Nicotinic Acid Adenine Dinucleotide 2'-Phosphate (NAADP) Binding Proteins in T-Lymphocytes.
Nicotinic Acid Adenine Dinucleotide 2'-Phosphate (NAADP) Binding Proteins in T-Lymphocytes.
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T 淋巴细胞中的烟酸腺嘌呤二核苷酸 2-磷酸 (NAADP) 结合蛋白。
DOI：
10.1166/msr.2012.1008
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Guse,AndreasH
中科院分区：
文献类型：
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作者：
Walseth,TimothyF;Lin-Moshier,Yaping;Weber,Karin;Marchant,JonathanS;Slama,JamesT;Guse,AndreasH
烟酸腺嘌呤二核苷酸磷酸 (NAADP) 是调节细胞内酸性储备钙释放的信使。尽管包括二孔通道 (TPC)、兰尼碱受体 (RYR) 和粘脂蛋白 (TRP-ML1) 在内的多种通道与 NAADP 钙信号传导调节有关，但 NAADP 受体尚未被鉴定。在本研究中，使用光亲和探针[32P]–5-azido-NAADP ([32P]–5-N3-NAADP)来研究NAADP反应性Jurkat T淋巴细胞提取物中的NAADP结合蛋白。 Jurkat S100 胞质级分的 [32P]–5-N3-NAADP 光标记导致至少十种不同蛋白质的标记。其中一些 S100 蛋白（包括 22/23 kDa 的双联体和 15 kDa 的小蛋白）显示出对 NAADP 的选择性，因为通过包含未标记的 NAADP 来保护标记，而结构相似的 NADP 需要更高的浓度来进行保护。有趣的是，几种 S100 蛋白（60、45、33 和 28 kDa）的标记受到低浓度未标记 NAADP 的刺激，但不被 NADP 刺激。 NAADP 对 60 kDa 蛋白质标记的影响是双相的，在 100 μM 时达到峰值，增加了五倍，在 1 M NAADP 时没有变化。当检查 Jurkat 细胞的 P100 膜组分时，一些蛋白质也被光标记。与 S100 的结果类似，22/23 kDa 双联体和 15 kDa 蛋白质似乎被选择性标记。 NAADP 不会像在 S100 组分中那样增加任何 P100 蛋白的标记。通过二维凝胶电泳成功解析了光标记的 S100 和 P100 蛋白。 [32P]–5-N3-NAADP 光标记和二维电泳应该代表一种识别和表征 NAADP 结合蛋白的合适策略。
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Although several channels, including two-pore channels (TPC), ryanodine receptor (RYR) and mucolipin (TRP-ML1) have been implicated in NAADP regulation of calcium signaling, the NAADP receptor has not been identified. In this study, the photoaffinity probe, [32P]–5-azido-NAADP ([32P]–5-N3-NAADP), was used to study NAADP binding proteins in extracts from NAADP responsive Jurkat T-lymphocytes. [32P]–5-N3-NAADP photolabeling of Jurkat S100 cytosolic fractions resulted in the labeling of at least ten distinct proteins. Several of these S100 proteins, including a doublet at 22/23 kDa and small protein at 15 kDa displayed selectivity for NAADP as the labeling was protected by inclusion of unlabeled NAADP, whereas the structurally similar NADP required much higher concentrations for protection. Interestingly, the labeling of several S100 proteins (60, 45, 33 and 28 kDa) was stimulated by low concentrations of unlabeled NAADP, but not by NADP. The effect of NAADP on the labeling of the 60 kDa protein was biphasic, peaking at 100 μM with a five-fold increase and displaying no change at 1 M NAADP. Several proteins were also photolabeled when the P100 membrane fraction from Jurkat cells was examined. Similar to the results with S100, a 22/23 kDa doublet and a 15 kDa protein appeared to be selectively labeled. NAADP did not increase the labeling of any P100 proteins as it did in the S100 fraction. The photolabeled S100 and P100 proteins were successfully resolved by two-dimensional gel electrophoresis. [32P]–5-N3-NAADP photolabeling and two-dimensional electrophoresis should represent a suitable strategy in which to identify and characterize NAADP binding proteins.