Structure/activity relationship of thapsigargin inhibition on the purified Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a)
Structure/activity relationship of thapsigargin inhibition on the purified Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a)
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DOI：
10.1074/jbc.m117.778431
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发表时间：
2017-04-28
影响因子：
4.8
通讯作者：
Vangheluwe, Peter
中科院分区：
文献类型：
--
作者：
Chen, Jialin;De Raeymaecker, Joren;Vangheluwe, Peter
高尔基体/分泌途径Ca 2 +/Mn 2+转运ATP酶（SPCA 1a）与乳腺癌和Hailey-Hailey病有关。在这里，我们纯化的重组人SPCA 1a从酿酒酵母和测量Ca 2+依赖的ATP酶活性后，重组蛋白脂质体。纯化的SPCA 1a比纯化的肌浆网Ca ~（2+）-ATP酶（SERCA 1a）表现出更高的表观Ca ~（2+）亲和力和更低的最大周转率。脂质胆固醇半琥珀酸酯、亚油酰胺/油酰胺和磷脂酰乙醇胺抑制SPCA 1a活性，磷脂酸和鞘磷脂增强SPCA 1a活性。此外，SPCA 1a被微摩尔浓度的常用SERCA 1a抑制剂毒胡萝卜素（Tg）、环匹阿尼酸和2，5-二叔丁基对苯二酚阻断。由于Tg类似物对SERCA 2b的组织特异性靶向作用被认为用于前列腺癌治疗，因此Tg对SPCA 1a的抑制可能代表脱靶风险。我们通过计算机模拟、定点突变和测量一系列Tg类似物的效价来评估SPCA 1a的Tg的结构-活性关系（SAR）。这些表明Tg和类似物通过Tg支架结合，但与SERCA 1a膜表面上相同的同源腔的亲和力较低。较低的Tg亲和力可能取决于SPCA 1a中更灵活的结合腔，Tg O-3、O-8和O-10链对结合能的贡献较低。相反，Tg O-2侧链与SPCA 1a的蛋白质相互作用似乎与SERCA 1a相当。这些差异定义了SPCA 1a与SERCA 1a不同的Tg SAR，表明可能开发出对SPCA 1a具有更高特异性的Tg类似物。
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.