CYCLIC ADP/RIBOSE-DEPENDENT CALCIUM RELEASE PATHWAY

循环 ADP/核糖依赖性钙释放途径

• 批准号: 2204937
• 负责人: HONCHEUNG LEE
• 金额: $ 16.49万
• 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/2204937
• 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.

Ca+2从细胞内库中的动员是一个重要的信号传导过程， 在细胞中的机制，并介导的两个主要机制，肌醇 三磷酸（IP 3）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 它完全独立于IP 3通路。这个系统被激活了 我们称之为环ADP核糖（cADPR）。此外 到海胆卵，几种哺乳动物细胞类型已经被证明是 响应于cADPR，表明该机制的通用性。 越来越多的证据表明，cADPR可能是一种内源性调节因子， 细胞内的CICR。最近的三项进展表明，cADPR机制是 严格监管。首先，cADPR的合成途径已被证明是 受到cGMP依赖性机制的刺激。第二，淋巴细胞蛋白 CD 38是一种双功能酶，可以催化 cADPR的合成和水解以及第三，可溶性蛋白质 已经证明，赋予cADPR敏感性需要一个因子 到微粒体 这项研究将探讨cADPR的调控机制， 通路(l)脑和海胆卵中的可溶性蛋白因子 将纯化赋予cADPR对卵微粒体敏感性的蛋白， 表征了我们将研究可溶性 因子作为cADPR受体对cADPR的敏化剂和/或 Ca +2释放机制。(2)cGMP依赖性调节 ADP-核糖基环化酶和CD 38的作用机制将被阐明。我们将使用 直接光亲和标记和蛋白质测序来鉴定 参与介导cGMP刺激作用的组分对 cADPR合成途径。(3)ADP-核糖基的酶学机制 环化酶和CD 38将被研究。我们将研究 ADP-核糖基化酶中间体，并确定催化位点 的酶的光亲和标记。(4)最后，我们将扩展 从卵微粒体到完整卵和哺乳动物脑获得的结果 微粒体