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Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein
Authors:Ruth Greussing  Hermann Unterluggauer  Rafal Koziel  Andrea B. Maier  Pidder Jansen-Dürr
Affiliation:Molecular and Cell Biology, Institute for Biomedical Aging Research;Department of Gerontology and Geriatrics, Netherlands Consortium for Healthy Aging, Leiden University Medical Center
Abstract:
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)–dgn fusion protein (GFP–dgn, unstable) and a variant carrying a frameshift mutation (GFP–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–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.
Keywords:Cellular Biology   Issue 69   Molecular Biology   Medicine   Biomedical Engineering   Virology   proteasome activity   lentiviral particles   GFP-dgn   GFP-dgnFS   GFP   human diploid fibroblasts   flow cytometry   plasmid   vector
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