Estamos muito gratos a Victor GJ Rodgers para o conselho valioso. Agradecemos a todos os membros do grupo de Liao de muito perto o trabalho colaborativo e para ajudar com este estudo. Este estudo foi financiado pelos Institutos Nacionais de Saúde (Grant AI076504 a JL).

1. Construções de plasmídeos <ol><li> Amplificar os quadros de leitura abertos de genes por PCR, e clonar os produtos de PCR em pCRII-TOPO vector. </li><li> Confirmar os produtos de sequenciação e clonagem do cDNA que codifica CyPet (pré-SUMO1)-YPet, CyPet-SUMO1, YPet e domínios catalíticos de SENP1 para o vector pET28 (b) com um etiqueta de hexa-histidina N-terminal. </li></ol> 2. Protein Expression and Purification <ol><li> Transformar células de Escherichia coli da estirpe BL21 (DE3) com pET28 (b) vectores que codificam CyPet (pré-SUMO1)-YPet, CyPet-SUMO1, YPet e os domínios catalíticos de SENP1. </li><li> Cultivadas as bactérias transformadas em meio 2xYT durante 3 horas a 37 ° C (com agitação a 250 rpm) para atingir uma densidade óptica de 0,5 a 600 nm. </li><li> Adicione 100 ^ M isopropil-β-D-tiogalactósido (IPTG) (concentração final) para induzir a expressão da proteína e agitar durante 16 horas a 25 ° C a 200 rpm. </li><li> Colheita das bactérias por centrifugção a 10.000 rpm a 4 ° C durante 5 min e ressuspender os em um tampão de 20 mM Tris-HCl (pH 7,4), NaCl 50 mM e 5 mM de imidazole. </li><li> Sonicar as células durante 10 minutos em intervalos de 5 s com um ajuste de potência de 25 W, utilizando MiSonics sonicador 4000 e recolhê-las por meio de centrifugação a 35.000 xg, a 4 ° C durante 30 min. </li><li> Defina-se a coluna com 500 ml de pérolas de Ni-NTA de uma cultura de L e transferir o sobrenadante para a coluna. </li><li> Lava-se a resina com tampão de 20 mM Tris-HCl (pH 7,4), NaCl 500 mM, imidazol 10 mM e duas vezes. </li><li> Proteína elui-se com tampão de 20 mM Tris-HCl (pH 7,4), NaCl 500 mM, e 500 mM de imidazole e diálise num tampão de 20 mM Tris-HCl (pH 7,4), NaCl 50 mM e 1 mM de ditiotreitol (DTT) a 4 ° C durante a noite. </li><li> Determinar as concentrações das proteínas purificadas pelo ensaio de Bradford. Método alternativo para a medição da concentração de proteína será SDS-PAGE electroforese em gel e, em seguida, coradas com azul de Coomassie seguido de imagiologiaquantitativo. </li></ol> 3. Quantitativa FRET Análise de Espectro <ol><li> A estratégia geral para o ensaio foi baseado em FRET de sinalização (Figura 1). O par FRET, CyPet e YPet, foi etiquetada com o N-e C-terminais, respectivamente, da pré-SUMO1. SENP1 cliva a proteína de fusão CyPet (pré-SUMO1) YPet-no local Gly-Gly, em SUMO1 do C-terminal e, deste modo, liberta a cauda SUMO com YPet. O sinal FRET é interrompido, resultando num aumento da emissão de CyPet e uma diminuição dramática das emissões de YPet no comprimento de onda de excitação CyPet. </li><li> Quando excitado por luz de comprimento de onda de 414 nm, a emissão total de fluorescência de CyPet-(pré-SUMO1)-YPet a 530 nm pode ser obtido a partir de três fontes: emissões absoluto FRET induzida YPet, a CyPet directo emissão e YPet directa de emissão (figura 2). </li></ol><img alt="Equação 1" src="/files/ftp_upload/4430/4430eq1.jpg" /> onde FL 530/414 &lt;/ Sub&gt; é o total de emissões de fluorescência a 530 nm quando excitado a 414 nm, FL FRET é o absoluto FRET sinal, FL CyPet (cont) é a emissão direta CyPet quando excitado a 414 nm, e FL YPet (cont) é o YPet dirigir emissão quando excitado a 414 nm. O índice de (cont) representa contribuição. <ol start="3"><li> A emissão directa de CyPet a 530 nm foi proporcional à sua emissão a 475 nm quando excitado a 414 nm, com uma proporção constante de α. CyPet-SUMO1 foi preparada em concentrações de 50, 100, 200, 500, e 750 nM e 1 mM , e as emissões de 475 e 530 nm foi medida após a excitação a 414 nm para determinar α (Figura 3-1). </li><li> A emissão directa de YPet a 530 nm sob excitação em 414 nm foi proporcional à sua emissão a 530 nm quando excitado a 475 nm, com uma proporção constante de β. YPet foi preparada em concentrações de 50, 100, 200, 500, 750 And 1000 nM, e a emissão a 530 nm foi medida em que as amostras foram excitados por comprimentos de onda de 414 e 475 nm para determinar β (Figura 3-2). </li><li> Quando CyPet (pré-SUMO1)-YPet foi digerido por SENP1, a clivagem libertado CyPet-SUMO1 ea cauda SUMO1 com YPet. Quando o composto foi excitada a 414 nm, a emissão de fluorescência a 530 nm (FL &#39;530/414) foi reduzida, mas ainda podem ser divididas em três partes como: </li></ol><img alt="Equação 2" src="/files/ftp_upload/4430/4430eq2.jpg" /> onde FL &#39;530/414 é a emissão total de fluorescência a 530 nm após a digestão, quando excitado a 414 nm, FL&#39; FRET é o restante sinal FRET absoluto, CyPet FL &#39;(475/414) é a emissão a 475 nm CyPet após a digestão quando excitada a 414 nm (a emissão CyPet aqui é a partir de duas partes: CyPet não digerido (pré-SUMO1)-YPet e digerido CyPet-SUMO1) e FL YPet(530/475) é a emissão YPet quando excitado a 475 nm, que é constante se CyPet (pré-SUMO1)-YPet é digerido ou não. <ol start="6"><li> Após a digestão por SENP1, o restante FRET emissão (FL &#39;FRET) é a seguinte: </li></ol><img alt="Equação 3" src="/files/ftp_upload/4430/4430eq3.jpg" /> em que C é a concentração total de CyPet (pré-SUMO1)-YPet e x é a concentração de digerido CyPet (pré-SUMO1)-YPet. <ol start="7"><li> Através da combinação de todos os elementos, a emissão de fluorescência detectada a 530 nm sob excitação de 414 nm (530 FL &#39;/ 414) é o seguinte: </li></ol><img alt="Equação 4" src="/files/ftp_upload/4430/4430eq4.jpg" /> 4. FRET baseada Protease Assay para Estudo Cinético Enzyme <ol><li> CyPet (pré-SUMO1)-YPet foi incubada com o domínio catalítico de SENP1 a 37 ° C num tampão de 20 mM Tris-HCl (pH 7,4), NaCl 50 mM, 0,1% (v /v) de Tween-20 e 1 mM de DTT a um volume total de 80 ml e transferidas para placas de 384 poços. </li><li> Ensaios foram realizados através da medição da emissão de fluorescência a 475 e 530 nm, após uma excitação a 414 nm num leitor de fluorescência de placas com múltiplas cavidades, durante 5 min com intervalos de 15 seg. </li><li> A velocidade da reacção (v) foi correlacionado com a alteração da quantidade de substrato (S), como: </li></ol><img alt="Equação 5" src="/files/ftp_upload/4430/4430eq5.jpg" /><ol start="4"><li> A concentração do produto aumentou exponencialmente a partir de 0, tal como [S] 0 (concentração de substrato original) quando t = 0: </li></ol><img alt="Equação 6" src="/files/ftp_upload/4430/4430eq6.jpg" /><ol start="5"><li> Quando t = 0, a velocidade inicial (V 0) é a seguinte: </li></ol><img alt="Equação 7" src="/files/ftp_upload/4430/4430eq7.jpg" /><ol start="6"><li> O concentration de SENP1 foi fixado, e a concentração de CyPet (pré-SUMO1)-YPet foi aumentada de 100 vezes mais do que a concentração de enzima, o que é exigido por equação de Michaelis-Menten. </li><li> Todas as leituras de fluorescência foram analisados ​​pelo método de análise quantitativa FRET e representado no GraphPad Software Prism V para ajustar a equação de Michaelis Menten. A análise de regressão não-linear também pode ser realizada com o auxílio de pacotes de software mais comuns, tais como programas de folha de cálculo (tais como o Microsoft Excel). </li></ol> 5. Resultados representativos Maturação de pré-SUMO1 por SENP1 pode ser determinada por monitorização das alterações do sinal de fluorescência a 475 e 530 nm durante o processo. O resultado mostrou que a velocidade de pré-SUMO1 digestão por SENP1 de um modo de substrato dependente da dose (Figura 4). Isto sugere que o domínio catalítico de SENP1 exibe uma actividade excelente para a maturação de pré-SUMO1. A reação inicialção velocidades foram calculadas através da análise acima, com diferentes concentrações de substrato (Tabela 1). O k cat / K M é geralmente usado para comparar a eficiência de enzimas diferentes com um substrato de uma enzima específica ou com diferentes substratos. K M e V max pode ser obtido a partir da equação de Michaelis-Menten, traçando as velocidades iniciais diferentes, correspondentes para as diferentes concentrações de CyPet (pré-SUMO1)-YPet (Figura 5) k cat foi obtido como.: <img alt="Equação 8" src="/files/ftp_upload/4430/4430eq8.jpg" /> De acordo com a análise acima, o K calculado M foi de 0,21 ± 0,04 mM, o gato k foi 6,90 ± 0,28 s -1, e razão k cat / K M foi de (3,2 ± 0,55) x10 7 M -1 </sup<html> &gt; S -1. <img alt="Figura 1" src="/files/ftp_upload/4430/4430fig1.jpg" /> Figura 1. Gráfico de FRET ensaio baseado em protease para a maturação de pré-SENP sumos. <img alt="Figura 2" src="/files/ftp_upload/4430/4430fig2.jpg" /> Figura 2. Análise quantitativa de sinal fluorescente como contribuições pelo dador-aceitador e FRET. Dissecação de espectros de emissão a partir de CyPet (pré-SUMO1) YPet sob excitação a 414 nm. CyPet FL (530/414) é emissão CyPet de a 530 nm, com excitação de 414 nm, FL FRET é a FRET induzida emissão YPet a 530 nm sob excitação de 414 nm, e FL YPet (530/414) é a emissão YPet de a 530 nm, com excitação de 414 nm. load/4430/4430fig3.jpg &quot;/&gt; Figura 3. Cálculo de direção α fator de emissão e β. <img alt="Figura 4" src="/files/ftp_upload/4430/4430fig4.jpg" /> Figura 4. Análise quantitativa de CyPet (pré-SUMO1) YPet digerido por razões diferentes do domínio catalítico de SENP1. As reações foram monitoradas dentro primeiros 5 min. <img alt="Figura 5" src="/files/ftp_upload/4430/4430fig5.jpg" /> Figura 5. Michaelis-Menten análise gráfica de digestão CyPet (pré-SUMO1)-YPet do por SENP1. Os dados foram traçados e analisados ​​por GraphPad Prism V e de regressão não-linear. <table border="1"><tbody><tr><td> [S] (uM) </td><td> V 0 (^ M / s) </td></tr><tr><td> 0,115 </td><td> 0,0023 ± 0,00005 </td></tr&gt; <tr><td> 0,214 </td><td> 0,0028 ± 0,00004 </td></tr><tr><td> 0,407 </td><td> 0,0033 ± 0,00007 </td></tr><tr><td> 0,594 </td><td> 0,0037 ± 0,00013 </td></tr><tr><td> 0,725 </td><td> 0,0043 ± 0,00012 </td></tr><tr><td> 1,471 </td><td> 0,0051 ± 0,00036 </td></tr><tr><td> 1,899 </td><td> 0,0050 ± 0,00031 </td></tr><tr><td> 2,300 </td><td> 0,0050 ± 0,00062 </td></tr></tbody></table> Tabela 1. Determinação velocidades iniciais de maturação de pré-SUMO1 por SENP1. Em cada concentração de substrato, quatro amostras foram utilizadas para medir a digestão. O desvio padrão foi de variações destas quatro amostras. <table border="1"><tbody><tr><td> K M (| iM) </td><td> k cat (s-1) </td><td> k cat / K m (M -1 • s-1) </td></tr><tr><td> 0,21 ± 0,04 </td><td> 6,90 ± 0,28 </td><td> 3,2 ± 0,55 x 10 7 </td></tr></tbody></table> Tabela 2. Parâmetros cinéticos de maturação de pré-SUMO1 por SENP1 por análise quantitativa FRET. O desvio padrão foi de quatro amostras de cada concentração de substrato.

<ol>
	<li>Johnson, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+modification+By+SUMO.">Protein modification By SUMO.</a> Annual Review of Biochemistry. 73, 355-382 (2004).</li><li>Gill, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO+and+ubiquitin+in+the+nucleus:+different+functions,+similar+mechanisms.">SUMO and ubiquitin in the nucleus: different functions, similar mechanisms.</a> Genes &amp; Development. 18, 2046-2059 (2004).</li><li>Hay, R. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DeSUMOylating+enzymes-SENPs.">DeSUMOylating enzymes-SENPs.</a> IUBMB Life. 60, 734-742 (2008).</li><li>Mukhopadhyay, D., Dasso, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+in+reverse:+the+SUMO+proteases.">Modification in reverse: the SUMO proteases.</a> Trends in Biochemical Sciences. 32, 286-295 (2007).</li><li>Reverter, D., Lima, C. 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FRET tecnologia tem sido utilizada para estudar a maturação de pré-SUMO1 por SENP1 9. CFP-YFP foi utilizado como o par de FRET e análise raciométrica, que é a relação entre as emissões de aceitador de doadores, foi utilizada para caracterizar as propriedades cinéticas. No entanto, não há qualquer consideração de dador e aceitador de auto-fluorescência na raciométrica tradicional FRET análise. A relação não é directamente correlacionada com a quantidade de substrato digerido. <p class=...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> PCR II Kit TOPO </td><td> Invitrogen </td><td> K466040 </td></tr><tr><td> 2xYT </td><td> Pesquisa de Produtos Internationa.l Corp </td><td> X15600 </td></tr><tr><td> Ni-NTA Agrose </td><td> Thermo Scientific </td><td> 88222 </td></tr><tr><td> Coomassie plus (Bradford) Regent Assay </td><td> Thermo Scientific </td><td> 23238 </td></tr><tr><td> 384-bem placa (fundo de vidro) </td><td> Greiner </td><td> 781892 </td></tr><tr><td> FlexStation II 384 leitor de placas </td><td> Dispositivo Molecular </td><td></td></tr></tbody></table>

quantitative fret (f&#246;rster resonance energy transfer) analysis for senp1 protease kinetics determination

Reversíveis modificações pós-tradução de proteínas com proteínas ubiquitina ou ubiquitina-like (Ubls) são amplamente usados ​​para regular a actividade da proteína de forma dinâmica e têm diversos papéis em diversos processos biológicos. Por exemplo, SUMO covalentemente modifica um grande número de proteínas ou com papéis importantes em muitos processos celulares, incluindo a regulação do ciclo celular, sobrevivência e morte celular, resposta a danos no ADN, e resposta ao estresse 1-5. SENP, como SUMO-protease específico, funciona como uma endopeptidase na maturação dos precursores SUMO ou como uma isopeptidase para remover SUMO de proteínas-alvo e refrescar o ciclo SUMOilação 1,3,6,7. A eficiência catalítica ou especificidade de uma enzima é melhor caracterizada por a razão entre as constantes cinéticas, k cat / K M. Em diversos estudos, os parâmetros cinéticos da SUMO-SENP pares foram determinadas por vários métodos, incluindo em gel de poliacrilamida-based western-blot, radIOActive marcado com composto de substrato, fluorescente ou proteína marcada substrato 8-13. No entanto, as técnicas de gel de poliacrilamida-base, que utilizaram os &quot;nativos&quot; proteínas, mas são trabalhosos e tecnicamente exigente, que não se prestam facilmente à análise quantitativa detalhada. A obtido k cat / K M a partir de estudos que utilizam tetrapéptidos ou proteínas com um ACC (ácido 7-amino-4-carbamoylmetylcoumarin) ou AMC (7-amino-4-metilcumarina) fluoróforo ou eram até duas ordens de grandeza mais baixa do que os substratos naturais ou não pode diferenciar claramente as iso-endopeptidase e atividades de SENPs. Os ensaios de protease recentemente, FRET base foram utilizados para estudar as enzimas deubiquitinating (Dubs) ou SENPs com o par FRET de proteína fluorescente ciano (PCP) e proteína fluorescente amarela (YFP) 9,10,14,15. A proporção de emissão de aceitador para emissão de dador foi utilizado como parâmetro quantitativo para FRET monitor de sinal para a protease acdade determinação. No entanto, este método ignorado sinal contaminações cruzadas, no comprimento de onda de emissão de aceitador e dador de aceitador e dador de auto-fluorescência e, portanto, não era correcto. Desenvolveu-se um novo ensaio altamente sensível e quantitativo da protease FRET com base para a determinação dos parâmetros cinéticos de uma pré-maturação por SUMO1 SENP1. Um par de engenharia FRET CyPet e YPet melhorou significativamente a eficiência com FRET e rendimento quântico de fluorescência, foram utilizadas para gerar o CyPet (pré-SUMO1)-substrato YPet 16. Nós diferenciados e quantificados absolutos sinais de fluorescência contribuídos pelo dador e aceitador FRET e nos comprimentos de onda de emissão e de aceitador, respectivamente. O valor de k cat / K M foi obtido como (3,2 ± 0,55) x10 7 M -1 s -1 de SENP1 para pré-SUMO1, o que está de acordo com os parâmetros cinéticos enzimáticos gerais. Assim, este método é válido e pode ser utilizado umsa geral aproximar para caracterizar outras proteases bem.

Watch this Scientific Journal Video about Quantitative FRET FÃ¶rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination at JoVE.com

Quantitative FRET F&amp;#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

Um novo método que envolve a análise quantitativa de FRET (Förster Resonance Energy Transfer) sinais é descrito para o estudo de cinética enzimática.<em&gt; K<sub&gt; M</sub</em&gt; E<em&gt; K<sub&gt; Gato</sub</em&gt; Foram obtidos por hidrólise do domínio catalítico de SENP1 (SUMO / Sentrin protease específica 1) para pré-SUMO1 (modificador ubiquitina-like Pequeno). Os princípios gerais deste estudo cinético quantitativo-FRET baseada protease pode ser aplicado a outras proteases.

Quantitativa FRET (Förster Resonance Energy Transfer) Análise para SENP1 Determinação Cinética Protease

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

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Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

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18.9K visualizações.  University of California, Riverside. Um novo método que envolve a análise quantitativa de FRET (Förster Resonance Energy Transfer) sinais é descrito para o estudo de cinética enzimática. K M

Vídeo: Quantitative FRET F&#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Visualização melhorada e Análise Quantitativa dos Efeitos de Drogas Usando Células micropatterned

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Bioengenharia

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

Ensaio de aderência de freqüência para In Situ Análise Cinética de Cross-juncional interações moleculares na interface célula-célula

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

Electrotaxis baseados em microfluídica para análise quantitativa On-demand de<em&gt; Caenorhabditis elegans</em&gt; &#39;Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Induzida por drogas Sensibilização de adenililciclase: Racionalização Ensaio e miniaturização para pequenas moléculas e siRNA Triagem Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Luminescência Ressonância transferência de energia para estudar mudanças conformacionais na membrana proteínas expressas em células de mamíferos

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

Medição de TCR-pMHC Binding<em&gt; In Situ</em&gt; Usando a microscopia Ensaio de FRET

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

Detecção sensível de Proteopathic Sementeira Atividade com FRET Citometria de Fluxo

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Multicolor detecção de fluorescência para Gota microfluídica Utilizando Fibras Ópticas

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A Protocol for Using F&amp;#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

Um protocolo para usar a transferência de Förster Ressonância Energia (FRET) Biossensores -force medir forças mecânicas em todo o Complexo LINC Nuclear

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Reconhecimento Molecular de engenharia com Polymers Bio-mimético em únicos Walled nanotubos de carbono

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...