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

ArticleinCancer Research 73(7) · February 2013with19 Reads
DOI: 10.1158/0008-5472.CAN-12-4400 · Source: PubMed
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.
    • "In addition, several studies indicated a role for DPP III in the mammalian pain-modulatory system (Chiba et al., 2003; Baršun et al., 2007 ), and its participation in the endogenous defense against oxidative stress is well supported (Liu et al., 2007). It has been shown that DPP III is an activator in the Keap1-Nrf2 signaling pathway (Hast et al., 2013), which is the major regulator of cytoprotective responses to oxidative and electrophilic stress (Kansanen et al., 2013). In search for plant homologues of the M49 family, we have found a predicted protein of 770 amino acids from the moss Physcomitrella patens (UniProtKB code: A9TLP4), and an Arabidopsis thaliana uncharacterized protein of 772 amino acids, designated Nudix hydrolase 3 in UniProtKB (entry code Q8L831; gene: NUDT3). "
    [Show abstract] [Hide abstract] ABSTRACT: In a search for plant homologues of dipeptidyl peptidase III (DPP III) family, we found a predicted protein from the moss Physcomitrella patens (UniProt entry: A9TLP4), which shared 61% sequence identity with the Arabidopsis thaliana uncharacterized protein, designated Nudix hydrolase 3. Both proteins contained all conserved regions of the DPP III family, but instead of the characteristic hexapeptide HEXXGH zinc-binding motif, they possessed a pentapeptide HEXXH, and at the N-terminus, a Nudix box, a hallmark of Nudix hydrolases, known to act upon a variety of nucleoside diphosphate derivatives. To investigate their biochemical properties, we expressed heterologously and purified Physcomitrella (PpND) and Arabidopsis (AtND) protein. Both hydrolyzed, with comparable catalytic efficiency, the isopentenyl diphosphate (IPP), a universal precursor for the biosynthesis of isoprenoid compounds. In addition, PpND dephosphorylated four purine nucleotides (ADP, dGDP, dGTP, and 8-oxo-dATP) with strong preference for oxidized dATP. Furthermore, PpND and AtND showed DPP III activity against dipeptidyl-2-arylamide substrates, which they cleaved with different specificity. This is the first report of a dual activity enzyme, highly conserved in land plants, which catalyses the hydrolysis of a peptide bond and of a phosphate bond, acting both as a dipeptidyl peptidase III and an atypical Nudix hydrolase.
    Article · Jul 2016
    • "This uncontrolled activation may result in either constitutive or prolonged activation of Nrf2. The constitutive activation of Nrf2 observed in numerous cancer cell lines and patient tumor samples confers them a more malignant phenotype, as assessed by higher proliferative rates, increased migration, and chemo-or radio-resistance [15, 33, 34, 59, 70, 84, 91, 96] . Prolonged Nrf2 activation occurs when autophagy is dysregulated , either by deletion of Atg5 and Atg7, or when autophagosome-lysosome fusion is blocked by arsenic treatment , causing the formation of p62-Keap1 aggregates and Nrf2 activation [44]. "
    [Show abstract] [Hide abstract] ABSTRACT: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are the clinical manifestations of severe lung damage and respiratory failure. Characterized by severe inflammation and compromised lung function, ALI/ARDS result in very high mortality of affected individuals. Currently, there are no effective treatments for ALI/ARDS, and ironically, therapies intended to aid patients (specifically mechanical ventilation, MV) may aggravate the symptoms. Key events contributing to the development of ALI/ARDS are increased oxidative and proteotoxic stresses, unresolved inflammation, and compromised alveolar-capillary barrier function. Since the airways and lung tissues are constantly exposed to gaseous oxygen and airborne toxicants, the bronchial and alveolar epithelial cells are under higher oxidative stress than other tissues. Cellular protection against oxidative stress and xenobiotics is mainly conferred by Nrf2, a transcription factor that promotes the expression of genes that regulate oxidative stress, xenobiotic metabolism and excretion, inflammation, apoptosis, autophagy, and cellular bioenergetics. Numerous studies have demonstrated the importance of Nrf2 activation in the protection against ALI/ARDS, as pharmacological activation of Nrf2 prevents the occurrence or mitigates the severity of ALI/ARDS. Another promising new therapeutic strategy in the prevention and treatment of ALI/ARDS is the activation of autophagy, a bulk protein and organelle degradation pathway. In this review, we will discuss the strategy of concerted activation of Nrf2 and autophagy as a preventive and therapeutic intervention to ameliorate ALI/ARDS.
    Full-text · Article · Feb 2016
    • "Thus, the importance of GSH system relative to other components of the protective machinery must be taken into account. It has already been shown that enzymatic activity of human DPP III is not required for Keap-1 binding, and subsequent Nrf-2-dependent transcriptional activation, since enzymatically inactive mutant Y318F bound Keap1, and had similar effect on Nrf2-dependent transcription as well as the wild type DPP III [8]. In this study we have shown that E 2 administration exerted protective effect by lowering both LPO and GSSG and through enhancement of DPP III protein level in the cytosol, and its nuclear accumulation. "
    [Show abstract] [Hide abstract] 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. In this study we address the question whether the E2-induced protective effect against hyperoxia is mediated by the Nrf-2/Keap-1 signaling pathway. In particular, we investigate the E2-induced expression and cellular distribution of DPP III monozinc exopeptidase, a member of the Nrf-2/Keap-1 pathway, upon hyperoxia treatment. We find that DPP III accumulates in the nucleus in response to hyperoxia. Further, we show that combined induction of hyperoxia and E2 administration have an additive effect on the nuclear accumulation of DPP III. The level of nuclear accumulation of DPP III is comparable to nuclear accumulation of Nrf-2 in healthy female mice exposed to hyperoxia. In ovariectomized females exposed to hyperoxia, supplementation of E2 induced upregulation of DPP III, Ho-1, Sirt-1 and downregulation of Ppar-γ. While other cytoprotective mechanisms cannot be excluded, these findings demonstrate a prominent role of DPP III, along with Sirt-1, in the E2-mediated protection against hyperoxia.
    Full-text · Article · Jan 2016
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