Transcription factor NF-E2 p45-related factor 2 (Nrf2) orchestrates defenses against oxidants and thiol-reactive electrophiles. It is controlled at the protein stability level by several E3 ubiquitin ligases (CRL3^Keap1, CRL4^DCAF11, SCF^β-TrCP, and Hrd1). CRL3^Keap1 is of the greatest importance because it constitutively targets Nrf2 for proteasomal degradation under homeostatic conditions but is prevented from doing so by oxidative stressors. Repression of Nrf2 by CRL3^Keap1 is attenuated by SQSTM1/p62, and this is reinforced by phosphorylation of SQSTM1/p62. Repression by SCF^β-TrCP requires phosphorylation of Nrf2 by GSK3, the activity of which is inhibited by PKB/Akt and other kinases. We discuss how Nrf2 activity is controlled by the ubiquitin ligases under different circumstances. We also describe endogenous signaling molecules that inactivate CRL3^Keap1 to alleviate stress and restore homeostasis.

Cruciferous vegetables are rich sources of glucosinolates which are the biogenic precursor molecules of isothiocyanates (ITCs). The relationship between the consumption of cruciferous vegetables and chemoprotection has been widely documented in epidemiological studies. Phenethyl isothiocyanate (PEITC) occurs as its glucosinolate precursor gluconasturtiin in the cruciferous vegetable watercress (Nasturtium officinale). PEITC has multiple biological effects, including activation of cytoprotective pathways, such as those mediated by the transcription factor nuclear factor erythroid 2 p45‐related factor 2 (NRF2) and the transcription factor heat shock factor 1 (HSF1), and can cause changes in the epigenome. However, at high concentrations, PEITC leads to accumulation of reactive oxygen species and cytoskeletal changes, resulting in cytotoxicity. Underlying these activities is the sulfhydryl reactivity of PEITC with cysteine residues in its protein targets. This chemical reactivity highlights the critical importance of the dose of PEITC for achieving on‐target selectivity, which should be carefully considered in the design of future clinical trials.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease that leads to skin fibrosis. Altered metabolism has recently been described in autoimmune diseases and SSc. Itaconate is a product of the Krebs cycle intermediate cis-aconitate and is an immunomodulator. This work examines the role of the cell-permeable derivative of itaconate, 4-octyl itaconate (4-OI), in SSc. SSc and healthy dermal fibroblasts were exposed to 4-OI. The levels of collagen Nrf2-target genes and pro-inflammatory cytokines interleukin 6 (IL-6) and monocyte chemotactic protein 1 (MCP-1) were determined. Levels of reactive oxygen species (ROS) as well as the gene expression of collagen and Cellular Communication Network Factor 2 (CCN2) were measured after transforming growth factor beta 1 (TGF-β1) stimulation in the presence or absence of 4-OI. Wild-type or Nrf2-knockout (Nrf2-KO) mouse embryonic fibroblasts (MEFs) were also treated with 4-OI to determine the role of Nrf2 in 4-OI-mediated effects. 4-OI reduced the levels of collagen in SSc dermal fibroblasts. Incubation with 4-OI led to activation of Nrf2 and its target genes heme oxygenase 1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). 4-OI activated antioxidant response element (ARE)-dependent gene expression, reduced inflammatory cytokine release and reduced TGF-β1-induced collagen and ROS production in dermal fibroblasts. The effects of 4-OI are dependent on Nrf2. The cell-permeable derivative of itaconate 4-OI is anti-fibrotic through upregulation of Nrf2 and could be a potential therapeutic option in an intractable disease.

The evolutionary conserved non-heme Fe-containing protein pirin has been implicated as an important factor in cell proliferation, migration, invasion, and tumour progression of melanoma, breast, lung, cervical, prostate, and oral cancers. Here we found that pirin is overexpressed in human colorectal cancer in comparison with matched normal tissue. The overexpression of pirin correlates with activation of transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2) and increased expression of the classical Nrf2 target NAD(P)H:quinone oxidoreductase 1 (NQO1), but interestingly and unexpectedly, not with expression of the aldo-keto reductase (AKR) family members AKR1B10 and AKR1C1, which are considered to be the most overexpressed genes in response to Nrf2 activation in humans. Using pharmacologic and genetic approaches to either downregulate or upregulate Nrf2, we show that pirin is regulated by Nrf2 in human and mouse cells and in the mouse colon in vivo. The small molecule pirin inhibitor TPhA decreased the viability of human colorectal cancer (DLD1) cells, but this decrease was independent of the levels of pirin. Our study demonstrates the Nrf2-dependent regulation of pirin and encourages the pursuit for specific pirin inhibitors.

Pharmacologic inhibition of the protein-protein interaction (PPI) interface of the Keap1:Nrf2 complex, which leads to Nrf2 activation and cytoprotective gene expression, offers a promising strategy for disease prevention and treatment. To facilitate identification and validation of small-molecule Keap1:Nrf2 PPI inhibitors in the cellular environment in a low- and medium-throughput manner, we detail two adapted cellular thermal shift assay (CETSA) protocols, Keap1-CETSA, an immunoblotting-based methodology for detecting endogenous Keap1, and Keap1-Glow CETSA, a microtiter plate assay of overexpressed fluorescently-tagged Keap1.

Keap1-Kelch-targeting protein–protein interaction inhibitors (left) increase the thermostability of Keap1-mCherry, whereas double Michael acceptors (right) decrease the thermostability of Keap1-mCherry in the cellular environment.