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 fromSaccharomyces cerevisiaeand 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 byin silicomodeling, 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.
authors.affiliation: From the Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, and.
authors.forename: Jialin
authors.initials: J
authors.lastname: Chen
authors.affiliation: Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven, 3000 Leuven, Belgium.
authors.forename: Joren
authors.initials: J
authors.lastname: De Raeymaecker
authors.affiliation: the Department of Drug Design and Pharmacology, University of Copenhagen, DK-2100 Copenhagen, Denmark, and.
authors.forename: Jannik Brøndsted
authors.initials: JB
authors.lastname: Hovgaard
authors.affiliation: From the Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, and.
authors.forename: Susanne
authors.initials: S
authors.lastname: Smaardijk
authors.affiliation: From the Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, and.
authors.forename: Ilse
authors.initials: I
authors.lastname: Vandecaetsbeek
authors.affiliation: From the Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, and.
authors.forename: Frank
authors.initials: F
authors.lastname: Wuytack
authors.affiliation: the Department of Biomedicine, University of Aarhus, 8000 Aarhus, Denmark.
authors.forename: Jesper Vuust
authors.initials: JV
authors.lastname: Møller
authors.affiliation: From the Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, and.
authors.forename: Jan
authors.initials: J
authors.lastname: Eggermont
authors.affiliation: Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven, 3000 Leuven, Belgium.
authors.forename: Marc
authors.initials: M
authors.lastname: De Maeyer
authors.affiliation: the Department of Drug Design and Pharmacology, University of Copenhagen, DK-2100 Copenhagen, Denmark, and.
authors.forename: Søren Brøgger
authors.initials: SB
authors.lastname: Christensen
authors.affiliation: From the Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, and peter.vangheluwe@kuleuven.be.
authors.forename: Peter
authors.initials: P
authors.lastname: Vangheluwe
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use; D001943:Breast Neoplasms / Q000188:drug therapy; D002118:Calcium / Q000737:chemistry; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D002784:Cholesterol / Q000737:chemistry; D015195:Drug Design; D005260:Female; D006801:Humans; D006873:Hydroquinones / Q000737:chemistry; D007211:Indoles / Q000737:chemistry; D008041:Linoleic Acids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D008297:Male; D016297:Mutagenesis, Site-Directed; D009829:Oleic Acids / Q000737:chemistry; D010712:Phosphatidic Acids / Q000737:chemistry; D011471:Prostatic Neoplasms / Q000188:drug therapy; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D013109:Sphingomyelins / Q000737:chemistry; D013329:Structure-Activity Relationship; D019284:Thapsigargin / Q000737:chemistry*
chemical_list: D000970:Antineoplastic Agents; D006873:Hydroquinones; D007211:Indoles; D008041:Linoleic Acids; D008081:Liposomes; D009829:Oleic Acids; D010712:Phosphatidic Acids; D011994:Recombinant Proteins; D013109:Sphingomyelins; C019359:2,5-di-tert-butylhydroquinone; C420162:linoleamide; D019284:Thapsigargin; C029407:oleylamide; D002784:Cholesterol; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506946:ATP2A2 protein, human; C403624:ATP2C1 protein, human; D000252:Calcium-Transporting ATPases; D002118:Calcium; C000543:cyclopiazonic acid
keywords: Golgi; calcium transport; cholesterol; cyclopiazonic acid; membrane transporter reconstitution; molecular modeling; prostate cancer; protein purification; thapsigargin