Natural allelic variations in glutathione peroxidase-1 affect its subcellular localization and function

Abstract

Glutathione peroxidase 1 (GPx-1) has been implicated in the etiology of several common diseases due to the association between specific allelic variations and cancer risk. The most common among these variations are the codon 198 polymorphism that results in either a leucine or proline and the number of alanine repeat codons in the coding sequence. The molecular and biologic consequences of these variations remain to be characterized. Toward achieving this goal, we have examined the cellular location of GPx-1 encoded by allelic variants by ectopically expressing these genes in MCF-7 human breast carcinoma cells that produce undetectable levels of GPx-1, thus achieving exclusive expression in the same cellular environment. A differential distribution between the cytoplasm and mitochondria was observed, with the allele expressing the leucine-198 polymorphism and 7 alanine repeats being more cytoplasmically located than the other alleles examined. To assess whether the distribution of GPx-1 between the cytoplasm and mitochondria had a biologic consequence, we engineered derivative GPx-1 proteins that were targeted to the mitochondria by the addition of a mitochondria targeting sequence and expressed these proteins in MCF-7 cells. These cells were examined for their response to oxidative stress, energy metabolism, and impact on cancer-associated signaling molecules. The results obtained indicated that both primary GPx-1 sequence and cellular location have a profound impact on cellular biology and offer feasible hypotheses about how expression of distinct GPx-1 alleles can affect cancer risk. Cancer Res; 74(18); 5118-26. ©2014 AACR.

Figure 1

GPx-1 gene polymorphisms affect the cellular distribution between cytosolic and mitochondrial compartments. (A) Protein extracts were prepared from cells expressing different GPx-1 alleles and separated into cytoplasmic and mitochondrial fractions. Equal amounts of proteins were electrophoresed and visualized by western blotting with anti-GPx-1 monoclonal antibodies as well as antibodies directed against GAPDH and COX-1 as indicators of cytoplasmic and mitochondrial proteins, respectively. (B) Densitometry was performed on independently prepared extracts with the filled bar representing the signals obtained from the nuclear fraction and the open bars the signals from the cytoplasmic fractions (left panel) and the ratio of the signals obtained from the cytoplasmic to the mitochondrial fractions was determined (right panel). (C) The data was pooled by either the codon 198 polymorphism (proline vs. leucine) or by the number of alanine codons. *P<0.005 for A7L vs. A5P in panel B, of 198P vs. 198L in panel C and 5 alanine repeats vs. 7 repeats with an n=6.

Figure 2

GPx activity in the cytoplasmic and mitochondrial fractions reflects the distribution pattern of the protein expressed from different GPx-1 alleles using the same extracts used to determine the levels of the GPx-1 protein in Fig. 1. (A) Enzyme activity was determined and the ratio of the activity obtained from the cytoplasmic to mitochondrial fractions is presented. The data was pooled by either the number of alanine codons in (B) or the codon 198 polymorphism (proline vs. leucine) in (C). *P<0.005 for A7L and A7P vs. A5P in panel A, the alanine repeats in panel B or of 198P vs. 198L in panel C and the number of alanine repeats with an n=6.

Figure 3

GPx-1 allelic identity affects ROS generation. MCF-7 cells either transfected with vector or either A5P or A7L were used to assess mitochondrial and cytoplasmic ROS levels using mito-roGFP (A) and cyto-roGFP (B). ro-GFP oxidation was determined by flow cytometry as described in the methods section. *P<0.005 vs. MCF vector control with an n=6.

Figure 4

GPx-1 allelic identity affects viability after challenge with ROS. Cells expressing either A5P, A7L, mitochondria-targeted A5P (mitoA5P) or mitochondria-targeted A7L (mitoA7L) were challenged with 100 μM t-BOOH for 48 hrs and viability determined. The difference between A5P and A7L following exposure to t-BOOH was significant at *P<0.05. The error bars indicate the S.D. with an n=6.

Figure 5

Molecular targeting of the A5P and A7L GPx-1 to the mitochondria. Constructs engineered to express the A5P or A7L GPx-1 alleles were targeted to the mitochondria by attaching the MLS sequence encoded by the mitochondrial MnSOD gene and these constructs were transfected into GPx-1 null MCF-7 cells, selected in antibiotic and expanded for analysis. (A) Protein extracts were prepared, separated into mitochondrial and cytoplasmic fractions and analyzed by western blotting with anti-GPx-1 antibodies. (B) The localization of mitochondrially-directed GPx-1 was confirmed in individual transfectants by immunhistochemistry and subsequent confocal imaging, where GPx-1 is seen to be exclusively co-localized with mitotracker (red fluorescence) in the transfectants. Nuclei were stained with DAP1 (blue fluorescence).

Figure 6

GPx-1 allelic identity and cellular localization affect mitochondrial metabolism. (A) Oxygen consumption rates were determined in cells expressing either GPx-1 variants or vector using the Sea Horse XF analyzer platform. Data was calculated using the XF Wave software supplied with the analyzer. *P<0.005, Ψ P<0.005 and § P<0.0005 from MCF7 with n=3. (B) The levels of superoxide in MCF-7 transfectants expressing A5P, A7L, mitoA5P or mitoA7L was determined by measuring the oxidation of DHE. The differences between A5P and mitoA5P, and between A7L and mitoA5L was significant at a P<0.05 with a n=3

Figure 7

Expression of GPx-1 isozymes affects the levels of signaling and selenium-containing proteins. Protein extracts were prepared from the indicated cells and analyzed using protein specific antibodies. Actin levels were determined as an indication of equal loading of protein on the gel.