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Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells

Dantuma, Nico P., Lindsten, Kristina, Glas, Rickard, Jellne, Marianne, Masucci, Maria G.·2000·Nature Biotechnology
DOI 10.1038/75406
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Abstract

The ubiquitin/proteasome-dependent proteolytic pathway is an attractive target for therapeutics because of its critical involvement in cell cycle progression and antigen presentation. However, dissection of the pathway and development of modulators are hampered by the complexity of the system and the lack of easily detectable authentic substrates. We have developed a convenient reporter system by producing N-end rule and ubiquitin fusion degradation (UFD)-targeted green fluorescent proteins that allow quantification of ubiquitin/proteasome-dependent proteolysis in living cells. Accumulation of these reporters serves as an early predictor of G2/M arrest and apoptosis in cells treated with proteasome inhibitors. Comparison of reporter accumulation and cleavage of fluorogenic substrates demonstrates that the rate-limiting chymotrypsin-like activity of the proteasome can be substantially curtailed without significant effect on ubiquitin-dependent proteolysis. These reporters provide a new powerful tool for elucidation of the ubiquitin/proteasome pathway and for high throughput screening of compounds that selectively modify proteolysis in vivo.

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  • Evaluation of the ratio between SQSTM1 aggregates/dimers and SQSTM1 monomers is likely a better measurement of changes in SQSTM1 dynamics linked to autophagy or the UPS.

    “Although the accumulation of this protein complex can be related to the accumulation of ubiquitinated SQSTM1-bound proteins, or the dimerization/inactivation of SQSTM1 [561, 607], evaluation of the ratio between SQSTM1 aggregates/dimers and SQSTM1 monomers is likely a better measurement of changes in SQSTM1 dynamics linked to autophagy or the UPS.”

    From Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition) 1 (2021) · cites this paper as [607]

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Metadata

Authors
Dantuma, N. P., Lindsten, K., Glas, R., Jellne, M., & Masucci, M. G.
Venue
Nature Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2000-05
Volume
18
Issue
5
Pages
538-543
ISSN
  • 1087-0156
  • 1546-1696
DOI
10.1038/75406
Cited by
553
References
0
Language
en
Licence
www.springer.com/tdm (tdm)

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