Este estudo foi financiado por: Rede Nacional de Pesquisa do Envelhecimento (NFN S93) pelo austríaco Science Foundation (FWF), Comissão Europeia e projetos integrados de mimage PROTEOMAGE, Holanda Genomics Initiative / Organização Holandesa para Pesquisa Científica (NGI / NWO; 05040202 e 050 - 060-810 NCHA), a UE financiou Rede de Excelência Longevidade (FP6 036.894), Programa de Pesquisa e Inovação Orientada em Genômica (SenterNovem; IGE01014 e IGE5007).

1. Construção do plasmídeo <ol><li> Ordem personalizada codificação oligonucleótidos para dgn (ACKNWFSSLSHFVIHL 11) e para dgnFS (HARTGSLACPTSSSICE) e ligar-lo para o vector pEGFP-C1 para obter a fusão da GFP com dgn / dgnFS (Figura 1). </li><li> Amplificar a sequência que codifica para GFP-dgn e GFP-dgnFS por PCR de acordo com o protocolo do kit de clonagem TOPO pENTR direccional e continuar com o pLenti6/V5 Directional TOPO Cloning Kit (Figura 6). </li></ol> 2. Produção de vírus <ol><li> Um dia antes da transfecção (dia 1) para fora da semente HEK 293FT células em frascos T75 a fim de que eles serão de 90-95% confluentes no dia da transfecção. </li><li> No dia da transfecção dilua 36 ul de reagente de lipofectamina 2000 em 1,5 ml de DMEM sem soro e antibióticos em 15 ml Falcon. Use outro 15 ml e diluir falcon 3 ug pLenti6/V5 portador do DNA complementar para construir ou GFP<html> -Dgn ou GFP-dgnFS, 2,5 ug do plasmídeo envelope pMD2.G codificação e 7,5 ug psPAX2 esqueleto do vector em 1,5 ml de DMEM sem soro e antibióticos. Depois de 5 min o DNA diluída é combinada com o reagente de lipofectamina 2000 diluída. </li><li> Incubar a mistura durante 20 min à temperatura ambiente para permitir que os complexos de ADN-lipofectamina a forma. </li><li> Remover o meio de HEK 293FT células, substitui-lo por cuidadosamente 7 ml de meio (sem antibióticos) e adicionar a mistura de ADN-lipofectamina ao balão e rocha frente e para trás para misturar (Dia 2). Incubar o balão durante a noite a 37 ° C em um humidificada de 5% CO 2 incubadora. </li><li> Alterar meio do dia seguinte (10 ml de meio sem antibióticos; Dia 3). </li><li> Após 48 horas (dia 5) sobrenadante da colheita, centrifugar a 300 xg durante 5 min à temperatura ambiente e filtrar o sobrenadante através de um filtro de PVDF de 0,45 um. </li></ol> O meio de cultura - DMEM (HEK 293FT) tenda &quot;&gt; DMEM FBS a 10% 0,1 mM MEM NEAA 6 mM de L-glutamina 1 mM de piruvato de sódio MEM 1% Pen Strep (opcional) 500 ug / ml de geneticina (opcional) 3. A concentração de vírus por polietileno glicol (PEG) Precipitação <ol><li> Adicionar um volume de solução de polietileno-glicol (50 Polietilenoglicol mM, 41 mM NaCl, autoclave, pH = 7,2; PEG) para quatro volumes de sobrenadante e incubar durante 2 horas a 4 ° C. Cuidadosamente misture todos os 20-30 minutos por inversão. </li><li> Girar a 1500 xg durante 30 min a 4 ° C. Um pó branco deve ser visível. </li><li> Aspirar o sobrenadante e centrifugar novamente a 1500 xg durante 5 min a 4 ° C. Aspirar a solução de PEG restante. </li><li> Ressuspender o sedimento em meio ou tampão fosfato salino (PBS), pipetando para cima e para baixo vigorosamente e vortex durante 20 a 30 segundos. A título de orientação usam 500 ul para um frasco T75 e alíquota a 100 ul. Armazenar o vírus a -80 ° C. </li></ol><ol><li> Semente fora 5 × 10 4 U2-OS células para cada poço de uma placa de 6 poços um dia antes da transfecção. </li><li> No dia da transfecção remover o meio de cultura e substitui-lo por 1 ml de DMEM contendo 8 ug / ml de polibreno (brometo Hexadimetrina). Dilui-se o vírus concentrado sobrenadante 1:10 e 1:100 e adicionar 1 ul de cada uma de um poço. Para concentrações mais elevadas de vírus usar 1 ul, 5 ul e 10 ul de sobrenadante concentrado de vírus para as cavidades remanescentes. Um poço é deixada como um controlo positivo. </li><li> No dia seguinte, substituir o meio por 2 ml de DMEM. </li><li> Comece com a selecção do dia seguinte. Aplicar 10 blasticidina ug / mL em cada poço. Substitua o meio antibiótico contendo cada segundo dia. </li><li> Cerca de 6-7 dias após o início da seleção das células untransduced estão mortos. Para a coloração de lavar as células três vezes com PBS e cobrir as células com violeta de cristal (10 mg / l de cristal violetaem etanol a 20%) e incuba-se durante 5-10 minutos à temperatura ambiente. Aspirado de cristal violeta (pode ser reutilizado várias vezes) e lavar os poços duas vezes com DDQ 2 O e secar a placa. </li><li> Contar as colónias e se multiplicam em 1000 e a diluição correspondente (Figura 7). Este procedimento permite calcular as unidades de transfecção (TU) do vírus / ml. </li></ol> Meio de cultura - DMEM (HFF-2/U2-OS) DMEM FBS a 10% 6 mM de L-glutamina 1% Pen Strep- 5. Transdução de fibroblastos diplóides humanos (Este procedimento pode ser usado para qualquer tipo de células.) <ol><li> Descendência a 5 × 10 4 fibroblastos humanos diplóides (HDFS) a 6 poços no dia antes de transdução. </li><li> Usar uma multiplicidade de infecção de dois em conjunto com 8 ug / ml de polibreno como transdução potenciador, num total de um mililitro. </li><li> Alterar meio no dia seguinte. </li><li> Quandoas células estão a 70-80% de confluência início a selecção com 10 ng / ml blasticidina. Após a selecção estar completa (ausência de células não transf ectadas morrem mais) a expansão das células pode iniciar e as células estão prontas para experiências. Seleção crônica com blasticidina permite gerar uma linha celular estável superexpressão GFP-dgn ou proteínas GFP-dgnFS fusão, pronto para medir a atividade do proteassoma. Este procedimento garante uma eficiência de transfecção elevada, independentemente do tipo de célula. </li></ol> 6. Medição por citometria de fluxo <ol><li> Semente out 1 × 10 5 células (transporte ou GFP-dgn ou GFP-dgnFS) um dia antes do experimento. </li><li> Tratar as células de controlo 3 horas antes da medição com o inibidor de proteassoma, por exemplo, 100 ug / ml LLNL. </li><li> Lavar duas vezes com PBS e colheita das células utilizando-se 0,5 ml de tripsina. Para parar a tripsinização utilizar 4,5 ml de meio de cultura e transferência das células para uma de 15 ml Falcon. </li><li> Spin-tele células a 300 xg durante 5 min à temperatura ambiente. </li><li> Aspirar o meio, ressuspender as células em PBS e girar para baixo outra vez a 300 xg durante 5 min a 37 ° C. </li><li> Aspirar o PBS, ressuspender em 400 ul de tampão de FACS (10 mM de Hepes, 140 mM de NaCl, 2,5 mM de CaCl2, pH = 7,4) e transferir as células em tubos de FACS. </li><li> Manter as células em amostras de gelo e medida por citometria de fluxo. As células não deve ser armazenado por mais de 30 min a 4 ° C. </li></ol> 7. Resultados representativos A proteína de fusão GFP-dgn transporta uma sequência que é voltado para o proteassoma e, portanto, a proteína é imediatamente degradada, que corresponde à diminuição do sinal de fluorescência da GFP. O mutante de frameshift (GFP-dgnFS) transporta uma versão mutada desta sequência e não é degradada pelo proteassoma, que leva a uma maior fluorescência verde. Por estas razões, o jovem esperava HDFs com alta atividade do proteassoma e transduzidas com GFP-dgn apresentam um sinal de (6% de células positivas) baixa fluorescência na medida tanto em citometria de fluxo e de epifluorescência (Figura 2A e B, Figura 3). O mesmo HDFs transduzidas com GFP-dgnFS exibir 39,7% de células positivas. O tratamento das células com inibidor de proteassoma LLNL (N-acetil-L-leucil-L-leucil-L-norleucinal) elevou o sinal a um máximo de 62,9% em ambos, GFP-dgn e GFP-dgnFS células (Figura 2 A e B ). Para determinar possível declínio da actividade do proteassoma em idade as amostras de pele humana, os fibroblastos da derme isolados de doadores jovens, de meia idade e de idade foram infectadas com GFP-dgn e GFP-dgnFS, como descrito acima e cultivadas à passagem número mesmo antes da análise por fluxo citometria. Nesses experimentos, um aumento nítido na GFP sinal entre indivíduos jovens (11,2 ± 0,88% de células GFP-positivo) e de meia-idade doadores (20,4 ± 2,27% GFP células positivas, p = 0,003) foi observada, indicando uma diminuir em proteasatividade ome em amostras obtidas de doadores com idades entre 7 (Figura 4). Nenhuma diminuição adicional na actividade do proteassoma foi observada em fibroblastos isolados a partir de indivíduos mais idosos (Figura 4). A intensidade da fluorescência para GFP-dgnFS foi em todos os casos, a 90% (dados não apresentados) o que indica uma alta eficiência de transfecção para os três grupos etários distintos. <img src="/files/ftp_upload/3327/3327fig1.jpg" alt="Figura 1" /> Figura 1. A sequência de GFP-dgn e GFP-dgnFS. Displayed é a extremidade 3 &#39;de GFP (verde) no local de clonagem múltipla do vector pEGFP-CL1 (cinzento) e a sequência de dgn / dgnFS (vermelho). <img src="/files/ftp_upload/3327/3327fig2.jpg" alt="Figura 2" /> Figura 2. A análise de citometria de fluxo de GFP-dgn e GFP-dgnFS fibroblastos diplóides humanos. A. Young fibroblastos de prepúcio humano (HFF-2) foram infectadas com os vectores lentivirais que transportam a proteína verde fluorescente (GFP)-dgn gene ou um GFP-dgnFS (frameshift) construção, tal como indicado e analisadas por fluorescência de GFP por citometria de fluxo (FACS Canto II, Becton Dickinson). Quando indicado, as células foram também tratados, durante 3 h com inibidor de proteassoma de N-acetil-L-leucil-L-leucil-L-norleucinal (LLNL). As células não infectadas foram utilizadas como um controlo. As experiências foram realizadas em triplicado. B. Os dados são representativos de três experiências independentes. Os números refletem a quantidade de células GFP positivas. <a href="http://www.jove.com/files/ftp_upload/3327/3327fig2large.jpg" target="_blank">Clique aqui para ver maior figura</a> . <img src="/files/ftp_upload/3327/3327fig3.jpg" alt="Figura 3" /> Figura 3. A análise por microscopia de fluorescência de GFP-dgn e GFP-dgnFS HFF-2 células. Jovem HFF-2 foram tratados como na Figura 2. Aos 9 dias após a infecção, as células foram visualized por fluorescência e microscopia de contraste de fase. <img src="/files/ftp_upload/3327/3327fig4.jpg" alt="Figura 4" /> Figura 4. Mudanças na atividade do proteassoma no envelhecimento da pele humana. Os fibroblastos de prepúcio humano de nove diferentes doadores nas faixas etárias indicadas foram minimamente expandidos e infectados com construções lentivirais codificam GFP-DGN. Aos 9 dias após a infecção, as células foram analisadas por citometria de fluxo. Os dados foram obtidos em três amostras por grupo de idade em duplicados (± SE). <img src="/files/ftp_upload/3327/3327fig5.jpg" alt="Figura 5" /> Figura 5. Abordagem experimental. Custom-oligo nucleótidos para dgn e dgnFS são clonados no vector pEGFP-C1 e os vírus são produzidos para cada constructo utilizando células HEK 293FT células. O título do vírus foi determinado. As células são transfectadas com o vírus e expandido. Após o tratamento favorecido as células são analisadaspara o sinal de fluorescência por citometria de fluxo. <img src="/files/ftp_upload/3327/3327fig6.jpg" alt="Figura 6" /> Figura 6. Mapa de Plenti GFP-dgn. O mapa da pLenti6/V5-DEST vector incluindo a sequência de GFP-dgn é exibida. Abreviaturas: pCMV (promotor CMV), GFP-dgn (sequência de GFP-dgn), P SV40 (SV40 promotor precoce), EM7 (EM7 promotor), blasticidina (blasticidina gene de resistência), ΔU3 / LTR 3 &#39;(LTR 3&#39; com excluído região U3), SV40 pA (sinal de poliadenilação de SV40), ampicilina (gene de resistência à ampicilina), pUC ori (origem pUC), PRSV / 5 &#39;LTR (RSV / LTR 5&#39; do promotor híbrido), Ψ (HIV-1 do sinal de embalagem Ψ ), RRE (HIV-1 elemento de resposta rev). <img src="/files/ftp_upload/3327/3327fig7.jpg" alt="Figura 7" /> Figura 7. U2-OS placa titulação. U2-OS foram semeadas em uma placa de 6 poços e transfectadas com concentrações de vírus diferentes (diluição de 1/100 e 1/10, 1, 5 e 10 ul de concentrated sobrenadante viral). Um bem foi utilizado como controle não transfectados (NT). Após 6-7 dias, as células foram coradas com violeta de cristal e seco ao ar.

<ol>
	<li>Coux, O., Tanaka, K., Goldberg, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+functions+of+the+20S+and+26S+proteasomes.">Structure and functions of the 20S and 26S proteasomes.</a> Annu. Rev. Biochem. 65, 801-847 (1996).</li><li>Davies, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+oxidized+proteins+by+the+20S+proteasome.">Degradation of oxidized proteins by the 20S proteasome.</a> Biochimi. 83, 301-310 (2001).</li><li>Stangl, K., Stangl, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin-proteasome+pathway+and+endothelial+(dys)function.">The ubiquitin-proteasome pathway and endothelial (dys)function.</a> Cardiovasc. Res. 85, 281-290 (2009).</li><li>Tuoc, T. C., Stoykova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+the+ubiquitin-proteosome+system+in+neurogenesis.">Roles of the ubiquitin-proteosome system in neurogenesis.</a> Cell Cycle. 9, 3174-3180 (2010).</li><li>Rock, K. 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=Inhibitors+of+the+proteasome+block+the+degradation+of+most+cell+proteins+and+the+generation+of+peptides+presented+on+MHC+class+I+molecules.">Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules.</a> Cell. 78, 761-771 (1994).</li><li>Lehman, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin+proteasome+system+in+neuropathology.">The ubiquitin proteasome system in neuropathology.</a> Acta Neuropathol. 118, 329-347 (2009).</li><li>Koziel, R., Greussing, R., Maier, A. B., Declercq, L., Jansen-Durr, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Interplay+between+mitochondrial+and+proteasome+activity+in+skin+aging.">Functional Interplay between mitochondrial and proteasome activity in skin aging.</a> J. Invest. Dermatol. 131, 594-603 (2010).</li><li>Grillari, J., Grillari-Voglauer, R., Jansen-Durr, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modification+of+cellular+proteins+by+ubiquitin+and+ubiquitin-like+molecules:+role+in+cellular+senescence+and+aging.">Post-translational modification of cellular proteins by ubiquitin and ubiquitin-like molecules: role in cellular senescence and aging.</a> Adv. Exp. Med. Biol. 694, 172-196 (2010).</li><li>Bulteau, A. L., Szweda, L. I., Friguet, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+declines+in+proteasome+activity+in+the+heart.">Age-dependent declines in proteasome activity in the heart.</a> Arch. Biochem. Biophys. 397, 298-304 (2002).</li><li>Strucksberg, K. H., Tangavelou, K., Schroder, R., Clemen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasomal+activity+in+skeletal+muscle:+A+matter+of+assay+design,+muscle+type,+and+age.">Proteasomal activity in skeletal muscle: A matter of assay design, muscle type, and age.</a> Anal. Biochem. , (2009).</li><li>Bence, N. F., Sampat, R. M., Kopito, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+the+ubiquitin-proteasome+system+by+protein+aggregation.">Impairment of the ubiquitin-proteasome system by protein aggregation.</a> Science. 292, 1552-1555 (2001).</li><li>Dantuma, N. P., Lindsten, K., Glas, R., Jellne, M., Masucci, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-lived+green+fluorescent+proteins+for+quantifying+ubiquitin/proteasome-dependent+proteolysis+in+living+cells.">Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells.</a> Nat. Biotechnol. 18, 538-543 (2000).</li><li>Lindsten, K., Menendez-Benito, V., Masucci, M. G., Dantuma, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+mouse+model+of+the+ubiquitin/proteasome+system.">A transgenic mouse model of the ubiquitin/proteasome system.</a> Nat. Biotechnol. 21, 897-902 (2003).</li><li>Liu, 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=Impairment+of+the+ubiquitin-proteasome+system+in+desminopathy+mouse+hearts.">Impairment of the ubiquitin-proteasome system in desminopathy mouse hearts.</a> FASEB J. 20, 362-364 (2006).</li><li>Bowman, A. B., Yoo, S. Y., Dantuma, N. P., Zoghbi, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+dysfunction+in+a+polyglutamine+disease+model+occurs+in+the+absence+of+ubiquitin-proteasome+system+impairment+and+inversely+correlates+with+the+degree+of+nuclear+inclusion+formation.">Neuronal dysfunction in a polyglutamine disease model occurs in the absence of ubiquitin-proteasome system impairment and inversely correlates with the degree of nuclear inclusion formation.</a> Hum. Mol. Genet. 14, 679-691 (2005).</li><li>Myung, J., Kim, K. B., Lindsten, K., Dantuma, N. P., Crews, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+proteasome+active+site+allostery+as+revealed+by+subunit-specific+inhibitors.">Lack of proteasome active site allostery as revealed by subunit-specific inhibitors.</a> Mol. Cell. 7, 411-420 (2001).</li><li>Menendez-Benito, V., Heessen, S., Dantuma, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+ubiquitin-dependent+proteolysis+with+green+fluorescent+protein+substrates.">Monitoring of ubiquitin-dependent proteolysis with green fluorescent protein substrates.</a> Methods Enzymol. 399, 490-511 (2005).</li><li>Lener, B., 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=The+NADPH+oxidase+Nox4+restricts+the+replicative+lifespan+of+human+endothelial+cells.">The NADPH oxidase Nox4 restricts the replicative lifespan of human endothelial cells.</a> Biochem. J. 423, 363-374 (2009).</li></ol>

Não há conflitos de interesse declarados.

A primeira publicação utilizando a proteína fluorescente verde (GFP) como substrato repórter para a actividade de ubiquitina-proteassoma, foi publicado em 2000 12. Desde então, a GFP tem sido uma ferramenta comum para visualizar as actividades celulares, especialmente o processo de ubiquitina-proteassoma. Para monitorar a ubiquitina-proteassoma actividade in vivo num modelo de ratinho transgénico com um repórter GFP baseada foi introduzido 13. Também, em pesquisa in vi...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> pEGFP-C1 Vector </td><td> BD Bioscience Clontech </td><td> 6084-1 </td></tr><tr><td> pENTR Direcional TOPO Cloning Kit </td><td> Invitrogen </td><td> K2400-20 </td></tr><tr><td> pLenti6/V5 Direcional TOPO Kit Clonagem </td><td> Invitrogen </td><td> V496-10 </td></tr><tr><td> Lipofectamine Reagente 2000 </td><td> Invitrogen </td><td> 11668019 </td></tr><tr><td> DMEM </td><td> Sigma </td><td> D5546 </td></tr><tr><td> PVDF filtro (Rotilabo-Spritzenfilter) </td><td> Roth </td><td> P667.1 </td></tr><tr><td> Polietileno glicol </td><td> Sigma </td><td> P2139 </td></tr><tr><td> NaCl </td><td> Merck </td><td> 1.06404.1000 </td></tr><tr><td> Fosfato de Dulbecco tamponada Saline 1x (PBS) </td><td> Invitrogen </td><td> 14190 </td></tr><tr><td> Hexadimetrina brometo </td><td> Sigma </td><td> 10,768-9 </td></tr><tr><td> Blasticidina </td><td> Invitrogen </td><td> R21001 </td></tr><tr><td> O violeta de cristal </td><td> Sigma </td><td> C3886 </td></tr><tr><td> FACS tubos </td><td> BD Biosciences </td><td></td></tr><tr><td> Penicilina, estreptomicina (Pen-Strep) </td><td> Invitrogen </td><td> 15140130 </td></tr><tr><td> L-glutamina 200 mM </td><td> Invitrogen </td><td> 25030024 </td></tr><tr><td> Soro fetal bovino (FBS) </td><td> Biochrom AG </td><td> S0115 </td></tr><tr><td> MAM não-aminoácidos essenciais (NEAA) 100x </td><td> Invitrogen </td><td> 11140035 </td></tr><tr><td> MEM de piruvato de sódio 100 mM </td><td> Invitrogen </td><td> 11360039 </td></tr><tr><td> D-(+)-Glucose (45%) </td><td> Sigma </td><td> G8769 </td></tr><tr><td> Geneticina </td><td> Invitrogen </td><td> 11811023 </td></tr><tr><td> CaCl2 </td><td> Merck </td><td> C5080 </td></tr><tr><td> Hepes </td><td> Sigma </td><td> H3375 </td></tr><tr><td> Tripsina-EDTA (0,05%) </td><td> Invitrogen </td><td> 25300054 </td></tr></tbody></table>

monitoring of ubiquitin-proteasome activity in living cells using a degron (dgn)-destabilized green fluorescent protein (gfp)-based reporter protein

Proteassoma é o principal organelo intracelular envolvida na degradação proteolítica da anormais, deformadas, proteínas danificadas ou oxidadas 1, 2. Manutenção da atividade do proteassoma foi implicado em vários processos celulares, como resposta celular ao estresse 3, regulação do ciclo celular e diferenciação celular em 4 ou 5 resposta do sistema imunológico. A disfunção do sistema ubiquitina-proteassoma tem sido relacionado com o desenvolvimento de tumores e doenças neurodegenerativas 4, 6. Além disso, a diminuição da actividade do proteassoma foi encontrada como uma característica de senescência celular e envelhecimento organismal 7, 8, 9, 10. Aqui, apresentamos um método para medir a atividade da ubiquitina-proteassoma em células vivas usando uma proteína de fusão GFP-dgn. Para ser capaz de monitorizar actividade de ubiquitina-proteassoma nas células vivas primárias, constrói DNA complementar que codifica para uma proteína fluorescente verde (GFP), proteína de fusão dgn (GFP-dgn, instável) e uma variante de realização de uma mutação frameshift (GFP-dgnFS, estável 11) são inseridos em vectores de expressão lentiviral. Nós preferimos desta técnica em relação às técnicas tradicionais de transfecção, porque garante uma eficiência de transfecção elevada, independentemente do tipo de célula ou a idade do dador. A diferença entre a fluorescência exibida pelo GFP-dgnFS (estável) da proteína e da proteína desestabilizado (GFP-dgn) na ausência ou na presença de inibidor de proteassoma podem ser utilizados para estimar a actividade de ubiquitina-proteassoma em cada linhagem celular particular. Estas diferenças podem ser monitorizadas através de microscopia de epifluorescência, ou pode ser medido por citometria de fluxo.

Watch this Scientific Journal Video about Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein at JoVE.com

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

Um método para monitorizar a actividade de ubiquitina-proteassoma nas células vivas é descrito. A degron-GFP-desestabilizado (GFP-dgn) e uma proteína de fusão GFP estável dgnFS-são gerados e transduzidos para a célula utilizando um vector de expressão lentiviral. Esta técnica permite a geração de um estábulo GFP-dgn/GFP-dgnFS linha celular que expressa em que a actividade de ubiquitina-proteassoma podem ser facilmente avaliados utilizando epifluorescência ou citometria de fluxo.

Monitoramento da ubiquitina-proteassoma Atividade em células vivas Usando um Degron (dgn) desestabilizado Green Fluorescent Protein Protein Repórter (GFP) com base em

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

monitoring-ubiquitin-proteasome-activity-living-cells-using-degron

Research

JoVE Journal

Biology

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

16.6K visualizações.  Institute for Biomedical Aging Research. Um método para monitorizar a actividade de ubiquitina-proteassoma nas células vivas é descrito. A degron-GFP-desestabilizado (GFP-dgn) e uma proteína de fusão GFP estável dgnFS-são gerados e transduzidos para a célula utilizando um vector de expressão lentiviral. Esta técnica permite a geração de um estábulo GFP-dgn/GFP-dgnFS linha celular que expressa em que a actividade de ubiquitina-proteassoma podem ser facilmente avaliados utilizando epifluorescência ou...

Vídeo: Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Biologia

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Usando o sistema de fracionamento GELFREE 8100 para Fracionamento baseada no peso molecular com Recovery Fase Líquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Bimolecular de fluorescência de Complementação Assay (BiFC) para Interação Proteína-Proteína em células de cebola Utilizando o Gene Gun Helios

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Fotobranqueamento Ensaios (FRAP &amp; FLIP) a Medida Dynamics Protein Chromatin em Viver Células-Tronco Embrionárias

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Ensaios para Monitoramento juntamente redobramento de proteínas em<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

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 ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

Baseada Reporter ensaio de crescimento para análise sistemática de degradação de proteínas

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Genome-wide proteína-proteína Interaction Triagem por proteína-fragmento de Complementação Assay (PCA) em células vivas

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Verde Fluorescente à base de proteínas Expressão Rastreio de Proteínas de Membrana em<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

Um Protocolo de Avaliação Funcional da Whole-Protein saturação mutagênese Bibliotecas Utilizando-High Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...