Structure/activity Relationship of Thapsigargin Inhibition on the Purified Golgi/secretory Pathway Ca2+/Mn2+-Transport ATPase (SPCA1a)

Chen Jialin; Joren  de Raeymaecker; Jannik Brøndsted Hovgaard; Susanne Smaardijk; Ilse Vandecaetsbeek; Frank Wuytack; Jesper Vuust Møller; Jan Eggermont; Marc de Meyer; Søren Brøgger Christensen; Peter Vangheluwe

doi:10.1074/jbc.m117.778431

Structure/activity Relationship of Thapsigargin Inhibition on the Purified Golgi/secretory Pathway Ca²⁺/Mn²⁺-Transport ATPase (SPCA1a)

Chen Jialin, Joren de Raeymaecker, Jannik Brøndsted Hovgaard, Susanne Smaardijk, Ilse Vandecaetsbeek, Frank Wuytack, Jesper Vuust Møller, Jan Eggermont, Marc de Meyer, Søren Brøgger Christensen, Peter Vangheluwe

Department of Drug Design and Pharmacology

15 Citations (Scopus)

Abstract

The Golgi/secretory pathway Ca²⁺/Mn²⁺-transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca²⁺-dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca²⁺ affinity and a lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linoleamide/oleamide, and phosphatidylethanolamine inhibit and phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by micromolar concentrations of the commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid, and 2,5-ditert-butylhydroquinone. Because tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8, and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed.

Original language	English
Journal	Journal of Biological Chemistry
Volume	292
Issue number	17
Pages (from-to)	6938-6951
Number of pages	14
ISSN	0021-9258
DOIs	https://doi.org/10.1074/jbc.m117.778431
Publication status	Published - 28 Apr 2017

Keywords

Former Faculty of Pharmaceutical Sciences

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10.1074/jbc.m117.778431

Cite this

Jialin, C., de Raeymaecker, J., Hovgaard, J. B., Smaardijk, S., Vandecaetsbeek, I., Wuytack, F., Møller, J. V., Eggermont, J., de Meyer, M., Christensen, S. B., & Vangheluwe, P. (2017). Structure/activity Relationship of Thapsigargin Inhibition on the Purified Golgi/secretory Pathway Ca²⁺/Mn²⁺-Transport ATPase (SPCA1a). Journal of Biological Chemistry, 292(17), 6938-6951. https://doi.org/10.1074/jbc.m117.778431

Structure/activity Relationship of Thapsigargin Inhibition on the Purified Golgi/secretory Pathway Ca²⁺/Mn²⁺-Transport ATPase (SPCA1a). / Jialin, Chen; de Raeymaecker, Joren ; Hovgaard, Jannik Brøndsted et al.

In: Journal of Biological Chemistry, Vol. 292, No. 17, 28.04.2017, p. 6938-6951.

Research output: Contribution to journal › Journal article › Research › peer-review

Jialin, C, de Raeymaecker, J, Hovgaard, JB, Smaardijk, S, Vandecaetsbeek, I, Wuytack, F, Møller, JV, Eggermont, J, de Meyer, M, Christensen, SB & Vangheluwe, P 2017, 'Structure/activity Relationship of Thapsigargin Inhibition on the Purified Golgi/secretory Pathway Ca²⁺/Mn²⁺-Transport ATPase (SPCA1a)', Journal of Biological Chemistry, vol. 292, no. 17, pp. 6938-6951. https://doi.org/10.1074/jbc.m117.778431

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abstract = "The Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca2+-dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca2+ affinity and a lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linoleamide/oleamide, and phosphatidylethanolamine inhibit and phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by micromolar concentrations of the commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid, and 2,5-ditert-butylhydroquinone. Because tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8, and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed.",

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AU - de Raeymaecker, Joren

AU - Hovgaard, Jannik Brøndsted

AU - Smaardijk, Susanne

AU - Vandecaetsbeek, Ilse

AU - Wuytack, Frank

AU - Møller, Jesper Vuust

AU - Eggermont, Jan

AU - de Meyer, Marc

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