Analysis of ubiquitination in vivo using a transgenic mouse model.
The primary pathway for the proteolytic destruction of cellular proteins is through ubiquitin-mediated targeting to the proteasome. This pathway is pivotal not only in the elimination of damaged or misfolded proteins but also in the temporal, developmental, or signal-mediated destruction of normal cellular substrates. The list of known substrates of the ubiquitin/proteasome pathway is long, but most substrates have been identified in yeast or, more recently, in cultured mammalian cells. It is likely that many mammalian substrates with developmental or disease relevance have yet to be identified because their ubiquitination occurs in tissue or organ systems that cannot be adequately modeled in vitro. We have developed a transgenic mouse model that will allow the isolation and identification of these substrates. The human UbC promoter was used to drive expression of a hexahistidine-tagged version of human ubiquitin in a variety of mouse tissues from early embryonic stages, as assessed by a green fluorescent protein marker. Cleavage of the fusion protein by endogenous enzymes produced epitope-tagged ubiquitin that was detected both in monomeric form and conjugated to cellular proteins. This mouse model should facilitate in the analysis of normal and disease-related ubiquitination events in vivo.
Tsirigotis
M
M
Ottawa Regional Cancer Centre, Ottawa, ON, Canada.
Thurig
S
S
Dubé
M
M
Vanderhyden
B C
BC
Zhang
M
M
Gray
D A
DA
D000818:Animals; D001921:Brain; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D000939:Epitopes / Q000235:genetics; D005260:Female; D005838:Genotype; D049452:Green Fluorescent Proteins; D006639:Histidine / Q000235:genetics; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D009097:Multienzyme Complexes / Q000378:metabolism; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic / Q000235:genetics; D046988:Proteasome Endopeptidase Complex; D013623:Tail; D014162:Transfection; D019076:Transgenes / Q000235:genetics; D025801:Ubiquitin / Q000378:metabolism*
D000939:Epitopes; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D006639:Histidine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex