Department of Medicinal and Biological Chemistry, University of Toledo College of Pharmacy and Pharmaceutical Sciences, 3000 Arlington Avenue, Toledo, OH, 43614, United States.
Department of Pharmacology, University of Minnesota Medical School, 312 Church St., Minneapolis, MN, 55455-0217, United States.
Cell Calcium. 2019 Nov;83:102060. doi: 10.1016/j.ceca.2019.102060. Epub 2019 Aug 1.
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca mobilizing second messenger which triggers Ca release in both sea urchin egg homogenates and in mammalian cells. The NAADP binding protein has not been identified and the regulation of NAADP mediated Ca release remains controversial. To address this issue, we have synthesized an NAADP analog in which 3-azido-5-azidomethylbenzoic acid is attached to the amino group of 5-(3-aminopropyl)-NAADP to produce an NAADP analog which is both a photoaffinity label and clickable. This 'all-in-one-clickable' NAADP (AIOC-NAADP) elicited Ca release when microinjected into cultured human SKBR3 cells at low concentrations. In contrast, it displayed little activity in sea urchin egg homogenates where very high concentrations were required to elicit Ca release. In mammalian cell homogenates, incubation with low concentrations of [P]AIOC-NAADP followed by irradiation with UV light resulted in labeling 23 kDa protein(s). Competition between [P]AIOC-NAADP and increasing concentrations of NAADP demonstrated that the labeling was selective. We show that this label recognizes and selectively photodervatizes the 23 kDa NAADP binding protein(s) in cultured human cells identified in previous studies using [P]5-N-NAADP.
烟酰胺腺嘌呤二核苷酸磷酸（NAADP）是一种有效的钙动员第二信使，可在海胆卵匀浆和哺乳动物细胞中引发钙释放。尚未鉴定出 NAADP 结合蛋白，NAADP 介导的钙释放的调节仍然存在争议。为了解决这个问题，我们合成了一种 NAADP 类似物，其中 3-叠氮-5-叠氮甲基苯甲酸连接到 5-(3-氨基丙基)-NAADP 的氨基上，产生一种既是光亲和标记又是可点击的 NAADP 类似物。这种“一体式可点击”的 NAADP（AIOC-NAADP）在低浓度时微注射到培养的人 SKBR3 细胞中会引发钙释放。相比之下，它在海胆卵匀浆中的活性很小，需要非常高的浓度才能引发钙释放。在哺乳动物细胞匀浆中，用低浓度的 [P]AIOC-NAADP 孵育，然后用紫外光照射，导致标记 23 kDa 蛋白（s）。[P]AIOC-NAADP 与不断增加的 NAADP 浓度之间的竞争表明标记是选择性的。我们表明，这种标记可识别并选择性地光修饰先前使用 [P]5-N-NAADP 研究中在培养的人细胞中鉴定出的 23 kDa NAADP 结合蛋白（s）。
