An Autoregulatory Loop between Nrf2 and Cul3-Rbx1 Controls Their Cellular Abundance

Abstract

The cellular defense system protects against oxidative stress caused by a vast range of xenobiotics, inflammation, and ionizing radiation (1-3). Disruption of these protective systems causes the accumulation of reactive oxygen species (ROS) 2 and electrophiles that can contribute to diseases such as cancer, cardiovascular complications, acute and chronic inflammation, and neurodegenerative diseases (4). Therefore, it is obvious that cells must constantly labor to control levels of ROS, preventing them from accumulating. Cells have mechanisms to activate over two hundred defensive genes that protect against ROS and the diseases they contribute to (1, 5-7).The antioxidant response element (ARE) was first identified as cis-element in the upstream regulatory region of the GSTA2 gene (8) and was found in the promoters of detoxifying enzyme genes such as glutathione S-transferases, NAD(P)H:quinone oxidoreductases, gastrointestinal glutathione peroxidase, and peroxiredoxin1 (9 -13). The ARE is recognized by the family of Cap'n'Collar containing basic leucine zipper proteins including Nrf2. Among the family, Nrf2 is the most potent transcription factor in regulating the basal and inducible expression of antioxidant enzyme genes (14). Gene deletion studies also supported the important function of Nrf2 in cellular protection against oxidative stress and neoplasia (15).At basal levels, Nrf2 resides within the cytoplasm of the cells by an interaction with an actin-bound cytosolic protein, INrf2 (inhibitor of Nrf2) or Keap1 (Kelch-like ECH-associated protein 1) (16 -18). INrf2 functions as a substrate adaptor protein for a Cullin 3 (Cul3)-dependent ubiquitin-protein ligase complex to maintain the steady-state levels of Nrf2 (19). Covalent conjugation of proteins by ubiquitin usually involve three enzymatic activities for activating (E1), conjugating (E2), and ligating (E3) ubiquitin to a substrate (20). In this case, Nrf2 serves as the substrate, while Cul3 serves as a scaffold protein that forms the E3 ligase complex with Ring Box1 (Rbx1) that recruits a cognate E2 enzyme (6). INrf2, via its N-terminal BTB/POZ domain, binds to Cul3 (20, 22) and via its C-terminal Kelch domain binds to the substrate Nrf2, leading to the ubiquitination and subsequent degradation of Nrf2 through the 26 S proteasome (23-27).Cellular exposure to oxidative stress leads to dissociation of Nrf2 from the INrf2/Cul3-Rbx1 complex (1, 5-7). Nrf2 escapes proteolysis and stabilizes, translocates into the nucleus, and causes activation of ARE-mediated genes leading to cytoprotection. Several reports suggest that persistent accumulation of Nrf2 in the nucleus is harmful. Nrf2 regulates the expression of several multidrug resistance-associated protein (MRP) efflux transporters in responses to oxidative stress (28) which could lead to chemotherapeutic drug resistance. INrf2-null mice demonstrated persistent accumulation of Nrf2 in the nucleus that led to postnatal death from malnutrition resulting from hyperkeratosis in the esophagus and forestomach (29

Src Subfamily Kinases Regulate Nuclear Export and Degradation of Transcription Factor Nrf2 to Switch Off Nrf2-mediated Antioxidant Activation of Cytoprotective Gene Expression

“…Therefore, it appears that cells contain mechanisms that autoregulate cellular abundance of Nrf2 (27,28). Based on the reported studies, it is suggested that the Nrf2 up-regulation of ARE-mediated gene expression is an early response to antioxidants (1,2).…”

Dynamics of unfolded protein response in recombinant CHO cells

“…It is induced on day 3 in HP and reaches a maximum of 8.2-folds on day 6. NRF2 is is phosphorylated by PERK, and it later auto-regulates its own mRNA expression (Kaspar and Jaiswal 2010;Stępkowski and Kruszewski 2011). NRF2 also induces the expression of anti-oxidant responsive genes (Chen and Kunsch 2004).…”

Cardiomyocyte-specific perilipin 5 overexpression leads to myocardial steatosis and modest cardiac dysfunction

“…Immunoblot analyses were performed as previously described ( 23 ). For whole extract, 2 mg equivalents of heart tissue homogenates were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and probed with specifi c antibodies against Plin2-5 and Nrf2 ( 23,27 ). GAPDH (1:1000) (Cell Signaling, Beverly, MA) and ␤ -actin (1:5000) (Sigma, St. Louis, MO) were used as the housekeeping genes.…”