## 3 Keep1-Nrf2-ARE Signaling System  
Nrf2 is a member of the Cap 'n' collar transcription factor family that contains a distinct CNC domain that is connected to the N-terminal side of a greatly protected leucine zipper (bZIP) motif. Nrf2 performs its function through pairing with other bZip proteins, such as small MAF proteins, facilitating dimerization, DNA binding, and nuclear export-import. Nrf2 modulates antioxidant and detoxification gene expression and has been established as a tumor suppressor [53]. In different experiments, Nrf2-knockout mice demonstrated enhanced carcinogen susceptibility and increased metastasis of the lung, which was guided by elevated ROS levels [54,55]. Keep1, the major Nrf2 repressor, is an adaptor subunit of Cullin3-based E3 ubiquitin ligase that works as a sensor element for oxidative stresses [56]. The Keep1-Nrf2-ARE signaling system is critical for maintaining a healthy balance between the ROS and the redox state of cells [57]. Nrf2 binds to the endogenous AREs to form the Nrf2-ARE complex, which needs to be activated when needed. Activity of this complex is restricted during the period in which it binds with Keep1 [58]. Under basal conditions, the Nrf2-Keap1 complex is anchored to cytosolic actin [59] and allows the Nrf2 to react with ubiquitin. Ubiquitination subsequently hydrolyzes it to its subcellular organelles and halt DNA transcription [60]. On the contrary, the ubiquitination can be prevented by breaking down the Nrf2-Keap1 complex [58, 59, 60]. This usually happens when cells undergo oxidative stress due to the presence of excessive intracellular ROS. Here, Keep1 works as a redox sensor, as it contains a sulfhydryl group in the cysteine residues that can sense oxidative stress. Due to the electrophilic nature of ROS, they tend to react with the cysteine residues of Keep1 protein, and subsequently break the Keep1-Nrf2 complex. Free Nrf2 becomes phosphorylated at Ser40 and translocates into the nucleus from cytosol to bind with the ARE region of DNA [61]. Thus, it activates the transcription of genes encoding for antioxidant elements and accordingly triggers several downstream enzymes including HO-1, thioredoxin (TXN), thioredoxin reductase (TXNRD), superoxide dismutase (SOD), NQO1, glutathione reductase (GR), GCL, glutathione-S-transferase (GST), UDP-glucuronosyltransferase(UGT), etc., [62,63]. Therefore, this overall system is known as the Keep1-Nrf2-ARE signaling system. Over 500 genes are regulated by the Nrf2-ARE complex, including phase I and II detoxification enzymes, proteasome subunits, transport proteins, growth factors and some other transcription factors [64]. The indirect activation of the Nrf2 signaling pathway involves stress-response protein kinases including PKC, mitogen activated protein kinase (MAPK) cascade, PI3K, extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), etc., [65, 66, 67, 68, 69]. Transcription factors like heat shock factor 1 (Hsf1), p53, p65, nuclear factor-\(\kappa\)B (NF-\(\kappa\)B), peroxisome proliferator activated receptor \(\gamma\) (PPAR\(\gamma\)), etc., are either positively or negatively involved in the regulation of Nrf2 activity [70, 71, 72, 73, 74]. A new but poorly studied Nrf2 activation mechanism has been established of late, the non-canonical pathway, where proteins like p62, p21, dipeptidyl peptidase III (DPP3), wilms tumor gene on X chromosome (WTX), etc., abrogate the Nrf2-Keap1 complex by directly interacting with Keep1, in turn disrupts Nrf2 ubiquitination and thereby facilitates Nrf2 nuclear translocation and its subsequent activation [75]. Figure 3 represents how Nrf2 works intracellularly under both basal and stress conditions, and Figure 4 summarizes the factors that are involved in Nrf2 regulation. The \(\alpha\),\(\beta\)-unsaturated carbonyl group in ZB forms a covalent bond with Keap1 to activate Nrf2 [77]. ZB suppressed the migration and invasion of colorectal cancer cells in vitro by inhibiting the focal adhesion kinase (FAK)/PI3K/NF-kB signaling pathway [78].