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

This study was funded by: National Research Network on Aging (NFN S93) by the Austrian Science Foundation (FWF), European Commission Integrated Projects MiMAGE and PROTEOMAGE, Netherlands Genomics Initiative/Netherlands Organization for Scientific Research (NGI/NWO; 05040202 and 050-060-810 NCHA), the EU funded Network of Excellence Lifespan (FP6 036894), and Innovation Oriented Research Program on Genomics (SenterNovem; IGE01014 and IGE5007).

1. Plasmid Construction
<ol>
 <li>Order custom oligo-nucleotides encoding for dgn (ACKNWFSSLSHFVIHL11) and for dgnFS (HARTGSLACPTSSSICE) and ligate it into the pEGFP-C1 vector to obtain the fusion of the GFP with dgn/dgnFS (Figure 1). </li>
 <li>Amplify the coding sequence for GFP-dgn and GFP-dgnFS by PCR according to the protocol of the pENTR Directional TOPO Cloning Kit and continue with the pLenti6/V5 Directional TOPO Cloning Kit (Figure 6). </li>
</ol>
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<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>

The first publication using green fluorescent protein (GFP) as a reporter substrate for ubiquitin-proteasome activity was published in 2000 12. Since then, GFP has become a common tool to visualize cellular activities, especially the ubiquitin-proteasome process. To monitor ubiquitin-proteasome activity in vivo a transgenic mouse model with a GFP-based reporter has been introduced 13. Additional in vivo research established another transgenic mouse model with a similar degron-desta...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</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>Lipofectamine 2000 Reagent</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>Polyethylene glycol</td>
 <td>Sigma</td>
 <td>P2139</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Merck</td>
 <td>1.06404.1000</td>
 </tr>
 <tr>
 <td>Dulbecco's Phosphate Buffered Saline 1x (PBS)</td>
 <td>Invitrogen</td>
 <td>14190</td>
 </tr>
 <tr>
 <td>hexadimethrine bromide</td>
 <td>Sigma</td>
 <td>10,768-9</td>
 </tr>
 <tr>
 <td>Blasticidin</td>
 <td>Invitrogen</td>
 <td>R21001</td>
 </tr>
 <tr>
 <td>Crystal violet</td>
 <td>Sigma</td>
 <td>C3886</td>
 </tr>
 <tr>
 <td>FACS tubes</td>
 <td>BD Biosciences</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin Streptomycin (Pen-Strep)</td>
 <td>Invitrogen</td>
 <td>15140130</td>
 </tr>
 <tr>
 <td>L-glutamine 200 mM</td>
 <td>Invitrogen</td>
 <td>25030024</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum (FBS)</td>
 <td>Biochrom AG</td>
 <td>S0115</td>
 </tr>
 <tr>
 <td>MEM Non-Essential Amino Acids (NEAA) 100x</td>
 <td>Invitrogen</td>
 <td>11140035</td>
 </tr>
 <tr>
 <td>MEM Sodium Pyruvate 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

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

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

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

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