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. 2005 Dec;25(24):10895-906.
doi: 10.1128/MCB.25.24.10895-10906.2005.

The carboxy-terminal Neh3 domain of Nrf2 is required for transcriptional activation (V体育官网入口)

Paul Nioi  1 , Truyen Nguyen, Philip J Sherratt, Cecil B Pickett
Affiliations

The carboxy-terminal Neh3 domain of Nrf2 is required for transcriptional activation

Paul Nioi et al. Mol Cell Biol. 2005 Dec.

"VSports app下载" Abstract

Nrf2 is a transcription factor critical for the maintenance of cellular redox homeostasis. We have previously found that Nrf2 is a labile protein, and its activation in cells under stress involves mechanisms leading to its stabilization. As a modular protein, Nrf2 possesses distinct transactivation and DNA binding domains essential for its transcriptional activity. In this study, we found that the C-terminal "Neh3" domain of Nrf2 is also important for its activity. Deletion of the last 16 amino acids of the protein completely abolishes its ability to activate both reporter and endogenous gene expression. Using site-directed mutagenesis, we have identified a stretch of amino acids within this region that are essential for its activity and that are found to be conserved across species and among other members of the CNC-bZIP family. Importantly, deletion of the final 16 amino acids of Nrf2 does not influence its dimerizing capability, DNA binding activity, or subcellular localization, although it does increase the half-life of the protein. In addition, this region was found to be important for interaction with CHD6 (a chromo-ATPase/helicase DNA binding protein) in a yeast two-hybrid screen VSports手机版. RNA interference-mediated knockdown of CHD6 reduced both the basal and tert-butylhydroquinone-inducible expression of NQO1, a prototypical Nrf2 target gene. These data suggest that the Neh3 domain may act as a transactivation domain and that it is possibly involved in interaction with components of the transcriptional apparatus to affect its transcriptional activity. .

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V体育官网入口 - Figures

FIG. 1.
FIG. 1.
Nrf2CTΔ16 is unable to activate transcription of ARE reporter genes. (A) Domain structure of Nrf2. The numbers shown represent the positions in the rat protein sequence. (B) Alignment of Neh3 domains from the indicated species. The position of the first amino acid in each sequence is shown in parentheses. The 16-amino-acid deletion of Nrf2 is shown below the aligned sequences. (C) Q293 cells were cotransfected with either GSTA2-ARE-CAT (left; 6-well plates) or −1016/nqo15′-luc (right; 96-well plates) and the indicated dose of HA-Nrf2 or HA-Nrf2CTΔ16 plasmid. CAT and luciferase assays were performed using standard procedures. Activity is shown as activation (n-fold) over the level obtained when reporter constructs were transfected alone (Ctrl), and results are the mean plus standard deviation of three independent experiments. (D) CAT lysates from panel C were resolved by SDS-polyacrylamide gel electrophoresis, blotted, and then probed with antibodies against either the HA epitope or GAPDH. (E) Q293 cells were cotransfected with either GSTA2-ARE-CAT (left; 6-well plates) or −1016/nqo15′-luc (right; 96-well plates), and the indicated dose of HA-Nrf2 plasmid in either the presence or absence of increasing doses of vector encoding HA-Nrf2CTΔ16, as indicated. Assays were performed and results are presented as described for panel C.
FIG. 2.
FIG. 2.
Nrf2CTΔ16 interacts with MafK and binds DNA with affinity similar to that of wild-type Nrf2. (A) Lysates were prepared from Q293 cells expressing HA-tagged wild-type Nrf2, Nrf2CTΔ16, or Nrf2ΔbZip in the presence or absence of V5-tagged MafK, as indicated. Lysates were immunoprecipitated with anti-V5 antibody, and immune complexes were blotted and then probed with either HA or V5 antibodies as shown. Samples of lysates prior to IP were also retained, blotted, and probed with HA or V5 antibodies (input samples). (B) HA-Nrf2, HA-Nrf2CTΔ16, and MafK-V5 were in vitro transcribed/translated and then mixed in the indicated combinations and incubated for 15 min at 30°C. The complexes were then immunoprecipitated with anti-V5 antibodies and blotted and then probed with anti-HA or anti-V5 antibodies. A portion of each sample (10%) was removed prior to IP (input) and was blotted and probed with either anti-HA or anti-V5 antibodies. (C) The indicated combinations of in vitro-transcribed/translated proteins were incubated with radiolabeled ARE probe before being resolved. The Nrf2-MafK and Nrf2CTΔ16-MafK shifted complexes are indicated, and a nonspecific complex is shown with an asterisk. (D) Nrf2-MafK or Nrf2CTΔ16-MafK complexes were preincubated with the indicated molar excess of unlabeled ARE probe prior to addition of radiolabeled DNA. Complexes were resolved, and the specific shifted bands are indicated along with a nonspecific complex shown with an asterisk next to it. (E) Quantitation of the results shown in panel D by densitometry. (F) Nrf2-MafK (wild type [WT]) or Nrf2CTΔ16-MafK (Δ16) complexes were preincubated with the indicated molar excess of unlabeled mutant ARE DNA prior to the addition of radiolabeled probe. Complexes were resolved, and the specific shifted bands are indicated, along with a nonspecific complex highlighted with an asterisk.
FIG. 3.
FIG. 3.
Nrf2 and Nrf2CTΔ16 are localized to the nucleus. Q293 cells were transfected with either HA-Nrf2 or HA-Nrf2CTΔ16, as indicated. Following fixation, the cells were stained with rat anti-HA primary, followed by anti-rat immunoglobulin G Alexa 488 secondary antibodies (A and B). Nuclei were visualized with propidium iodide (P.I.) (C and D), and merged images are shown in panels E and F.
FIG. 4.
FIG. 4.
Nrf2CTΔ16 cannot activate endogenous ARE-driven gene expression. (A) 293/HA-Nrf2 or 293/HA-Nrf2CTΔ16 cells were treated with Dox (1 μg/ml) for the indicated periods. Following treatments, lysates were prepared and Western blotting was performed using the indicated antibodies. (B) The indicated cell lines were treated with Dox (1 μg/ml) for 0, 4, 12, 24, or 48 h, and total RNA was subsequently isolated. The RNA was analyzed by TaqMan assay using the ABI Prism 7700 Sequence Detection System. Primers/probes used were specific for NQO1 (left) GCLM (right), and 23hbp (loading control). The values obtained for 23hbp mRNA were used to normalize those obtained for NQO1 and GCLM. Results are mean ± standard deviation of three independent experiments.
FIG. 5.
FIG. 5.
Nrf2CTΔ16 is more stable than wild-type Nrf2 and is regulated normally by Keap1. (A) Q293 cells were transiently transfected with plasmids encoding HA-Nrf2 or HA-Nrf2CTΔ16, as indicated. Subsequently, cycloheximide (30 μg/ml) was added to the media for the indicated times. Lysates were resolved by SDS-polyacrylamide gel electrophoresis, blotted, and probed with anti-HA antibodies. (B) Semilog graphical depiction of the results shown in panel A. (C) Q293 cells were transfected with 1 μg HA-Nrf2 or HA-Nrf2CTΔ16 plasmid in the presence or absence of 50 ng of Keap1 plasmid. Where indicated, cells were treated with 50 μM t-BHQ or 10 μM MG132 for 4 h prior to being harvested. The blots were probed with either anti-HA (top) or anti-GAPDH (bottom) antibodies.
FIG. 6.
FIG. 6.
Characterization of the last 16 amino acids of Nrf2. (A) Sequence of the Neh3 domain of Nrf2. The numbers shown represent the positions in the rat protein sequence, and the sequence separated into blocks highlights the final 16 amino acids of Nrf2. The mutations shown (M1 to M5) were introduced into the full-length protein by site-directed mutagenesis of an expression vector encoding wild-type Nrf2. (B) Q293 cells were cotransfected with an ARE-CAT reporter construct and the indicated HA-tagged Nrf2 expression construct: wild-type Nrf2 (WT), Nrf2CTΔ16 (Δ16), or Nrf2M1 to -M5. CAT assays were performed using standard procedures. Activity is shown as activation (n-fold) over the level obtained when reporter constructs were transfected alone (Ctrl), and results are the mean plus standard deviation of three independent experiments. (C) CAT lysates from panel B were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), blotted, and probed with anti-HA or anti-GAPDH. (D) Experiments were conducted exactly as described for panel B using the indicated HA-tagged Nrf2 expression constructs. (E) CAT lysates from panel D were resolved by SDS-PAGE, blotted, and probed with anti-HA or anti-GAPDH.
FIG. 7.
FIG. 7.
Nrf2 interacts with CHD6 in a yeast two-hybrid assay, and this is dependent upon the VFLVPK motif. (A) Depiction of constructs used in the yeast two-hybrid assay. (B) Yeasts transformed with the indicated plasmids were mated, and diploids were selected on SD-Leu-Trp plates and subsequently streaked onto SD-Leu-Trp-His-Ade-X-α-Gal plates as shown.
FIG. 8.
FIG. 8.
CHD6 interacts with Nrf2 in mammalian cells and is required for expression of NQO1. (A) Lysates were prepared from Q293 cells expressing HA-tagged Nrf2, Nrf2CTΔ16, Nrf2M1, Nrf2M2, or Nrf2M4 in the presence or absence of a V5-tagged CHD6 protein (amino acids 124 to 324), as indicated. Following IP with immobilized HA antibodies, immune complexes were blotted and probed with V5 or HA antibodies, as specified. A portion of each sample (10%) was removed prior to IP (input) and was blotted and probed with either anti-HA or anti-V5 antibodies. (B to D) HeLa cells were transfected with nonspecific siRNA (Ctrl) or siRNA specific for CHD6 or lamin A/C, as indicated. Subsequently, cells were treated for 24 h with either 0.1% (vol/vol) DMSO (white bars) or 50 μM t-BHQ (black bars). Total RNA was isolated and analyzed by TaqMan with a primer-probe set specific for NQO1 mRNA (B), CHD6 mRNA (C), or Nrf2 mRNA (D). The error bars indicate standard deviations.
FIG. 9.
FIG. 9.
Alignment of Neh3 domains from members of the CNC transcription factor family. The VFLVPK motif is highlighted by asterisks.
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