Paramonov AS , Shulepko MA , Makhonin AM , Bychkov ML , Kulbatskii DS , Chernikov AM , Myshkin MY , Shabelnikov SV , Shenkarev ZO , Kirpichnikov MP , Lyukmanova EN .

19-74-20176 Russian Science Foundation

	Figure 1. E-value and bit score heatmaps for putative TFPs from A. rubens and A. planci predicted by BLAST search vs. closest Ly6/uPAR proteins. Darker cells correspond to higher bit-scores and lower e-values of blast hit. The sequence of LyAr1 from A. rubens studied in this work (Lystar5) is designated by asterisk. Correspondence of putative TFPs from starfishes to the transcript IDs in the NCBI database is given in Table S2.
	Figure 2. Alignment of amino acid sequences of BLAST hits from A. rubens (a) and A. planci (b) with corresponding TFPs. Disulfide bonds are shown by orange brackets. The sequence of LyAr1 from A. rubens studied in this work (Lystar5) is designated by asterisk. Correspondence of putative TFPs from starfishes to the transcript IDs in the NCBI database is given in Table S2 (supplementary file).
	Figure 3. Analysis of TFPs clusters from different taxonomic groups including proteins from starfishes A. rubens and A. planci. The sequence of LyAr1 from A. rubens studied in this work (Lystar5) is designated by an asterisk. Correspondence of putative TFPs from starfishes to the transcript IDs in the NCBI database is given in Table S2.
	Figure 4. Alignment of amino acid sequences of TFPs found in A. rubens and A. planci. Disulfide bonds are shown by orange brackets. The sequence of LyAr1 from A. rubens studied in this work (Lystar5) is designated by an asterisk.
	Figure 5. 15N-HSQC NMR spectrum of 13C,15N-labelled Lystar5 (37 °C, pH 7.0, 800 MHz). Cross-peaks of the form I labeled in black, cross-peaks of the form II are labeled in red where they differ.
	Figure 6. NMR data on secondary structure and conformational heterogeneity of Lystar5. Data for form I and form II are shown by black and red symbols, respectively. (a) Δδ – Difference in 1H15N chemical shifts of backbone amide groups between form I and form II (Δδ=(ΔδHN)2+(ΔδNH5)2); Pα and Pβ–Probabilities of α-helix and β-structure formation calculated from chemical shifts in the TALOS-N software; protein sequence is given in the bottom of the Pβ panel. Disulfide connectivities are shown by black brackets; the determined secondary structure is shown above the Pα panel; Δδ1HN/ΔT–temperature coefficients of amide protons. The filled circles denote amide protons with absolute values of temperature gradients less than 4.5 ppb/K; 3JHNHα–coupling constants. The small (<5.5 Hz), large (>8.5 Hz), and medium (others) couplings are designated by open triangles, filled triangles and open squares, respectively; (b) Scheme of contacts between β-strands observed in the NOESY spectra. (c) Scheme of folding of secondary structure elements. β-Sheets formed by β1/ β2 and β3/β4/β6 strands are shown by red dotted rectangles.
	Figure 7. Comparison of structural features of Lystar5 and Lynx2. (a) Alignment of primary structures of Lystar5 and Lynx2. Disulfide bonds are shown by orange brackets. (b) Comparison of secondary structures of Lystar5 and Lynx2. (c) Ribbon representation of the Lystar5 spatial structure predicted by AlphaFold2 [45]. (d) Ribbon representation of the Lynx2 spatial structure obtained by NMR (PDB Id 6ZSS). Experimentally defined elements of the secondary structure are denoted by color: β-strands in blue and helical elements in red. Disulfide bonds are in gold.
	Figure 8. Lystar5 interaction with α4β2-nAChRs. (a,d) Composition of high selective (HS) and low selective (LS) subtypes of α4β2-nAChR. (b,e) Representative current traces through LS (b) and HS (e) α4β2-nAChRs expressed in X. laevis oocytes, evoked by 10 µM (HS receptors) or 100 µM (LS receptors) acetylcholine (ACh). The bars above the traces designate the application of specific compounds, the Lystar5 bar is off-scale along time axis (50 µM Lystar5 preincubation time 15s). (c,f) Average current amplitudes normalized to the control currents (before Lystar5 application) at LS (c) and HS (f) receptors, mean ± SEM, n = 4. * (p < 0.05) indicates significant differences between the group and control value (1.0) by one-sample two-sided t-test, n.s.—non-significant difference.
	Figure 9. Analysis of the Lystar5 influence on expression of α4 (a), α7 (b) nAChR subunits, acetylcholine esterase (c), synapsin (d), synaptophysin (e), and PSD95 (f) in rat hippocampal neurons upon 7-day incubation. The mRNA level was normalized to the β-actin level and presented as a relative level ± SEM (n = 4–6). ** (p < 0.01) and **** (p < 0.0001) indicate significant difference between the data groups according to two-sided t-test.

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