda empapando una gasa estéril con agua destilada y ponerlo en la placa de Petri para evitar que la muestra de la falta de agua. </li><li> Se incuban las células durante la noche a 37 ° C en un 5% de CO2 la atmósfera en la superficie micropatterned. </li><li> Antes de analizar las células en el microscopio, el medio de intercambio por solución salina tamponada Hank s como Ca 2 + y Mg 2 +. </li></ol> Microscopía: <ol><li> El cubreobjetos se coloca sobre un soporte adecuado y la morfología de las células se examinan bajo luz blanca. </li><li> La calidad de los BSA-Cy5 patrones está marcada por la excitación a 647 nm. </li><li> Micropatrones de presa de proteínas fluorescentes (GFP o YFP) se registran en 488 o 514 nm en reflexión interna total (TIR) ​​de excitación. </li></ol> Los resultados representativos: Si hay una interacción entre las proteínas diana en las células cultivadas en una superficie micropatterned, la presa seguirá la redistribución de cebo. El micropattern resultante puede ser visualizado por la etiqueta de fluorescencia de la proteína de la presa. Es importante destacar el contraste obtenido proporciona una medida directa de la fuerza de interacción (ver Figura 1). Por lo tanto, una simple evaluación de la interacción de dos proteínas es posible - sin necesidad de otro proceso de los datos primarios medidos. <img src="/files/ftp_upload/1969/1969fig1.jpg" alt="Figura 1" /> Figura 1. Reordenamiento del cebo en la membrana de células plasmáticas en vivo en diferentes fuerzas de interacción. TIR imágenes de las células transfectadas con T24 (a) CD71-GFP en CD71-anticuerpo, CD4 (b) y YFP citosólica de CD4-anticuerpo y (c) GPI-DAF-GFP en microbiochips CD59-anticuerpo se muestran. Los datos son característicos de una fuerte (a), no (b) o de la interacción débil (c). (A) se reproduce a partir de (Weghuber et al., En prensa), (b) de (Schwarzenbacher et al., 2008).

<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>

El vídeo contiene imágenes que fueron proporcionados por Olympus, tomada de la página principal del Laboratorio de Investigación de Visualización de la Universidad Brown o reproducido a partir de (Kim<em&gt; Et al</em&gt;., 2003).

El video que acompaña muestra un método para la detección de interacciones proteína-proteína en la membrana plasmática de las células vivas (Schwarzenbacher et al, 2008;. Brameshuber et al, 2009;.. Weghuber et al, en prensa). En principio, cualquier plataforma microscopía TIRF basado puede ser utilizado como sistema de lectura. Sólo cuando se desea una alta sensibilidad (por ejemplo, para la detección de moléculas individuales), los microscopios avanzados se requerirá. Para obtener los mejores resultados los siguientes puntos críticos durante el proceso de preparación requieren una atención especial: <ol class="lroman"><li> Tienda Cy5-de la solución madre en condiciones de oscuridad para evitar la decoloración del fluoróforo y preparar la BSA-Cy5 solución de trabajo poco antes de la impresión por microcontacto. </li><li> Tenga cuidado de no rayar la micropattern en el PDMS con la punta de la pipeta y se incuba la muestra en condiciones de oscuridad para evitar el blanqueo del fluoróforo. </li><li> Coloque el cubreobjetos sobre una gasa estéril para evitar la adherencia de la lámina de vidrio de la placa de Petri. Una vez que el sello de PDMS se ha adherido a la lámina de vidrio, no lo mueva. </li><li> Evite las burbujas de aire en la cámara de reacción e incubar la muestra en condiciones de oscuridad para evitar la decoloración del fluoróforo. </li><li> No permita que la muestra seca por más de 2 minutos para evitar la formación de cristales de sal. </li><li> Para el desprendimiento de las células no utilizan la tripsina, ya que se unirá al dominio extracelular de proteínas de la membrana hasta ahora expresados. Evitar la tripsina será útil para las proteínas con baja tasa de rotación para formar micropatrones inmediatamente después de fijación de las células en la superficie. </li><li> Controlar el número de células se incubaron en un microscopio y asegurarse de que las células no llegan a más de 30-50% de confluencia. Asegúrese de que las células se eliminan inmediatamente excesiva debido a la fijación rápida de las células en la superficie recubiertas de anticuerpos. </li><li> Sólo con diapositivas de alta calidad BSA-Cy5 patrones se pueden utilizar para su posterior análisis. </li><li> La expresión estable de la proteína fluorescente presa es preferible debido a un mayor número de células que pueden ser analizados por barrido. </li><li> Adecuado ajuste TIRF es indispensable para visualizar los patrones de fondo debido a la citosólica. </li></ol> La técnica de micro-patrones ofrece varias posibilidades para el análisis de las interacciones proteína-proteína. En primer lugar, junto con la cuantificación de los locales, resolución espacial interacciones proteína-proteína también la detección de interacciones débiles o indirecta es posible sin el inconveniente de dar un alto número de falsos positivos o negativos. En segundo lugar, permite al investigador para analizar interacciones proteína-proteína en la membrana plasmática de células vivas, lo cual es difícil de conseguir por métodos bioquímicos como la pantalla de dos híbridos. En tercer lugar el método permite la detección de interacciones presa-cebo que son moduladas por los cambios ambientales como la temperatura, la presencia de diferentes proteínas u otras moléculas o modificaciones posteriores a la traducción. Así, el ensayo permite moduladores de detección de un par de interacción dada en el contexto de las células vivas. Por otra parte, mediante la reducción de la densidad superficial del ligando de captura o el uso de ligandos monovalentes, el análisis del estado de reposo se hace posible. Por último, si suficientes plataformas de exploración se utilizan, el número de células analizadas es lo suficientemente alto como para satisfacer las demandas de alto rendimiento de las compañías farmacéuticas para la detección de drogas (Ramm, 2005).

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

Desentrañar la red de interacción de las moléculas<em&gt; En-vivo</em&gt; Es clave para entender los mecanismos que regulan la función celular y el metabolismo. Una multitud de opciones metodológicas para abordar las interacciones moleculares en las células se han desarrollado, pero la mayoría de estos métodos sufren de ser más bien indirecta y por lo tanto, no cuantitativa. Por el contrario, algunos de gama alta métodos cuantitativos se han introducido, que sin embargo son difíciles de extender a un alto rendimiento. Para combinar las capacidades de alto rendimiento con la posibilidad de extraer información cuantitativa, hemos desarrollado recientemente un nuevo concepto para la identificación de interacciones proteína-proteína (Schwarzenbacher<em&gt; Et al</em&gt;., 2008). Aquí se describe un protocolo detallado para el diseño y la construcción de este sistema que permite analizar las interacciones entre una proteína marcada con fluoróforo (&quot;presa&quot;) y una proteína de membrana (&quot;carnada&quot;)<em&gt; En-vivo</em&gt;. Las células se colocan sobre superficies micropatterned funcionalizados con anticuerpos contra el dominio de cebo exoplasmic. Presa de cebo se analizan las interacciones a través de la redistribución de la presa fluorescentes. El método se caracteriza por una alta sensibilidad al nivel de moléculas individuales, la capacidad de detectar las interacciones débiles, y la capacidad de alto rendimiento, por lo que es aplicable como herramienta de detección.

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

Este video muestra los experimentos con el posterior análisis de las interacciones proteína-proteína mediante el uso de micro-modelado de superficies. El enfoque ofrece la posibilidad de detectar las interacciones de proteínas en las células vivas y combina una alta capacidad de rendimiento con la posibilidad de extraer información cuantitativa.

En vivo-La detección de interacciones proteína-proteína en la Micro-patrón superficies

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 vistas.  Johannes Kepler Universitat Linz. Este video muestra los experimentos con el posterior análisis de las interacciones proteína-proteína mediante el uso de micro-modelado de superficies. El enfoque ofrece la posibilidad de detectar las interacciones de proteínas en las células vivas y combina una alta capacidad de rendimiento con la posibilidad de extraer información cuantitativa.

Video: 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-ubiquitina levadura membrana base de dos híbridos (MITO) del sistema: Una poderosa herramienta para identificar interacciones proteína-proteína

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

Investigación

Biología

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.

Imágenes de interacciones proteína-proteína 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.

En la cuantificación in vivo de interacciones de proteínas G del receptor acoplado con espectralmente resuelve de dos fotones de Microscopía

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.

El Protocolo para la identificación de las interacciones proteína-proteína base de<sup&gt; 15</sup&gt; N metabólico Etiquetado, inmunoprecipitación, cuantitativa Espectrometría de Masas y Modulación Affinity

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.

Interacciones proteína-proteína se visualizó por fluorescencia Bimolecular Complementación en protoplastos de tabaco y hojas

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.

La investigación de las interacciones proteína-proteína en las células vivas utilizando la transferencia de energía de resonancia de bioluminiscencia

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.

Sondeo de alta densidad microarrays de proteínas funcionales para detectar interacciones proteína-proteína

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.

El desarrollo de inhibidores de interacciones proteína-proteína a través REEMPLAZAR: Aplicación al Diseño y Desarrollo para no ATP inhibidores de CDK Competitiva

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.

Ensayo de inmunoprecipitación de la cromatina para la Identificación de Arabidopsis proteína-DNA<em&gt; En 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.

Estudio de las interacciones proteína-proteína en la autofagia investigación

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