Ragusa MA et al. (2019), A Survey on Tubulin and Arginine Methyltransfer...

ECB-ART-47166

Int J Mol Sci 2019 Apr 30;209:. doi: 10.3390/ijms20092136.

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A Survey on Tubulin and Arginine Methyltransferase Families Sheds Light on P. lividus Embryo as Model System for Antiproliferative Drug Development.

Ragusa MA , Nicosia A , Costa S , Casano C , Gianguzza F .

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Tubulins and microtubules (MTs) represent targets for taxane-based chemotherapy. To date, several lines of evidence suggest that effectiveness of compounds binding tubulin often relies on different post-translational modifications on tubulins. Among them, methylation was recently associated to drug resistance mechanisms impairing taxanes binding. The sea urchin is recognized as a research model in several fields including fertilization, embryo development and toxicology. To date, some α- and β-tubulin genes have been identified in P. lividus, while no data are available in echinoderms for arginine methyl transferases (PRMT). To evaluate the exploiting of the sea urchin embryo in the field of antiproliferative drug development, we carried out a survey of the expressed α- and β-tubulin gene sets, together with a comprehensive analysis of the PRMT gene family and of the methylable arginine residues in P. lividus tubulins. Because of their specificities, the sea urchin embryo may represent an interesting tool for dissecting mechanisms of tubulin targeting drug action. Therefore, results herein reported provide evidences supporting the P. lividus embryo as animal system for testing antiproliferative drugs.

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Species referenced: Echinodermata
Genes referenced: carm1 glis3 LOC100887844 LOC100893907 LOC574936 LOC579909 LOC586122 prmt5 tubgcp2

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	Figure 1. Multiple sequence alignment (MSA) of the α-tubulins of P. lividus. Alignment was performed with T-coffee and rendered by ESPript 3.0. Similar residues are written in black bold characters and boxed in yellow whereas conserved residues are in white bold characters and boxed in red. The sequence numbering on the top refers to the alignment. Tubulin domains are highlighted by long arrows also on the top. The principals PTMs are indicated on the bottom. Ac: acetylation; Me: methylation; P: phosphorylation; Glu: polyglutamylation and polyglycylation.
	Figure 2. MSA of the β-tubulins of P. lividus. Alignment was performed with T-coffee and rendered by ESPript 3.0. Similar residues are written in black bold characters and boxed in yellow whereas conserved residues are in white bold characters and boxed in red. The sequence numbering on the top refers to the alignment. Tubulin domains are highlighted by long arrows also on the top. The principals PTMs are indicated on the bottom. Me: methylation; P: phosphorylation; Glu: polyglutamylation and polyglycylation.
	Figure 3. α- and β-tubulin gene structure and core promoter analysis. Schematic gene structures of the Paracentrotus lividus α-tubulins (A), β-tubulins (B) and an α-tubulin gene of Eucidaris tribuloides (C), drawn to scale. The bent arrows indicate the putative transcription start sites (TSS). Boxes represent exons. In particular, white boxes indicate untranslated regions and coding regions are coloured. Core promoter elements are shown as indicated.
	Figure 4. P. lividus α- and β-tubulin NJ distance trees. The trees were generated using MEGA X. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the reliability of the branches of the analysed sequences. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) greater than 50% are shown in red next to the branches.
	Figure 5. Schematic representation of the seven P. lividus PRMT isoforms and their domains predicted by InterPro (drawn to scale). SH3: SRC homology 3 domain; Zn finger: Zinc finger C2H2 superfamily; CARM1/PH: Histone-arginine methyltransferase CARM1, N-terminal / PH-like domain superfamily; TIM barrel: PRMT5, TIM barrel domain; R-N-methyltransferase: SAM-dependent methyltransferase PRMT-type domain. The protein regions predicted as disordered are depicted in red.
	Figure 6. Amino acid sequences of P. lividus and Homo sapiens PRMTs were aligned and a distance tree was constructed. The tree was generated using MEGA X. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the reliability of the branches of the analysed sequences. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) greater than 50% are shown in red next to the branches. Pairwise amino acid sequence alignment of human and P. lividus PRMTs are shown in Supplementary Materials Figures S1 and S2.
	Figure 7. MSA of the methyltransferase domains (ProSite ID PS51678) of human and rat PRMT1 and P. lividus PRMT family performed with T-Coffee. Similar residues are written in black bold characters and boxed in yellow whereas conserved residues are in white bold characters and boxed in red. The sequence numbering on the top refers to the rat PRMT1 sequence (Q63009). PRMT1 domains and secondary structures are highlighted on the top; the colour coding is red for the N terminus (residues 41–51), green for the SAM binding domain (residues 52–176), yellow for the β barrel structure (residues 177–187 and 217–352), and blue for the dimerization arm (residues 188–216), as depicted in Zhang and Cheng [85].
	Figure 8. Molecular evolution of sea urchin PRMT1. Top: Superposition of three-dimensional models in ribbon representation of rat PRMT1 (1ORH; showed in pale) and P. lividus PRMT1 showed in light blue. Bottom: same superposition in surface (50% transparency) representation with their structures coloured according to the evolutionary conservation of amino acids. The P. lividus 3D structure was created via the Phyre 2 software [87] and rendered by using Chimera package [88]. Variable positions are presented in blue; while conserved amino acids are shown in red as defined in the colour-coding bar. In all the structures only the residues after amino acid 40 are shown, since the amino terminus is disordered.
	Figure 9. P. lividus PRMT1 dimer structure models obtained by ClusPro [89]. The colour coding is red/orange red for the N-terminal domains, green/light green for the SAM binding domains, orange/yellow for the β barrel structures, and blue/light blue for the dimerization arm. (A) the ring-like model similar to the model described in Zhang and Cheng [85]; (B) the best ranked compact model.
	Figure 10. Comparison between arginine methylation sites in human and P. lividus β-tubulins. Top, ribbon/surface diagrams of the human neural β-tubulin structure Tubb3 (5JCO). Bottom, ribbon/surface diagrams of the neural β-tubulin structure generated by homology modelling. The 3D structures were created via the Phyre 2 software [87] and rendered by using Chimera package [88]. The arginine residues that can be methylated were coloured according to their accessibility on the surface: buried in yellow, intermediate accessibility in cyan, exposed in blue. The last amino acid shown in the structures (Q426 for the human protein and D427 for the P. lividus protein) are coloured in red as reference.

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