The Nrf1 CNC-bZIP protein is regulated by the proteasome and activated by hypoxia.
BACKGROUND
Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is a transcription factor mediating cellular responses to xenobiotic and pro-oxidant stress. Nrf1 regulates the transcription of many stress-related genes through the electrophile response elements (EpREs) located in their promoter regions. Despite its potential importance in human health, the mechanisms controlling Nrf1 have not been addressed fully.


RESULTS
We found that proteasomal inhibitors MG-132 and clasto-lactacystin-β-lactone stabilized the protein expression of full-length Nrf1 in both COS7 and WFF2002 cells. Concomitantly, proteasomal inhibition decreased the expression of a smaller, N-terminal Nrf1 fragment, with an approximate molecular weight of 23 kDa. The EpRE-luciferase reporter assays revealed that proteasomal inhibition markedly inhibited the Nrf1 transactivational activity. These results support earlier hypotheses that the 26 S proteasome processes Nrf1 into its active form by removing its inhibitory N-terminal domain anchoring Nrf1 to the endoplasmic reticulum. Immunoprecipitation demonstrated that Nrf1 is ubiquitinated and that proteasomal inhibition increased the degree of Nrf1 ubiquitination. Furthermore, Nrf1 protein had a half-life of approximately 5 hours in COS7 cells. In contrast, hypoxia (1% O(2)) significantly increased the luciferase reporter activity of exogenous Nrf1 protein, while decreasing the protein expression of p65, a shorter form of Nrf1, known to act as a repressor of EpRE-controlled gene expression. Finally, the protein phosphatase inhibitor okadaic acid activated Nrf1 reporter activity, while the latter was repressed by the PKC inhibitor staurosporine.


CONCLUSIONS
Collectively, our data suggests that Nrf1 is controlled by several post-translational mechanisms, including ubiquitination, proteolytic processing and proteasomal-mediated degradation as well as by its phosphorylation status.
Department of Biology, Carleton University, Ottawa, Ontario, Canada.
Nikolai L
NL
Chepelev
Joshua D
JD
Bennitz
Ting
T
Huang
Skye
S
McBride
William G
WG
Willmore
D000818:Animals; D019556:COS Cells; D015687:Cell Hypoxia; D002460:Cell Line; D002522:Chlorocebus aethiops; D006801:Humans; D051266:Nuclear Respiratory Factor 1 / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D059748:Proteolysis; D054875:Ubiquitination
C495633:NRF1 protein, human; D051266:Nuclear Respiratory Factor 1; D046988:Proteasome Endopeptidase Complex