+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

Critical cysteine residues of Kelch-like ECH-associated protein 1 in arsenic sensing and suppression of nuclear factor erythroid 2-related factor 2



Critical cysteine residues of Kelch-like ECH-associated protein 1 in arsenic sensing and suppression of nuclear factor erythroid 2-related factor 2



Journal of Pharmacology and Experimental Therapeutics 332(1): 66-75



Arsenic activates nuclear factor erythroid 2-related factor 2 (Nrf2) to induce phase II and antioxidative genes. Here we analyzed arsenic-Kelch-like ECH-associated protein 1 (Keap1) cysteine thiol interaction in Nrf2 activation. Arsenic-based Nrf2 activators, fluorescent biarsenical labeling reagent (FlAsH) and phenylarsine oxide (PAO), were used to probe binding of arsenic to Keap1. Strong fluorescence was observed on binding of FlAsH to purified Keap1. Pretreatment with arsenic, tert-butylhydroquinone (tBHQ), or 2,3-dimercaptopropanol significantly reduced the fluorescent signal. PAO affinity beads effectively pulled down Keap1 in vitro and from hepa1c1c7 cells. Arsenic, tBHQ, free PAO, or cadmium blocked Keap1 pulldown. Furthermore, arsenic and free PAO significantly reduced the free thiol contents of purified or endogenous Keap1. Thus, arsenic, FlAsH, and PAO, as well as tBHQ and cadmium, bind to Keap1 cysteine thiols in a similar fashion. All the domains of Keap1 bound PAO, and the linker region exhibited the highest binding activity. The function of arsenic-Keap1 interaction was evaluated in a reconstituted system that mimics endogenous Nrf2 regulation. Mutation of Cys273 or Cys288 in the linker region resulted in high level basal expression of Nrf2 protein. Mutation of Cys151 abolished Nrf2 activation by arsenic. Overexpression of C273A, C288A, or C151A altered the basal and arsenic-induced expression of Nrf2 target genes. The study shows an important role of Cys273 and Cys288 in the suppression of Nrf2 by Keap1 and a critical function of Cys151 in arsenic responsiveness. Our findings support a model in which arsenic binds to different sets of Keap1 cysteine residues to regulate divergent functions in Nrf2 signal transduction.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 052385724

Download citation: RISBibTeXText

PMID: 19808700

DOI: 10.1124/jpet.109.160465


Related references

Expression of UDP-glucuronosyltransferase 1A, nuclear factor erythroid-E2-related factor 2 and Kelch-like ECH-associated protein 1 in colonic mucosa, adenoma and adenocarcinoma tissue. Oncology Letters 4(5): 925-930, 2012

Discovery and Development of Kelch-like ECH-Associated Protein 1. Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1:NRF2) Protein-Protein Interaction Inhibitors: Achievements, Challenges, and Future Directions. Journal of Medicinal Chemistry 59(24): 10837-10858, 2016

Monoacidic Inhibitors of the Kelch-like ECH-Associated Protein 1: Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1:NRF2) Protein-Protein Interaction with High Cell Potency Identified by Fragment-Based Discovery. Journal of Medicinal Chemistry 59(8): 3991-4006, 2016

Overexpression of miR-200a protects cardiomyocytes against hypoxia-induced apoptosis by modulating the kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 signaling axis. International Journal of Molecular Medicine 38(4): 1303-1311, 2016

Non-covalent Small-Molecule Kelch-like ECH-Associated Protein 1-Nuclear Factor Erythroid 2-Related Factor 2 (Keap1-Nrf2) Inhibitors and Their Potential for Targeting Central Nervous System Diseases. Journal of Medicinal Chemistry 61(18): 8088-8103, 2018

Induction of metallothionein I by arsenic via metal-activated transcription factor 1: critical role of C-terminal cysteine residues in arsenic sensing. Journal of Biological Chemistry 284(19): 12609-12621, 2009

Structure-Activity and Structure-Conformation Relationships of Aryl Propionic Acid Inhibitors of the Kelch-like ECH-Associated Protein 1/Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1/NRF2) Protein-Protein Interaction. Journal of Medicinal Chemistry 62(9): 4683-4702, 2019

Kelch-like ECH-associated protein 1 (KEAP1) differentially regulates nuclear factor erythroid-2-related factors 1 and 2 (NRF1 and NRF2). Journal of Biological Chemistry 293(6): 2029-2040, 2018

NRF2 cysteine residues are critical for oxidant/electrophile-sensing, Kelch-like ECH-associated protein-1-dependent ubiquitination-proteasomal degradation, and transcription activation. Molecular Pharmacology 76(6): 1265-1278, 2009

Nuclear factor erythroid 2-related factor gene variants and susceptibility of arsenic-related skin lesions. Human and Experimental Toxicology 33(6): 582-589, 2014

Nobiletin inhibits breast cancer via p38 mitogen-activated protein kinase, nuclear transcription factor-κB, and nuclear factor erythroid 2-related factor 2 pathways in MCF-7 cells. Food and Nutrition Research 62:, 2018

Human age‑related cataracts: epigenetic suppression of the nuclear factor erythroid 2‑related factor 2‑mediated antioxidant system. Molecular Medicine Reports 11(2): 1442-1447, 2015

Osthole protects against inflammation in a rat model of chronic kidney failure via suppression of nuclear factor-κB, transforming growth factor-β1 and activation of phosphoinositide 3-kinase/protein kinase B/nuclear factor (erythroid-derived 2)-like 2 signaling. Molecular Medicine Reports 16(4): 4915-4921, 2017

Kelch-like ECH-associated Protein 1-dependent Nuclear Factor-E2-related Factor 2 Activation in Relation to Antioxidation Induced by Sevoflurane Preconditioning. Anesthesiology 126(3): 507-521, 2017

Replacement of a Naphthalene Scaffold in Kelch-like ECH-Associated Protein 1 (KEAP1)/Nuclear Factor (Erythroid-derived 2)-like 2 (NRF2) Inhibitors. Journal of Medicinal Chemistry 61(17): 8029-8047, 2018