Sensitivity of mammalian cells expressing mutant ubiquitin to protein-damaging agents.
There is convincing evidence from studies in yeast that a functional ubiquitin/proteasome pathway is required to degrade misfolded or oxidatively damaged proteins but for technical reasons, it has been difficult to perform comparable studies in mammalian cells. To investigate the possibility that the ubiquitin/proteasome pathway is cytoprotective for mammalian cells, we have introduced epitope-tagged wild-type ubiquitin or dominant-negative mutant versions of ubiquitin into mouse HT4 neuroblastoma cells. Cells expressing mutant versions of ubiquitin were found to be sensitive to cadmium, an agent that causes oxidative damage to cellular components, and to canavanine, an amino acid analog that generates misfolded proteins. The greatest sensitivity to canavanine was observed in cells expressing a mutant version of ubiquitin unable to support the formation of Lys(48) linkages. Substrates of the proteasome were found to accumulate in these cells, suggesting a general deficit in proteolysis. Our data suggest that defects in the ubiquitin-mediated proteolytic system predispose mammalian cells to the toxic effects of abnormal protein.
Tsirigotis
M
M
Ottawa Regional Cancer Centre, Ottawa, Ontario K1H 1C4, Canada.
Zhang
M
M
Chiu
R K
RK
Wouters
B G
BG
Gray
D A
DA
D000818:Animals; D001483:Base Sequence; D002104:Cadmium / Q000494:pharmacology*; D002172:Canavanine / Q000494:pharmacology*; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D017931:DNA Primers; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D046988:Proteasome Endopeptidase Complex; D013379:Substrate Specificity; D014162:Transfection; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D009097:Multienzyme Complexes; D025801:Ubiquitin; D002104:Cadmium; D002172:Canavanine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex