Gostaríamos de agradecer o Dr. Scott Henderson para ajudar com microscopia confocal. Esta pesquisa foi suportada por concessões do National Institutes of Health 1RO1CA127501 de WAB, bem como o financiamento de projectos-piloto a partir do Centro de Câncer Massey e Faculdade de Medicina (VCU) para Microscopia WAB foi realizada na VCU-Dept. Facilidade de Neurobiologia e Anatomia Microscopia, apoiado, em parte, com financiamento do Centro de NIHNINDS núcleo conceder 5P30NS047463.

O protocolo consiste em três etapas principais. A clonagem, primeira etapa do seu gene de interesse em um vetor de expressão de mamífero amino-terminais para as versões monoméricas de PCP e / ou YFP só será discutido brevemente. A segunda e terceira etapas; transfecção de DNA vetor em EA.hy926 células endoteliais, e de imagem confocal com FRET, são descritos com mais profundidade a seguir. 1. Construção de PCP e YFP Receptores Quimérico Receptores de interesse devem ser clonado amino-terminal para monomérica versões aprimoradas do PCP e YFP. Determinação da FRET é empírica e é extremamente dependente do comprimento de vinculador entre os transmembrana abrangendo região e início do fluoróforo 1. Por conseguinte, várias diferentes comprimentos de linker deve ser explorado para cada par receptor novo sob investigação. <ol><li> No pcDNA3.1 R higrómetro ou vetor contendo R neo-C ou monomérico YFP (este vetor pode ser obtido em nosso laboratório), clone seu receptor de interesse para os sites NheI / EcoRI. * Nota-Se necessário, NheI é compatível várias outras enzimas incluindo SPEI e XbaI. </li><li> Transformar a sua reação em uma ligadura recA endA1 apropriado linha de células competentes (que usam rotineiramente Mach1). Colônias de tela para a presença de inserção seguida por seqüenciamento de DNA para verificar a ausência de mutações. </li><li> Após a obtenção de conformação de clonagem molecular de sucesso, prepare-transfecção grade DNA vetor em água esterilizada ou TE usando Qiagen, ou similar kits, DNA. Avaliar a concentração e pureza usando espectroscopia UV. Rendimentos típicos de DNA deve ser na faixa de 100-200 mg, que é rotineiramente diluída para um estoque de trabalho de 1 mg / mL. </li></ol> 2. Transfecção de células EA.hy 926 <ol><li> Dividir EA.hy 926 células 4, cultivadas em DMEM completo-(DMEM +10% soro fetal bovino + Pen. / Strep.) Em Mattek lamela (n º 0) 35 mm com dois pratos médio mL. Você pode obter conservadora ~ 15 pratos (~ 90% confluentes no dia seguinte) de um prato de 15 centímetros confluentes. Prepare um prato para avaliar a expressão de cada receptor individual, bem como um prato para cada par receptor. </li><li> Permitir que as células para incubar durante a noite em atmosfera de 5% CO 2 a 37 ° C. No dia seguinte, as culturas devem ser confluentes ~ 70-90%. </li><li> No dia da transfecção, preparar os complexos nucleolipid pela mistura de 2 mg de DNA quiméricos receptor vector (2 mL) com 8 mL de Fugene6 em 200 mL de pré-aquecido OptiMEM. Para pares receptor, transfecção com 1 mg de cada receptor, para um total de 2 mg (Veja também a discussão abaixo). </li><li> Misture delicadamente por inversão. </li><li> Continuam a incubar a temperatura ambiente por 30 minutos. </li><li> Durante o período de incubação, preparar as células para transfecção. Aspirar off mídia fresco e lavar cada placa com cuidado e brevemente com PBS quente antes da adição de 2 mL pré-aquecido complete-DMEM sem antibióticos (DMEM + FBS). É importante evitar a presença de penicilina e estreptomicina, como eles são consideravelmente mais tóxico para as células durante o tratamento com Fugene6. </li><li> Adicionar a queda complexo sábio células e incubar por um adicional de 20-24 horas a 37 ° C em uma atmosfera de 5% CO 2. </li><li> Após 24 horas, aspirar cuidadosamente os meios de comunicação das células e pipeta fresco complete-DMEM com antibióticos. Após transfecção, viabilidade celular deverá permanecer relativamente alta, ou seja, pouco ou nenhum células de flutuação devem ser observadas. </li><li> Expressão do gene é tipicamente máxima após um adicional de 48-60 horas após a transfecção. No entanto, imaging é normalmente possível em tão curto quanto 24 horas. Experimentos o tempo do curso para a expressão do gene ideal deve ser realizada. </li></ol> 3. Imagem confocal e FRET Análise Ao vivo imagens de células é realizada em um TCS-SP2 AOBS Leica microscópio laser confocal equipados com diodo azul (405 nm), Argon (458, 476, 488, 514 nm). HeNe verde (543 nm), laranja HeNe (594 nm) e vermelho HeNe (633 nm) lasers, um HCX PI Apo 63x/1.3 nd lente objetiva de imersão de glicerina, uma motorizada XY palco (Märzhäuser), e um ambiente controlado ( temperatura, umidade e CO 2) estágio incubadora (Pecon). Controles experimentais são fundamentais para eliminar fluoróforo cross-talk entre os canais de emissão, bem como para avaliar as questões de expressão e não-específica multimerization fluoróforo. Como tal, transfections fluoróforo única são utilizados para a criação de cada sessão de imagem. Negativos FRET controles (usando conhecida receptores não interagem) terá de ser realizada, a fim de determinar o fundo FRET que ocorre devido à expressão mais. <ol><li> Para eliminar a interferência entre os canais de emissão de PCP e YFP, coloque o controle de expressão PCP na incubadora palco. Usando luz branca, ajustar o Z-prato para se concentrar empara as células. </li><li> Definir as configurações de janela de SP para a detecção de emissão de PCP para 465-505 nm e 525-600 nm para YFP. </li><li> Enquanto monitora dois canais de emissão, excitar o PCP em 458 nm. Usando o AOTF, defina a potência do laser para eliminar sangrar apreciável ao longo do PCP para o canal de emissão YFP. Salvar esta configuração. </li><li> Realizar o mesmo controle para YFP crosstalk (usando o controle de expressão YFP) no canal PCP, ajustando a força de excitação a 514 nm para células que expressam somente YFP. Salvar esta configuração. </li><li> Células próximo lugar co-expressando PCP e YFP na incubadora palco. Utilizando a definição de seqüência para o software Leica confocal, localizar células que expressam níveis semelhantes de PCP e YFP com localização adequada da membrana. </li><li> Usando o software Leica aceitador foto-branqueamento programa, definir as configurações de doador e receptor para o PCP salvos e configurações YFP, respeitável. </li><li> Zoom para a célula, destaque um ROI (região de interesse) em que a foto-destruição de YFP deve ocorrer na membrana celular e iniciar o programa. </li><li> Para a foto-destruição do aceitante, ajustar o AOTF a 100% a 514 nm e permitir que o clareamento a laser para continuar até 70% da emissão YFP foi esgotado. Para acelerar o branqueamento, ROIs escolhidos são tipicamente no eixo de varredura do laser (eixo X). </li><li> Durante foto-branqueamento, monitorar o movimento celular e rejeitar medidas obtidas com o movimento apreciável no plano XY, uma vez que estas medidas podem relatório falso FRET eficiências. </li><li> Imagens pré e pós-bleach são registrados tanto para doadores (PCP) e receptor (YFP) e FRET eficiência é calculado como: FRET Eff = post (D pós-D pre) / D para todos os pós-D&gt; D, onde D pré pre e D pós é a intensidade dos doadores antes e depois fotobranqueamento respectivamente. </li><li> JMP Software (SAS) é usado para determinar a extensão da variabilidade experimental. </li></ol> 4. Resultados representante Eficiências de transfecção são geralmente entre 30 a 40%. Apesar de descobertas anteriores por outros, não temos visto que a transfecção e expressão de um vetor de favores que do outro. De fato, é freqüente a observação expressão exclusiva de um fluoróforo quimera ou outro. Típico FRET eficiências variam entre os sistemas de receptor. Para o sistema receptor Tie, valores típicos são 20-28% para células epiteliais e 19-23% em células endoteliais. Dos controlos negativos, as eficiências típicas estão abaixo de 2-3%. A extensão da variabilidade irá diminuir consideravelmente com a experiência. <img src="/files/ftp_upload/2149/2149fig1.jpg" alt="Figura 1" /> Figura 1. Imagens representativas de 926 células transfectadas EA.hy. Uma imagem) DIC de EA.hy monocamada de células 926. B) imagem de fluorescência de células exibido em (A). C) Sobreposição de (A) e (B) demonstrando a eficiência de transfecção ~ 20-30%. <img src="/files/ftp_upload/2149/2149fig2.jpg" alt="Figura 2" /> Figura 2. Aceitador representante foto-branqueamento análise de FRET ocorrendo entre PCP e YFP em uma célula EA.hy926. O PCP de emissão do canal (painéis superiores) e YFP emissão canal (bottom dois painéis) foram monitorados em separado, antes e pós fotobranqueamento aceitador. Fotobranqueamento experimentos foram restritas a, e FRET valores calculados a partir, a região dentro da caixa verde. FRET eficiência é apresentado como uma gama absoluta de alta (red-1.0) para baixo (roxo-0.0) em um overlay ampliado de um CFP / YFP fusão de imagens para fins de orientação apenas.

<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>

Não há conflitos de interesse declarados.

Há várias etapas que são críticos para o sucesso. O mais proeminente entre eles é os níveis relativos de expressão entre os dois receptores quiméricos. Para contornar esse problema, pode-se fazer linhas celulares estáveis ​​expressando ambas as proteínas de interesse, ou identificar média ideal de DNA de vetor para permitir a expressão equivalente. Da mesma forma, devido à não-uniforme transfecção através de um prato, os níveis de proteína raramente será equivalente entre as células. Portanto, a atenção deve ser dada para distinguir &#39;alta&#39; expressors de &#39;low&#39;. Aqueles que os níveis &quot;médio&quot; exibição de proteínas normalmente produzem confiável e reprodutível FRET eficiências. Um método nosso laboratório tem desenvolvido para aliviar este problema é o uso de adeno e lenti-vírus para o &#39;mesmo&#39; a expressão do gene. Além disso, um método alternativo para foto-clareamento aceitador para determinar FRET eficiência é a emissão sensibilizados. Embora, apesar do potencial de emissão sensibilizados para monitorar as células individuais em tempo real, temos encontrado foto-branqueamento aceitador a ser mais sensíveis e mais confiável. Finalmente, usando FRET para monitorar interações proteína pode ser um desafio, e requer cuidadosa seleção de comprimentos de vinculador para os receptores de membrana ligados. Além disso, a adição de C / YFP muitas vezes influencia os níveis de proteína expressão e de resultados na agregação no retículo endoplasmático e complexo de Golgi. No entanto, para interações transitório ou instável, FRET é a metodologia ideal para utilizar.

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

Interações proteína-proteína são uma marca registrada de todos os processos essenciais celular. No entanto, muitas dessas interações são transitórias, ou energeticamente fracos, impedindo a sua identificação e análise através de métodos bioquímicos tradicionais, tais como co-imunoprecipitação. A este respeito, as proteínas geneticamente codificável fluorescente (GFP, RFP, etc) e seus associados se sobrepõem espectro de fluorescência revolucionaram a nossa capacidade de monitorar interações fracas in vivo utilizando Förster transferência de energia de ressonância (FRET) 1-3. Aqui, detalhe que o uso de um ensaio de proximidade FRET baseado receptor para o monitoramento de interações receptor na superfície das células endoteliais.

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

Imaging Protein-protein Interactions in vivo

Este protocolo descreve como imagem de interações proteína-proteína através de um ensaio de proximidade FRET-based.

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, ...

imaging-protein-protein-interactions-in-vivo

Research

JoVE Journal

Biology

Imaging Protein-protein Interactions in vivo

21.3K visualizações.  Virginia Commonwealth University. Este protocolo descreve como imagem de interações proteína-proteína através de um ensaio de proximidade FRET-based.

Vídeo: 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 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

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

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

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

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

In vivo 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 ...

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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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. ...

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.

Avançadas técnicas de microscopia Confocal para estudar interações da proteína-proteína e cinética de lesões do ADN

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

Imagem interações proteína-proteína In vivo

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