Adaptation of phenylalanine and tyrosine catabolic pathway to hibernation in bats

Abstract

Some mammals hibernate in response to harsh environments. Although hibernating mammals may metabolize proteins, the nitrogen metabolic pathways commonly activated during hibernation are not fully characterized. In contrast to the hypothesis of amino acid preservation, we found evidence of amino acid metabolism as three of five key enzymes, including phenylalanine hydroxylase (PAH), homogentisate 1,2-dioxygenase (HGD), fumarylacetoacetase (FAH), involved in phenylalanine and tyrosine catabolism were co-upregulated during hibernation in two distantly related species of bats, Myotis ricketti and Rhinolophus ferrumequinum. In addition, the levels of phenylalanine in the livers of these bats were significantly decreased during hibernation. Because phenylalanine and tyrosine are both glucogenic and ketogenic, these results indicate the role of this catabolic pathway in energy supply. Since any deficiency in the catabolism of these two amino acids can cause accumulations of toxic metabolites, these results also suggest the detoxification role of these enzymes during hibernation. A higher selective constraint on PAH, HPD, and HGD in hibernators than in non-hibernators was observed, and hibernators had more conserved amino acid residues in each of these enzymes than non-hibernators. These conserved amino acid residues are mostly located in positions critical for the structure and activity of the enzymes. Taken together, results of this work provide novel insights in nitrogen metabolism and removal of harmful metabolites during bat hibernation.

Figure 1. Two-dimensional (2D) gel electrophoresis of liver proteins of Myotis ricketti.

(A) The 2D gels of the first pair of male bats in hibernation (left panel, 1LH) and active states (right panel, 1LA) are shown. The numbers on the left of the gel indicate the positions and approximate molecular mass (in kDa). The numbers below the gel denote the approximate pH gradient across the gel. Protein spots identified are labeled with ID numbers shown in Table 1. (B) Expression levels of PAH, HPD, HGD, and FAH were determined by the ImageMaster^™2D Platinum software. V% indicates spot volume normalized as percentages of the total volume of all spots in the gel. The average levels of each protein in hibernation (H) and active (A) states are represented by light-gray and black bars, respectively. Each bar represents results of nine gels. Experimental data are mean ± SD. A P value <0.05 (one-way ANOVA test) is considered significant: *P<0.05 and **P<0.001. (The 2D gels of other bat pairs are shown in Figure S1.)

Figure 2. IPA analyses of transcription regulators.

The interaction between transcription factors (inner circle) and both upregulated and downregulated proteins listed in Table 1 (outer circle) is shown. Upregulated and down-regulated expressions are indicated in red and green, respectively, the darker the red color the higher level of protein expression. Numbers indicate the protein IDs shown in Table 1. Arrows denote activation, and T-bars represent inhibition. Solid and dashed arrows represent direct and indirect interactions, respectively. All P values of overlap are <10⁻³. Asterisks * and ** indicate P values of overlap <10⁻⁵ and <10⁻⁶, respectively.

Figure 3. Expression patterns of the enzymes responsible for the catabolism of Phe and Tyr.

Protein expressions of PAH, HPD, HGD, and FAH in the livers of pigs, rats, mice, and male (♂, A) bats were determined by Western blotting. H and A indicate bats in hibernation and active states, respectively. Arrow heads indicate predicted molecular weight (kDa) of the proteins; the numbers in parentheses denote observed molecular weights. (B) Relative protein levels (y-axis) are represented as mean ± SD. The lowest level of a detectable protein is considered as 1. Hibernation (H) and active (A) states are represented by light-gray and black bars, respectively. Phylogenetic trees are drawn at the bottom of the panel. Statistical significance: *P<0.05, **P<0.001.

Figure 4. Selective pressure on PAH, HPD, HGD, and FAH.

The selective pressure (ω value) of each gene was determined by comparing the ratio of synonymous to nonsynonymous nucleotide changes of each protein from several species of hibernators (gray bars) and non-hibernators (black bars) of bats. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA: *P<0.05, **P<0.001.

Figure 5. Stereoview of PAH, HPD, HGD, and FAH.

Amino acid residues that are conserved in all hibernators of bats but are different or diverged among non-hibernators of bats are indicated in red or blue, respectively. Orange color denotes positions that are conserved in all non-hibernating bats but diverged among hibernating bats. N-terminus and C-terminus are labeled as N and C, respectively. (A) The catalytic domain of PAH consisting of residues R131-L137, L250-R253, D316-W327, and E377-I380 is colored in yellow. The autoregulatory region is located between G20 and G34 at the N-terminus. Residues R112-T118 located in the hinge region are essential for phenylalanine-modulated proteolytic cleavage, and residues L431-K453 are required for the formation of a fully active enzyme tetramer. (B) Activity cave of HPD is indicated with a purple asterisk. Green color denotes residues and side chains of H183, H266, and E349 for binding metal ions and water molecule. (C) The active site of HGD composed of residues F282-T299, P320-K327, and M368-K385 is colored in yellow. Residue H269 is critical for trimerization of HGD and activation of the enzyme. (D) The purple asterisk denotes the activity cave of FAH. Green color indicates residues and side chains of D126, E199, E201, and D233 for Ca²⁺ binding. The H133/E364 dyad is critical for the catabolism of enzyme substrate.