Duplication and diversification of the spermidine/spermine N1-acetyltransferase 1 genes in zebrafish

Abstract

Spermidine/spermine N(1)-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.

Figure 1. Phylogenetic analysis of ssat-like genes.

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.

Figure 2. Primary structures of zebrafish family of Ssat1 proteins and the constructs of chimeric proteins used in this study.

(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.

Figure 3. Temporal and spatial expression patterns of zebrafish ssat1 homologous genes.

(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.

Figure 4. Translational regulation inside the ORF of zebrafish family of Ssat1 proteins.

(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.

Figure 5. Protein stability of Ssat1b was regulated by polyamine.

(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.

Figure 6. Protein-protein interactions of zebrafish family of Ssat1 proteins.

(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.