Russo R et al. (2024), Gene, Protein, and in Silico Analyses of FoxO, ...

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Genes (Basel) 2024 Aug 15;158:. doi: 10.3390/genes15081078.

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Gene, Protein, and in Silico Analyses of FoxO, an Evolutionary Conserved Transcription Factor in the Sea Urchin Paracentrotus lividus.

Russo R , Ragusa MA , Arancio W , Zito F .

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FoxO is a member of the evolutionary conserved family of transcription factors containing a Forkhead box, involved in many signaling pathways of physiological and pathological processes. In mammals, mutations or dysfunctions of the FoxO gene have been implicated in diverse diseases. FoxO homologs have been found in some invertebrates, including echinoderms. We have isolated the FoxO cDNA from the sea urchin Paracentrotus lividus (Pl-foxo) and characterized the corresponding gene and mRNA. In silico studies showed that secondary and tertiary structures of Pl-foxo protein corresponded to the vertebrate FoxO3 isoform, with highly conserved regions, especially in the DNA-binding domain. A phylogenetic analysis compared the Pl-foxo deduced protein with proteins from different animal species and confirmed its evolutionary conservation between vertebrates and invertebrates. The increased expression of Pl-foxo mRNA following the inhibition of the PI3K signaling pathway paralleled the upregulation of Pl-foxo target genes involved in apoptosis or cell-cycle arrest events (BI-1, Bax, MnSod). In silico studies comparing molecular data from sea urchins and other organisms predicted a network of Pl-foxo protein-protein interactions, as well as identified potential miRNAs involved in Pl-foxo gene regulation. Our data may provide new perspectives on the knowledge of the signaling pathways underlying sea urchin development.

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	Figure 1. Pl-foxo gene analysis. (A) Intron–exon structure of the Pl-foxo gene showing the two possible alternative splicings that result in short and long mRNA isoforms. Rectangles represent exons: white rectangles represent 5′UTR and 3′UTR sequences, and colored rectangles represent coding sequences. Numbers below the black lines and rectangles indicate the size (kb/bp) of the corresponding introns or exons, respectively. Pink diamonds represent the putative polyadenylation sites. (B) The regulatory region around the Pl-foxo gene promoter is visualized as tracks in the UCSC genome browser. The first exon of Pl-foxo is shown in purple, ATAC-seq signals and peaks are shown in blue, transcription factor binding sites (TFBS) are in green, conserved sequences in Echinoidea are in dark blue and repeats are in black.
	Figure 2. Pl-foxo protein analysis. (A) Diagram of the Pl-foxo protein obtained by Blast program, showing the most conserved regions of the FoxO superfamily and specific regions. (B) Clustal W alignment of the Forkhead DNA-binding domain of FoxO proteins from different organisms: Pl, P. lividus; Sp, S. purpuratus; Hl, H. leucospilota; Ar, A. rubens; Sk, S. kowaleskii; Hs, H. sapiens; Cc, C. charcarias; Bb, B. floridae; Dm, D. melanogaster; Ce, C. elegans. Asterisk = fully conserved amino acids; colon = conservative substitutions; period = semi-conservative substitutions.
	Figure 3. A 2/3D analysis of Pl-foxo. (A) The secondary structure of the DNA-binding domain of Pl-foxo transcription factor obtained by the Phyre2 program: the Forkhead domain is highlighted with two red rectangles. (B) The 3D structure predicted for Pl-foxo DBD obtained by I-Tasser software. (C) Scheme of the phosphorylation sites and their positions inside the Pl-foxo protein, revealed by the Netphos program 3.1.
	Figure 4. Evolutionary relationships of taxa for the Pl-foxo protein. Evolutionary analyses were conducted in ETE3 3.1.2. The tree is drawn to scale, with branch lengths proportional to the evolutionary distances used to infer the phylogenetic tree. The analysis involved 10 amino acid sequences. Ar, A. rubens, Bb, B. floridae; Dm, D. melanogaster; Cc, C. charcarias; Ce, C. elegans; Hl, H. leucospilota; Hs, H. sapiens; Pl, P. lividus; Sp, S. purpuratus; Sk, S. kowaleskii.
	Figure 5. qPCR analyses of the Pl-foxo transcription levels in normal or PI3K-inhibited P. lividus embryos. (A) Pl-foxo expression levels throughout P. lividus sea urchin embryogenesis: E, Eggs (used as reference samples and assumed as 1 in the histogram); 8C, 8 cells; M, morula; B, blastula; EG, early gastrula; LG, cate gastrula; Pr, prism; Pl, pluteus. Mean values were significantly different. The asterisks () indicate statistically significative variations to the relative reference sample. Each bar represents the mean of three independent experiments ± SD ( p < 0.05, p < 0.01, * p < 0.001). (B) Expression of PI3K and FoxO mRNAs, in addition to target genes of Pl-foxo (Pl-Bi-1, Pl-Bax, Pl-MnSod) in PI3K-inhibited embryos.
	Figure 6. Predicted interaction networks between FoxO proteins and the proteins encoded by the genes analyzed in PI3K-inhibted embryos, i.e., Bax, BI-1, Pi3k, Mnsod, together with Akt, Sirt1 and 14.3.3 e. Protein–protein interaction networks were simulated by the STRING database (confidence interaction score 0.300) in four different organisms, i.e., S. purpuratus, C. elegans, D. melanogaster and H. sapiens. Lines linking nodes represent the sources of active interactions, including known (databases, experiments) and predicted (gene neighborhood, fusion, co-occurrence) interactions, as well as text mining (proteins that are frequently mentioned together), co-expression and protein homology. The table shows synonyms for the proteins analyzed by the STRING database for each organism.
	Figure 7. Complete predicted interaction networks of FoxO proteins (Sp-foxo1 and homologs). Protein–protein interaction networks were simulated by searching all predicted interactions using the STRING database (highest confidence interaction score 0.900) in four different organisms, i.e., S. purpuratus (A), C. elegans (B), D. melanogaster (C) and H. sapiens (D). Lines linking nodes represent the sources of active interactions, including known (databases, experiments) and predicted (gene neighborhood, fusion, co-occurrence) interactions, as well as text mining (proteins that are frequently mentioned together), co-expression and protein homology.
	Figure 8. Diagram showing a summary of the experimental and prediction data described in this study. Genes analyzed by qPCR (green); proteins interacting with Pl-foxo, predicted by STRING analyses (turquoise); TFs, annotated in the S. purpuratus genome, binding the putative regulatory region of the Pl-foxo promoter, found by the footprint approach (fuchsia; in bold TFs characterized in P. lividus); potential miRNAs for the 3′UTR (orange) and the coding region of Pl-foxo (blue), according to database analyses.