Kulbatskii D , Shenkarev Z , Bychkov M , Loktyushov E , Shulepko M , Koshelev S , Povarov I , Popov A , Peigneur S , Chugunov A , Kozlov S , Sharonova I , Efremov R , Skrebitsky V , Tytgat J , Kirpichnikov M , Lyukmanova E .

             cholinergic synapse

           PARKINSON-DEMENTIA SYNDROME

	Figure 1. Effect of ws-Lypd6 on heteromeric nAChRs expressed in X. laevis oocytes. (A) Representative responses to 100 ms pulses of ACh (100 μM for α1β1γδ, α1β1δε, α3β2, α3β4; 10 μM for α4β2, and α4β4) recorded in the presence or absence of 30 μM ws-Lypd6. Pre-incubation time of oocytes with ws-Lypd6 was 5 min. (B) Dose-response curve for ws-Lypd6 inhibition of ACh-evoked currents at α3β4-nAChRs. The data are normalized to the peak amplitude of current recorded without ws-Lypd6 (100%) and presented as mean ± SEM (n = 6 oocytes). The data were fitted by the Hill’s equation resulted in IC50 for ws-Lypd6 of 34.9 ± 1.2 μM, A0 of 33 ± 5%. (C) Dose-response curves of ACh at α3β4-nAChRs in absence (open symbols) and presence of 35 μM ws-Lypd6 (closed symbols). The peak inward current amplitudes were normalized to the peak current obtained by the application of 1 mM ACh and plotted against the different concentrations of ACh applied in control. The concentration-response curve in presence of ws-Lypd6 was normalized to the control. The obtained EC50 values for ACh are 29.8 ± 4.7 μM and 31.3 ± 6.2 μM in absence and presence of ws-Lypd6, respectively. * (p < 0.05) indicates significant difference from the control value (100%, max. response for ACh without ws-Lypd6) (one sample t-test).
	Figure 2. Ws-Lypd6 inhibits homomeric α7-nAChR expressed in X. laevis oocytes. (A,B) Representative responses to 100 ms pulses of 100 μM ACh recorded in the presence or absence of 30 μM ws-Lypd6 or ws-Lypd6b. Pre-incubation time of oocytes with ws-Lypd6 or ws-Lypd6b was 15 s. (C) Dose-response curve for the ws-Lypd6 inhibition of ACh-evoked currents at α7-nAChRs (closed symbols). The data are normalized to the peak amplitude of current recorded without ws-Lypd6 and ws-Lypd6b (100%) and presented as mean ± SEM (n = 8 oocytes from 3 animals). The data were fitted by the Hill equation resulted in IC50 for Lypd6 of 10.9 ± 1.8 μM, nH of 1.6 ± 0.4, and A0 of 46 ± 4%. Response to 30 μM ws-Lypd6b is shown by open symbol (n = 4 oocytes from 2 animals). * (p < 0.05) indicates significant difference of ws-Lypd6 and ws-Lypd6b effects (t-test).
	Figure 3. Co-localization of endogenous Lypd6 and nAChRs containing α7 subunit in primary cortical and hippocampal neurons. Cells were sequentially incubated with the mouse anti-α7/rabbit anti-Lypd6 antibodies and secondary anti-mouse Alexa-488 (green)/anti-rabbit TRITC (red) labeled antibodies, respectively. (A,C) Colocalization of Lypd6 and the α7 subunit in the cortical neurons. (B,D) Colocalization of Lypd6 and the α7 subunit in the hippocampal neurons. Scale bar 10 μm. (C,D) Enlarged fragments showed by white rectangles on (A,B), respectively. Points with the different Lypd6 and α7 expression are shown by arrows. (E) Pearson’s correlation coefficients of the Lypd6 and α7 colocalization in primary cortical and hippocampal neurons (n = 4, microphotographs).
	Figure 4. Co-localization of endogenous Lypd6 and nAChRs containing α3 subunit in primary cortical and hippocampal neurons. Cells were sequentially incubated with the mouse anti-α3/rabbit anti-Lypd6 antibodies and secondary anti-mouse Alexa-488 (green)/anti-rabbit TRITC (red) labeled antibodies, respectively. (A,C) Colocalization of Lypd6 and the α3 subunit in the cortical neurons. (B,D) Colocalization of Lypd6 and the α3 subunit in hippocampal neurons. Scale bar 10 μm. (C,D) Correspond to the enlarged fragments showed by white rectangles on (A,B), respectively. Points with the different Lypd6 and α3 expression are shown by arrows. (E) Pearson’s correlation coefficients of the Lypd6 and α3 colocalization in primary cortical and hippocampal neurons (n = 4, microphotographs).
	Figure 5. Ws-Lypd6 inhibits choline-evoked currents at nAChRs in the hippocampus. (A) Representative responses to 1 mM choline pulses in the absence and presence of 8 μM DhβE or 10 nM α-Bgtx. (B) Representative responses to 25 ms pulses of 1 mM choline (Ch) recorded in one cell in the absence and presence of 1 μM ws-Lypd6. (C) Peak amplitude of the choline-evoked currents in the presence of 1 μM ws-Lypd6 and after wash-out recorded in four different cells taken from different animals (n = 4 slices, four rats). Four responses (like shown in B) were averaged for each slice and normalized to the average control amplitude (100%, shown by dotted line). The data are presented as mean ± SEM. * (p < 0.05) indicates significant difference from the control value (t-test). Experiments in (B,C) were performed in presence of 8 μM DhβE.
	Figure 6. Ws-Lypd6 suppresses long-term potentiation (LTP) in the mice hippocampal slices. (A) Averaged 1 h LTP responses in hippocampal slices after 1 h preincubation in ACSF containing 1 μM ws-Lypd6 or 10 nM α-Bgtx (in each case n = 6 slices, six mice). The same compounds were present in ACSF during recording. Representative normalized fEPSP traces are shown above: gray—baseline, black—post-tetanic recording. (B) Normalized fEPSP slopes averaged over 0–10 and 50–60 min after HFS. The data are presented as mean ± SEM. * (p < 0.05), ** (p < 0.01), and *** (p < 0.001) indicate significant difference (t-test).
	Figure 7. Spatial structures and molecular surfaces of ws-Lypd6 (A,B) and Lypd6b (C,D). (A,C) The disulfide bonds are in orange. Only one conformation of the flexible C-terminal “tail” is shown for each protein. The C-termini of the structured LU-domains (Leu85) are marked with asterisks. (B,D) Two-sided view of the molecular surfaces. Only LU-domains without C-tails (terminated at Leu85) are shown. The hydrophobic (Ala, Met, Ile, Leu, Val, Cys, and Pro), aromatic (Phe, Trp, Tyr), polar (Asn, Gln, Gly, Ser, and Thr), positively charged (Arg and Lys), negatively charged (Asp and Glu), and His residues are colored in yellow, green, violet, blue, red, and cyan, respectively. Two possible receptor-binding sites responsible for the Lypd6 interaction with nAChRs are highlighted by dashed ellipses. Orientation of the molecular surfaces in the left parts of the panels (B,D) is identical to the molecules orientation in (A,C) PDB codes: ws-Lypd6—6IB6, ws-Lypd6B—6ZSO (Paramonov et al., 2020).
	Figure 8. In silico modeling of the α7-ECD/ws-Lypd6 complex. (A) General view of homopentameric nAChR in the membrane. For clarity, the subunits are colored differently. (B,D) The top view on α7-ECD with the bound ws-Lypd6 molecules. Ligand-binding sites of the receptor have different conformations of the loop C. In the sites 1, 3, and 4 the ligand-binding pockets are “closed” by the loop C, while in the sites 2 and 5 the ligand-binding pockets are “open”. (C,E) The side view on the ligand-binding sites 1 and 5 of α7-ECD. Each binding site is formed at the interface between the primary (+) and complementary (–) α7 subunits. (B,C) Representative solution, where ws-Lypd6 contacts the “closed” α7-nAChR binding site with the loops I, II and III (complex #1, Supplementary Table 1). For clarity, the agonist (epibatidine) molecule (green spheres) is placed into the orthosteric ligand-binding site according to PDB id 3SQ6. (D,E) Representative solution, where ws-Lypd6 interacts with the “open” α7-nAChR binding site by the “head” and loop III (complex #2, Supplementary Table 1). The putative position of an agonist in the orthosteric binding site is shown by blue dashed-dot ellipses. C loop of the primary α7 subunit and ws-Lypd6 loops are shown by red and orange/magenta/blue colors, respectively. Disulfide bonds are in yellow. N- and C-termini of the Lypd6 LU-domain are shown with black and red spheres, respectively. The “favorable” contacts observed in the complexes are listed in Supplementary Table 1.
	Figure 9. Comparison of the ws-Lypd6 and ws-Lypd6b amino acid sequences and their possible complexes with α7-ECD. (A) The “favorable” contacts in the α7-ECD/ws-Lypd6 complex (Figure 8C). The sidechains participating in the electrostatic and hydrophobic interactions are shown. (B) The possible structure of the α7-ECD/ws-Lypd6b complex. The ws-Lypd6b molecule is shown in the same orientation as ws-Lypd6 molecule in (A). Ws-Lypd6b cannot form as many “favorable” contacts with the receptor as ws-Lypd6. (C) Sequence alignment of the LU-domains of ws-Lypd6 and ws-Lypd6b. The similarity (%) is independently shown for each region of the proteins. The total number of residues in the loops is given in brackets. The differing residues are underlined. The aromatic (Phe, Trp, Tyr), positively charged (Arg and Lys), negatively charged (Asp and Glu), and His residues are colored in green, blue, red, and cyan, respectively. The residues of ws-Lypd6 forming contacts with α7-ECD in the proposed model are highlighted by violet background.

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