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Comparative Study
, 8, 13

The OXR Domain Defines a Conserved Family of Eukaryotic Oxidation Resistance Proteins

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Comparative Study

The OXR Domain Defines a Conserved Family of Eukaryotic Oxidation Resistance Proteins

Mathieu Durand et al. BMC Cell Biol.

Abstract

Background: The NCOA7 gene product is an estrogen receptor associated protein that is highly similar to the human OXR1 gene product, which functions in oxidation resistance. OXR genes are conserved among all sequenced eukaryotes from yeast to humans. In this study we examine if NCOA7 has an oxidation resistance function similar to that demonstrated for OXR1. We also examine NCOA7 expression in response to oxidative stress and its subcellular localization in human cells, comparing these properties with those of OXR1.

Results: We find that NCOA7, like OXR1 can suppress the oxidative mutator phenotype when expressed in an E. coli strain that exhibits an oxidation specific mutator phenotype. Moreover, NCOA7's oxidation resistance function requires expression of only its carboxyl-terminal domain and is similar in this regard to OXR1. We find that, in human cells, NCOA7 is constitutively expressed and is not induced by oxidative stress and appears to localize to the nucleus following estradiol stimulation. These properties of NCOA7 are in striking contrast to those of OXR1, which is induced by oxidative stress, localizes to mitochondria, and appears to be excluded, or largely absent from nuclei.

Conclusion: NCOA7 most likely arose from duplication. Like its homologue, OXR1, it is capable of reducing the DNA damaging effects of reactive oxygen species when expressed in bacteria, indicating the protein has an activity that can contribute to oxidation resistance. Unlike OXR1, it appears to localize to nuclei and interacts with the estrogen receptor. This raises the possibility that NCOA7 encodes the nuclear counterpart of the mitochondrial OXR1 protein and in mammalian cells it may reduce the oxidative by-products of estrogen metabolite-mediated DNA damage.

Figures

Figure 1
Figure 1
A Organization of OXR1 and NCOA7 genes. Exon-intron structures both genes are shown. OXR1 is located on Chromosome 8q22, NCOA7 is located on Chromosome 6q22.33. The black boxes represent exons comprising the minimal OXR domains. Exons shown in gray are those regions that are similar in OXR1 and NCOA7. Areas in white are unique to, either NCOA7, or OXR1. The striped exons are exons 10 and 11, which are also unique to OXR1. The length of the lines connecting exons is an indication of the relative size of the intron. B. Shows the comparison of the extent of identity of individual exons (black boxes) and similarity (gray boxes). The exon numbers listed are those of OXR1.
Figure 2
Figure 2
Bacterial Papillation Assay. Individual colonies of the white E. coli lacZ cc104 mutant colonies containing the dark blue microcolonies which are the GC→TA revertants. Panel A shows the high spontaneous mutation frequency of the mutM mutY strain carrying only the vector. The remaining panels show the reduction in LacZ papillation in the mutM mutY strain resulting from the expression of either full length, (B) OXR1C; (C), full length NCOA7; (D), NCOA7 (657–942).
Figure 3
Figure 3
Expression and stability of full length and truncated NCOA7 proteins in E. coli. Cells were either induced or not with IPTG. Proteins from induced or uninduced exponential phase cells were labeled with 35 [S] Met and chased, then harvested either immediately, or after 15, or 30 min further incubation as indicated in the figure. The arrows indicate the positions of the full length and truncated (657–942) forms of NCOA7 protein.
Figure 4
Figure 4
Subcellular localization of full length FLAG-tagged NCOA7 protein in human MCF-7 cells. MCF-7 cells were cultured in hormone-free medium and transiently transfected with FLAG-tagged full-length NCOA7. Two days after transfection, cells were treated without or with 100 nM E2 for 2 hours. Cellular localization of NCOA7 was detected by immunofluorescence using an anti-FLAG antibody (red stain). Cell nuclei were indicated by the blue DAPI stain. -E2, no estrogen, +E2, estrogen treated cells.
Figure 5
Figure 5
Protein expression of NCOA7 after treatment with hydrogen peroxide. MCF-7 cells were treated with indicated concentrations of hydrogen peroxide (H2O2) for 1, 4, 8, or 16 hours. Whole cell lysates were prepared for western analysis. The protein band corresponding to NCOA7 was indicated by its loss after siRNA-mediated inhibition. Calnexin is a loading control.

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