Deseamos agradecer a la Dra. Scott Henderson en busca de ayuda con la microscopía confocal. Esta investigación fue financiada por becas de los Institutos Nacionales de Salud 1RO1CA127501 de WAB, así como la financiación del proyecto piloto del Centro de Cáncer de Massey y la Facultad de Medicina (UCV) de Microscopía WAB se llevó a cabo en la UCV-Dept. de Neurobiología y Anatomía Servicio de Microscopía, apoyada, en parte, con fondos del Centro de NIHNINDS núcleo conceder 5P30NS047463.

El protocolo consta de tres pasos principales. La primera clonación, los pasos de su gen de interés en un vector de expresión de mamíferos amino-terminal de las versiones de monómero de la PPC y / o YFP sólo se discutirán brevemente. Los pasos segundo y tercero, la transfección de ADN vector en EA.hy926 las células endoteliales, y la imagen confocal con FRET, se describen con más detalle a continuación. 1. La construcción de los receptores PPC y YFP quiméricos Receptores de interés debe ser clonado amino-terminal de monómero versiones mejoradas de la PPC y YFP. Determinación de FRET es empírica y depende fundamentalmente de la longitud del enlazador entre la transmembrana que abarca la región y el inicio de la fluoróforo 1. Por lo tanto, diferentes longitudes de enlazador debe ser explorado para cada par de receptores de nuevo bajo investigación. <ol><li> En la R pcDNA3.1 Hygro o neo vector R que contiene monómero C-o YFP (este vector se puede obtener de nuestro laboratorio), el clon de su receptor de interés en los sitios NheI / EcoRI. * Nota: Si es necesario, NheI es compatible varias enzimas incluyendo otros SpeI y XbaI. </li><li> Transforme su reacción de ligación en una adecuada línea de células endA1 Reca competente (nosotros usamos Mach1). Colonias de detectar la presencia de Insert seguida de la secuenciación del ADN para verificar la ausencia de las mutaciones. </li><li> Tras la obtención de la conformación de la clonación molecular éxito, preparar la transfección de grado vector de ADN en agua esterilizada o TE utilizando Qiagen, o kits similares, el ADN. Evaluar la concentración y la pureza utilizando espectrometría UV. Los rendimientos típicos de ADN debe estar en el rango de 100-200 mg, que es habitualmente diluida a una solución de trabajo de 1 mg / mL. </li></ol> 2. La transfección de células EA.hy 926 <ol><li> Dividir EA.hy 926 células 4, que se cultiva en total-DMEM (DMEM +10% de suero fetal bovino + pen. / Strep.) En MatTek cubreobjetos (N º 0) 35 mm platos con 2 ml de medio. Usted puede obtener de forma conservadora ~ 15 platos (~ 90% confluentes al día siguiente) de un plato de 15 cm confluente. Preparar un plato para evaluar la expresión de cada receptor individual, así como un plato para cada par de los receptores. </li><li> Permitir que las células se incuban durante la noche en el 5% atmósfera de CO 2 a 37 ° C. Al día siguiente, las culturas deben ser confluentes ~ 70-90%. </li><li> En el día de la transfección, preparar los complejos nucleolipid mediante la mezcla de 2 g de ADN de los receptores de vectores quiméricos (2 l) con 8 L de Fugene6 en 200 l de pre-calentado OptiMEM. Para los pares de receptores, transfectar con 1 g de cada receptor, para un total de 2 mg (ver también más adelante). </li><li> Mezclar suavemente por inversión. </li><li> Continuar a incubar a temperatura ambiente durante 30 minutos. </li><li> Durante el período de incubación, preparar las células para la transfección. Aspirar fuera de los medios de comunicación fresca y lave cada plato suave y brevemente con PBS caliente antes de la adición de 2 ml precalentado completa-DMEM sin antibióticos (DMEM + FBS). Es importante evitar la presencia de penicilina y estreptomicina, ya que son mucho más tóxicos para las células durante el tratamiento con Fugene6. </li><li> Añadir la caída complejo sabio a las células e incubar durante otros 20 a 24 horas a 37 ° C en un 5% de CO2 la atmósfera. </li><li> Después de 24 horas, con cuidado aspirar los medios de comunicación de las células y pipeta nueva completa-DMEM con antibióticos. Después de la transfección, la viabilidad celular se mantienen relativamente altos, es decir, poco o nada de las células flotantes deben ser observados. </li><li> La expresión de genes es por lo general después de un máximo adicional de 48-60 horas después de la transfección. Sin embargo, la imagen suele ser factible en tan corto como 24 horas. Experimentos de tiempo de los cursos de la expresión génica óptima debe ser realizada. </li></ol> 3. Imagen confocal y análisis de FRET En vivo de imágenes de células se realiza en una Leica TCS-SP2 microscopio láser confocal de barrido AOBS equipada con diodo azul (405 nm), argón (458, 476, 488, 514 nm). verde HeNe (543 nm), naranja HeNe (594 nm) y rojo HeNe (633 nm), un HCX PI Apo 63x/1.3 na glicerina lente objetivo de inmersión, una motorizada XY etapa (Märzhäuser), y un control del medio ambiente ( temperatura, humedad y CO 2) incubadora (PECON). Controles experimentales son fundamentales para eliminar fluoróforo diafonía entre los canales de emisión, así como para evaluar los problemas de expresión y no específicos multimerización fluoróforo. Como tal, transfecciones único fluoróforo se utilizan para la creación de cada sesión de imagen. FRET controles negativos (no interactúan con los receptores conocidos) tendrá que llevarse a cabo, con el fin de determinar el fondo, temen que se produce debido a la sobre-expresión. <ol><li> Para eliminar la diafonía entre los canales de emisión de la PPC y YFP, coloque el control de la expresión política pesquera común en la incubadora etapa. Con luz blanca, ajustar la placa Z-para centrarse ena las células. </li><li> Establecer la configuración de SP ventana para la detección de emisión de 465-505 nm para PPC y YFP a 525-600 nm. </li><li> Durante la supervisión de los dos canales de emisión, excitar la PPC a 458 nm. Utilizando el AOTF, ajustar la potencia de láser para eliminar el sangrado apreciable sobre la PPC en el canal de emisión de YFP. Guardar esta configuración. </li><li> Realizar el mismo control para YFP crosstalk (usando el control de la expresión YFP) en el canal de la PPC mediante el ajuste de la potencia de excitación a 514 nm de las células que expresan sólo YFP. Guardar esta configuración. </li><li> Celdas contiguas a cabo co-expresando PPC y YFP en la incubadora etapa. Uso de la secuencia de configuración para el software de Leica confocal, localizar las células que expresan niveles similares de la PPC y YFP con la localización de la membrana apropiada. </li><li> Utilizando el software Leica aceptor foto-blanqueamiento del programa, establecer la configuración de donante y el receptor de la PPC guardados y las configuraciones YFP, respetable. </li><li> Zoom de las células, resalte un ROI (región de interés) en el que la foto-destrucción de YFP se va a producir en la membrana celular y comenzar el programa. </li><li> Para la foto-destrucción del aceptante, ajustar el AOTF al 100% a 514 nm y permitir que el blanqueamiento con láser para continuar hasta el 70% de la emisión YFP se ha agotado. Para acelerar la decoloración, rendimiento de la inversión son por lo general elegido en el eje de la trama láser (eje X). </li><li> En la foto-blanqueamiento, vigilar el movimiento celular y rechazar las mediciones obtenidas con el movimiento apreciable en el plano XY, ya que estas medidas pueden reportar falsos FRET eficiencia. </li><li> Imágenes pre-y post-cloro-se registran para el donante (PPC) y aceptor (YFP) y FRET eficiencia se calcula como: FRET Ef = (D post-D pre) / D después de todos los mensajes D&gt; D donde D pre pre y D no está en la intensidad de los donantes antes y después de photobleaching respectivamente. </li><li> El software JMP (SAS) se utiliza para determinar el grado de variabilidad experimental. </li></ol> 4. Resultados representante Eficiencia de transfección son por lo general entre 30 a 40%. A pesar de los resultados anteriores por otros, no hemos visto que la transfección y la expresión de un vector a favor de la otra. De hecho, es frecuente observar la expresión exclusiva de un fluoróforo quimera o la otra. FRET eficiencia típica varían entre los sistemas de receptor. Para que el sistema receptor Tie, valores típicos son 20-28% de las células epiteliales y 19-23% en las células endoteliales. Para los controles negativos, las eficiencias típicas están por debajo de 2.3%. El grado de variabilidad se reducirá considerablemente con la experiencia. <img src="/files/ftp_upload/2149/2149fig1.jpg" alt="Figura 1" /> Figura 1. Imágenes representativas de las células transfectadas EA.hy 926. A la imagen) DIC de monocapa celular EA.hy 926. B) Imagen de fluorescencia de células se muestra en (A). C) Superposición de (A) y (B) que demuestra la eficiencia ~ 20-30% de transfección. <img src="/files/ftp_upload/2149/2149fig2.jpg" alt="Figura 2" /> Figura 2. Aceptor representante foto-blanqueamiento análisis de FRET se produce entre PPC y YFP en una celda de EA.hy926. El PPC de emisión del canal (paneles superiores) y YFP emisión de canales (dos paneles inferiores) se efectuará por separado antes y después de photobleaching aceptor. Photobleaching experimentos se limitaron a, y FRET valores calculados de la región dentro de la caja verde. FRET eficiencia se muestra como un rango absoluto de alta (rojo-1.0) a baja (púrpura-0.0) en una plantilla ampliada de un PPC / YFP fusión de imágenes con fines de orientación únicamente.

<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>

Hay varios pasos que son críticos para el éxito. El más destacado entre ellos es el nivel relativo de expresión entre los dos receptores quiméricos. Para evitar este problema, uno puede hacer que las líneas celulares estables que expresan tanto las proteínas de interés, o identificar proporciones óptimas de vector de ADN para permitir la expresión equivalente. Del mismo modo, debido a la falta de uniformidad de la transfección a través de una antena, los niveles de proteína rara vez será equivalente entre las células. Por lo tanto, se debe prestar atención para distinguir &quot;alto&quot; expresores de &quot;baja&quot;. Los que los niveles &quot;promedio&quot; de visualización de proteínas por lo general el rendimiento fiable y reproducible FRET eficiencia. Uno de los métodos nuestro laboratorio ha llevado a cabo para aliviar este problema es el uso de adenovirus y los virus lenti-a la expresión génica &quot;, incluso&quot;. Además, un método alternativo para la foto-receptor de blanqueo para la determinación de la eficiencia de FRET es la emisión sensibilizada. Aunque, a pesar del potencial de la emisión de sensibilización para controlar las células individuales en tiempo real, hemos encontrado aceptor foto-blanqueamiento a ser más sensibles y más confiable. Por último, utilizando FRET para controlar las interacciones de proteínas puede ser difícil, y requiere de una cuidadosa selección de las longitudes enlazador para los receptores unidos a la membrana. Además, la adición de C / YFP a menudo una gran influencia en los niveles de expresión de la proteína y los resultados de la agregación en el retículo endoplasmático y el aparato de Golgi. Sin embargo, para las interacciones transitorias o inestable, FRET es la metodología ideal para utilizar.

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<td>DMEM</td>
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imaging protein-protein interactions in vivo

Interacciones proteína-proteína son un sello distintivo de todos los procesos celulares esenciales. Sin embargo, muchas de estas interacciones son transitorios, o débil energía, la prevención de su identificación y el análisis a través de tradicionales métodos bioquímicos, como co-inmunoprecipitación. En este sentido, las proteínas fluorescentes genéticamente codificable (GFP, RFP, etc) y sus asociados se superponen espectro de fluorescencia han revolucionado nuestra capacidad de monitorear las interacciones débiles en vivo con la transferencia de energía por resonancia de Förster (FRET) 1-3. Aquí, le detallamos el uso de un ensayo de proximidad FRET para el monitoreo del receptor de los receptores de las interacciones en la superficie de las células endoteliales.

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

Imaging Protein-protein Interactions in vivo

Este protocolo describe cómo la imagen de interacciones proteína-proteína mediante un ensayo basado en la proximidad FRET.

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, ...

imaging-protein-protein-interactions-in-vivo

Research

JoVE Journal

Biology

Imaging Protein-protein Interactions in vivo

21.2K vistas.  Virginia Commonwealth University. Este protocolo describe cómo la imagen de interacciones proteína-proteína mediante un ensayo basado en la proximidad FRET.

Video: 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-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

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.

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 ...

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. ...

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.

Avanzadas técnicas de Microscopía Confocal para estudiar las interacciones proteína-proteína y cinética en las lesiones del ADN

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.

Complementación de luciferase imagen ensayo en hojas de Nicotiana benthamiana para determinar transitoriamente proteínas interacción dinámica

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