Proteasome inhibitors reduce luciferase and beta-galactosidase activity in tissue culture cells.
Reporter enzymes are commonly used in cell biology to study transcriptional activity of genes. Recently, reporter enzymes in combination with compounds that inhibit proteasome function have been used to study the effect of blocking transcription factor degradation on gene activation. While investigating the effect of proteasome inhibition on steroid receptor activation of the mouse mammary tumor virus (MMTV) promoter, we found that treatment with proteasome inhibitors enhanced glucocorticoid activation of the promoter attached to a chloramphenicol acetyltransferase (CAT) reporter, but inhibited activation of MMTV attached to a firefly luciferase or beta-galactosidase reporter. MMTV RNA levels under these conditions correlated with the promoter activity observed using the CAT reporter, suggesting that proteasome inhibitor treatment interfered with luciferase or beta-galactosidase reporter assays. Washout experiments demonstrated that the majority of luciferase activity was lost if the proteasome inhibitor was added at the same time luciferase was produced, not once the functional protein was made, suggesting that proteasome inhibition interferes with production of luciferase protein. Indeed, we found that proteasome inhibitor treatment dramatically reduced the levels of luciferase and beta-galactosidase protein produced, as determined by Western blot. Thus, treatment with proteasome inhibitors interferes with luciferase and beta-galactosidase reporter assays, possibly by inhibiting production of a functional reporter protein.
Deroo
Bonnie J
BJ
Chromatin and Gene Expression Section, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Archer
Trevor K
TK
D003546:Cysteine Endopeptidases / Q000187:drug effects*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics; D009097:Multienzyme Complexes / Q000187:drug effects*; D046988:Proteasome Endopeptidase Complex; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics
D015853:Cysteine Proteinase Inhibitors; D009097:Multienzyme Complexes; D008156:Luciferases; D001616:beta-Galactosidase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex