NRP/B mutations impair Nrf2-dependent NQO1 induction in human primary brain tumors

Abstract

Brain tumors are associated with genetic alterations of oncogenes and tumor suppressor genes. Accumulation of reactive oxygen species (ROS) in cells leads to oxidative stress-induced damage, resulting in tumorigenesis. Here, we showed that the nuclear matrix protein nuclear restricted protein in brain (NRP/B) was colocalized and interacted with NF-E2-related factor 2 (Nrf2). During oxidative stress response, NRP/B expression and its interaction with Nrf2 were upregulated in SH-SY5Y cells. Association of NRP/B with Nrf2 was crucial for NAD(P)H:quinone oxidoreductase 1 (NQO1) expression. NRP/B was localized predominantly in the nucleus of normal brain cells, whereas in primary brain tumors NRP/B was almost exclusively contained in the cytoplasm. In addition, unlike wild-type NRP/B, the expression of NRP/B mutants isolated from primary brain tumors was found in the cytoplasm, and these mutants failed to induce Nrf2-dependent NQO1 transcription. Thus, NRP/B mutations and their altered localization resulted in changes in NRP/B function and deregulation of Nrf2-dependent NQO1 activation in brain tumors. This study provides insights into the mechanism by which the NRP/B modulates Nrf2-dependent NQO1 induction in cellular protection against ROS in brain tumors.

Nuclear Matrix Protein (NRP/B) Modulates the Nuclear Factor (Erythroid-derived 2)-related 2 (NRF2)-dependent Oxidative Stress Response

“…GST-NRF2 truncation proteins were expressed in Escherichia coli BL21(DE3) cells by induction with isopropyl 1-thio-␤-D-galactopyranoside at 37°C for 3 h, purified on gluthatione-Sepharose beads (GE Healthcare) according to the manufacturer's instructions, and stored at Ϫ80°C. FLAG-tagged constructs corresponding to the BTB domain, intervening sequence (IVS), BTB-IVS, and IVS regions of each of these domains were generated by PCR using a NRP/B cDNA template (17,20) and specific primers ( Table 1). The PCR products were purified, digested with HindIII and EcoRV restriction enzymes, ligated into pFLAG-CMV4 (catalog number E1775, Sigma), and designated pCMV4-NRP/B BTB, pCMV4-NRP/B BTB-IVS, and pCMV4-NRP/B IVS (Table 1).…”

“…Neh4 and Neh5 interact with the CREB-binding protein (cAMP responsive elementbinding protein) to synergistically induce strong NRF2 transcriptional activation (22). Under conditions of oxidative stress, NRF2 is phosphorylated and released by KEAP1 into the nucleus where it associates with NRP/B and activates phase II detoxifying and antioxidant genes (17,20). The molecular mechanism by which NRF2 activates ARE-driven genes has remained elusive.…”

“…The cytoplasmic protein Kelch-like ECH-associated protein 1 (KEAP1) and the nuclear NRP/B are both members of the Kelch-related family (8,19). The localization of these proteins is important for regulation of the NRF2 pathway in which KEAP1 and NRP/B/ENC1 suppress and enhance, respectively, NRF2-mediated phase II detoxifying and antioxidant enzymes in response to oxidative stress (8,17,20).…”

“…The cytoplasmic protein Kelch-like ECH-associated protein 1 (KEAP1) and the nuclear NRP/B are both members of the Kelch-related family (8,19). The localization of these proteins is important for regulation of the NRF2 pathway in which KEAP1 and NRP/B/ENC1 suppress and enhance, respectively, NRF2-mediated phase II detoxifying and antioxidant enzymes in response to oxidative stress (8,17,20).Six functional domains of NRF2, termed NRF2 ECH homology (Neh) 1-6, have been identified and found to be highly conserved in various species (8). Neh1 is a bZip motif that mediates DNA binding and participates in heterodimerization with 2 The abbreviations used are: NF, nuclear factor; NRF2, nuclear factor erythroid-derived 2-related factor 2; ARE, antioxidant response element; BTB, Broad-complex, Tramtrack, Bric-a-brac; CSK, c-src tyrosine kinase; HO1, heme oxygenase 1; KEAP1, Kelch-like ECH-associated protein; MRP2, MAYVEN-related protein 2; NRP/B, nuclear-restricted protein in brain; NQO1, NAD(P)H:quinine oxidoreductase 1 (nicotinamide quinine oxidoreductase); CREB, cAMP responsive element-binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Neh, NRF2 ECH homology; GST, glutathione S-transferase; IVS, intervening sequence; PBS, phosphate-buffered saline; Pipes, 1,4-piperazinediethanesulfonic acid; RT, reverse transcription; siRNA, small interfering RNA; WT, wild-type.…”

“…Multiprotein complexes formed through contact sites in ␤-propeller domains (14) are believed to be important for gene expression, cytoskeletal maintenance (15,16), and controlling cellular organization and morphology (15,16). Importantly, alterations or mutations in Kelch-related proteins have been found in numerous tumors (14,17) and neurodegenerative disorders (18). The cytoplasmic protein Kelch-like ECH-associated protein 1 (KEAP1) and the nuclear NRP/B are both members of the Kelch-related family (8,19).…”