通过光 afinity 蛋白质 - 连接物标记方法识别甘 3-酸脱酶作为循环腺二糖结合蛋白
Kehui Zhang1,2, Wei Sun2,3, Lihong Huang2
1State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University , Beijing 100191, China.
Journal of the American Chemical Society
|December 13, 2016
概括
糖三脱酶 (GAPDH) 结合循环腺二酶 (cADPR) 并调解其释放的功能. 这项研究确定GAPDH是cADPR和通过受体调动之间的关键联系.
科学领域:
- 细胞生物学
- 生物化学
- 分子信号
背景情况:
- 循环腺二酶 (cADPR) 是从尼古丁胺氨基二核酸 (NAD+) 衍生的关键细胞内调节剂.
- 精确的分子机制和蛋白质中间体将cADPR与通过受体 (RyRs) 从内网 (ER) 释放的仍然难以捉摸.
- 了解这些相互作用对于解读细胞平衡和信号通路至关重要.
研究的目的:
- 识别与cADPR结合并调解其调动活性的蛋白质.
- 描述cADPR及其结合蛋白之间的相互作用.
- 阐明已识别的蛋白质在cADPR介导的释放中的作用.
主要方法:
- 一种用于蛋白质识别的新型光亲和标记 (PAL) cADPR激动剂 (PAL-cIDPRE) 的合成.
- 从人类Jurkat T细胞中纯化cADPR结合蛋白.
- 表面等离子共振 (SPR) 测试以表征结合动力学和亲和力.
- 在体内和体外实验以评估蛋白质相互作用和功能后果.
主要成果:
- 甘3-酸脱酶 (GAPDH) 被确定为cADPR的直接结合蛋白.
- SPR分析证实了cADPR和GAPDH之间的高亲和结合,在GAPDH上绘制了特定的结合位点.
- 在体内,cADPR 诱导了 GAPDH 和 RyRs 之间的短暂相互作用,并且 GAPDH 降低了 cADPR 诱导的释放.
结论:
- GAPDH是长期寻找的与cADPR结合的蛋白.
- GAPDH对于调解cADPR诱导的 (Ca2+) 从ER通过RyRs至关重要.
- 这一发现确定了GAPDH在细胞内信号传递中的新角色.
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