TY - JOUR
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
Y1 - 2017
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 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.
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 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.
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
VL - 292
SP - 6938
EP - 6951
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 17
ER -