Monitoring of ubiquitin-proteasome activity in living cells using a Degron (dgn)-destabilized green fluorescent protein (GFP)-based reporter protein

Greussing, R.; Unterluggauer, H.; Koziel, R.; Maier, A.B.; Jansen-Duurr, P.

doi:doi:10.3791/3327

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...Show moreProteasome 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.Show less

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Monitoring of ubiquitin-proteasome activity in living cells using a Degron (dgn)-destabilized green fluorescent protein (GFP)-based reporter protein