Spermidine/spermine N1-acetyltransferase 1 (Ssat1) is a key enzyme in the polyamine interconversion pathway, which maintains polyamine homeostasis. In addition, mammalian Ssat1 is also involved in many physiological and pathological events such as hypoxia, cell migration, and carcinogenesis. Using cross-genomic bioinformatic analysis in 10 deuterostomes, we found that ssat1 only exists in vertebrates. Comparing with mammalian, zebrafish, an evolutionarily distant vertebrate, contains 3 homologous ssat1 genes, named ssat1a, ssat1b, and ssat1c. All zebrafish homologues could be transcribed and produce active enzymes. Despite the long history since their evolutionary diversification, some features of human SSAT1 are conserved and subfunctionalized in the zebrafish family of Ssat1 proteins. The polyamine-dependent protein synthesis was only found in Ssat1b and Ssat1c, not in Ssat1a. Further study indicated that both 5′ and 3′ sequences of ssat1b mediate such kind of translational regulation inside the open reading frame (ORF). The polyamine-dependent protein stabilization was only observed in Ssat1b. The last 70 residues of Ssat1b were crucial for its rapid degradation and polyamine-induced stabilization. It is worth noting that only Ssat1b and Ssat1c, but not the polyamine-insensitive Ssat1a, were able to interact with integrin α9 and Hif-1α. Thus, Ssat1b and Ssat1c might not only be a polyamine metabolic enzyme but also simultaneously respond to polyamine levels and engage in cross-talk with other signaling pathways. Our data revealed some correlations between the sequences and functions of the zebrafish family of Ssat1 proteins, which may provide valuable information for studies of their translational regulatory mechanism, protein stability, and physiological functions.
Citation: Lien Y-C, Ou T-Y, Lin Y-T, Kuo P-C, Lin H-J (2013) Duplication and Diversification of the Spermidine/Spermine N1-acetyltransferase 1 Genes in Zebrafish. PLoS ONE 8(1): e54017. https://doi.org/10.1371/journal.pone.0054017
Editor: Vladimir Korzh, Institute of Molecular and Cell Biology, Singapore
Received: July 11, 2012; Accepted: December 5, 2012; Published: January 11, 2013
Copyright: © 2013 Lien et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by grants NSC 99-2313-B-019-010-MY3 and NSC 100-2627-B-019-005 from the National Science Council (Taipei, Taiwan). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Polyamines, found in the cells of most species, play vital roles in cell proliferation and many physiological functions . Thus, cellular polyamine homeostasis is strictly maintained by regulation of its anabolic and catabolic pathways . In mammals, the interconversion pathway enhances control of cellular polyamine. Spermidine/spermine N1-acetyltransferase 1 (Ssat1) is the key enzyme in the rate-determining reactions of this pathway, by which spermine or spermidine accepts the acetyl group from acetyl-CoA to produce N1-acetylspermine or N1-acetylspermidine . Ssat1 effectively reverses the biosynthetic reactions and alters cellular polyamine equivalence because the acetylated derivatives are readily excreted and are good substrates for acetylpolyamine oxidase (Apao), a peroxisomal enzyme that readily converts these molecules to smaller polyamines , .
Accumulating evidence has indicated that mammalian Ssat1 is also involved various physiological and pathological events, including liver regeneration , ischemia-reperfusion injury , , , pancreatitis , , lipid metabolism , , carcinogenesis , cell migration , and hypoxia signaling , through its ability to modulate polyamine content or by directly interacting with other protein effecters, such as hypoxia inducible factor 1-α (Hif-1α) and integrin α9. Moreover, the regulation of Ssat1 is as versatile as its functions, occurring at multiple levels including transcription , , mRNA processing , , , translation , and protein stabilization , , . Curiously, we know rather little about the properties of Ssat1 from non-mammalian species. Here, we found that ssat1 is only present in the vertebrate lineage. Comparing with mammalians, zebrafish, an evolutionarily distant vertebrate, contains not one but three ssat1 genes. To understand whether these ssat1 genes have evolved distinct structural and functional properties, their spatial and temporal expression, translational regulation inside the ORF, protein stability, enzyme kinetics, and protein-protein interactions were extensively investigated. Our data suggest that these zebrafish ssat1 homologues might be paralogous genes which underwent subfunctionalization in their regulatory mechanisms and physiological functions.
Materials and Methods
All chemicals, including 5,5′-dithio-bis-(2-nitrobenzoic acid), coenzyme A, acetyl-CoA, cycloheximide, DMSO, isopropyl β-D-1-thiogalactopyranoside, and polyamines were purchased from Sigma-Aldrich Chemical Co. and were of the highest purity available. N1,N11-diethylnorspermine (DENSPM) was from Tocris Bioscience. Enzymes used in molecular cloning were obtained from New England Biolabs. The pGEX-2T expression vector was from GE Healthcare Bioscience; the pcDNA3.1/myc-His vector was from Invitrogen; the pET28 expression vector and the Escherichia coli BL21 (DE3) host cells were from Novagen.
Database Searches and Phylogenetic Analysis
In order to investigate the presence of SSAT1-related genes in invertebrate and vertebrate deuterostomes, human SSAT1 (NM_002970) and SSAT2 (NM_133491) were used as templates for TBlastN searches at the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/mapview/) for sea urchin (Strongylocentrotus purpuratus), sea squirt (Ciona intestinalis), zebrafish (Danio rerio), and mouse (Mus musculus) genomes, or in Ensembl (http://www.ensembl.org/index.html) for medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), Takifugu (Takifugu rubripes), and Tetraodon (Tetraodon nigroviridis) genomes. The database hits were verified by BlastP queries of the NCBI nonredundant (nr) protein database and sequence alignments, before a putative homolog (accession numbers provided in Fig. 1) was considered.
The accession number of each ssat-like gene from the deuterostomia is denoted and the bars represent their evolutionary distance. The scale bar is 0.2 expected changes per amino acid site. The reliability of the tree was measured by bootstrap analysis. Bootstrap values of 1,000 replicates larger than 50 were labeled on branches.
The amino acid sequences of ssat1-related genes were aligned by ClustalW  and edited manually using the BioEdit software  to prepare for the phylogenetic analysis. The alignment is available upon request. Phylogenetic trees were constructed by the neighbor joining (NJ) method using the PHYLIP package  with the substitution model and represented with the FigTree software (http://tree.bio.ed.ac.uk/software/figtree/). Tree topology was evaluated by bootstrap resampling with 1000 replicates.
Reverse Transcription (RT)-PCR and Molecular Cloning
Total RNA was isolated from human HEK293T cells (CRL-11268), adult zebrafish, and embryos with TRIzol reagent (Invitrogen). After DNase treatment, total RNA (500 ng) was reverse-transcribed to single-strand cDNA using SuperScript II reverse transcriptase (Clontech) according to the manufacturer’s instructions. Transcription of each gene was detected by RT-PCR with specific primers (see Table S1 in the supplemental material). The PCR reaction was initiated with DreamTaq (Fermentas) according to the manufacturer’s protocol. The reactions were separated by 1% agarose gel electrophoresis and visualized by staining with ethidium bromide.
To prepare expression plasmids for zebrafish ZF4 (ATCC CRL-2050) or HEK293T cells, the full-length ORF of human SSAT1, zebrafish ssat1a, ssat1b, and ssat1c, or DNA fragments encoding the cytosolic domain of zebrafish integrin 9α and the PAS-B domain of hif-1α were amplified by RT-PCR with specific primer sets (Table S1) and Pfu DNA polymerase (Stratagene). These amplified inserts were ligated into the EcoRI/XhoI sites of pcDNA3.1/myc-His. The recombinant plasmids were transformed into E. coli for preservation and amplification.
The chimeric ssat1a and ssat1b genes were prepared by using the megaprimer PCR technique . We designed 4 chimeric primers, which could pair to the same region of ssat1a and ssat1b at nucleotides 235–248, 319–332, 374–389 and 452–467 (Table S1). To make ssat1a248b, for example, the chimeric primer (primer 11 in Table S1) and ssat1a forward primer (primer 3 in Table S1) were used to amplify the target DNA fragment (nucleotides 1–248 of ssat1a). Then, the 2 strands of newly synthesized PCR fragments were used as megaprimers with Pfu DNA polymerase to synthesize the whole plasmid (pcDNA3.1/myc-His-ssat1b) and incorporate the chimeric target DNA fragments. The original plasmid DNA was digested by DpnI, and the DpnI-resistant chimeric DNA was recovered by transformation into competent bacteria. The chimeric gene was named ssat1a248b, as it contains nucleotides 1–248 of ssat1a and the remainder of ssat1b. Chimeric genes ssat1a332b, ssat1a374b, ssat1a453b, ssat1b248a, ssat1b332a, ssat1b389a, and ssat1b467a were also prepared in this manner (Fig. 2B). The chimeric gene ssat1aba was prepared by using nucleotides 389–513 of ssat1a as the megaprimer and a plasmid containing ssat1a332b as template. Therefore, ssat1aba and ssat1bab contain most of the ssat1a and ssat1b sequences, but their 332–389 nucleotide regions are from ssat1b and ssat1a.
(A) The amino acid sequences of human SSAT1 and zebrafish family of Ssat1 proteins were aligned by MegAlign (Lasergene) with the ClustalW method. The conserved residues are shaded black. The secondary structures are denoted according to the structure of human SSAT1 . (B) In each chimeric construct, fragments from Ssat1a are labeled in blue and fragments from Ssat1b are in red. Nucleotide positions and the corresponding amino acid residues are labeled on the top and the bottom of each construct, respectively.
Preparation of Recombinant Protein and Enzyme Activity Assay
The coding sequence of human SSAT1, zebrafish ssat1 homologues, and the integrin α9 cytosolic, and hif-1α PAS-B domains were amplified (Table S1) from the cDNA of HEK293T cells or zebrafish embryos. PCR products were inserted into the EcoRI and XhoI sites of pET28a or pGEX2T and transformed into E. coli BL21 to express recombinant proteins with N-terminal His6-tag or GST fusions. Following cell lysis, the His-tagged and GST-fused enzymes were purified by affinity chromatography on a HisTrap FF column (GE Healthcare) and a GSTrap FF column (GE Healthcare), respectively. The SSAT1 activity assay was described in our previous work .
Cell Culture, Gene Transfection, and Cellular Protein Extraction
Zebrafish ZF4 cells were cultured in 45% DMEM (Gibco), 45% Ham’s F12 medium (Gibco), and 10% FBS (Gibco) at 28°C with humidified air/CO2 (19∶1 v/v). Human HEK293T cells (CRL-11268) were cultured in DMEM with 10% FBS at 37°C with humidified air/CO2 (19∶1 v/v). The HEK293T cells were transfected by using standard calcium phosphate precipitation and transfection of ZF4 cells was performed as described previously . To prepare cellular proteins for analysis, cells were collected by centrifugation at 300×g, washed twice with PBS, and extracted with M-PER reagent (Thermo Scientific) according to the manufacturer’s instructions. The crude extract was dialyzed against 50 mM Tris-HCl (pH 7.5) with 1× Protease inhibitor (complete protease inhibitor cocktail, Roche) with an Amicon ultra centrifugal filter device (Millipore).
Detection of Protein Expression and Degradation by Western Blotting
HEK293T cells were seeded at a density of 3 × 105/well (6-well plate) and transfected with 2 µg pcDNA3.1/myc-His plasmids containing the ORFs of human SSAT1, zebrafish ssat1a, ssat1b, ssat1c, or chimeric genes. After 24 h culture, cells were treated with 10 µM DENSPM, 5 µM MG132 or vehicle (DMSO), and incubated for another 24 h before harvest and detection of translated proteins. To assess protein stability, cells were transfected with 2 µg plasmid encoding Ssat1a, Ssat1aba, Ssat1a453b, or Ssat1b332a, or with 4 µg plasmid encoding Ssat1b, Ssat1c, Ssat1bab, Ssat1b332a, or Ssat1b467a. After 12 h culture, cells were treated with 200 mM cycloheximide or left untreated. Before harvest, cells were treated with 2 mM spermidine for 2–6 h in the presence of 10 µM MG132 or vehicle (DMSO).
Cell lysates were resolved by 12% SDS–PAGE and transferred to a PVDF membrane. Proteins were immunodetected with anti-myc primary (1∶2000, Cell Signaling) and anti-mouse IgG secondary antibodies (1∶5000, Promega). Signals were detected with ECL Plus chemiluminescence reagent (GE Healthcare) and an imaging system (UVP Biospectrum).
GST Pull-down Assay
HEK293T cells (3×106 cells/10-cm plate) were transiently transfected with expression vectors encoding myc-tagged Ssat1a, Ssat1b, Ssat1c, or the PAS-B domain of Hif-1α. Cells transfected with Ssat1a, Ssat1b and Ssat1c expression vectors were cultured in the medium with 10 µM DENSPM. After 48 h culture, cell lysates (100 µg) were harvested and mixed with 10 µg GST or GST fusion proteins in 500 µl PBS buffer at 4°C for 2 h, followed by addition of 20 µl of glutathione-Sepharose 4B beads (GE Healthcare). After mixing for 30 min, the beads were washed with PBS. The proteins were eluted in Laemmli sample buffer and analyzed by SDS-PAGE and western blotting.
Identification of ssat1-like Genes in Deuterostomes
Several ssat-like genes were found across the deuterostomia, including sea urchin, sea squirt, amphioxus, mouse, human, and 5 kinds of ray-finned fish. Phylogenetic analysis divided these genes into 3 clusters (Fig. 1). Cluster 1 was composed of human SSAT2, mouse Ssat2, and invertebrate ssat-like genes. Genes in cluster 2 were ssat2 orthologues from ray-finned fish. At least 2 ssat2 homologous genes were found in all ray-finned fish species analyzed in this study. These ssat2 homologues were further divided into 2 sub-groups that suggested an early duplication event at ssat2 in the common ancestor of ray-finned fishes. Cluster 3 included human SSAT1 and its cognate genes from vertebrates. Compared with the first 2 clusters, the ssat1 orthologues were more closely conserved. No ssat1 orthologue was found in invertebrates and only 1 ssat1 was found in most vertebrates except that there were 3 ssat1 homologues in zebrafish. The encoded amino acid sequences (Fig. 2A) and cDNA sequences (Fig. S2) of zebrafish ssat1 homologous genes were highly similar to each other, and they were clustered together in the phylogenitic analysis (Fig. 1).
Human SSAT1 is located on the X chromosome between the genes for peroxiredoxin 4 (PRDX4), acryl-CoA thioesterase 9 (ACOT9), and apolipoprotein O (APOO) (Fig. S1). The Ssat1 genes of evolutionarily distant vertebrates including medaka, stickleback, takifugu, and tetraodon are located between acot9 and apoo (data not shown). One of the zebrafish ssat1-like genes (NM_001093748) is also located between acot9 and apoo on chromosome 24; we therefore named it ssat1a. The other zebrafish genes (NM_001030199 and NM_001002169) are closely clustered together and located next to prdx4 on chromosome 5. We named them ssat1b and ssat1c, respectively. The ssat-like genes of invertebrates and ssat2 homologous genes of vertebrates are not grouped like ssat1 (Fig. 1) and their genomic localization also differ (data not shown).
The Expression Pattern of Zebrafish ssat1 Homologous Genes
The expression patterns of zebrafish ssat1 genes were analyzed by RT-PCR. During normal embryogenesis, ssat1c mRNA was the most abundant in every stage and was stably expressed from 12 to 96 hours post fertilization (hpf). The mRNA of ssat1a and ssat1b were not detected until 24 hpf (Fig. 3A, control). A previous study indicated that treatment of human cells with DENSPM, a spermine analog, enhances SSAT1 expression up to 20 fold . Another group of zebrafish embryos were developed with 10 µM DENSPM added immediately after fertilization. All embryos survived and displayed no obvious abnormalities through 96 hpf. Neither the expression nor mRNA abundance of these ssat1 genes was changed (Fig. 3A, DENSPM).
(A) After fertilization, zebrafish embryos were incubated with or without (control) 10 µM DENSPM. Embryos were collected at different developmental stages (shown on the top, hpf, hour post fertilization) and expression of ssat1a, ssat1b, ssat11c, and β-actin were analyzed by RT-PCR. (B) The expression patterns of zebrafish ssat1 homologous genes were also analyzed in the major organs (shown on the top) of adult zebrafish.
The expression profiles of zebrafish ssat1 genes in the major organs of adult fish were also studied. ssat1a mRNA was mainly expressed in the heart, spleen and kidney, and was also detectable in the brain, eye, liver, and fin. ssat1b mRNA was detectable in the heart, liver, gut, and kidney, and weakly in the brain, eye, gill, spleen, muscle, fin and testis. ssat1c mRNA was detectable in every organ we tested and was the most abundant among these three homologues (Fig. 3B).
The Translational Regulation Inside the ORF of Zebrafish ssat1 Homologues
It has been reported that the translation of human SSAT1 is strictly controlled by polyamine and the SSAT1 ORF region is responsible for such regulation , . To test this mechanism, the ORF of each gene was ligated into pcDNA3.1/myc-His, which allows the mRNA of each gene to be stably and abundantly expressed. Human SSAT1 protein was extensively expressed in transfected HEK293T cells incubated with DENSPM but not in cells cultured in normal medium or medium with MG132 (Fig. 4A). Addition of MG132, a proteasome inhibitor, may increase SSAT1 protein stability but did not increase protein abundance in the absence of DENSPM. This is similar to previous studies, which suggested that the activity of SSAT1 is mainly regulated by polyamine and polyamine analogs in the translation level , .
(A) HEK 293T cells were transiently transfected with the plasmid encoding myc-tagged full-length human SSAT1, zebrafish Ssat1a, Ssat1b, or Ssat1c. After incubation for 12 h, transfected cells were treated with 10 µM DENSPM, 20 µM MG132, or vehicle for 24 h. (B) HEK 293T cells were transiently transfected with the plasmid encoding myc-tagged zebrafish Ssat1 chimeric enzymes (detail in Materials and Methods). After incubation for 12 h, transfected cells were treated with vehicle (lane 1 and 3) or 10 µM DENSPM (lane 2 and 4) for 24 h. Cell lysates (5 µg total protein in each lane) were prepared and the protein content of Ssat1 and β-actin in each sample was detected by western blotting with anti-myc and anti-β-actin antibody.
We investigated the translational regulation inside the ORF of zebrafish ssat1 under the same experimental conditions. It is interesting to note that background expression of Ssat1a was detectable in cells cultured without DENSPM and its expression increased by approximately 3-fold in the presence of DENSPM. On the other hand, the translational regulation inside the ORF of ssat1b and ssat1c were as stringent as that of human SSAT1 (Fig. 4A). We performed the same experiment in zebrafish ZF4 cells that also obtained the same result (Fig. S3). Thus, the ssat1 translational regulation machinery appears to be conserved in human and fish cells.
In order to identify the key region for translational regulation inside the ORF, a series of chimeric genes (ssat1a248b, ssat1a332b, ssat1a374b and ssat1a453b), which contained the 5′ region of ssat1a ORF and the 3′ region of ssat1b, were prepared and tested (Fig. 2B). The results indicated that the last 181 nucleotides of ssat1b are important for translational inhibition, since chimeric genes containing more than 181 nucleotides from the 3′ region of ssat1b retained the translational regulation pattern of ssat1b, such as ssat1a248b and ssat1a332b (Fig. 4B, lanes 1 and 2). The results of another series of chimeric mRNA with the 5′ region of ssat1b and the 3′ region of ssat1a (ssat1b248a, ssat1b332a, ssat1b389a and ssat1b467a shown in Fig. 2B) showed that the first 389 nucleotides of ssat1b are also important for regulation (Fig. 4B, lanes 3 and 4). Because the 332∼389 nucleotide region of ssat1b is present in both ssat1a332b and ssat1b389a, the importance of this region was further investigated.
The chimera ssat1aba, which contains the 332∼389 region of ssat1b and the remainder of ssat1a, still retained the ssat1a regulatory pattern that suggests the 332∼389 region of ssat1b alone is not sufficient to inhibit the background protein translation (Figure 4B). Further, ssat1bab, which contains the 332∼374 nucleotide region of ssat1a and the remainder of ssat1b, behaved like ssat1b. These results, which are summarized in table 1, indicate that both the 5′ and 3′ regions of ssat1b are important for translational regulation inside the ORF.
The Stability of Zebrafish Ssat1 Proteins
By increasing the transfected plasmid by 2-fold and protein loading by 10-fold, the expression of zebrafish Ssat1b and Ssat1c in cells cultured in normal medium could be observed by western blotting. We used these conditions to observe protein stability inside cells. After adding cycloheximine to the culture medium, translation of Ssat1 was stopped and the protein was subsequently turned over. As shown in Figure 4A, the Ssat1b protein was quickly turned over after translation was stopped (Fig. 5A, lanes 3–5). Protein degradation was mediated by proteasome, since the addition of MG132 prevented degradation (Fig. 5A, lane 2). In addition, the presence of spermidine improved the stability of Ssat1b up to 6 hr (Fig. 5A, lanes 6–8). On the other hand, Ssat1a and Ssat1c were more stable and no obvious protein degradation occurred within 6 h (Fig. 5).
(A) HEK 293T cells were transiently transfected with the plasmid encoding full-length zebrafish Ssat1a, Ssat1b, or Ssat1c. After 24 h, cells were treated with 200 mM cycloheximide (CHX) or left untreated for 30 min (lane 1). Then cells were treated with 10 µM MG132 (lane 2), vehicle (lanes 3–5), or 2 mM spermidine (lanes 6–8) for 1 h (lanes 3 and 6), 2 h (lanes 4 and 7), or 4 h (lanes 2, 5 and 8). (B) HEK 293T cells were transiently transfected with the plasmid encoding myc-tagged zebrafish Ssat1 chimeric enzymes (details in Materials and Methods). After incubation for 24 h, transfected cells were treated with 200 mM cycloheximide (CHX) for 30 min (lanes 1 and 4) and then with 2 mM spermidine for 1 h (lanes 2 and 5) or 2 h (lanes 3 and 6). Cell lysates (50 µg total protein in the Ssat1b, Ssat1c, b467a, a332b, and bab samples; 5 µg of total protein in the remaining samples) were prepared and the Ssat1 and β-actin protein content in each sample was detected by western blotting with anti-myc and anti-β-actin antibody.
The 10 ssat1a and ssat1b chimeric genes were also applied to identify the critical regions responsible for rapid degradation (Fig. 2B). Our results indicated that none of the chimeric proteins, which contain the C-terminal regions of Ssat1a, turned over rapidly without spermidine treatment, such as Ssat1b467a, Ssat1b332a, and Ssat1aba shown here (Fig. 5B, lanes 1–3). Moreover, the chimeric proteins, which contain the C-terminal regions of Ssat1b, did not undergo rapid degradation without spermidine treatment, unless they contain more than 70 residues from the Ssat1b C-terminal region, such as Ssat1a332b (Fig. 5B, lanes 4–6) or Ssat1a248b (data not shown). The results, which are summarized in Table 1, indicated the last 70 residues of Ssat1b are important for the regulation of protein stability.
The Enzyme Activities of Zebrafish Ssat1
The kinetic studies indicated all 3 Ssat1 isoenzymes were bioactive and could use both spermidine and spermine as substrates. However, the substrate preference of these isoenzymes was different. Ssat1b had similar Km values for spermidine and spermine, while Ssat1a had a smaller Km toward spermidine and Ssat1c had a smaller Km for spermine (Table 2). Ssat1a and Ssatb had a better kcat/Km value for spermidine than that for spermine, indicating these enzymes were more efficient in spermidine catabolism. In contrast, Ssat1c had a similar kcat/Km ratio for spermidine and spermine.
Protein-protein Interactions of Zebrafish Family of Ssat1 Proteins
The structures of mammalian Ssat1s indicate the homodimer structure is essential for enzyme activity , . Since Ssat1a, Ssat1b, and Ssat1c are co-expressed in several zebrafish organs and their primary sequences are largely identical, the formation of heterodimers is possible. Here, GST pull-down experiments were applied to test this hypothesis. The GST-Ssat1a fusion protein was able to pull down Ssat1b-myc and Ssat1c-myc while GST itself could not (Fig. 6A). In addition, GST-Ssat1b interacted with Ssat1c-myc, suggesting the 3 zebrafish family of Ssat1 proteins could assemble into homodimers or heterodimers.
(A) GST, GST-Ssat1a, GST-Ssat1b, and GST-Ssat1c were purified from bacteria. Lysates of cells transfected with the plasmid encoding Ssat1b were incubated with GST (Lane 1) or GST-Ssat1a (Lane 2). Lysates of cells transfected with the plasmid encoding Ssat1c were incubated with GST (Lane 3), GST-Ssat1a (Lane 4), or GST-Ssat1b (Lane 5). (B) GST and GST fused with the cytosolic domain of zebrafish Integrin 9α (GST-Intg9α_c) were purified from bacteria. Lysates of cells transfected with the plasmid encoding Ssat1a (lanes 1 and 2), Ssat1b (lanes 3 and 4), or Ssat1c (lanes 5 and 6) were incubated with GST (lanes 1, 3 and 5) or GST-Intg9α_c (lanes 2, 4 and 6). (C) Lysates of cells transfected with the plasmid encoding the myc-tagged PAS-B domain of Zebrafish Hif-1α was incubated with GST (lane 1), GST-Ssat1a (lane 2), GST-Ssat1b (lane 3), or GST-Ssat1c (lane 4). Bound proteins in each sample were pulled down with glutathione Sepharose 4B beads and analyzed by western blotting.
It has been reported that human SSAT1 interacts with the cytosolic domain of integrin α9 to enhance cell migration . A recombinant GST fusion to the cytosolic domain of zebrafish integrin α9 (GST-Intg α9) was used to study interactions with zebrafish family of Ssat1 proteins. Ssat1b and Ssat1c interacted with integrin α9, but Ssat1a did not (Fig. 6B).
Human SSAT1 also binds to the PAS-B (Per-ARNT-Sim) domain of HIF-1α, facilitating its degradation . A DNA fragment encoding zebrafish Hif-1α PAS-B was cloned into pcDNA3.1/myc-His and transfected into HEK293T cells. Cell lysates were extracted and incubated with GST-fused Ssat1 proteins. The results of GST pull-down experiments indicated that Ssat1b and Ssat1c interacted with the Hif-1α PAS-B domain but Ssat1a did not (Fig. 6C).
Despite reports of several polyamine acetyltransferases in microbes, their sequences were neither similar to each other nor to animal ssat-like genes , , , , , suggesting different evolutionary origins. In this work, 29 ssat homologs from 10 deuterostomia species were identified and analyzed. Comparing with ssat2 genes and invertebrates’ ssat-like genes, ssat1 genes are present only in vertebrates and highly conserved to each other (Fig. 1). Previous studies indicated that human SSAT2 and Ssat-like enzymes from several organisms do not involve polyamine catabolism, because these enzymes prefer to use thialysine as their substrate , , . Therefore, the polyamine interconversion pathway might only be evolved in the vertebrate lineage.
In humans, SSAT1 mRNA is transcribed in almost every tissue with 2 splicing forms . In addition to the normal mRNA, one transcriptional variant (SSAT-X mRNA), which cannot produce bioactive SSAT1 due to its incorporation of an extra exon from intron 3, is accumulated in hypoxic or iron-deficient cells . When cellular polyamine level increasing, the transcription of SSAT1 is induced ,  while the alternative spliced SSAT-X mRNA is reduced . In zebrafish, none of these ssat1 genes was induced by polyamine analog (Fig. 3A) and RT-PCR found no sign of alternative transcript splicing (Fig. 3). Zebrafish thus does not appear to regulate Ssat1 activity through mRNA transcription and alternative splicing.
A previous report indicated that an RNA binding protein, which might bind to the stem-loop structures in the 5′ and 3′ regions of the human SSAT1 ORF, strictly inhibits SSAT1 mRNA translation . A nucleolin isoform was recently identified as a key factor to stabilize the 5′ stem-loop structure of SSAT1 ORF. Addition of polyamine enhances the turnover of nucleolin thus significantly restore translation . In zebrafish, the stringent inhibition of background translation was only observed in ssat1b and ssat1c (Fig. 4A). By assaying a series of chimeric zebrafish ssat1 genes, we found that the both 5′ and 3′ regions of ssat1b were important for such regulation (Fig. 4B) that seems to be in accordance with the previous study . However, it is difficult to explain that altering the 5′ or 3′ mRNA structures by replacing the first 332 or the last 124 nucleotides of ssat1b with that of ssat1a did not affect the regulatory pattern of ssat1b (Fig. 4B, a332b and b389a). Moreover, attempts to search similar stem-loop structures of human SSAT1 in zebrafish ssat1b and ssat1c were also failed.
A recent study of yeast antizyme (Az) might provide another explanation. Like Ssat1, Az is also a homeostatic feedback regulator of polyamines, since the activity of ornithine decarboxylase, a key enzyme in polyamine biosynthesis pathway, is readily down-regulated in the presence of Az . It is interesting to note that the mRNA translation of Az is regulated in response to the cellular polyamine level, too. A unique polyamine-dependent +1 ribosomal frameshifting regulatory mechanism involving the 5′ region of Az mRNA is conserved in many Az orthologues . In addition, Kurian et al. recently found another regulatory mechanism of Az synthesis involving the 3′ region of its mRNA. In fact, it is the encoded amino acid sequence responsible for such kind of regulation, since the nascent Az will act as a sensor of polyamine and regulate the completion of its own translation . Until now, it has been unclear whether nascent Ssat1 serves as a polyamine sensor and regulates its own translation. However, it was known that the amino acid residues in the C-terminus of human SSAT1 are also important for translational regulation . Deleting more than 5 residues from the C-terminus has a significant impact, but silent mutations, in which several nucleotides in the 3′ end were modified without changing the coding sequence, retain polyamine responsiveness . Nevertheless, further study is needed to test this hypothesis.
The fourth regulatory mechanism of human SSAT1 is modulation of protein stability. Binding of polyamines or polyamine analogs changes the configuration of the SSAT1 protein. Therefore, SSAT1 is prevented from ubiquitination and becomes stable , . Extensive effort has led to the identification of key residues responsible for this regulation . The MATEE sequence in the C-terminus is important. SSAT1 stabilizes in the absence of polyamine when M167, E170, and E171 are substituted or the last 2 residues (E170 and E171) are deleted. SSAT1, however, carries on rapid degradation even in the presence of polyamine when point mutations are made at R7, C14, R19, H126, K141, E152, or R155. These residues in human SSAT1 are largely conserved in zebrafish Ssat1b, except the MATEE sequence is replaced by MESEE in the C-terminus (Fig. 2A). Note that M167, E170, and E171 are conserved in zebrafish Ssat1a and Ssat1b, but Ssat1a does not turn over rapidly (Fig. 5A). The chimeric enzyme Ssat1a453b, which preserved the last 20 residues of Ssat1b, was as stable as Ssat1a, suggesting more residues may be involved in this mechanism. The results of Ssat1a332b indicated that the last 70 residues of Ssat1b were important for its rapid degradation (Fig. 5B). Only 14 residues differ in the last 70 amino acids of Ssat1a and Ssat1b. Further study is needed to identify the residues responsible for regulating protein stability.
All zebrafish family of Ssat1 proteins were active enzymes, though their substrate preferences and catalytic efficiencies differed. Moreover, our data also indicated these zebrafish Ssat1 proteins form heterodimers (Fig. 6A). Their kinetics may be more complex due to heterodimerization. Considering the differences of these Ssat1 proteins in their expression profile, regulation patterns and enzyme activities, zebrafish might be able to fine-tune the metabolism of polyamine to fit the physiological requirements in different organs by expressing different Ssat1 proteins.
Integrins are cell surface proteins that mediate cell-cell communication and cell morphology. Integrin α9, a mammalian specific form , is stimulated by extracellular signals, such as tenascin C , osteopontin , and vascular cell adhesion molecules-1 , and involved in embryogenesis , lymphangiogenesis, and wound healing . It has been reported that overexpression of human SSAT1 enhances cell migration mediated by integrin α9 . The first 20 amino acids of SSAT1 is crucial, since they could bind to the cytosolic domain of integrin α9 thus regulates the migration signaling . In this study, we identified an integrin α9 orthologue in zebrafish. Although the length of the extracellular region differs significantly between human and zebrafish integrin α9, the sequences of their cytosolic domains are largely identical (Fig. S4). By using GST-pull down experiments, we confirmed that zebrafish integrin α9 interacts with Ssat1b and Ssat1c, but not Ssat1a (Fig. 6B). It is worth noting that Ser15 of Ssat1b, Ssat1c, and human SSAT1 was replaced by Pro15 in Ssat1a. The structures of SSAT1 reveal a conserved α-helix located between residues 12 and 28 (Fig. 2A) . Pro15 in Ssat1a may break the helix structure and thus interfere with the interaction between Ssat1a and integrin α9.
Hif-1α, a key regulator of oxygen homeostasis in all metazoans, is mainly regulated by an oxygen-sensing prolyl hydroxylase, which facilitates its rapidly degradation in proteosome . A previous study has shown another oxygen- independent Hif-1α regulation mechanism that is triggered by the binding of human SSAT1 with the PAS-B domain of HIF-1α . PAS domains, found in many proteins in all kingdoms of life, are structurally conserved protein-protein interaction modules . Although the amino acid sequences in PAS-B domains of human and zebrafish Hif-1α are highly conserved (Fig. S5), it is interesting to note that only Ssat1b and Ssat1c, but not Ssat1a, were able to interact with the PAS-B domain of zebrafish Hif-1α (Fig. 6C). The sequence variants between these homologues may provide clues to identify the critical regions responsible for Hif-1α binding in the future.
Gene duplication is considered to be the major force of evolution , because new copies may acquire new functions by mutation (known as neofunctionalization) . However, the fates of redundant genes might also include becoming pseudogenes (nonfunctionalization) or being preserved in a complementary partitioning of subfunctions (subfunctionalization) . It is generally believed that 2 rounds of whole-genome duplication occurred during the intergradation of vertebrates from their deuterostome ancestors . Interestingly, we noticed that not only ssat1 but also hif-1α  and integrin α9  were evolved simultaneously in the vertebrate lineage. They might experience neofunctionalization to meet the physiological requirements of vertebrates.
In comparison with mammals, the ray-finned fishes underwent an extra round of whole-genome duplication, which caused the teleost radiation . It seems that nonfunctionalization is the fate of the majority of duplicated ssat1 genes, as is the case in medaka, stickleback, takifugu, and tetraodon. Thus there is only one ssat1 left in these species. However, zebrafish, which contains not one but three ssat1 homologues, is an exception. The phylogenetic analysis indicates that all zebrafish ssat1 homologues are derived from a common ancestor (Fig. 1). The remnants of vertebrates’ ssat1 syntenic genes are found scattering in the loci of each homologue. For example, acot9 and apoo are clustered with ssat1a in chromosome 24, and prdx4 is closely located near ssat1b and ssat1c in chromosome 5 (Fig. S1). It is worth to note that the sequences between ssat1b and ssat1c are more similar to each other (Fig. S2). Further, Ssat1b and Ssat1c also have similar translational regulation pattern and protein-protein interaction relationships with Hif-1α and Integrin α9. These observations suggest that ssat1a might be one of the products from the teleosts’ whole genome duplication while the other one underwent a local duplication to form ssat1b and ssat1c later on.
Our results suggest that zebrafish ssat1 homologues are paralogous genes which experienced subfunctionalization in their function and regulation. It is worth noting that only Ssat1b and Ssat1c, but not the polyamine-insensitive Ssat1a, are able to interact with integrin α9 and Hif-1α. Thus these signal pathways could be regulated by Ssat1 in response to cellular polyamine level. Besides polyamine catabolism, it might be the key feature that allows Ssat1 to coordinate certain physiological responses in vertebrates, such as fine-tuning the advanced immune system and the homeostasis of polyamine and hypoxia.
By characterizing properties of zebrafish family of Ssat1 proteins and the artificial chimeric enzymes, our data revealed some correlations between their sequences and functions that may provide valuable information for studies of the translational regulatory mechanism, protein stability, and physiological functions of Ssat1 in the future.
Chromosomal localizations of human SSAT1 and zebrafish ssat1 homologues.
Alignment of zebrafish ssat1 homologues cDNA sequence. The cDNA sequences were aligned by MegAlign (Lasergene) with the ClustalW method. The conserved residues are shaded black. The denoted amino acid sequences underneath cDNA sequences are consensus residues in all three homologues, while the encoded amino acids which are not conserved in all three homologues are denoted by dash symbols.
Translational regulation of ssat1 genes in zebrafish cells. ZF4 cells were transiently transfected with the plasmid encoding myc-tagged full-length human SSAT1, zebrafish Ssat1a, or Ssat1b. After incubation for 12 h, transfected cells were treated with 10 µM DENSPM, 20 µM MG132, or vehicle for 24 h. Cell lysates (5 µg total protein in each lane) were prepared and the Ssat1 protein content in each sample was detected by western blotting with anti-myc antibody.
Sequence alignment of integrin α9. The amino acid sequences of human (NP_002198), mouse (NP_598482), and zebrafish integrin α9 (XP_003199805) were aligned by MegAlign (Lasergene) with the ClustalW method. The conserved residues are shaded in black. The cytosolic domains are marked with a red box.
Sequence alignment of human, mouse and zebrafish Hif-1α. The amino acid sequences of human (NP_001521), mouse (NP_034561), and zebrafish Hif-1α (AAQ91619) were aligned by MegAlign (Lasergene) with the ClustalW method. The conserved residues are shaded in black. The PAS-B domain is underlined in red.
- 1. Agostinelli E (2012) Role of polyamines, their analogs and transglutaminases in biological and clinical perspectives. Amino Acids 42: 397–409.
- 2. Perez-Leal O, Merali S (2012) Regulation of polyamine metabolism by translational control. Amino Acids 42: 611–617.
- 3. Pegg AE (2008) Spermidine/spermine-N(1)-acetyltransferase: a key metabolic regulator. Am J Physiol Endocrinol Metab 294: E995–1010.
- 4. Agostinelli E, Tempera G, Viceconte N, Saccoccio S, Battaglia V, et al. (2010) Potential anticancer application of polyamine oxidation products formed by amine oxidase: a new therapeutic approach. Amino Acids 38: 353–368.
- 5. Vujcic S, Diegelman P, Bacchi CJ, Kramer DL, Porter CW (2002) Identification and characterization of a novel flavin-containing spermine oxidase of mammalian cell origin. Biochem J 367: 665–675.
- 6. Alhonen L, Räsänen T-L, Sinervirta R, Parkkinen JJ, Korhonen V-P, et al. (2002) Polyamines are required for the initiation of rat liver regeneration. Biochem J 362: 149–153.
- 7. Han L, Xu C, Guo Y, Li H, Jiang C, et al. (2009) Polyamine metabolism in rat myocardial ischemia-reperfusion injury. Int J Cardiol 132: 142–144.
- 8. Rao AM, Hatcher JF, Doğan A, Dempsey RJ (2000) Elevated N1-acetylspermidine levels in gerbil and rat brains after CNS injury. J Neurochem 74: 1106–1111.
- 9. Zahedi K, Wang Z, Barone S, Prada AE, Kelly CN, et al. (2003) Expression of SSAT, a novel biomarker of tubular cell damage, increases in kidney ischemia-reperfusion injury. Am J Physiol Renal Physiol 284: F1046–1055.
- 10. Uimari A, Merentie M, Sironen R, Pirnes-Karhu S, Peräniemi S, et al. (2012) Overexpression of spermidine/spermine N(1)-acetyltransferase or treatment with N(1)-N(11)diethylnorspermine attenuates the severity of zinc-induced pancreatitis in mouse. Amino Acids 42: 461–471.
- 11. Hyvönen MT, Merentie M, Uimari A, Keinänen TA, Jänne J, et al. (2007) Mechanisms of polyamine catabolism-induced acute pancreatitis. Biochem Soc Trans 35: 326–330.
- 12. Jell J, Merali S, Hensen ML, Mazurchuk R, Spernyak JA, et al. (2007) Genetically altered expression of spermidine/spermine N1-acetyltransferase affects fat metabolism in mice via acetyl-CoA. J Biol Chem 282: 8404–8413.
- 13. Niiranen K, Keinänen TA, Pirinen E, Heikkinen S, Tusa M, et al. (2006) Mice with targeted disruption of spermidine/spermine N1-acetyltransferase gene maintain nearly normal tissue polyamine homeostasis but show signs of insulin resistance upon aging. J Cell Mol Med 10: 933–945.
- 14. Casero RA, Pegg AE (2009) Polyamine catabolism and disease. Biochem J 421: 323–338.
- 15. Chen C, Young BA, Coleman CS, Pegg AE, Sheppard D (2004) Spermidine/spermine N1-acetyltransferase specifically binds to the integrin alpha9 subunit cytoplasmic domain and enhances cell migration. J Cell Biol 167: 161–170.
- 16. Baek JH, Liu YV, McDonald KR, Wesley JB, Zhang H, et al. (2007) Spermidine/spermine N(1)-acetyltransferase-1 binds to hypoxia-inducible factor-1alpha (HIF-1alpha) and RACK1 and promotes ubiquitination and degradation of HIF-1alpha. J Biol Chem 282: 33358–33366.
- 17. Wang Y, Devereux W, Stewart TM, Casero RA (1999) Cloning and characterization of human polyamine-modulated factor-1, a transcriptional cofactor that regulates the transcription of the spermidine/spermine N(1)-acetyltransferase gene. J Biol Chem 274: 22095–22101.
- 18. Wang Y, Xiao L, Thiagalingam A, Nelkin BD, Casero RA (1998) The identification of a cis-element and a trans-acting factor involved in the response to polyamines and polyamine analogues in the regulation of the human spermidine/spermine N1-acetyltransferase gene transcription. J Biol Chem 273: 34623–34630.
- 19. Hyvönen MT, Uimari A, Keinänen TA, Heikkinen S, Pellinen R, et al. (2006) Polyamine-regulated unproductive splicing and translation of spermidine/spermine N1-acetyltransferase. RNA 12: 1569–1582.
- 20. Kim K, Ryu J-H, Park J-W, Kim M-S, Chun Y-S (2005) Induction of a SSAT isoform in response to hypoxia or iron deficiency and its protective effects on cell death. Biochem Biophys Res Commun 331: 78–85.
- 21. Hyvönen MT, Uimari A, Vepsäläinen J, Khomutov AR, Keinänen TA, et al. (2012) Tissue-specific alternative splicing of spermidine/spermine N (1)-acetyltransferase. Amino Acids 42: 485–493.
- 22. Butcher NJ, Broadhurst GM, Minchin RF (2007) Polyamine-dependent regulation of spermidine-spermine N1-acetyltransferase mRNA translation. J Biol Chem 282: 28530–28539.
- 23. Coleman CS, Pegg AE (2001) Polyamine analogues inhibit the ubiquitination of spermidine/spermine N1-acetyltransferase and prevent its targeting to the proteasome for degradation. Biochem J 358: 137–145.
- 24. Coleman CS, Pegg AE (1997) Proteasomal degradation of spermidine/spermine N1-acetyltransferase requires the carboxyl-terminal glutamic acid residues. J Biol Chem 272: 12164–12169.
- 25. Coleman CS, Huang H, Pegg AE (1995) Role of the carboxyl terminal MATEE sequence of spermidine/spermine N1-acetyltransferase in the activity and stabilization by the polyamine analog N1,N12-bis(ethyl)spermine. Biochemistry 34: 13423–13430.
- 26. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673–4680.
- 27. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 14: 95–98.
- 28. Felsenstein J (1989) PHYLIP - Phylogeny Inference Package (Version 3.2). Cladistics 5: 164–166.
- 29. Kammann M, Laufs J, Schell J, Gronenborn B (1989) Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR). Nucleic Acids Res 17: 5404.
- 30. Lin H-J, Lien Y-C, Hsu C-H (2010) A high-throughput colorimetric assay to characterize the enzyme kinetic and cellular activity of spermidine/spermine N(1)-acetyltransferase 1. Anal Biochem 407: 226–232.
- 31. Fogel-Petrovic M, Vujcic S, Brown PJ, Haddox MK, Porter CW (1996) Effects of polyamines, polyamine analogs, and inhibitors of protein synthesis on spermidine-spermine N1-acetyltransferase gene expression. Biochemistry 35: 14436–14444.
- 32. Lee SB, Park JH, Woster PM, Casero RA, Park MH (2010) Suppression of exogenous gene expression by spermidine/spermine N1-acetyltransferase 1 (SSAT1) cotransfection. J Biol Chem 285: 15548–15556.
- 33. Montemayor EJ, Hoffman DW (2008) The Crystal Structure of Spermidine/Spermine N1-Acetyltransferase in Complex with Spermine Provides Insights into Substrate Binding and Catalysis. Biochemistry 47: 9145–9153.
- 34. Bewley M, Graziano V, Jiang J, Matz E, Studier F, et al. (2006) Structures of wild-type and mutant human spermidine/spermine N 1-acetyltransferase, a potential therapeutic drug target. Proc Natl Acad Sci USA 103: 2063–2068.
- 35. Bai L, Chang M, Shan J, Jiang R, Zhang Y, et al. (2011) Identification and characterization of a novel spermidine/spermine acetyltransferase encoded by gene ste26 from Streptomyces sp. 139. Biochimie 93: 1401–1407.
- 36. Liu B, Sutton A, Sternglanz R (2005) A yeast polyamine acetyltransferase. J Biol Chem 280: 16659–16664.
- 37. Forouhar F, Lee I-S, Vujcic J, Vujcic S, Shen J, et al. (2005) Structural and functional evidence for Bacillus subtilis PaiA as a novel N1-spermidine/spermine acetyltransferase. J Biol Chem 280: 40328–40336.
- 38. Woolridge DP, Martinez JD, Stringer DE, Gerner EW (1999) Characterization of a novel spermidine/spermine acetyltransferase, BltD, from Bacillus subtilis. Biochem J 340 (Pt 3): 753–758.
- 39. Fukuchi J, Kashiwagi K, Takio K, Igarashi K (1994) Properties and structure of spermidine acetyltransferase in Escherichia coli. J Biol Chem 269: 22581–22585.
- 40. Lüersen K (2005) Leishmania major thialysine N [epsilon]-acetyltransferase: Identification of amino acid residues crucial for substrate binding. FEBS letters 579: 5347–5352.
- 41. Coleman CS, Stanley BA, Jones AD, Pegg AE (2004) Spermidine/spermine-N1-acetyltransferase-2 (SSAT2) acetylates thialysine and is not involved in polyamine metabolism. Biochem J 384: 139–148.
- 42. Abo-Dalo B, Ndjonka D, Pinnen F, Liebau E, Lüersen K (2004) A novel member of the GCN5-related N-acetyltransferase superfamily from Caenorhabditis elegans preferentially catalyses the N-acetylation of thialysine [S-(2-aminoethyl)-L-cysteine]. Biochem J 384: 129.
- 43. Perez-Leal O, Barrero CA, Clarkson AB, Casero RA, Merali S (2012) Polyamine-Regulated Translation of Spermidine/Spermine-N1-Acetyltransferase. Molecular and Cellular Biology 32: 1453–1467.
- 44. Rom E, Kahana C (1994) Polyamines regulate the expression of ornithine decarboxylase antizyme in vitro by inducing ribosomal frame-shifting. Proceedings of the National Academy of Sciences of the United States of America 91: 3959–3963.
- 45. Kurian L, Palanimurugan R, Gödderz D, Dohmen RJ (2011) Polyamine sensing by nascent ornithine decarboxylase antizyme stimulates decoding of its mRNA. Nature 477: 490–494.
- 46. Johnson MS, Lu N, Denessiouk K, Heino J, Gullberg D (2009) Integrins during evolution: evolutionary trees and model organisms. Biochim Biophys Acta 1788: 779–789.
- 47. Yokosaki Y, Matsuura N, Higashiyama S, Murakami I, Obara M, et al. (1998) Identification of the ligand binding site for the integrin alpha9 beta1 in the third fibronectin type III repeat of tenascin-C. J Biol Chem 273: 11423–11428.
- 48. Yokosaki Y, Matsuura N, Sasaki T, Murakami I, Schneider H, et al. (1999) The integrin alpha(9)beta(1) binds to a novel recognition sequence (SVVYGLR) in the thrombin-cleaved amino-terminal fragment of osteopontin. J Biol Chem 274: 36328–36334.
- 49. Taooka Y, Chen J, Yednock T, Sheppard D (1999) The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1. J Cell Biol 145: 413–420.
- 50. Huang XZ, Wu JF, Ferrando R, Lee JH, Wang YL, et al. (2000) Fatal bilateral chylothorax in mice lacking the integrin alpha9beta1. Mol Cell Biol 20: 5208–5215.
- 51. Singh P, Chen C, Pal-Ghosh S, Stepp MA, Sheppard D, et al. (2009) Loss of integrin alpha9beta1 results in defects in proliferation, causing poor re-epithelialization during cutaneous wound healing. J Investig Dermatol 129: 217–228.
- 52. deHart GW, Jin T, McCloskey DE, Pegg AE, Sheppard D (2008) The alpha9beta1 integrin enhances cell migration by polyamine-mediated modulation of an inward-rectifier potassium channel. Proc Natl Acad Sci USA 105: 7188–7193.
- 53. Loenarz C, Coleman M, Boleininger A, Schierwater B, Holland P, et al. (2010) The hypoxia-inducible transcription factor pathway regulates oxygen sensing in the simplest animal, Trichoplax adhaerens. EMBO reports 12: 63–70.
- 54. Vreede J, van der Horst MA, Hellingwerf KJ, Crielaard W, van Aalten DMF (2003) PAS domains. Common structure and common flexibility. J Biol Chem 278: 18434–18439.
- 55. Zhang J (2003) Evolution by gene duplication: an update. Trends Ecol Evol 18: 292–298.
- 56. Force A, Lynch M, Pickett FB, Amores A, Yan YL, et al. (1999) Preservation of duplicate genes by complementary, degenerative mutations. Genetics 151: 1531–1545.
- 57. Meyer A, Schartl M (1999) Gene and genome duplications in vertebrates: the one-to-four (-to-eight in fish) rule and the evolution of novel gene functions. Curr Opin Cell Biol 11: 699–704.
- 58. Postlethwait J, Amores A, Cresko W, Singer A, Yan Y-L (2004) Subfunction partitioning, the teleost radiation and the annotation of the human genome. Trends Genet 20: 481–490.