Subcellular compartmentalization of glutathione peroxidase 1 allelic isoforms differentially impact parameters of energy metabolism

Abstract

Specific genetic variations in the gene for the selenium-containing antioxidant protein glutathione peroxidase 1 (GPX1) are associated with the risk of a variety of common diseases, including cancer, diabetes, and cardiovascular disorders. Two common variations have been focused upon, one resulting in leucine or proline at codon 198 and another resulting in 5, 6, or 7 alanine repeats were previously shown to affect the distribution of GPX1 between the cytoplasm and mitochondria. Human MCF7 cells engineered to exclusively express GPX1 with five alanine repeats at amino terminus and proline at codon 198 (A5P) and seven alanine repeats at amino terminus and leucine at codon 198 (A7L), as well as derivatives targeted to the mitochondria by the addition of a mitochondrial localization sequence (mA5P and mA7L) were used to assess the consequences of the expression of these proteins on the cellular redox state and bioenergetics. Ectopic expression of A5P and A7L reduced the levels of reactive oxygen species, and the mitochondrially targeted derivatives exhibited better activity in these assays. Bioenergetics and mitochondrial integrity were assessed by measuring mitochondrial membrane potential, oxygen consumption, adenosine triphosphate (ATP) levels, and the levels of lactate dehydrogenase. The results of these assays indicated distinctively, and sometimes opposing, patterns with regard to differences between the consequences of the expression of A5P, A7L, mA5P, and mA7L. These data provide new information on the consequences of differences in the primary structure and cellular location of GPX1 proteins and contribute to the understanding of how these effects might contribute to human disease.

Keywords: gene polymorphisms; glutathione peroxidase (GPX); mitochondria; oxidative stress; respiration.

Figure 1.. Ectopic GPx 1 expression in MCF7 cells.

GPx1 allelic variants and the mitochondrially-directed isoforms were expressed in MCF7 and analyzed for (A) GPx activity in the whole cell lysates and (B) in the sub-cellular fractions to confirm the mitochondrial targeting of the proteins. The statistical significances in (A) were calculated from the similarly treated vector-only transfected control and the values are the means ± sd, n = 3. *P < 0.05; ** P < 0.001

Figure 2.. Estimation of cellular ROS levels.

Transfected cells were treated with either 5μM MitoSOX to determine mitochondrial superoxide levels (A) or 100nM DCFH-DA for estimation total cellular ROS levels (B). The statistical significances were calculated from the vector-transfected control and the values are the means ± sd, n = 3. *P < 0.05; ** P < 0.001.

Figure 3.. Cellular proliferation under oxidative stress.

Transfected were treated with menadione (A) or hydrogen peroxide (B) for 48hrs and the MTT assay was performed post-treatment. The statistical significances were calculated from the similarly treated vector transfected control and from the non-targeted isoforms, the values are the means ± sd, n = 3. *P < 0.05; ** P < 0.001.

Figure 4.. The effect of GPX1 isoforms on mitochondrial membrane potential.

The mitochondrial membrane potential of transfected cells was determined using JC-1 fluorescence detection described in the Methods. The data is the ratio of red fluorescence (595 nm) which is proportional to the membrane potential and the green fluorescence (535 nm) which is emitted in necrotic or apoptotic cells. The statistical significances were calculated from the similarly treated vector transfected control and the values are the means ± sd, n = 3. ** P < 0.001.

Figure 5.. Oxygen consumption and ATP levels in MCF7 cells expressing different GPX1 isoforms.

(A) NaCN sensitive oxygen consumption was measured in the presence of reduced levels (Figure 5) in absence of 3mM glucose as described in the Methods. The statistical significances were calculated from the similarly treated vector transfected control and the values are the means ± sd, n = 3. ^## P < 0.001 versus non-treated vector, * P<0.05, **P<0.001 versus glucose treated vector, P<0.05 versus non-treated A5P, P<0.001 versus non-treated A7L. (B) ATP levels were estimated in the presence or in absence of 3mM glucose. The statistical significances were calculated from the similarly treated vector transfected control and the values are the means ± sd, n = 3. ^# P < 0.05 versus non-treated vector ^## P < 0.001 versus non-treated vector, * P<0.001 versus glucose treated vector, ^#P<0.001 versus non-treated A5P, P<0.001 versus non-treated A7L.

Figure 6.. GPx1 affects LDH activity.

LDH was assayed using lysates as described in the Methods section. The statistical significances were calculated compared to the vector-only transfected controls and the values are the means ± sd, n = 3. * P<0.001.