A functional link between housekeeping selenoproteins and phase II enzymes

Abstract

Sec (selenocysteine) is biosynthesized on its tRNA and incorporated into selenium-containing proteins (selenoproteins) as the 21st amino acid residue. Selenoprotein synthesis is dependent on Sec tRNA and the expression of this class of proteins can be modulated by altering Sec tRNA expression. The gene encoding Sec tRNA (Trsp) is a single-copy gene and its targeted removal in liver demonstrated that selenoproteins are essential for proper function wherein their absence leads to necrosis and hepatocellular degeneration. In the present study, we found that the complete loss of selenoproteins in liver was compensated for by an enhanced expression of several phase II response genes and their corresponding gene products. The replacement of selenoprotein synthesis in mice carrying mutant Trsp transgenes, wherein housekeeping, but not stress-related selenoproteins are expressed, led to normal expression of phase II response genes. Thus the present study provides evidence for a functional link between housekeeping selenoproteins and phase II enzymes.

Figure 1. Hierarchical clustering analysis of gene alterations following Trsp removal in liver

Hierarchical dendrogram representing the expression profiles of significantly altered genes, following data filtering as described in Results. (A) upregulated or (B) downregulated in knockout mice. The genes are ordered by clustering tightness, with a distance measure of 1 - Pearson correlation coefficient and a P-value threshold of 0.05. Each column represents data from one experimental set, and rows indicate individual genes. Increases and decreases in transcript expression levels are represented by shades of red and green, respectively.

Figure 1. Hierarchical clustering analysis of gene alterations following Trsp removal in liver

Hierarchical dendrogram representing the expression profiles of significantly altered genes, following data filtering as described in Results. (A) upregulated or (B) downregulated in knockout mice. The genes are ordered by clustering tightness, with a distance measure of 1 - Pearson correlation coefficient and a P-value threshold of 0.05. Each column represents data from one experimental set, and rows indicate individual genes. Increases and decreases in transcript expression levels are represented by shades of red and green, respectively.

Figure 2. Quantitative PCR of upregulated genes following Trsp removal in liver

The relative expression of genes upregulated in ΔTrsp mice (Table 3) was examined by Q-PCR, normalized to the expression of Gusb as described in Experimental Procedures. The normalized value for each mRNA in liver of ΔTrsp, A34, G37L and G37H mice was then compared to Trsp mice and plotted along with error bars. Upregulated genes analyzed are grouped according to their function (see Table 3): (A) metabolism; (B) defense stress and detoxification; (C) intracellular communication/signal transduction; and (D) cell cycle/growth and differentiation. Results represent 3-4 independent experiments, each carried out in triplicate.

Figure 2. Quantitative PCR of upregulated genes following Trsp removal in liver

The relative expression of genes upregulated in ΔTrsp mice (Table 3) was examined by Q-PCR, normalized to the expression of Gusb as described in Experimental Procedures. The normalized value for each mRNA in liver of ΔTrsp, A34, G37L and G37H mice was then compared to Trsp mice and plotted along with error bars. Upregulated genes analyzed are grouped according to their function (see Table 3): (A) metabolism; (B) defense stress and detoxification; (C) intracellular communication/signal transduction; and (D) cell cycle/growth and differentiation. Results represent 3-4 independent experiments, each carried out in triplicate.

Figure 3. Western blot analysis of phase II enzymes

Protein extracts were prepared from liver of Trsp, ΔTrsp, A34, G37L and G37H mice and electrophoresed on 10% polyacrylamide gels. (A) Coomassie Blue stained gel. The elevated band corresponding to GST in ΔTrsp mice is indicated by an arrow on the right side of the gel and molecular weight (MW) markers were run in lane 1 and their sizes indicated on the left. (B) The proteins were transferred to PVDF membranes and the membranes probed with antibodies specific for the indicated phase II enzymes and β-actin as described in Experimental Procedures. β-Actin served as a loading control.