REGULATION OF CYCLIC ADP RIBOSE SIGNALING SYSTEM

拟议研究的总体目标是深入了解 环ADP-核糖（cADPR）动员钙离子的机制 细胞内储存，并阐明cADPR水平 在哺乳动物细胞中受到调节。cADPR是一种天然存在的 β-烟酰胺腺嘌呤二核苷酸（β-NAD）的代谢物，其具有 已经发现在多种细胞中从细胞内储存中动员钙 细胞类型。具体目的是：（1）鉴定和表征cADPR 哺乳动物系统中的结合蛋白，（2）检查 哺乳动物系统中cADPR的内源性水平，以及（3）表征 cADPR代谢酶的调控机制。 cADPR结合蛋白将通过常规结合以及 通过光亲和标记技术。3-deaza-cADPR，一种有效的，非 cADPR的可水解类似物，最近已经被开发出来，并且将是一种 在cADPR相互作用的蛋白质检查中的重要探针。 内源性cADPR水平的调节将在几个实验中进行研究。 哺乳动物细胞系使用放射免疫测定cADPR开发的， 实验室将特别关注已知的代理人 改变细胞内钙浓度， 内流或通过动员钙的内部储存。机制 将通过表征cADPR水平来检查cADPR水平的调节机制， 负责合成的酶（ADP-核糖基环化酶）和 降解（cADPR水解酶）的系统显示有改变， cADPR水平。细胞内钙离子浓度的调节 是所有细胞的关键过程钙在一种 许多过程，包括神经递质释放，细胞骨架 变化、肌肉收缩、基因表达等。 提示cADPR可能是钙诱导的内源性调节因子- 通过兰尼碱受体型钙释放的钙释放（CICR） 渠道cADPR似乎增加了CICR对钙的敏感性 与咖啡因非常相似。咖啡因有许多生理作用 它是世界上最受社会滥用的药物之一。的 拟议中的研究应该提供有关cADPR所起作用的有用信息 在调节钙稳态，也可能提供有价值的 深入了解咖啡因的一些生物作用。

The overall objectives of the proposed research are to gain insight into the mechanism by which cyclic ADP-ribose (cADPR) mobilizes calcium from intracellular stores and to elucidate the mechanisms by which cADPR levels are regulated in mammalian cells. cADPR is a naturally occurring metabolite of beta-nicotinamide adenine dinucleotide )beta-NAD) that has been found to mobilize calcium from intracellular stores in a variety of cell types. The specific aims are: (1) to identify and characterize cADPR binding proteins in mammalian systems, (2) to examine the regulation of endogenous levels of cADPR in mammalian systems, and (3) to characterize the mechanisms involved in the regulation of cADPR metabolic enzymes. cADPR binding proteins will be studied by conventional binding as well as by photoaffinity labeling techniques. 3-deaza-cADPR, a potent, non- hydrolyzable analog of cADPR, has recently been developed, and will be an important probe in the examination of proteins to which cADPR interacts. The regulation of endogenous levels of cADPR will be examined in several mammalian cell lines using a radioimmunoassay for cADPR developed in this laboratory. Particular attention will be focused on agents that are known to alter intracellular calcium concentrations, either through calcium influx or by mobilization of interal stores of calcium. The mechanism(s) by which cADPR levels are regulated will be examined by characterizing the enzymes responsible for the synthesis (ADP-ribosyl cyclase) and degradation (cADPR hydrolase) from systems shown to have alterations in cADPR levels. The regulation of the intracellular concentration of calcium is a critical process in all cells. Calcium plays an important role in a number of processes, including neurotransmitter release, cytoskeleton changes, muscle contraction, gene expression, etc. Accumulating evidence suggests that cADPR may be the endogenous regulator of calcium induced- calcium release (CICR) through ryanodine receptor type calcium release channels. cADPR appears to increase the sensitivity of CICR to calcium ions in a manner very similar to caffeine. Caffeine has many physiological actions and is one of the most socially abused drugs in the world. The proposed studies should provide useful information on the role cADPR plays in the regulation of calcium homeostasis and may also provide valuable insight into some of caffeine's biological actions.