Human SLURP-1 and SLURP-2 Proteins Acting on Nicotinic Acetylcholine Receptors Reduce Proliferation of Human Colorectal Adenocarcinoma HT-29 Cells

Cover Page

Abstract


Human secreted Ly-6/uPAR related proteins (SLURP-1 and SLURP-2) are produced by various cells, including the epithelium and immune system. These proteins act as autocrine/paracrine hormones regulating the growth and differentiation of keratinocytes and are also involved in the control of inflammation and malignant cell transformation. These effects are assumed to be mediated by the interactions of SLURP-1 and SLURP-2 with the α7 and α3β2 subtypes of nicotinic acetylcholine receptors (nAChRs), respectively. Available knowledge about the molecular mechanism underling the SLURP-1 and SLURP-2 effects is very limited. SLURP-2 remains one of the most poorly studied proteins of the Ly-6/uPAR family. In this study, we designed for the first time a bacterial system for SLURP-2 expression and a protocol for refolding of the protein from cytoplasmic inclusion bodies. Milligram quantities of recombinant SLURP-2 and its 13С-15N-labeled analog were obtained. The recombinant protein was characterized by NMR spectroscopy, and a structural model was developed. A comparative study of the SLURP-1 and SLURP-2 effects on the epithelial cell growth was conducted using human colorectal adenocarcinoma HT-29 cells, which express only α7-nAChRs. A pronounced antiproliferative effect of both proteins was observed. Incubation of cells with 1 μM SLURP-1 and 1 μM SLURP-2 during 48 h led to a reduction in the cell number down to ~ 54 and 63% relative to the control, respectively. Fluorescent microscopy did not reveal either apoptotic or necrotic cell death. An analysis of the dose-response curve revealed the concentration-dependent mode of the SLURP-1 and SLURP-2 action with EC50 ~ 0.1 and 0.2 nM, respectively. These findings suggest that the α7-nAChR is the main receptor responsible for the antiproliferative effect of SLURP proteins in epithelial cells.


E. N. Lyukmanova

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry; Lomonosov Moscow State University

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

Russian Federation

M. A. Shulepko

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry; Lomonosov Moscow State University

Email: ekaterina-lyukmanova@yandex.ru

Russian Federation

M. L. Bychkov

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry; Lomonosov Moscow State University

Email: ekaterina-lyukmanova@yandex.ru

Russian Federation

Z. O. Shenkarev

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry; Lomonosov Moscow State University

Email: ekaterina-lyukmanova@yandex.ru

Russian Federation

A. S. Paramonov

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry; Lomonosov Moscow State University

Email: ekaterina-lyukmanova@yandex.ru

Russian Federation

A. O. Chugunov

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry; Lomonosov Moscow State University

Email: ekaterina-lyukmanova@yandex.ru

Russian Federation

A. S. Arseniev

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry; Moscow Institute of Physics and Technology (State University)

Email: ekaterina-lyukmanova@yandex.ru

Russian Federation

D. A. Dolgikh

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry; Lomonosov Moscow State University

Email: ekaterina-lyukmanova@yandex.ru

Russian Federation

M. P. Kirpichnikov

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry; Lomonosov Moscow State University

Email: ekaterina-lyukmanova@yandex.ru

Russian Federation

  1. Papke R.L. // Biochem. Pharmacol. 2014, V.89, №1, P.1-11
  2. Sharma G., Vijayaraghavan S. // J. Neurobiol. 2002, V.53, №4, P.524-534
  3. Miwa J.M., Ibanez-Tallon I., Crabtree G.W., Sánchez R., Sali A., Role L.W., Heintz N. // Neuron. 1999, V.23, №1, P.105-114
  4. Tekinay A.B., Nong Y., Miwa J.M., Lieberam I., Ibanez-Tallon I., Greengard P., Heintz N. // Proc. Natl. Acad. Sci. USA. 2009, V.106, P.4477-4482
  5. Chimienti F., Hogg R.C., Plantard L., Lehmann C., Brakch N., Fischer J., Huber M., Bertrand D., Hohl D. // Human Molecular Genetics 2003, V.12, P.3017-3024
  6. Arredondo J., Chernyavsky A.I., Jolkovsky D.L., Webber R.J., Grando S.A. // J. Cell. Physiol. 2006, V.208, P.238-245
  7. Darvas M., Morsch M., Racz I., Ahmadi S., Swandulla D., Zimmer A. // Eur. Neuropsychopharmacol. 2009, V.19, P.670-681
  8. Tsetlin V., Utkin Y., Kasheverov I. // Biochem. Pharmacol. 2009, V.78, P.720-731
  9. Moriwaki Y., Watanabe Y., Shinagawa T., Kai M., Miyazawa M., Okuda T., Kawashima K., Yabashi A., Waguri S., Misawa H. // Neurosci. Res. 2009, V.64, P.403-412
  10. Moriwaki Y., Yoshikawa K., Fukuda H., Fujii Y.X., Misawa H., Kawashima K. // Life Sci. 2007, V.80, P.2365-2368
  11. Arredondo J., Chernyavsky A.I., Webber R.J., Grando S.A. // J. Invest. Dermatol. 2005, V.125, P.1236-1241
  12. Chernyavsky A.I., Galitovskiy V., Shchepotin I.B., Grando S.A. // Biomed. Res. Int. 2014, V.2014, P.609086
  13. Chernyavsky A.I., Kalantari-Dehaghi M., Phillips C., Marchenko S., Grando S.A. // Wound Repair Regen. 2012, V.20, №1, P.103-113
  14. Arredondo J., Chernyavsky A.I., Grando S.A. // Life Sci. 2007, V.80, P.2243-2247
  15. Arredondo J., Chernyavsky A.I., Grando S.A. // Biochem. Pharmacol. 2007, V.74, №8, P.1315-1319
  16. Chernyavsky A.I., Marchenko S., Phillips C., Grando S.A. // Dermatoendocrinol. 2012, V.4, №3, P.324-330
  17. Pettersson A., Nylund G., Khorram-Manesh A., Nordgren S., Delbro D.S. // Auton. Neurosci. 2009, V.148, P.97-100
  18. Summers A.E., Whelan C.J., Parsons M.E. // Life Sci. 2003, V.72, №18-19, P.2091-2094
  19. Shulepko M.A., Lyukmanova E.N., Paramonov A.S., Lobas A.A., Shenkarev Z.O., Kasheverov I.E., Dolgikh D.A., Tsetlin V.I., Arseniev A.S., Kirpichnikov M.P. // Biochemistry (Mosc). 2013, V.78, P.204-211
  20. Webb B., Sali A. // Current Protocols in Bioinformatics. 2014, V.47, №5-6, P.1-32
  21. Lyukmanova E.N., Shulepko M.A., Tikhonov R.V., Shenkarev Z.O., Paramonov A.S., Wulfson A.N., Kasheverov I.E., Ustich T.L., Utkin Y.N., Arseniev A.S. // Biochemistry (Mosc). 2009, V.74, P.1142-1149
  22. Shulepko M.A., Lyukmanova E N., Kasheverov I.E., Dolgikh D.A., Tsetlin V.I., Kirpichnikov M.P. // Russian J. Bioorg. Chem. 2011, V.37, P.43-549
  23. Lyukmanova E.N., Shenkarev Z.O., Shulepko M.A., Mineev K.S., D’Hoedt D., Kasheverov I.E., Filkin S., Janickova H., Dolezal V., Dolgikh D.A. // J. Biol.Chem. 2011, V.286, P.1061-810627
  24. Grando S.A. // J. Investig. Dermatol. Symp. Proc. 1997, V.2, №1, P.41-48

Views

Abstract - 295

PDF (English) - 107

PDF (Russian) - 108

Cited-By


Article Metrics

Metrics Loading ...

Metrics powered by PLOS ALM

PlumX

Dimensions

Refbacks

  • There are currently no refbacks.

Copyright (c) 2014 Lyukmanova E.N., Shulepko M.A., Bychkov M.L., Shenkarev Z.O., Paramonov A.S., Chugunov A.O., Arseniev A.S., Dolgikh D.A., Kirpichnikov M.P.

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies