Human epithelial protein SLURP-2 as a prototype of drugs for wound healing

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Abstract

Wound healing is a complex process based on the regulation of proliferation and migration of epithelial cells. Chronic wounds are characterized by increased proliferation and lack of migration of epidermal cells. The secreted human protein SLURP-2 regulates the growth and differentiation of epithelial cells. It has previously been shown that the targets of SLURP-2 are various types of nicotinic acetylcholine receptors (nAChRs), as well as muscarinic acetylcholine receptors involved in the regulation of epithelial cell homeostasis. In this work, we found that the previously demonstrated acceleration of keratinocyte migration under the incubation with SLURP-2 is due to its interaction with α7 type nAChR. Using alanine scanning mutagenesis, we showed that the R20A mutation of the SLURP-2 molecule increases the inhibitory activity of SLURP-2 towards α7-nAChR and leads to an even greater stimulation of Het-1A keratinocyte migration, while, in contrast to SLURP-2, does not stimulate, but suppresses the proliferation of Het-1A cells. At the same time, other SLURP-2 mutations simultaneously lead to inhibition of α7-nAChR, proliferation and migration of keratinocytes. Thus, new information was obtained about the localization of regions of the SLURP-2 molecule, the replacement of which can lead to a targeted change in the biological activity of SLURP-2. Further research into the possibility of regulating the activity of SLURP-2 and the creation of targeted drugs based on it may be useful for the development of new drugs that stimulate wound healing.

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M. L. Bychkov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: ekaterina-lyukmanova@yandex.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

O. V. Shlepova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS; Moscow Institute of Physics and Technology (National Research University)

Email: ekaterina-lyukmanova@yandex.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; Institutskiy per. 9, Dolgoprudny, Moscow Region, 141701

M. A. Shulepko

Shenzhen MSU-BIT University

Email: ekaterina-lyukmanova@yandex.ru
China, International University Park Road 1, Dayun New Town, Longgang District, Shenzhen, Guangdong Province, 518172 PRC

D. S. Kulbatskii

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: ekaterina-lyukmanova@yandex.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

D. Bertrand

HiQScreen Sàrl

Email: ekaterina-lyukmanova@yandex.ru
Switzerland, 6 rte de Compois, 1222, Vésenaz, Geneva

A. V. Kirichenko

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS; Moscow Institute of Physics and Technology (National Research University)

Email: ekaterina-lyukmanova@yandex.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; Institutskiy per. 9, Dolgoprudny, Moscow Region, 141701

Z. O. Shenkarev

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: ekaterina-lyukmanova@yandex.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

M. P Kirpichnikov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS; Lomonosov Moscow State University

Email: ekaterina-lyukmanova@yandex.ru

Interdisciplinary Scientific and Educational School of Moscow University “Molecular Technologies of the Living Systems and Synthetic Biology”, Faculty of Biology, Lomonosov Moscow State University

Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; Leninskiye Gory 1/12, Moscow, 119234

E. N. Lyukmanova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS; Moscow Institute of Physics and Technology (National Research University); Shenzhen MSU-BIT University; Lomonosov Moscow State University

Author for correspondence.
Email: ekaterina-lyukmanova@yandex.ru

Interdisciplinary Scientific and Educational School of Moscow University “Molecular Technologies of the Living Systems and Synthetic Biology”, Faculty of Biology, Lomonosov Moscow State University

Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; Institutskiy per. 9, Dolgoprudny, Moscow Region, 141701; International University Park Road 1, Dayun New Town, Longgang District, Shenzhen, Guangdong Province, 518172 PRC; Leninskiye Gory 1/12, Moscow, 119234

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2. Fig. 1. Effect of recombinant protein SLURP-2 and α-Bgtx on Het-1A cell migration: (a) effect of different concentrations of SLURP-2 on the area of ​​migration after 24, 48, and 72 hours of incubation. Data are presented as percentage of control ± SEM (n = 8–30), 100% corresponds to the area of ​​migration of untreated cells. The data were approximated by the Hill equation (y = A1 + (100% – A1)/(1 + ([SLURP-2]/EC50)nH). The calculated EC50, nH and A1 parameters were 95.1 ± 21.6 nM, 2.9 ± 1.2 and 168 ± 11% for 24 h, 72.0 ± 15.8 nM, 3.9 ± 1.7 and 140 ± 6% for 48 h and 99.5 ± 17.4 nM, 3.6 ± 2.1 and 120 ± 4% for 72 h; (b) – the area occupied by migrating Het-1A cells after 24, 48 and 72 h incubation. Data are presented as percentages from control ± SEM (n = 13–30), 100% corresponds to the area of ​​migration of untreated cells. ## p < 0.01 and #### p < 0.0001 indicate a significant difference from the control (100%) by one-tailed Student's t-test followed by Holm-Sidak/hoc-test; ** p < 0.01 and *** p < 0.001 mean a significant difference from the “SLURP-2” group by one-way ANOVA test followed by Dunnet's/hoc-test; (c) Representative images of the “wound healing” test for Het-1A cells incubated with 100 nM SLURP-2, 1 μM α-Bgtx and 100 nM SLURP-2 together with 1 μM α-Bgtx for 0, 24, 48 and 72 hours.

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3. Fig. 2. Effect of SLURP-2 and its mutants on acetylcholine-induced currents through α7-nAChR channels expressed in Xenopus laevis oocytes: (a) amino acid sequence of SLURP-2. Cysteine ​​residues are highlighted in yellow, disulfide bonds are shown in brackets. Mutated amino acid residues are labeled at the bottom; (b) spatial structure of SLURP-2 (PDB: 2n99) with residues substituted with alanine indicated on it; (c) amplitude of currents evoked by 40 μM ACh in Xenopus laevis oocytes expressing α7-nAChR in the presence of 1 μM SLURP-2 (SL-2) or its mutants. Data are presented as percentage of control ± standard error of the mean (n = 3), 100% corresponds to the amplitude of the current evoked by 40 μM ACh in untreated oocytes. * p < 0.05 and ** p < 0.01 indicate a significant difference from the “SLURP-2” group by one-way ANOVA test followed by Dunnet’s/hoc test.

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4. Fig. 3. Effect of SLURP-2 and its mutants on Het-1A cell proliferation. Cells were incubated for 48 h with 1 μM SLURP-2 (SL-2) or its mutants. The number of viable cells was determined using the WST1 assay. Data are presented as a percentage of the control ± SEM (n = 4–6), 100% of viable cells correspond to untreated cells. * p < 0.05, ** p < 0.01, and **** p < 0.0001 indicate a significant difference from the control (100%) according to the one-tailed Student's t-test followed by the Holm-Sidak/hoc test.

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5. Fig. 4. Effect of SLURP-2 and its mutants on Het-1A cell migration: (a) representative images of the wound healing assay for Het-1A cells incubated with 1 μM SLURP-2 (SL-2) or its mutants for 0, 24, 48, and 72 h; (b) area occupied by migrating Het-1A cells after 24, 48, and 72 h of incubation. Data are presented as percentage of control ± SEM (n = 16), 100% corresponds to the area of ​​migration of untreated cells. #### p < 0.0001 indicates a significant difference from control (100%) by one-tailed Student's t-test followed by Holm-Sidak/hoc test; * p < 0.05 and *** p < 0.001 indicate significant difference from the “SLURP-2” group according to the one-way ANOVA test followed by Dunnet’s/hoc test.

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