Gostaríamos de agradecer Quentina Beatty, da Universidade de Linz, na Áustria, por sua ajuda tipo, Katharina Strub, Universidade de Genebra, na Suíça, para a construção hCD71-GFP, e Legler Daniel, Universidade de Konstanz, na Suíça, para o GPI-GFP DAF-construir. Este trabalho foi financiado pelo Fundo de Ciência Austríaco (FWF; projeto Y250-B03) eo projeto GEN-AU do Ministério Federal Austríaco da Ciência e Investigação.

Impressão microcontact: <ol><li> Cortar campo de micropattern do selo PDMS utilizando um bisturi </li><li> Lave o campo que contém o micropattern através de lavagem com etanol (100%) e água destilada. </li><li> Seque o selo PDMS com nitrogênio ou gás argônio. </li><li> Pipetar 50 ul BSA-Cy5 solução de trabalho (0,67 mg / ml) para o selo de PDMS para que o campo micropattern inteiro é coberto com a solução. Incubar por 30 min em temperatura ambiente. </li><li> Lavar o campo micropattern através de lavagem com Tampão fosfato salino (PBS) e água destilada. </li><li> Seque o PDMS com nitrogênio ou gás argônio. </li><li> Coloque o selo PDMS sob seu próprio peso sobre o centro de uma lamela epoxy revestido e incubar por 30 min à temperatura ambiente numa placa de Petri. </li><li> Rotular a posição do carimbo PDMS na parte traseira da lamela e tira o selo do slide com uma pinça. </li><li> Vara uma câmara de hibridação Seguro Selo sobre o campo microcontact-impresso na lamela. O rótulo de ajuda para localizar o centro do campo. </li><li> Pipetar 60 mL de solução de trabalho estreptavidina (50 mcg / ml) para a câmara de reação e incubar a amostra por 60 min em temperatura ambiente. </li><li> Lavar a amostra com 1 ml de PBS, adicionando o buffer em uma porta da câmara e removê-lo simultaneamente a segunda porta com uma bomba. </li><li> Pipetar 60 mL de solução de trabalho biotinilado anticorpo (10 mg / ml em PBS suplementado com 0,1% Tween 20; &quot;PBST&quot;) para a câmara de reação e incubar por 60 min em temperatura ambiente. </li><li> Lavar a amostra com 1 ml PBST e, posteriormente, 1 ml de PBS, adicionando o buffer em uma porta da câmara e removê-lo novamente na segunda porta com uma bomba. </li><li> Armazenar as superfícies micropatterned em PBS no escuro à temperatura ambiente até que as células estão prontos para a semeadura. </li></ol> Incubação de células na superfície micropatterned: <ol><li> Cultivar células aderentes à confluência de 50% em um 3 centímetros placa de cultura de tecidos. </li><li> Separar células que expressam a isca presa e proteínas de interesse, com solução de ácido etilenodiaminotetracético (EDTA) e centrifugar 5 min a 1.000 rpm. </li><li> Desprezar o sobrenadante e dissolver o celular em meio de crescimento de acordo. </li><li> Centrifugar 5 min a 1.000 rpm. </li><li> Desprezar o sobrenadante e dissolver o pellet celular em meio de crescimento adequado. </li><li> Trocar o PBS da câmara de reação na lamela micropatterned em 60 mL da suspensão de células. </li><li> Criar uma câmara úmida por imersão uma almofada em água destilada estéril e colocá-lo na placa de Petri para evitar que a amostra de funcionamento a seco. </li><li> Incubar as células ao longo da noite a 37 ° C em uma atmosfera de 5% CO 2 na superfície micropatterned. </li><li> Antes de analisar as células no microscópio, a troca do meio de solução de Hank s sal tamponado incluindo Ca 2 + e Mg 2 +. </li></ol> Microscopia: <ol><li> A lamínula é colocada sobre um suporte adequado ea morfologia das células é verificada em branco-luz. </li><li> A qualidade da BSA-Cy5 é marcada por padrões de excitação em 647 nm. </li><li> Micropadrões de proteína presa fluorescente (GFP ou YFP) são registrados a 488 ou 514 nm sob Total Internal Reflection excitação (TIR). </li></ol> Resultados representativos: Se não houver interação entre as proteínas-alvo em células cultivadas em uma superfície micropatterned, a presa vai seguir a redistribuição isca. O micropattern resultante pode ser visualizado pelo rótulo de fluorescência da proteína presa. Importante é que o contraste obtido fornece uma medida direta da força de interação (ver Figura 1). Portanto, uma simples avaliação da interação de duas proteínas torna-se possível - sem a necessidade de continuar a processar os dados medidos primária. <img src="/files/ftp_upload/1969/1969fig1.jpg" alt="Figura 1" /> Figura 1. Rearranjo da isca na membrana plasmática de células vivas em dosagens diferentes de interação. Imagens TIR de células transfectadas com T24 (a) CD71-GFP em anticorpos CD71, (b) CD4 e YFP citosólica em CD4-anticorpo e (c) GPI-GFP-DAF em anticorpos CD59 microbiochips são mostrados. Os dados são característicos para a forte (a), não (b) ou interação fraca (c). (A) é reproduzida a partir de (Weghuber et al., No prelo), (b) a partir 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>

O vídeo contém imagens que foram fornecidos pela Olympus, a partir da homepage do Laboratório de Pesquisa Visualization, da Universidade Brown ou reproduzidas a partir de (Kim et al., 2003).

O vídeo que acompanha demonstra um método para a detecção de interações proteína-proteína na membrana plasmática de células vivas (Schwarzenbacher et al, 2008;. Brameshuber et al, 2009;.. Weghuber et al, in press). Em princípio, qualquer plataforma de microscopia TIRF baseada pode ser usado como sistema de leitura. Somente quando de alta sensibilidade é desejada (por exemplo, para a detecção de moléculas simples), microscópios avançados serão necessários. Para alcançar melhores resultados os seguintes pontos críticos durante o processo de preparação requerem uma atenção especial: <ol class="lroman"><li> Solução de loja Cy5 estoque em condições escuras, para evitar o branqueamento do fluoróforo e preparar o BSA-Cy5 solução de trabalho imediatamente antes da impressão microcontact. </li><li> Tenha cuidado para não arranhar a micropattern no PDMS com a ponta da pipeta e incube a amostra em condições escuras, para evitar o branqueamento do fluoróforo. </li><li> Coloque a lamínula sobre uma almofada esterilizada para evitar que grudem da lâmina de vidro para a placa de Petri. Uma vez que o carimbo de PDMS se adere à lâmina de vidro, não movê-lo. </li><li> Evite bolhas de ar na câmara de reação e incubar a amostra em condições escuras, para evitar o branqueamento do fluoróforo. </li><li> Não deixe que a amostra seca por mais de 2 minutos para evitar a formação de cristais de sal. </li><li> Para o desprendimento das células não utilizar de tripsina, uma vez que irá unir o domínio extracelular de proteínas da membrana, até agora manifestado. Evitando tripsina será útil para proteínas com baixo índice de rotatividade para formar micropadrões imediatamente após fixação das células na superfície. </li><li> Controlar o número de células incubadas em um microscópio e certifique-se que as células não alcançam mais do que 30-50% confluência. Certifique-se que as células excessivo são removidos imediatamente, devido à fixação rápida de células na superfície revestidos com anticorpo. </li><li> Slides apenas com alta qualidade BSA-Cy5 padrões podem ser usados ​​para análise posterior. </li><li> Expressão estável da proteína fluorescente presa é preferível devido à maior número de células que podem ser analisados ​​por varredura. </li><li> Ajuste TIRF adequada é indispensável para visualizar padrões devido ao fundo citosólica. </li></ol> A técnica de micro-padronização oferece diversas possibilidades para a análise das interações proteína-proteína. Em primeiro lugar, juntamente com a quantificação do local, espacialmente resolvidos interações proteína-proteína também a detecção de interações fracas ou indirecta é possível sem a desvantagem de dar um elevado número de falsos positivos ou negativos. Em segundo lugar, permite ao pesquisador analisar interações proteína-proteína na membrana plasmática de células vivas, que é difícil de conseguir por abordagens bioquímicas como a tela de 2 híbridos. Em terceiro lugar a abordagem permite a detecção de isca presa interações que são modulados por mudanças ambientais, como a temperatura, a presença de diferentes proteínas ou outras moléculas ou modificações pós-translacionais. Assim, o ensaio permite a moduladores de triagem de um par de interação dada no contexto de células vivas. Além disso, ao reduzir a densidade de superfície do ligante captura ou o uso de ligantes monovalentes, a análise do estado de repouso se torna possível. Finalmente, se adequado plataformas de digitalização são utilizados, o número de células analisadas é alta o suficiente para atender as demandas de alto rendimento das empresas farmacêuticas para a triagem de drogas (Ramm, 2005).

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

Desvendar a rede de interação de moléculas<em&gt; In-vivo</em&gt; É a chave para a compreensão dos mecanismos que regulam a função celular e metabolismo. Uma multidão de opções metodológicas para tratar as interações moleculares nas células têm sido desenvolvidas, mas a maioria destes métodos sofrem de ser bastante indireta e, portanto, dificilmente quantitativa. Pelo contrário, alguns high-end abordagens quantitativas foram introduzidas, que no entanto são difíceis de estender a alta taxa de transferência. Para combinar as capacidades de alto rendimento com a possibilidade de extrair informações quantitativas, que recentemente desenvolveu um novo conceito para a identificação de interações proteína-proteína (Schwarzenbacher<em&gt; Et al</em&gt;., 2008). Aqui, nós descrevemos um protocolo detalhado para a concepção e construção deste sistema que permite analisar as interações entre um fluoróforo marcado com proteína (&quot;presa&quot;) e uma proteína de membrana (&quot;isca&quot;)<em&gt; In-vivo</em&gt;. Células são semeadas em superfícies micropatterned funcionalizados com anticorpos contra o domínio isca exoplasmic. Isca presa interações são analisadas através da redistribuição da presa fluorescente. O método é caracterizado pela alta sensibilidade para o nível de moléculas individuais, a capacidade de detectar interações fracas, e capacidade de produção elevada, tornando-o aplicável como ferramenta de triagem.

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 vídeo mostra experimentos com posterior análise de interações proteína-proteína através da utilização de micro-padrão superfícies. A abordagem oferece a possibilidade de detectar as interações de proteínas em células vivas e combina alta capacidade de processamento com a possibilidade de extrair informações quantitativas.

In vivo Detecção de interações proteína-proteína em Micro-padrão Superfícies

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

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Research

JoVE Journal

Biology

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

10.7K visualizações.  Johannes Kepler Universitat Linz. Este vídeo mostra experimentos com posterior análise de interações proteína-proteína através da utilização de micro-padrão superfícies. A abordagem oferece a possibilidade de detectar as interações de proteínas em células vivas e combina alta capacidade de processamento com a possibilidade de extrair informações quantitativas.

Vídeo: 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 Yeast Membrana Baseado Two-Hybrid (MITO) do sistema: uma ferramenta poderosa para identificação de interações proteína-proteína

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

Biologia

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.

Imagem interações 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.

Em Quantificação in vivo de proteínas G Interações Receptor Acoplado usando espectral Resolvido Microscopia de dois fótons

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.

Um protocolo para a identificação de interações proteína-proteína Baseado<sup&gt; 15</sup&gt; Etiquetagem N Metabólica, imunoprecipitação, Quantitativa Espectrometria de Massa e Affinity Modulação

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.

Interações proteína-proteína visualizado por Bimolecular Fluorescência de Complementação em tabaco Protoplastos e folhas

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.

Investigando interações proteína-proteína em células vivas usando a transferência de Bioluminescência Resonance Energy

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.

Sondagem de alta densidade microarrays proteína funcional para detectar interacções 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.

Desenvolvimento de inibidores de interacções proteína-proteína através REPLACE: Aplicação para a concepção e desenvolvimento não-ATP inibidores 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.

Ensaio para imunoprecipitação da cromatina a identificação de Arabidopsis interações proteína-ADN<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.

Estudo das interações proteína-proteína em pesquisa de autofagia

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