Direct and Specific Functional Evaluation of the Nrf2 and MafG Heterodimer by Introducing a Tethered Dimer into Small Maf-Deficient Cells

Abstract

A group of cytoprotective genes is regulated by heterodimers composed of the cap’n’collar (CNC) family member Nrf2 and one of the small Maf (sMaf) proteins (MafF, MafG, or MafK) through the antioxidant response element (ARE, also referred to as the CNC-sMaf binding element [CsMBE]). Many lines of evidence support this model; however, a direct and specific evaluation of the Nrf2-sMaf heterodimer remains to be executed. To address this issue, we constructed a tethered Nrf2-MafG (T-N2G) heterodimer using a flexible linker peptide. We then introduced the T-N2G construct into cells lacking all three sMaf proteins to specifically evaluate the function of the tethered heterodimer without interference from other endogenous CNC-sMaf heterodimers or sMaf homodimers. In response to an Nrf2 activator, diethyl maleate, the T-N2G protein can widely activate the target genes of Nrf2 but not those of Nrf1, such as proteasome subunit genes. Genome-wide binding analysis showed that the T-N2G protein preferentially bound to the CsMBE motifs in the regulatory regions of the Nrf2 target genes. These results provide direct evidence that the Nrf2-MafG heterodimer acts as a transcriptional activator of Nrf2-dependent genes and show that this assay system will be a powerful tool to specifically examine the function of other CNC-sMaf heterodimers.

The Molecular Mechanisms Regulating the KEAP1-NRF2 Pathway

“…The global analysis of gene expression in different model systems confirmed that NRF2 directly regulates both the basal and inducible expression of a broad range of genes involved in the oxidative stress response (Table 2) (24,(32)(33)(34)(35)(36)(37). Furthermore, a comparison of chromatin immunoprecipitation sequencing (ChIP-Seq) analyses carried out on the CNC transcription factors revealed that although all family members bind to the same TGACNNNGC consensus motif, they mostly regulate the expression of nonoverlapping sets of genes, confirming the specific function of NRF2 in the oxidative stress response (38)(39)(40)(41)(42)(43)(44)(45).…”

Pyrroloquinoline quinone regulates the redox status in vitro and in vivo of weaned pigs via the Nrf2/HO-1 pathway

“…The Nrf2 pathway is a master regulator of cell resistance to oxidative injury [ 53 ] Keap1 is a cytoplasmic junction protein that may downregulate Nrf2. Normally, under physiological conditions, of low the activity of Nrf2 activity is maintained at a low level via its distribution in the cytoplasm in the Nrf2-keap1 complex [ 54 ]. After activation, Nrf2 is phosphorylated, dissociates from the complex, and is then released to translocate into the nucleus, where it induces downstream expression of HO-1 and SOD, which eliminate ROS [ 55 ].…”

Pyrroloquinoline quinone regulates the redox status in vitro and vivo of weaned pigs via the Nrf2/HO-1 pathway

“…The Nrf2 pathway is a master regulator of cell resistance to oxidative injury [53] Keap1 is a cytoplasmic junction protein that may downregulate Nrf2. Normally, under physiological conditions, of low the activity of Nrf2 activity is maintained at a low level via its distribution in the cytoplasm in the Nrf2-keap1 complex [54]. After activation, Nrf2 is phosphorylated, dissociates from the complex, and is then released to translocate into the nucleus, where it induces downstream expression of HO-1 and SOD, which eliminate ROS [55].…”