Nous sommes très reconnaissants à Victor GJ Rodgers pour ses conseils précieux. Nous tenons à remercier tous les membres du groupe Liao pour le travail collaboratif très proche et de l&#39;aide pour cette étude. Cette étude a été financée par le National Institutes of Health (Grant AI076504 à JL).

1. Constructions plasmidiques <ol><li> Amplifier les cadres ouverts de lecture des gènes par PCR et le clonage des produits PCR dans pCRII-TOPO vecteur. </li><li> Confirmer les produits de séquençage, et cloner l&#39;ADNc codant pour CyPet-(pré-SUMO1)-YPet, CyPet-SUMO1, et YPet domaines catalytiques de SENP1 dans l&#39;pET28 (b) avec un vecteur étiquette hexahistidine N-terminale. </li></ol> 2. Expression et purification des protéines <ol><li> Transformer des cellules d&#39;Escherichia coli de la souche BL21 (DE3) avec pET28 (b) des vecteurs codant CyPet-(pré-SUMO1)-YPet, CyPet-SUMO1, YPet et les domaines catalytiques de SENP1. </li><li> Cultivé des bactéries transformées dans un milieu 2xYT pendant 3 heures à 37 ° C (agitation à 250 tours par minute) pour atteindre une densité optique de 0,5 à 600 nm. </li><li> Ajouter 100 uM isopropyl-β-D-galactoside (IPTG) (concentration finale) pour induire l&#39;expression des protéines et agiter pendant 16 heures à 25 ° C à 200 tpm. </li><li> Récolter les bactéries par centrifugtion à 10000 rpm à 4 ° C pendant 5 min et les remettre en suspension dans un tampon de 20 mM Tris-HCl (pH 7,4), 50 mM de NaCl et 5 mM imidazole. </li><li> Soniquer les cellules pendant 10 min dans 5 sec intervalles à une puissance de 25 W en utilisant MiSonics 4000 sonicateur et les récupérer par centrifugation à 35.000 xg à 4 ° C pendant 30 min. </li><li> Mettre en place la colonne avec 500 ml de Ni-NTA perles de culture 1 L et transférer le surnageant dans la colonne. </li><li> Laver la résine avec un tampon de 20 mM Tris-HCl (pH 7,4), 500 mM de NaCl, et 10 mM d&#39;imidazole deux fois. </li><li> Protéines élue avec un tampon de 20 mM Tris-HCl (pH 7,4), NaCl 500 mM et 500 mM d&#39;imidazole et la dialyse dans un tampon de 20 mM Tris-HCl (pH 7,4), NaCl 50 mM et 1 mM de dithiothréitol (DTT) à 4 ° C pendant la nuit. </li><li> Déterminer les concentrations des protéines purifiées par la méthode de Bradford. Autre méthode pour la mesure des concentrations de protéines sera électrophorèse sur gel SDS-PAGE, puis colorées au bleu de Coomassie suivie par imageriequantitative. </li></ol> 3. FRET quantitative d&#39;analyse spectrale <ol><li> La stratégie générale pour le test était basé sur le FRET de signalisation (Figure 1). La paire FRET, et CyPet YPet, a été marqué à l&#39;extrémité N-et C-terminales, respectivement, de pré-SUMO1. SENP1 clive la protéine de fusion CyPet-(pré-SUMO1)-YPet sur le site Gly-Gly dans SUMO1 C-terminale et, ainsi, libère la queue avec YPet SUMO. Le signal de FRET est perturbé, ce qui entraîne une augmentation de l&#39;émission de CyPet et une diminution spectaculaire des émissions de YPet de la longueur d&#39;onde d&#39;excitation CyPet. </li><li> Lorsqu&#39;il est excité par de la lumière de longueur d&#39;onde 414 nm, l&#39;émission de fluorescence totale de CyPet-(pré-SUMO1)-YPet à 530 nm peuvent être dérivés à partir de trois sources: des émissions de l&#39;absolu FRET induite YPet, la CyPet directe d&#39;émission et YPet directe d&#39;émission (Figure 2). </li></ol><img alt="L&#39;équation 1" src="/files/ftp_upload/4430/4430eq1.jpg" /> où FL 530/414 &lt;/ Sub&gt; est l&#39;émission de fluorescence totale à 530 nm lorsqu&#39;il est excité à 414 nm, FL FRET est l&#39;absolu signal de FRET, FL CyPet (cont) est l&#39;émission CyPet directe lorsqu&#39;il est excité à 414 nm, et FL YPet (cont) est le YPet diriger des émissions lorsqu&#39;il est excité à 414 nm. L&#39;indice de (cont) représente la contribution. <ol start="3"><li> L&#39;émission directe de CyPet à 530 nm est proportionnelle à son émission à 475 nm lorsqu&#39;il est excité à 414 nm avec un rapport constant de α. CyPet-SUMO1 été préparées à des concentrations de 50, 100, 200, 500 et 750 nM et 1 mM , et les émissions à 475 et 530 nm ont été mesurées après excitation à 414 nm pour déterminer α (figure 3-1). </li><li> L&#39;émission directe de YPet à 530 nm sous excitation à 414 nm est proportionnelle à son émission à 530 nm lorsqu&#39;il est excité à 475 nm avec un rapport constant de β. YPet été préparées à des concentrations de 50, 100, 200, 500, 750 Ae 1.000 nM, et l&#39;émission à 530 nm a été mesurée lorsque les échantillons sont excités par des longueurs d&#39;onde de 414 et 475 nm pour déterminer β (figure 3-2). </li><li> Lorsque CyPet-(pré-SUMO1)-YPet a été digéré par SENP1, le clivage publié CyPet-SUMO1 et la queue SUMO1 avec YPet. Lorsque le composé est excité à 414 nm, l&#39;émission de fluorescence à 530 nm (FL &#39;530/414) a été diminué, mais peut encore être divisé en trois parties: </li></ol><img alt="Équation 2" src="/files/ftp_upload/4430/4430eq2.jpg" /> où FL &#39;530/414 est l&#39;émission de fluorescence totale à 530 nm après digestion lorsqu&#39;il est excité à 414 nm, FL&#39; FRET est le reste absolue signal de FRET, CyPet FL »(475/414) est l&#39;émission CyPet à 475 nm après digestion quand excité à 414 nm (ici l&#39;émission CyPet est en deux parties: non digérés CyPet-(pré-SUMO1)-YPet et digéré CyPet-SUMO1), et FL YPet(530/475) est l&#39;émission YPet lorsqu&#39;il est excité à 475 nm, qui est constante si CyPet-(pré-SUMO1)-YPet est digéré ou non. <ol start="6"><li> Après digestion par SENP1, le reste FRET émission (FL &#39;FRET) est la suivante: </li></ol><img alt="L&#39;équation 3" src="/files/ftp_upload/4430/4430eq3.jpg" /> où C est la concentration totale de CyPet-(pré-SUMO1)-YPet et x est la concentration de digéré CyPet-(pré-SUMO1)-YPet. <ol start="7"><li> En combinant tous les éléments, l&#39;émission de fluorescence détectée à 530 nm sous excitation de 414 nm (FL &#39;530/414) est la suivante: </li></ol><img alt="L&#39;équation 4" src="/files/ftp_upload/4430/4430eq4.jpg" /> 4. Essai de la protéase à base de FRET pour l&#39;étude cinétique enzymatique <ol><li> CyPet-(pré-SUMO1)-YPet a été incubé avec le domaine catalytique de SENP1 à 37 ° C dans un tampon de 20 mM Tris-HCl (pH 7,4), 50 mM NaCl, 0,1% (v /v) de Tween-20 et 1 mM de DTT dans un volume total de 80 ml et transférée dans la plaque 384-puits. </li><li> Essais ont été réalisés en mesurant l&#39;émission de fluorescence à 475 et 530 nm après une excitation à 414 nm dans un lecteur de fluorescence de plaque multi puits pendant 5 min à 15-Sec intervalles. </li><li> La vitesse de la réaction (V) est en corrélation avec la variation de la quantité de substrat (S) en tant que: </li></ol><img alt="L&#39;équation 5" src="/files/ftp_upload/4430/4430eq5.jpg" /><ol start="4"><li> La concentration du produit augmente de façon exponentielle à partir de 0 à [S] 0 (concentration en substrat d&#39;origine) quand t = 0: </li></ol><img alt="L&#39;équation 6" src="/files/ftp_upload/4430/4430eq6.jpg" /><ol start="5"><li> Quand t = 0, la vitesse initiale (V 0) est la suivante: </li></ol><img alt="Équation 7" src="/files/ftp_upload/4430/4430eq7.jpg" /><ol start="6"><li> Le concentréentration de SENP1 est fixée, et la concentration de CyPet-(pré-SUMO1)-YPet a été augmentée de 100 fois supérieure à la concentration de l&#39;enzyme, ce qui est requis par l&#39;équation de Michaelis-Menten. </li><li> Toutes les lectures fluorescentes ont été analysés par la méthode d&#39;analyse FRET quantitative et tracés dans GraphPad Prism V Logiciel pour s&#39;adapter à l&#39;équation de Michaelis-Menten. La régression non linéaire peut également être effectuée à l&#39;aide d&#39;autres logiciels courants tels que des tableurs (comme Microsoft Excel). </li></ol> 5. Les résultats représentatifs Maturation de la pré-SUMO1 par SENP1 peut être déterminée en contrôlant les variations du signal de fluorescence à 475 nm et 530 au cours du processus. Les résultats ont montré que la vitesse de pré-SUMO1 digestion par SENP1 substrat d&#39;une manière dose-dépendante (Figure 4). Ceci suggère que le domaine catalytique de SENP1 présente une activité excellente pour la maturation du pré-SUMO1. La réaction initialetion vitesses ont été calculées par l&#39;analyse ci-dessus avec des concentrations de substrat différentes (tableau 1). Le k cat / K M est généralement utilisé pour comparer l&#39;efficacité des différentes enzymes avec un substrat ou une enzyme particulière avec différents substrats. K M et V max peut être obtenu à partir de l&#39;équation de Michaelis-Menten en traçant les différentes vitesses initiales, correspondant pour les différentes concentrations de CyPet-(pré-SUMO1)-YPet (figure 5) k cat a été obtenue comme.: <img alt="Équation 8" src="/files/ftp_upload/4430/4430eq8.jpg" /> Selon l&#39;analyse ci-dessus, la valeur calculée est de 0,21 M K ± 0,04 uM, le chat était k 6,90 ± 0,28 s -1 et k cat / K M ratio était de (3,2 ± 0,55) x 10 7 M -1 </sup<html> &gt; S -1. <img alt="Figure 1" src="/files/ftp_upload/4430/4430fig1.jpg" /> Figure 1. Graphe de test FRET protéase à base de SENP pré-sumos maturation. <img alt="Figure 2" src="/files/ftp_upload/4430/4430fig2.jpg" /> Figure 2. Analyse quantitative de signal fluorescent à titre de contributions par le donneur, accepteur et FRET. La dissection des spectres d&#39;émission de CyPet-(pré-SUMO1)-YPet sous excitation à 414 nm. CyPet FL (530/414) est des émissions CyPet de à 530 nm sous excitation de 414 nm, FL FRET est l&#39;émission FRET induite YPet à 530 nm sous excitation de 414 nm, et FL YPet (530/414) est une émission YPet de à 530 nm sous excitation de 414 nm. load/4430/4430fig3.jpg &quot;/&gt; Figure 3. Calcul du facteur α et β direction d&#39;émission. <img alt="Figure 4" src="/files/ftp_upload/4430/4430fig4.jpg" /> La figure 4. L&#39;analyse quantitative de CyPet-(pré-SUMO1)-YPet digéré par différentes proportions du domaine catalytique de SENP1. Les réactions ont été suivies dans les 5 premières minutes. <img alt="Figure 5" src="/files/ftp_upload/4430/4430fig5.jpg" /> Figure 5. Michaelis-Menten analyse graphique de la digestion CyPet-(pré-SUMO1)-YPet par SENP1. Les données ont été tracées et analysées par GraphPad Prism V et la régression non linéaire. <table border="1"><tbody><tr><td> [S] (M) </td><td> V 0 (pM / 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> Tableau 1. Initiale de détermination des vitesses de maturation pré-SUMO1 par SENP1. Dans chaque concentration de substrat, quatre échantillons ont été utilisés pour mesurer la digestion. L&#39;écart-type est venu à partir des variations de ces quatre échantillons. <table border="1"><tbody><tr><td> K M (M) </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> Tableau 2. Paramètres cinétiques de maturation pré-SUMO1 par SENP1 par FRET quantitative d&#39;analyse. L&#39;écart-type est venu des quatre échantillons de chaque concentration de substrat.

<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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+SENP2+protease+interactions+with+SUMO+precursors+and+conjugated+substrates.">Structural basis for SENP2 protease interactions with SUMO precursors and conjugated substrates.</a> Nature Structural &amp; Molecular Biology. 13, 1060-1068 (2006).</li><li>Shen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO+protease+SENP1+induces+isomerization+of+the+scissile+peptide+bond.">SUMO protease SENP1 induces isomerization of the scissile peptide bond.</a> Nature Structural &amp; Molecular Biology. 13, 1069-1077 (2006).</li><li>Horton, R., Strachan, E., Vogel, K., Riddle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+substrate+for+deubiquitinating+enzymes+based+on+time-resolved+fluorescence+resonance+energy+transfer+between+terbium+and+yellow+fluorescent+protein.">A substrate for deubiquitinating enzymes based on time-resolved fluorescence resonance energy transfer between terbium and yellow fluorescent protein.</a> Analytical Biochemistry. 360, 138-143 (2007).</li><li>Mikolajczyk, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+Ubiquitin-related+Modifier+(SUMO)-specific+Proteases:+PROFILING+THE+SPECIFICITIES+AND+ACTIVITIES+OF+HUMAN+SENPs.">Small Ubiquitin-related Modifier (SUMO)-specific Proteases: PROFILING THE SPECIFICITIES AND ACTIVITIES OF HUMAN SENPs.</a> Journal of Biological Chemistry. 282, 26217-26224 (2007).</li><li>Kolli, N., Mikolajczyk, J., Drag, M., Mukhopadhyay, D., Moffatt, N., Dasso, M., Salvesen, G., Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distrubition+and+paralogue+specificity+of+mammalian+deSUMOylating+enzymes.">Distrubition and paralogue specificity of mammalian deSUMOylating enzymes.</a> Biochem. J. , 335-344 (2010).</li><li>Drag, M., Mikolajczyk, J., Krishnakumar, I. M., Huang, Z., Salvesen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+profiling+of+human+deSUMOylating+enzymes+(SENPs)+with+synthetic+substrates+suggests+an+unexpected+specificity+of+two+newly+characterized+members+of+the+family.">Activity profiling of human deSUMOylating enzymes (SENPs) with synthetic substrates suggests an unexpected specificity of two newly characterized members of the family.</a> Biochemical Journal. 409, 461 (2008).</li><li>Engels, I. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-resolved+fluorescence+resonance+energy+transfer-based+assay+for+DEN1+peptidase+activity.">A time-resolved fluorescence resonance energy transfer-based assay for DEN1 peptidase activity.</a> Analytical Biochemistry. 390, 85-87 (2009).</li><li>Martin, S., Hattersley, N., Samuel, I., Hay, R., Tatham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence-resonance-energy-transfer-based+protease+activity+assay+and+its+use+to+monitor+paralog-specific+small+ubiquitin-like+modifier+processing.">A fluorescence-resonance-energy-transfer-based protease activity assay and its use to monitor paralog-specific small ubiquitin-like modifier processing.</a> Analytical Biochemistry. 363, 83-90 (2007).</li><li>Nguyen, A. W., Daugherty, P. 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Aucun conflit d&#39;intérêt déclaré.

FRET technologie a été utilisée pour étudier la maturation pré-SUMO1 par SENP1 9. CFP-YFP a été utilisé comme la paire FRET et l&#39;analyse ratiométrique, qui est le rapport de l&#39;accepteur d&#39;émissions de bailleurs de fonds, a été utilisé pour caractériser les propriétés cinétiques. Cependant, il n&#39;ya aucune considération de donneur et accepteur d&#39;auto-fluorescence dans le ratiométrique traditionnelle analyse FRET. Le rapport n&#39;est pas directement corrélée avec la quan...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> II TOPO PCR Kit </td><td> Invitrogen </td><td> K466040 </td></tr><tr><td> 2xYT </td><td> Recherche sur les produits Internationa.l Corp </td><td> X15600 </td></tr><tr><td> Ni-NTA agarose </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> Plaque de 384 puits (à fond de verre) </td><td> Greiner </td><td> 781892 </td></tr><tr><td> FlexStation II 384 lecteur de plaque </td><td> Appareil moléculaire </td><td></td></tr></tbody></table>

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

Réversibles modifications post-traductionnelles des protéines avec des protéines ubiquitine ou l&#39;ubiquitine-like (UBLS) sont largement utilisés pour réguler dynamiquement l&#39;activité des protéines et jouent des rôles divers dans de nombreux processus biologiques. Par exemple, SUMO modifie de façon covalente un grand nombre ou des protéines avec des rôles importants dans de nombreux processus cellulaires, y compris les cellules cycle de régulation, la survie cellulaire et la mort, la réponse dommages à l&#39;ADN et de réponse au stress 1-5. SENP, comme SUMO protéase spécifique, fonctionne comme une endopeptidase dans la maturation de précurseurs ou sous forme SUMO isopeptidase pour supprimer SUMO de ses protéines cibles et actualiser le cycle 1,3,6,7 SUMOylation. L&#39;efficacité ou la spécificité catalytique d&#39;une enzyme est mieux caractérisée par le rapport des constantes cinétiques k cat / K m. Dans plusieurs études, les paramètres cinétiques de SUMO-SENP paires ont été déterminées par des méthodes différentes, y compris en gel de polyacrylamide à base de western-blot, radIOActive substrat marqué, un composé fluorescent ou une protéine marquée substrat 8-13. Cependant, les techniques de polyacrylamide à base de gel, qui étaient les «indigènes» des protéines, mais sont laborieux et techniquement exigeant, qui ne se prêtent pas facilement à une analyse quantitative détaillée. Le obtenues k cat / K M d&#39;études utilisant tétrapeptides ou des protéines avec un ACC (7-amino-4-carbamoylmetylcoumarin) ou AMC (7-amino-4-méthylcoumarine) fluorophore étaient soit jusqu&#39;à deux ordres de grandeur plus faible que les substrats naturels ou ne peut pas distinguer clairement les activités et les iso-endopeptidase de SENPs. Tests de protéase récemment, à base de FRET ont été utilisés pour étudier les enzymes désubiquitination (DUB) ou SENPs avec la paire FRET de la protéine fluorescente cyan (CFP) et la protéine fluorescente jaune (YFP) 9,10,14,15. Le taux d&#39;émission de l&#39;accepteur d&#39;émission du donneur a été utilisé en tant que paramètre quantitatif de FRET signal de contrôle en courant alternatif pour la protéasetivité détermination. Cependant, cette méthode de signaux ignorés contaminations croisées à l&#39;accepteur et donneur d&#39;onde d&#39;émission accepteur et donneur d&#39;auto-fluorescence et donc n&#39;était pas exacte. Nous avons développé un roman très sensible et quantitative dosage de protease FRET basée sur la détermination des paramètres cinétiques de la pré-SUMO1 maturation par SENP1. Une ingénierie FRET CyPet paire et avec YPet considérablement amélioré l&#39;efficacité et la fluorescence FRET rendement quantique, ont été utilisés pour générer le CyPet-(pré-SUMO1)-YPet substrat 16. Nous avons distingué et quantifiés absolues des signaux de fluorescence apportées par le donneur et l&#39;accepteur et FRET aux longueurs d&#39;onde d&#39;émission et accepteurs, respectivement. La valeur de k cat / K M a été obtenu comme (3,2 ± 0,55) x 10 7 M -1 s -1 SENP1 vers pré-SUMO1, qui est en accord avec les généraux paramètres cinétiques enzymatiques. Par conséquent, cette méthode est valable et peut être utilisésa approche générale pour caractériser d&#39;autres protéases ainsi.

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

Une nouvelle méthode qui analyse quantitative de FRET (Förster Resonance Energy Transfer) des signaux est décrite pour l&#39;étude de la cinétique enzymatique.<em&gt; K<sub&gt; M</sub</em&gt; Et<em&gt; K<sub&gt; Cat</sub</em&gt; Ont été obtenus pour l&#39;hydrolyse du domaine catalytique de SENP1 (SUMO / sentrine protéase spécifique 1) de pré-SUMO1 (Small Ubiquitin-comme modificateur). Les principes généraux de cette étude quantitative-FRET à base de protéase cinétique peut être appliquée à d&#39;autres protéases.

Quantitative FRET (transfert de Förster Resonance Energy) Analyse de la protéase SENP1 Kinetics Détermination

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 vues.  University of California, Riverside. Une nouvelle méthode qui analyse quantitative de FRET (Förster Resonance Energy Transfer) des signaux est décrite pour l'étude de la cinétique enzymatique. K M

Vidéo: 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.

Amélioration de la visualisation et l&#39;analyse quantitative des effets des drogues utilisant des cellules 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 ...

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Bio-ingénierie

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.

Essai Fréquence d&#39;adhérence pour In Situ Analyse cinétique de la Croix-jonctionnelle interactions moléculaires à l&#39;interface cellule-cellule

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.

Électrotaxie microfluidique à base d&#39;analyse quantitative à la demande 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.

Sensibilisation à la drogue induit des adénylylcyclase: Rationalisation de dosage et la miniaturisation des petites molécules et siARN préalable 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.

Luminescence Resonance Energy Transfer pour étudier les changements conformationnels dans des protéines membranaires exprimés dans les cellules de mammifères

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.

Mesure de TCR-pMHC Reliure<em&gt; In Situ</em&gt; L&#39;aide d&#39;un essai basé sur la microscopie 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.

Détection sensible de Proteopathic semis Activité FRET avec la cytométrie en flux

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 détection Fluorescence pour Droplet microfluidique utilisant des fibres optiques

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.

Un protocole pour l&#39;utilisation Förster Resonance Energy Transfer (FRET) -force biocapteurs pour mesurer les forces mécaniques à travers le complexe nucléaire LINC

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.

Reconnaissance moléculaire Ingénierie avec Polymères Bio-mimétiques sur simple 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 ...