CYCLIC ADP/RIBOSE-DEPENDENT CALCIUM RELEASE PATHWAY

循环 ADP/核糖依赖性钙释放途径

• 批准号: 2204936
• 负责人: HONCHEUNG LEE
• 金额: $ 15.14万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-15 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2204936
• 关键词: ADP ribosylation; CD antigens; affinity labeling; animal tissue; calcium channel; calcium flux; chickens; cyclic GMP; egg /ovum; enzyme activity; enzyme mechanism; high performance liquid chromatography; hydrolase; laboratory rat; microsomes; nicotinamide adenine dinucleotide; nitric oxide; nucleotide metabolism; phosphorylation; protein kinase; protein purification; protein sequence; second messengers; western blottings

Mobilization of Ca+2 from intracellular stores is an important signaling mechanism in cells and is mediated by two major mechanisms, the inositol trisphosphate (IP3) pathway and the Ca+2-induced Ca+2 release (CICR) process. We have identified a Ca+2 mobilization system in sea urchin eggs which is totally independent of the IP3 pathway. This system is activated by a metabolite of NAD+ we called cyclic ADP-ribose (cADPR). In addition to sea urchin eggs, several mammalian cell types have been shown to be responsive to cADPR, indicating the generality of the mechanism. Increasing evidence suggests that cADPR may be an endogenous regulator of CICR in cells. Three recent advances indicate that the cADPR mechanism is tightly regulated. First, the synthetic pathway of cADPR has been shown to be stimulated by a cGMP-dependent mechanism. Second, a lymphocyte protein called CD38 has been shown to be a bifunctional enzyme that can catalyze both the synthesis and hydrolysis of cADPR and third, a soluble protein factor has been shown to be required for conferring the cADPR-sensitivity to microsomes. The proposed research will examine the regulation mechanisms of the cADPR pathway. (l) The soluble protein factors from brain and sea urchin eggs that confer the cADPR sensitivity to egg microsomes will be purified and characterized. We will investigate the possibilities that the soluble factor functions as a sensitizer of the cADPR-receptor to cADPR and/or the Ca+2 release mechanism to Ca+2. (2) The cGMP-dependent regulation mechanism of ADP-ribosyl cyclase and CD38 will be elucidated. We will use direct photoaffinity labeling and protein sequencing to identify the components involved in mediating the stimulatory effect of cGMP on the synthetic pathway of cADPR. (3) The enzymatic mechanisms of ADP-ribosyl cyclase and CD38 will be investigated. We will examine the formation of the ADP-ribosylated enzyme intermediates and identify the catalytic sites of the enzymes by photoaffinity labeling. (4) Finally, we will extend the results obtained from egg microsomes to intact eggs and to mammalian brain microsomes.

来自细胞内存储的Ca+2的动员是一个重要的信号传导 细胞中的机理，并由两种主要机制介导肌醇 三磷酸（IP3）途径和Ca+2诱导的Ca+2释放（CICR） 过程。我们已经确定了海胆卵中的CA+2动员系统 这完全独立于IP3途径。该系统已激活 通过NAD+的代谢产物，我们称为环状ADP-核糖（CADPR）。此外 到海胆卵，已经证明了几种哺乳动物细胞类型 对CADPR的反应，表明机制的一般性。 越来越多的证据表明，CADPR可能是 细胞中的CICR。最近的三个进展表明，CADPR机制是 严格调节。首先，已证明CADPR的合成途径 由CGMP依赖性机制刺激。其次，淋巴细胞蛋白 称为CD38已显示为一种可以催化的双功能酶 CADPR的合成和水解，第三，可溶性蛋白 已经证明要赋予因子来赋予CADPR-敏感性 到微粒体。 拟议的研究将检查CADPR的调节机制 路径。 （l）大脑和海胆卵的可溶性蛋白质因子 赋予CADPR对鸡蛋微粒体的敏感性将被纯化，并且 特征。我们将调查可溶性的可能性 因子是CADPR受体对CADPR和/或 CA+2释放机制至CA+2。 （2）CGMP依赖性调节 ADP-核糖基环化酶和CD38的机制将被阐明。我们将使用 直接的光性标签和蛋白质测序以识别 参与介导CGMP对刺激作用的成分 CADPR的合成途径。 （3）ADP-核糖基的酶促机制 将研究循环酶和CD38。我们将研究 ADP-核糖基化酶中间体并识别催化位点 酶通过光性标记。 （4）最后，我们将扩展 从鸡蛋微粒体到完整卵和哺乳动物大脑获得的结果 微粒体。