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

Denne undersøgelse blev finansieret af: National Research Network on Aging (NFN S93) af den østrigske Science Foundation (FWF), Europa-Kommissionen integrerede projekter MiMAGE og PROTEOMAGE, Holland Genomics Initiative / Holland Organisation for Videnskabelig Forskning (NGI / NWO, 05.040.202 og 050 - 060-810 NCHA), EU-finansierede Network of Excellence Levetid (FP6 036.894), og Innovation Oriented Research Program på Genomics (SenterNovem, IGE01014 og IGE5007).

1. Plasmid Construction <ol><li> For custom oligonucleotider kodende for DGN (ACKNWFSSLSHFVIHL 11) og for dgnFS (HARTGSLACPTSSSICE) og ligere det ind i pEGFP-C1 vektoren til opnåelse af fusion af GFP med DGN / dgnFS (figur 1). </li><li> Forstærk den kodende sekvens for GFP-DGN og GFP-dgnFS ved PCR ifølge protokollen af pENTR Directional TOPO Cloning Kit og fortsætte med pLenti6/V5 Directional TOPO Cloning Kit (figur 6). </li></ol> 2. Virusproduktio...

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	<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>

Den første offentliggørelse ved hjælp af grønt fluorescerende protein (GFP) som en reporter substrat for ubiquitin-proteasom aktivitet blev offentliggjort i 2000 12. Siden da har GFP blive et fælles redskab til at visualisere cellulære aktiviteter, navnlig den ubiquitin-proteasom proces. At overvåge Ubiquitin-proteasom aktivitet in vivo en transgen musemodel med et GFP-baseret reporter er blevet indført 13. Yderligere in vivo forskning etableret en anden transgen ...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> pEGFP-C1 Vector </td><td> BD Bioscience Clontech </td><td> 6084-1 </td></tr><tr><td> pENTR Directional TOPO Cloning Kit </td><td> Invitrogen </td><td> K2400-20 </td></tr><tr><td> pLenti6/V5 Directional TOPO Cloning Kit </td><td> Invitrogen </td><td> V496-10 </td></tr><tr><td> Lipofectamin 2000 Reagens </td><td> Invitrogen </td><td> 11668019 </td></tr><tr><td> DMEM </td><td> Sigma </td><td> D5546 </td></tr><tr><td> PVDF filter (Rotilabo-Spritzenfilter) </td><td> Roth </td><td> P667.1 </td></tr><tr><td> Polyethylenglycol </td><td> Sigma </td><td> P2139 </td></tr><tr><td> NaCl </td><td> Merck </td><td> 1.06404.1000 </td></tr><tr><td> Dulbeccos phosphatbufrede Saline 1x (PBS) </td><td> Invitrogen </td><td> 14.190 </td></tr><tr><td> hexadimethrine bromid </td><td> Sigma </td><td> 10,768-9 </td></tr><tr><td> Blasticidin </td><td> Invitrogen </td><td> R21001 </td></tr><tr><td> Krystalviolet </td><td> Sigma </td><td> C3886 </td></tr><tr><td> FACS-rør </td><td> BD Biosciences </td><td></td></tr><tr><td> Penicillin Streptomycin (Pen-Strep) </td><td> Invitrogen </td><td> 15140130 </td></tr><tr><td> L-glutamin 200mM </td><td> Invitrogen </td><td> 25030024 </td></tr><tr><td> Føtalt bovint serum (FBS) </td><td> Biochrom AG </td><td> S0115 </td></tr><tr><td> MEM ikke-essentielle aminosyrer (NEAA) 100x </td><td> Invitrogen </td><td> 11140035 </td></tr><tr><td> MEM natriumpyruvat 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> 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> Trypsin-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

Proteasom er den vigtigste intracellulære organel involveret i den proteolytiske nedbrydning af unormale, fejlfoldede, beskadigede eller oxiderede proteiner 1, 2. Vedligeholdelse af proteasom aktivitet blev impliceret i mange vigtige cellulære processer, ligesom celle stress reaktion 3, cellecyklusregulering og cellulær differentiering 4 eller i immunsystemet respons 5. Dysfunktion af den ubiquitin-proteasom system er blevet forbundet med udviklingen af tumorer og neurodegenerative sygdomme 4, 6. Desuden blev et fald i proteasom aktivitet fundet som en funktion af cellulære ældning og organismal aldring 7, 8, 9, 10. Her præsenteres en metode til måling af ubiquitin-proteasom aktivitet i levende celler ved hjælp af en GFP-DGN fusionsprotein. At kunne overvåge Ubiquitin-proteasom aktivitet i levende primære celler, komplementære DNA-konstruktioner der koder for et grønt fluorescerende protein (GFP)-DGN fusionsprotein (GFP-DGN og ustabil) og en variant bærer en rammeskift mutation (GFP-dgnFS, stabile 11) indsættes i lentivirale ekspressionsvektorer. Vi foretrækker denne teknik over traditionelle transfektionsteknikker, fordi den sikrer en meget høj transfektionseffektivitet uafhængig af den celletype eller alder donoren. Forskellen mellem fluorescens vises af GFP-dgnFS (stabilt) protein og destabiliseret protein (GFP-DGN) i fravær eller nærvær af proteasom inhibitor kan anvendes til at estimere Ubiquitin-proteasom aktivitet i hvert enkelt cellestamme. Disse forskelle kan overvåges ved epifluorescens-mikroskopi eller kan måles ved flowcytometri.

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

Fremgangsmåde til overvågning Ubiquitin-proteasom aktivitet i levende celler er beskrevet. En degron-destabiliseret GFP-(GFP-DGN) og en stabil GFP-dgnFS fusionsprotein genereres og transduceres ind i cellen under anvendelse af en lentiviral ekspressionsvektor. Denne teknik gør det muligt at generere en stabil GFP-dgn/GFP-dgnFS udtrykkende cellelinie, hvori Ubiquitin-proteasom aktivitet kan let vurderes ved hjælp af epifluorescens eller flowcytometri.

Overvågning af Ubiquitin-proteasom aktivitet i levende celler ved hjælp af en Degron (DGN)-destabiliseret Green Fluorescent Protein (GFP)-baseret Reporter Protein

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

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

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