Keap1 Dependent Regulation of Nrf2

The Nrf2 protein is rapidly turned over; it has a short half-life under basal conditions (7–15 min) which is increased to 30–100 min in the presence of inducers [44] and [45]. The Nrf2 protein stability is primarily regulated by Keap1 [46] (Fig. 4). The ETGE and DLG motifs within Nrf2 interact with Keap1. Keap1 is a substrate adaptor protein for a Cullin 3 (Cul3)-dependent E3 ubiquitin ligase complex; hence binding of Keap1 with Nrf2 mediates ubiquitination and subsequent proteasomal degradation of Nrf2 [41], [46], [47], [48], [49] and [50].

 

Fig. 4. 

Keap1 dependent regulation of Nrf2. Under basal conditions, Nrf2 is sequestered in the cytosol by a Keap1 homodimer, which facilitates the ubiquitination and proteasomal degradation of Nrf2. Inducers react with specific cysteine residues in Keap1, leading to the release of Nrf2 and allowing its nuclear translocation. In the nucleus, Nrf2 heterodimerizes with small Maf proteins and binds to the antioxidant response element (ARE), activating the expression of a battery of cytoprotective genes.

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Sulfhydryl groups within Keap1 act as sensors for electrophiles and oxidants [51]. In the presence of reactive oxygen species, critical cysteine residues in Keap1 become oxidized leading to a conformational change of Keap1, which prevents its binding to Nrf2. As a consequence, Nrf2 ubiquitination and degradation is stopped, and its nuclear translocation promoted.

Among the 27 cysteine residues within Keap1, Cys151 seems to be the most critical sensor residue which mediates the formation of an intermolecular disulfide – Cys151–Cys151 – between two Keap1 molecules. Thus, in response to a diverse array of stimuli, redox-sensitive residues, especially Cys151 within Keap1, can be covalently modified, allowing Nrf2 to evade Keap1-mediated ubiquitination [52] and [53]. As a result, Nrf2 accumulates in the nucleus and heterodimerizes with a small Maf protein. The Nrf2-small Maf heterodimer binds to regulatory gene regions known as ARE sites with a consensus sequence 5′-TGACnnnGC-3′, also called electrophile response elements (EpRE) [54]. Substances able to induce a response at the ARE are derived from a variety of sources, including phytochemicals and derivatives such as genistein, quercetin, curcumin and sulforaphane; therapeutics such as oltipraz, auranofin and acetaminophen; environmental agents like paraquat and metals and endogenous inducers such as nitric oxide, nitro-fatty acids, hydrogen peroxide and 4-hydroxynonenal [55]. Nrf2 target genes mainly

 

Berberine;

 

BBR also activated nuclear erythroid 2-related factor 2 (Nrf2),

the key antioxidative transcription factor, which is accompanied with up-regulation of hemeoxygenase-1 (HO-1).

Furthermore, BBR markedly enhanced nerve growth factor (NGF) expression and promoted neurite outgrowth in high glucose-treated cells.

 

To further determine the role of the Nrf2 in BBR neuroprotection, RNA interference directed against Nrf2 was used. Results indicated Nrf2 siRNA abolished BBR-induced HO-1, NGF, neurite outgrowth and ROS decrease.

 

In conclusion, BBR attenuated high glucose-induced neurotoxicity, and we are the first to reveal this novel mechanism of BBR as an Nrf2 activator against glucose neurotoxicity, providing another potential therapeutic use of BBR on the treatment of diabetic complications.

 

 

 

Epalrestat As Nfr2 Activator 2015

 

Highlights

 

•

Epalrestat increases GSH levels in endothelial cells through transcription regulation.

•

Epalrestat increases Trx and HO-1 levels.

•

Nrf2 activation by epalrestat is involved in increases in GSH, Trx, and HO-1 levels.

•

Epalrestat enhances endothelial cell resistance to oxidative stress.

---

Abstract

Epalrestat (EPS) is the only aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy.

 

Recently, we found that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH) in rat Schwann cells. GSH plays a crucial role in protecting endothelial cells from oxidative stress, thereby preventing vascular diseases.

 

 

Here we show that EPS increases GSH levels in not only Schwann cells but also endothelial cells. Treatment of bovine aortic endothelial cells (BAECs), an in vitro model of the vascular endothelium, with EPS caused a dramatic increase in intracellular GSH levels.

 

 

This was concomitant with the up-regulation of glutamate cysteine ligase, an enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis. Moreover, EPS stimulated the expression of thioredoxin and heme oxygenase-1, which have important redox regulatory functions in endothelial cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that regulates the expression of antioxidant genes. EPS increased nuclear Nrf2 levels in BAECs. Nrf2 knockdown by siRNA suppressed the EPS-induced glutamate cysteine ligase, thioredoxin-1, and heme oxygenase-1 expression.

 

 

Interestingly, LY294002, an inhibitor of phosphatidylinositol 3-kinase, abolished the EPS-stimulated GSH synthesis, suggesting that the kinase is associated with Nrf2 activation induced by EPS.

 

 

Furthermore, EPS reduced the cytotoxicity induced by H2O2 and tert-butylhydroperoxide, indicating that EPS plays a role in protecting cells from oxidative stress.

 

 

Taken together, the results provide evidence that EPS exerts new beneficial effects on endothelial cells by increasing GSH, thioredoxin, and heme oxygenase-1 levels through the activation of Nrf2.

 

 

We suggest that EPS has the potential to prevent several vascular diseases caused by oxidative stress.

Nrf2 Activator As Meuroprotective Agent

Synthesis of Piperlongumine Analogues and Discovery of Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) Activators as Potential Neuroprotective Agents

Shoujiao Peng, Baoxin Zhang, Xianke Meng, Juan Yao, and Jianguo Fang^*

 

J. Med. Chem., 2015, 58 (13), pp 5242–5255

DOI: 10.1021/acs.jmedchem.5b00410

 

Abstract

 

 

 

The cellular antioxidant system plays key roles in blocking or retarding the pathogenesis of adult neurodegenerative disorders as elevated oxidative stress has been implicated in the pathophysiology of such diseases. Molecules with the ability in enhancing the antioxidant defense thus are promising candidates as neuroprotective agents. We reported herein the synthesis of piperlongumine analogues and evaluation of their cytoprotection against hydrogen peroxide- and 6-hydroxydopamine-induced neuronal cell oxidative damage in the neuron-like PC12 cells. The structure–activity relationship was delineated after the cytotoxicity and protection screening. Two compounds (4 and 5) displayed low cytotoxicity and confer potent protection of PC12 cells from the oxidative injury via upregulation of a panel of cellular antioxidant molecules. Genetically silencing the transcription factor Nrf2, a master regulator of the cellular stress responses, suppresses the cytoprotection, indicating the critical involvement of Nrf2 for the cellular action of compounds 4 and 5 in PC12 cells.

Details of the synthetic intermediates, in vitro free radical scavenging assay, HPLC chromatograms, and NMR spectra of the final compounds. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00410.

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