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

LUKKET: Institut for Lægemiddeldesign og Farmakologi

15 Citationer (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 lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linole-/oleamide and phosphatidyl ethanolamine inhibit, whereas phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by μM concentrations of commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid (CPA) and 2,5-di-tert-butyl hydroquinone (BHQ). Since 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 by 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.

Originalsprog	Engelsk
Tidsskrift	Journal of Biological Chemistry
Vol/bind	292
Udgave nummer	17
Sider (fra-til)	6938-6951
Antal sider	14
ISSN	0021-9258
DOI	https://doi.org/10.1074/jbc.m117.778431
Status	Udgivet - 28 apr. 2017

Emneord

Det tidligere Farmaceutiske Fakultet
Thapsigargin
SPCA1a
Golgi/secretory pathway Ca2+/Mn2+ transport ATPase

FN’s Verdensmål

Dette resultat bidrager til følgende verdensmål

Adgang til dokumentet

10.1074/jbc.m117.778431

Citationsformater

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.

I: Journal of Biological Chemistry, Bind 292, Nr. 17, 28.04.2017, s. 6938-6951.

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › 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, bind 292, nr. 17, s. 6938-6951. https://doi.org/10.1074/jbc.m117.778431

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

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.",

keywords = "Former Faculty of Pharmaceutical Sciences, Thapsigargin , SPCA1a, Golgi/secretory pathway Ca2+/Mn2+ transport ATPase",

author = "Chen Jialin and {de Raeymaecker}, Joren and Hovgaard, {Jannik Br{\o}ndsted} and Susanne Smaardijk and Ilse Vandecaetsbeek and Frank Wuytack and M{\o}ller, {Jesper Vuust} and Jan Eggermont and {de Meyer}, Marc and Christensen, {S{\o}ren Br{\o}gger} and Peter Vangheluwe",

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T1 - Structure/activity Relationship of Thapsigargin Inhibition on the Purified Golgi/secretory Pathway Ca2+/Mn2+-Transport ATPase (SPCA1a)

AU - Jialin, Chen

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

AU - Christensen, Søren Brøgger

AU - Vangheluwe, Peter

PY - 2017/4/28

Y1 - 2017/4/28

N2 - 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.

AB - 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.

KW - Former Faculty of Pharmaceutical Sciences

KW - Thapsigargin

KW - SPCA1a

KW - Golgi/secretory pathway Ca2+/Mn2+ transport ATPase

U2 - 10.1074/jbc.m117.778431

DO - 10.1074/jbc.m117.778431

M3 - Journal article

C2 - 28264934

SN - 0021-9258

VL - 292

SP - 6938

EP - 6951

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 17

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