Article

Proteomic Analysis of Ubiquitin Ligase KEAP1 Reveals Associated Proteins That Inhibit NRF2 Ubiquitination

Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill.
Cancer Research (Impact Factor: 9.33). 02/2013; 73(7). DOI: 10.1158/0008-5472.CAN-12-4400
Source: PubMed

ABSTRACT

Somatic mutations in the KEAP1 ubiquitin ligase or its substrate NRF2 (NFE2L2) commonly occur in human cancer, resulting in constitutive NRF2-mediated transcription of cytoprotective genes. However, many tumors display high NRF2 activity in the absence of mutation, supporting alternative mechanisms of pathway activation. Previously, we and others discovered that via a competitive binding mechanism, the proteins WTX (AMER1), PALB2 and SQSTM1 bind KEAP1 to activate NRF2. Proteomic analysis of the KEAP1 protein interaction network revealed a significant enrichment of associated proteins containing an ETGE amino acid motif, which matches the KEAP1 interaction motif found in NRF2. Like WTX, PALB2, and SQSTM1, we found that the dipeptidyl peptidase 3 (DPP3) protein binds KEAP1 via an 'ETGE' motif to displace NRF2, thus inhibiting NRF2 ubiquitination and driving NRF2-dependent transcription. Comparing the spectrum of KEAP1 interacting proteins with the genomic profile of 178 squamous cell lung carcinomas characterized by The Cancer Genome Atlas revealed amplification and mRNA over-expression of the DPP3 gene in tumors that have high NRF2 activity but lacking NRF2 stabilizing mutations. We further show that tumor-derived mutations in KEAP1 are hypomorphic with respect to NRF2 inhibition and that DPP3 over-expression in the presence of these mutants further promotes NRF2 activation. Collectively, our findings support the competition model of NRF2 activation and suggest that 'ETGE'-containing proteins like DPP3 contribute to NRF2 activity in cancer.

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    • "its substrates consisting of three or more amino acids [6]. DPP III is involved in the endogenous defense against oxidative stress, being a part of Nrf-2/Keap-1 signaling pathway [7] [8]. In mammalian tissues, DPP III is ubiquitously distributed and thought to contribute to the final steps of normal intracellular protein catabolism [9]. "
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    ABSTRACT: A number of age-related diseases have a low incidence in females, which is attributed to a protective effect of sex hormones. For instance, the female sex hormone estrogen (E2) has a well established cytoprotective effect against oxidative stress, which strongly contributes to ageing. However, the mechanism by which E2 exerts its protective activity remains elusive.
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    • "NRF2 stability is also regulated by the CR6-interacting factor 1 (CRIF1) under both reducing and oxidative stress conditions (Kang et al. 2010) and the glycogen synthase kinase 3b (GSK3b)/b-transducin repeat-containing protein (b-TrCP) axis (Chowdhry et al. 2013; Rada et al. 2011; Rada et al. 2012). It has been reported that stability of NRF2 is also regulated by competitive protein–protein interaction to inhibit NRF2-KEAP1 binding by various proteins such as p21 (Chen et al. 2009), the Wilms tumor gene on X chromosome (WTX) (Camp et al. 2012), p62 (Komatsu et al. 2010), the partner and localizer of BRCA2 (PALB2) (Ma et al. 2012), the dipeptidyl peptidase III (DPP3) (Hast et al. 2013), and the breast cancer susceptibility gene 1 (BRCA1) (Gorrini et al. 2013). NRF2 functions as either a protector against tumorigenesis or oncogene (DeNicola et al. 2011; Kensler and Wakabayashi 2010; Loboda et al. 2008; Muller and Hengstermann 2012). "
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    ABSTRACT: NRF2 is a nuclear transcription factor activated in response to oxidative stress and related with metabolizing of xenotoxic materials and ABC transporter mediated drug resistance. We studied the expression of mRNAs under the siRNA-mediated knockdown of NRF2 and tBHQ-treated condition in AsPC-1 metastatic pancreatic cancer cell line to understand the AsPC-1 specific role(s) of NRF2 and further to investigate the relationship between drug resistance and metastatic plasticity and mobility of AsPc1. Here we show that the genes of aldo–keto reductases, cytochrome P450 family, aldehyde dehydrogenase, thioredoxin reductase, ABC transporter and epoxide hydrolase responsible for drug metabolism or oxidative stress concisely responded to NRF2 stabilization and knockdown of NRF2. In addition the expression of PIR, a candidate of oncogene and KISS1, a suppressor of metastasis were affected by NRF2 stabilization and knockdown. Our result provide comprehensive understanding of NRF2 target genes of drug response, oxidative stress response and metastasis in AsPc-1 metastatic pancreatic cancer cell line. Electronic supplementary material The online version of this article (doi:10.1007/s13258-014-0253-2) contains supplementary material, which is available to authorized users.
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    • "This interaction represents a potential interface between chromatin regulation and DNA repair, as detailed in section 3.2. In addition, PALB2 also binds KEAP1[73,74], which is a regulator of Nrf2, a transcription factor for antioxidant genes. The interaction of PALB2 and KEAP1 will be examined in depth in section 2.3. "
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