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In the realm of molecular biology, the Keap1-Nrf2 pathway holds significant importance as a key regulatory in cellular defense against oxidative stress and inflammation. This intricate signaling pathway plays a vital role in maintaining cellular homeostasis and has garnered substantial interest due to its potential therapeutic implications in various diseases.
Kelch-like ECH-associated protein 1 (KEAP1) is a highly conserved cysteine-rich protein found in mammals. It was predicted before its discovery and later identified as a negative regulator of nuclear factor erythroid 2-related factor 2 (NRF2). Human KEAP1 has 27 cysteines, whereas mouse KEAP1 has 25, some of which are located near basic amino acids. Cysteine is a unique amino acid with a sulfhydryl group that has multiple functions. It can form bonds with other cysteine molecules, bind to metals, and undergo oxidation when exposed to oxidants. The reactivity of cysteine is determined by its pKa value, with lower pKa values indicating higher reactivity. When basic amino acids are near cysteine, the pKa value decreases, increasing its reactivity. The discovery of highly reactive cysteines in KEAP1 as inducer sensors explains why inducers can be effective at low concentrations despite high levels of cellular glutathione.
KEAP1 is a member of the BTB-Kelch protein family and forms homodimers. Structurally, Keap1 consists of five distinct domains: the Broad Complex, Tramtrack, and Bric-a-Brac (BTB) domain, the intervening region (IVR), the double glycine repeat (DGR) domain, the Kelch-repeat domain, and the C-terminal region. These domains together contribute to Keap1's function in the regulation of Nrf2 activity. Keap1 contains numerous cysteine residues, and modification of three specific cysteines (C151, C273, and C288) have been shown to play a functional role by altering the conformation of Keap1, resulting in nuclear translocation of Nrf2 and subsequent target gene expression.
Figure 1. Structures of Nrf2 and Keap1 and the cysteine code.
(Source: Kansanen, E. et al., 2013)
The Keap1-Nrf2 pathway plays a crucial role in protecting cells from damage caused by reactive oxygen species (ROS) and electrophiles. It is the primary regulatory system for cytoprotective responses to both internal and external stresses. Nrf2, a transcription factor, binds to the antioxidant response element (ARE) along with small Maf proteins to activate target gene expression. Keap1, a repressor protein, binds to Nrf2 and promotes its degradation.
In normal conditions, two Keap1 proteins bind to Nrf2 and facilitate its degradation through polyubiquitination by the Cul3-based E3 ligase complex. This process leads to the rapid breakdown of Nrf2 by the proteasome. However, a small fraction of Nrf2 manages to escape this inhibitory complex and accumulates in the nucleus. In the nucleus, Nrf2 mediates the expression of genes involved in maintaining cellular balance through the Antioxidant Response Element (ARE).
During times of stress, certain inducers modify specific cysteine residues in Keap1. This modification prevents Nrf2 from being ubiquitinated and degraded by causing the dissociation of the inhibitory complex. There are two proposed models to explain how this occurs. In the hinge and latch model, modification of certain cysteines in Keap1 induces conformational changes, resulting in the detachment of the Nrf2 DLG motif from Keap1. While Nrf2 is no longer ubiquitinated, it still retains binding with the ETGE motif of Keap1. In the Keap1-Cul3 dissociation model, electrophiles disrupt the binding between Keap1 and Cul3. This disruption allows Nrf2 to escape the ubiquitination system.
Regardless of the model, inducer-modified Keap1 and bound Nrf2 become inactive. Consequently, newly synthesized Nrf2 bypasses Keap1 and translocates into the nucleus. There, it binds to the ARE and drives the expression of target genes associated with Nrf2, such as heme oxygenase 1 (HMOX1), NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione S transferases (GSTs), and glutamate-cysteine ligase (GCL). These enzymes play a crucial role in neutralizing ROS and maintaining redox homeostasis within the cell. By enhancing the cellular antioxidant capacity, the Keap1-Nrf2 pathway helps to prevent oxidative damage and mitigate the risk of various oxidative stress-related diseases, including neurodegenerative disorders, cardiovascular diseases, and cancer.
The KEAP1-NRF2 system has garnered significant attention in both basic and clinical cancer research due to the dual role of NRF2 as an oncogene and tumor suppressor, depending on the specific context. NRF2-addicted cancers, which exhibit high levels of NRF2 expression, are particularly intriguing. In these cancers, somatic mutations in NRF2 or KEAP1 genes lead to the stabilization and accumulation of NRF2 protein. By exploiting NRF2's natural cytoprotective functions, including antioxidative and anti-electrophilic responses, as well as metabolic reprogramming, NRF2-addicted cancers gain a distinct advantage in surviving under challenging microenvironments.
Consequently, inhibiting NRF2 activity holds promise as a therapeutic strategy for patients with NRF2-addicted cancers, as it can disrupt the cancer cells' reliance on NRF2-mediated cytoprotection. On the other hand, it is worth noting that NRF2 activation in immune cells within the host can significantly suppress cancer cell growth. This suggests that NRF2 inducers might also have therapeutic potential for treating certain cancers.
References
| Target | Cat. No. | Product Name | Expression System | Tag/Conjugate | Application | |
| KEAP1 | DAG-KO379 | KEAP1 Knockout Cell Lysate | WB | Inquiry | ||
| HMOX1 | DAG-KO197 | HMOX1 Knockout Cell Lysate | WB | Inquiry | ||
| NQO1 | DAG-P1785 | Human NQO1 peptide | N/A | Unconjugated | ELISA | Inquiry |
| CDBP2087 | Human NQO1 blocking peptide | N/A | Unconjugated | Apuri, BL, ELISA | Inquiry | |
| GST | GST-01 | GST (Glutathione S-Transferase) | E. coli | Unconjugated | Immunogen, Control | Inquiry |
| DAG1663 | Recombinant GST (a.a. 1-242) | Wheat | Unconjugated | WB | Inquiry | |
| Cysteine | DAG3280 | L-Cysteine [G-BSA] | N/A | G-BSA | IHC, ICC | Inquiry |
| Target | Cat. No. | Product Name | Size | Species Reactivity | Application | Detection Sample | |
| KEAP1 | DEIA-FN758 | Human KEAP1 (kelch-like ECH-associated protein 1) ELISA Kit | 96T | Quantitative | serum, plasma, cell culture supernatants, tissue homogenate | Inquiry | |
| HMOX1 | DEIA-XY2257 | Human/Mouse Total HO-1/HMOX1 ELISA Kit | 96T | Qualitative | cultured cells | Inquiry | |
| DEIA-BJ2866 | Canine Heme Oxygenase 1, Decycling ELISA Kit | 96T | Quantitative | Serum, plasma, cell culture supernatants, body fluid and tissue homogenate | Inquiry | ||
| DEIA3574 | Human HO1(Heme Oxygenase 1) ELISA Kit | 96T | Human | Quantitative | Serum, plasma, tissue homogenates and other biological fluids. | Inquiry | |
| DEIA3575 | Rat HO-1(Heme Oxygenase 1) ELISA Kit | 96T | Rat | Quantitative | Serum, plasma, tissue homogenates and other biological fluids. | Inquiry | |
| DEIA6384 | Human HO-1 ELISA Kit | 96T | Human | Quantitative | lysates, tissues extracts | Inquiry | |
| NQO1 | ABPR-0631 | Human NQO1 ELISA Matched Antibody Pair | ELISA | Inquiry | |||
| DEIA8465 | Human NQO1(NAD(P)H dehydrogenase [quinone] 1) ELISA Kit | 96T | Human | Quantitative | Serum, plasma, tissue homogenates and other biological fluids | Inquiry | |
| DEIA7470 | Human NQO1 ELISA Kit | 96T | Human | Quantitative | Serum, plasma, tissue homogenates and other biological fluids. | Inquiry |
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