Thyroid hormone receptor orthologues from invertebrate species with emphasis on Schistosoma mansoni - PubMed (original) (raw)

Figure 1

Sequence alignment of the DNA binding domain (DBD) of TRs. There is a highly conserved motif found at the 3' end of A/B domain, termed the 'N-terminal signature sequence' (NTSS). In non-chordate TR homologues, it is PYIPSYMXXXGPEEP; while in chordate NTSS, three amino acids are deleted (represented by dashes) generating a sequence of GYIPYL(D/E)KE(P/Q/L). In the N-terminus of the DBD, a methionine at position 16 (highlighted in red) is non-chordate TR specific. In this position an isoleucine is conserved for all chordate TRs. Two amino acids (His and Pro for all species except Ciona) are inserted at positions 33–34 of the N-terminus of DBD in chordate TRs (highlighted in blue). An aspartic acid in vertebrate TRs at the 8th position of the T-box is highly conserved for vertebrate TRs, a glutamic acid at this position is conserved for other TRs (highlighted in red). Stars identify the conserved cysteine residues that comprise the zinc finger of the DBD. The conserved residues are highlighted in green. CiTR: Ciona intestinalis nuclear receptor 1, DpTR: water flea Daphnia pulex TR, hTRa: Human thyroid receptor alpha, hTRb: Human thyroid receptor beta, LgTR: owl limpet Lottia gigantean TR, SeTRa: turbellarian Schmidtea mediterranea TRα, SeTRb: S. mediterranea TRβ, SjTRα: blood fluke Schistosoma japonium TR alpha, SjTRβ: S. japonium TR beta, SmTRα: blood fluke S. mansoni TR alpha, SmTRβ: S. mansoni TR beta, SpTR: sea urchin Strongylocentrotu purpuratus TR. The accession numbers of the aligned human nuclear receptors can be found in Additional files 1 and 2.

Figure 2

Sequence alignment of the ligand binding domain (LBD) of S. mansoni TRα and SmTRβ. Alignment of sequences from Helices (H) 3–12 of the LBD domain of S. mansoni TRs (SmTRα and SmTRβ). Helices described in [53] are boxed. The autonomous activation domain (AF2-AD) is indicated. Numbers at the end of each line indicate residue positions in the original sequence, amino acids of CiTR 490–587 are not shown in the alignment. CiTR: Ciona intestinalis nuclear receptor 1, hTRa: Human thyroid receptor alpha, hTRb: Human thyroid receptor beta, SmTRa: blood fluke S. mansoni TR alpha, SmTRb: S. mansoni TR beta. The accession numbers of the aligned human nuclear receptors can be found in Additional file 2.

Figure 3

Gene organization of SmTRα and SmTRβ. A. SmTRα. B. SmTRβ. (a) Showing exons and size of introns; Roman numerals indicate exons. (b) Showing the size of exons and their correspondence to the different protein domains. A/B: A/B domain, DBD: DNA binding domain, D: D domain (hinge region), Tτ: signature sequence of the LBD, E: E domain (LBD) after Tτ.

Figure 4

Sequence alignment of motif I and motif II in the LBD. Alignment of motif I and motif II in the LBD of TRs shows that two amino acids in motif I (a glycine at position 21 and an aspartic acid at position 29) and three amino acids in motif II (an alanine at position 3, a leucine at position 9 and an isoleucine or leucine at postion 45) are invertebrate TR homologue-specific (all highlighted in red). The conserved residues are highlighted in green. DpTR: water flea Daphnia pulex TR, LgTR: owl limpet Lottia gigantean TR, SmTRα: blood fluke S. mansoni TR alpha, SmTRβ: S. mansoni TR beta, SpTR: sea urchin Strongylocentrotu purpuratus TR. The accession numbers of the aligned human nuclear receptors can be found in Additional files 1 and 2.

Figure 5

Maximum Likelihood phylogenetic tree derived from amino sequences of the DNA binding domain. A phylogenetic tree was constructed using the Maximum Likelihood (ML) method under Jones-Taylor-Thornton (JTT) substitution model with a gamma distribution of rates between sites (eight categories, parameter alpha, estimated by the program) using PHYML (v2.4.4)). Support values for the tree were obtained by bootstrapping a 100 replicates and are indicated above each branch. Branches under the bootstrap value of 50 were shown as polytomies. The same data set was also tested by Bayesian inference with a JTT amino acid replacement model + gamma rates. The trees were started randomly with four simultaneous Markov chains running for 5 million generations. Bayesian posterior probabilities (PPs) were calculated using a Markov chain Monte Carlo (MCMC) sampling approach implemented in MrBAYES v3.1.1, the PPs values are shown below each branch. Star indicates the node obtained by Bayesian inference which was different from that obtained by ML method. DpTR: water flea Daphnia pulex TR, LgTR: owl limpet Lottia gigantean TR, SeTRa: turbellarian Schmidtea mediterranea TRα, SeTRb: S. mediterranea TRβ, SjTRα: blood fluke Schistosoma japonium TR alpha, SjTRβ: S. japonium TR beta, SmTRα: blood fluke S. mansoni TR alpha, SmTRβ: S. mansoni TR beta, SpTR: sea urchin Strongylocentrotu purpuratus TR. The accession number of each sequence used for phylogenetic analysis can be found in Additional files 1 and 2.

Figure 6

Maximum Likelihood phylogenetic tree derived from amino sequences of DBD and LBD. The phylogenetic relationship among TRs was examined by the Maximum Likelihood method (ML) and Bayesian inference with the same methods as in Fig. 6. Support values for ML tree were obtained by bootstrapping a 100 replicates and are indicated above each branch. Branches under the bootstrap value of 50 were shown as polytomies. Bayesian inference was running 3 million generations. The PPs are shown below each branch or after the ML bootstrapping value separated by a slash. Star indicates the node obtained form by Bayesian inference which was different from that obtained by the ML method. SmTRα: blood fluke S. mansoni TR alpha, SmTRβ: S. mansoni TR beta. The accession number of each sequence used for phylogenetic analysis can be found in Additional files 1 and 2.

Figure 7

Conserved junction sites within TRs. A. Conserved junction site of TRs. JP1: Conserved junction position 1 which is within the DBD encoding region. JP2: Conserved junction position 2 which is at the end of the DBD coding region. JP3: Conserved junction position 3 which is within the LBD motif I coding region and is gene group specific. JP4: Conserved junction position 4 which is within the motif II of LBD encoding region. JP4 is conserved in all vertebrate NR genes. B. Shows the splice junction of invertebrate TRs within motif I which is at the same position as that found in all vertebrate TRs (NR1A) [20]. DBD: DNA binding domain, LBD: ligand binding domain. CiTR: Ciona NR1, DpTR: water flea Daphnia pulex TR, hTRα: Human thyroid receptor alpha, hTRβ: Human thyroid receptor beta, LgTR: owl limpet Lottia gigantean TR, SeTRα: turbellarian Schmidtea mediterranea TRα, SeTRβ: S. mediterranea TRβ, SjTRα: blood fluke Schistosoma japonium TR alpha, SjTRβ: S. japonium TR beta, SmTRα: blood fluke S. mansoni TR alpha, SmTRβ: S. mansoni TR beta, SpTR: sea urchin Strongylocentrotu purpuratus TR. H3: helix 3, H4: helix 4, H5: helix 5. The accession number of each sequence used for analysis can be found in Additional file 1.

Figure 8

Protein-protein interaction of SmTRs with SmRXR1. GST pull down shows that both SmTRα and SmTRβ can form a heterodimer with SmRXR1 in vitro. 35S-labeled SmRXR1 was synthesized in vitro using pCITE-SmRXR1 as template and then incubated with GST-SmTRα(LBD), GST-SmTRβ(LBD) or GST (negative control) protein affixed to glutathione-Sepharose beads. The beads were collected, washed and the bound protein was resolved on 10% SDS acrylamide gel and visualized by autoradiography.

Figure 9

DNA binding properties of SmTRs in vitro. Electrphoretic mobility shift assay (EMSAs) shows the DNA binding properties of SmTRs. A single protein or a combination of two proteins (SmTRα/SmRXR1 or SmTRβ/SmRXR1) were synthesized in a TNT quick coupled transcription/translation system (Promega) and allowed to bind to γ-32P labeled DNA elements containing a half-site, DR0-DR5 and Pal0. A. SmTRα. Lanes 1, 5, 9, 13, 17, 21, 25 and 29 contain lysate from the control transcription-translation reaction as negative controls. Lanes 2, 6, 10, 14, 18, 22, 26 and 30 contain lysate with in vitro translated SmTRα. Lanes 3, 7, 11, 15, 19, 23, 27 and 31 contain lysate with in vitro translated SmTRα and SmRXR1. Lanes 4, 8, 12, 16, 20, 24, 28 and 32 contain lysate with in vitro translated SmRXR1. NS: non-specific binding. B. SmTRβ. Lanes 1, 5, 9, 13, 17, 21, 25 and 29 contain lysate from the control transcription-translation reaction as negative controls. Lanes 2, 6, 10, 14, 18, 22, 26 and 30 contain lysate with in vitro translated SmTRβ. Lanes 3, 7, 11, 15, 19, 23, 27 and 31 contain lysate with in vitro translated SmTRβ and SmRXR1. Lanes 4, 8, 12, 16, 20, 24, 28 and 32 contain lysate with in vitro translated SmRXR1.. Ho: homodimer, Mo: monomer, NS: nonspecific binding.

Figure 10

DNA binding of SmTRα(Gln182-Ala288) in vitro. Electrphoretic mobility shift assay (EMSAs) shows the DNA binding properties of SmTRα(Gln182-Ala288). DNA binding of SmTRα(Gln182-Ala288) containing 20 aa at the 5' end of the DBD, the DBD and the 40 aa at 3' end of the DBD. Lanes 1, 5, 9, 13, 17, 21, 25 and 29: lysate from the control transcription-translation reaction as negative control. Lanes 2, 6, 10, 14, 18, 22, 26 and 30 contain with lysate with in vitro translated SmTRα (Gln182-Ala288). Lanes 3, 7, 11, 15, 19, 23, 27 and 31 contain lysate with in vitro translated SmTRα (Gln182-Ala288) with a 100 fold of cold specific competitor (unlabeled oligonucleiotides same as the labeled one). Lanes 4, 8, 12, 16, 20, 24, 28 and 32 contain lysate with in vitro translated SmTRα(Gln182-Ala288) with a 100 fold of cold non-specific competitor (5'-CGCGGATCCTGCAGCTCCAG-OH).