Name: monitoring of ubiquitin-proteasome activity in living cells using a degron (dgn)-destabilized green fluorescent protein (gfp)-based reporter protein
Uploaded: 2012-11-10T14:00:00
Description: 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

Questo studio è stato finanziato da: National Research Network on Aging (NFN S93) dalla Science Foundation austriaco (FWF), Commissione europea integrata Progetti MIMAGE e PROTEOMAGE, Netherlands Genomics Initiative / Organizzazione olandese per la ricerca scientifica (NGI / NWO, 05040202 e 050 - 060-810 LPN), finanziato dall&#39;UE, Rete di eccellenza Durata (6 ° PQ 036894), e il Programma Innovation Research Oriented sulla genomica (SenterNovem, IGE01014 e IGE5007).

1. Costruzione plasmide <ol><li> Ordine personalizzato oligo-nucleotidi codifica per DGN (ACKNWFSSLSHFVIHL 11) e per dgnFS (HARTGSLACPTSSSICE) e legare esso nel pEGFP-C1 vettore per ottenere la fusione della GFP con DGN / dgnFS (Figura 1). </li><li> Amplifica la sequenza codificante per GFP-DGN e GFP-dgnFS mediante PCR secondo il protocollo della clonazione pENTR direzionale Kit TOPO e continuare con il pLenti6/V5 direzionale TOPO Cloning Kit (Figura 6). </li></ol><p class=...

<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>

La prima pubblicazione con la proteina fluorescente verde (GFP) come substrato reporter per ubiquitina-proteasoma attività è stata pubblicata nel 2000 12. Da allora, la GFP è diventata uno strumento comune per visualizzare le attività cellulari, in particolare il sistema ubiquitina-proteasoma processo. Per monitorare ubiquitina-proteasoma attività in vivo un modello di topo transgenico con GFP-reporter è stato introdotto 13. Ulteriori ricerca in vivo stabilito un altro model...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> pEGFP-C1 Vector </td><td> BD Bioscience Clontech </td><td> 6084-1 </td></tr><tr><td> pENTR direzionale TOPO Cloning Kit </td><td> Invitrogen </td><td> K2400-20 </td></tr><tr><td> pLenti6/V5 direzionale TOPO Cloning Kit </td><td> Invitrogen </td><td> V496-10 </td></tr><tr><td> Lipofectamine 2000 Reagente </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> Polietilene glicole </td><td> Sigma </td><td> P2139 </td></tr><tr><td> NaCl </td><td> Merck </td><td> 1.06404.1000 </td></tr><tr><td> Fosfato Dulbecco Buffered Saline 1x (PBS) </td><td> Invitrogen </td><td> 14190 </td></tr><tr><td> hexadimethrine bromuro </td><td> Sigma </td><td> 10,768-9 </td></tr><tr><td> Blasticidina </td><td> Invitrogen </td><td> R21001 </td></tr><tr><td> Cristallo viola </td><td> Sigma </td><td> C3886 </td></tr><tr><td> FACS tubi </td><td> BD Biosciences </td><td></td></tr><tr><td> Penicillina Streptomicina (Pen-Strep) </td><td> Invitrogen </td><td> 15140130 </td></tr><tr><td> L-glutammina 200 mM </td><td> Invitrogen </td><td> 25030024 </td></tr><tr><td> Siero bovino fetale (FBS) </td><td> Biochrom AG </td><td> S0115 </td></tr><tr><td> MEM Aminoacidi non essenziali (NEAA) 100x </td><td> Invitrogen </td><td> 11140035 </td></tr><tr><td> MEM Piruvato di sodio 100 mM </td><td> Invitrogen </td><td> 11360039 </td></tr><tr><td> D-(+)-glucosio (45%) </td><td> Sigma </td><td> G8769 </td></tr><tr><td> Geneticin </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

Proteasoma è l&#39;organello intracellulare principale coinvolto nella degradazione proteolitica della anormali, proteine ​​misfolded, danneggiati o ossidati 1, 2. Manutenzione delle attività del proteasoma è stato coinvolto in molti processi cellulari chiave, come risposta allo stress cellulare 3, regolazione del ciclo cellulare e la differenziazione cellulare 4 o nella risposta del sistema immunitario 5. La disfunzione del sistema ubiquitina-proteasoma è stata correlata allo sviluppo di tumori e malattie neurodegenerative 4, 6. Inoltre, una diminuzione dell&#39;attività del proteasoma trovato è una caratteristica della senescenza cellulare e organismal invecchiamento 7, 8, 9, 10. Qui, vi presentiamo un metodo per misurare ubiquitina-proteasoma attività in cellule viventi usando un GFP-DGN proteina di fusione. Per poter controllare ubiquitina-proteasoma attività nelle cellule viventi primarie, DNA complementare costruisce codificante per una proteina fluorescente verde (GFP)-DGN proteina di fusione (GFP-DGN, instabile) e una variante portando una mutazione frameshift (GFP-dgnFS, stabile 11) sono inseriti in vettori di espressione lentivirali. Preferiamo questa tecnica rispetto alle tecniche tradizionali di transfezione in quanto garantisce una elevata efficienza di trasfezione molto indipendente dal tipo di cellula o l&#39;età del donatore. La differenza tra fluorescenza visualizzata dal GFP-dgnFS (stabile) proteine ​​e la proteina destabilizzato (GFP-dgn) in assenza o presenza di inibitori del proteasoma può essere usato per stimare ubiquitina-proteasoma attività in ciascun ceppo cella particolare. Queste differenze possono essere monitorati mediante microscopia in epifluorescenza o può essere misurata mediante citometria a flusso.

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

Un metodo per monitorare l&#39;attività ubiquitina-proteasoma in cellule viventi è descritto. Un degron-destabilizzato GFP-(GFP-dgn) e di una stalla GFP-dgnFS proteina di fusione vengono generati e trasdotto nella cella utilizzando un vettore lentivirale espressione. Questa tecnica permette di generare una linea cellulare stabile GFP-dgn/GFP-dgnFS esprimendo in cui ubiquitina-proteasoma attività può essere facilmente valutata usando epifluorescenza o citometria a flusso.

Monitoraggio del sistema ubiquitina-proteasoma attività nelle cellule viventi Utilizzando una degron (dgn)-destabilizzato proteina verde fluorescente (GFP) a base di proteine ​​Reporter

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

Research

JoVE Journal

Biology

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

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

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

Ricerca

Biologia

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

Utilizzando il sistema GELFREE 8100 Frazionamento per peso molecolare basata su Frazionamento con recupero della fase liquida

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

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

Biomolecolare fluorescenza complementazione (BiFC) Assay per interazione proteina-proteina nelle cellule Cipolla Utilizzando la Helios Gun Gene

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

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

Fotodecolorazione saggi (FRAP e FLIP) per misurare la dinamica delle proteine ​​della cromatina in Living cellule staminali embrionali

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

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Saggi accoppiati per il monitoraggio delle proteine ​​in ripiegamento<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

Indagare interazioni proteina-proteina in cellule vive utilizzando il trasferimento bioluminescenza Resonance Energy

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

siRNA screening per identificare ubiquitina e ubiquitina-come regolatori del sistema di percorsi biologici in cellule di mammifero coltivate

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Saggio di crescita a base Reporter, per l&#39;analisi sistematica di proteine ​​degradazione

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

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

Genome-wide proteina-proteina interazione Screening per Protein-frammento Complementation Assay (PCA) in cellule viventi

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

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

Green Fluorescent Protein-base Espressione Proiezione di proteine ​​di membrana in<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 ...

Monitoraggio del sistema ubiquitina-proteasoma attività nelle cellule viventi Utilizzando una degron (dgn)-destabilizzato proteina verde fluorescente (GFP) a base di proteine ​​Reporter

Monitoraggio del sistema ubiquitina-proteasoma attività nelle cellule viventi Utilizzando una degron (dgn)-destabilizzato proteina verde fluorescente (GFP) a base di proteine Reporter