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-di-tert-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.
Highlights
The Golgi/secretory pathway Ca2؉/Mn2؉-transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease
We purified for the first time the human Golgi/ secretory pathway Ca2ϩ/Mn2ϩ-ATPase SPCA1a from a yeast overexpression model
We demonstrate that SPCA1a is highly sensitive to the lipid environment and that several SERCA inhibitors, including Tg, block SPCA1a activity, at higher concentrations only
Summary
Inhibition of SPCA1a by thapsigargin largely unexplored [11]. The more restricted expression pattern of SPCA2 in cultured hippocampal neurons, colon, secretory acini, and luminal epithelial cells of mouse mammary tissue suggests that the second isoform may play a more specialized function, for instance in secretion [6, 12,13,14]. Independent of its transport activity, SPCA2 interacts with and activates the plasma membrane Ca2ϩ channel Orai via the N and C termini [18], leading to store-independent Ca2ϩ entry [18] and subsequent Ca2ϩ transfer into the secretory pathway [19] During lactation, this system contributes to the cellular uptake of Ca2ϩ in mammary gland epithelial cells for the subsequent release of Ca2ϩ into the milk [20]. The inhibition of SPCA1a by Tg may give rise to some concerns in connection with the clinical use of Tg analogues that target SERCA for prostate cancer therapy. The estimated 3 orders of magnitude difference in the binding affinity of Tg for SERCA and SPCA may be considered sufficiently discriminatory to enable the use of Tg analogues for selective SERCA inhibition in vitro and in vivo. We have established the SAR of Tg for SPCA1a to facilitate the design of more selective SPCA inhibitors
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