Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells.
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.
Dantuma
N P
NP
Microbiology and Tumor Biology Center, Karolinska Institutet, S-171 77 Stockholm Sweden.
Lindsten
K
K
Glas
R
R
Jellne
M
M
Masucci
M G
MG
D003546:Cysteine Endopeptidases / Q000187:drug effects* / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000187:drug effects* / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013450:Sulfones / Q000494:pharmacology; D014162:Transfection; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D013450:Sulfones; D014452:Ubiquitins; C106727:carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde