Fig 1.
Unconstrained Nrf2 Bayesian phylogenetic tree and species tree.
(A) Unconstrained Bayesian phylogenetic tree based on Nrf2 coding sequences, under the GTR+G model. Values on the nodes are posterior probabilities. (B) Species tree of the 21 mammals used in this study based on their accepted relationships. Four branches tested by the two-ratio model tests and the branch-site model tests are marked as A, B, C, and D. The four species with orange labels are Old World fruit bats. The seven amino acid substitutions specific to Old World fruit bats are shown (A100D, N133D, D205F, D224G, T279I, T350R and N424S).
Table 1.
Results of the branch model tests of selection pressure on Nrf2 in bats.
Fig 2.
Omega (dN/dS) values of sites along the Nrf2 protein sequence on selected branches.
(A) Ancestral branch of Yinpterochiroptera insectivorous bats, (B) ancestral branch of Yangochiroptera, (C) ancestral branch of Old World fruit bats (OWFBs), and (D) branch of New World fruit bat (NWFB). The red arrows indicate the positions of the seven amino acid replacements in OWFBs with elevated ω values.
Table 2.
Results of the branch-site model A tests for the detection of positively selected sites on bats lineages.
Fig 3.
Distribution of the seven OWFBs-specific amino acid replacements in the secondary structure of Nrf2 protein.
The locations of the seven amino acid replacements in OWFBs are labeled in red line. The seven Neh domains are shown, Neh1 to Neh7. Neh1 domain (residue 435–561) is the CNC-bZIP domain. Neh2 domain (residue 27–82) is a redox-sensitive regulatory domain that binds with the repressor Keap1, and also contains one of the three nuclear localization sequences (NLS: residue 42–53, 494–511 and 587–593). Neh3 (residue 562–605), Neh4 (residue 112–134) and Neh5 (residue 183–201) domains are involved in Nrf2 transactivation activity. Neh6 domain (residue 338–388) is involved in Nrf2 stability. Neh7 domain (residue 209–316) is involved in interactions with RXRα and functions as a transcriptional repressor.
Table 3.
PROVEAN and SIFT predictions of the functional consequences of the Nrf2 amino acid replacements in Old World fruit bats.
Fig 4.
The levels of Nrf2 protein and catalase (CAT) in the brain, heart and liver of four bats species.
Four bats species were used in this study and are two insectivorous bats (M.ric: Myotis ricketti; R.fer: Rhinolophus ferrumequinum) and two Old World fruit bats (R.les: Rousettus leschenaultia; C.sph: Cynopterus sphinx). Protein level of (A) Nrf2 protein and (B) catalase in representative bat species and rats (positive control) were determined by Western blotting. Relative levels are presented as mean ± SD. Arrows indicate the predicted molecular weights (kDa) of the proteins. Statistical significance of the differences among species were determined by one-way ANOVA with post hoc Holm-Sidak tests: *P < 0.05, **P < 0.001.