Ap4A as an Alarmone in Stress Physiology

说明（由申请人提供）：腺苷二核苷酸（ApxA）被称为警报素，因为它们在应激后从血小板中释放并调节基因或酶活性。为了支持这一假设，我们的实验室已经证明Ap 4A（最丰富和最好表征的Ap 4A）是一种信号传导装置，其触发将L-Arg递送到牛主动脉内皮细胞（BAEC）一氧化氮合酶以产生一氧化氮所需的蛋白质的合成。我们的实验室还证明，Ap 4A通过内吞作用被BAEC内化，并且这种内化的Ap 4A不被降解。这些数据支持了内化Ap 4A可能在血管生物学中具有生理作用的假设。我们还证明了Ap 4A作为ATP的部分激动剂。这些数据表明，Ap 4A可能是由ATP引起的生理反应的调节剂。因此，Ap 4A真正符合警报素的定义，它提醒细胞代谢应激的发生，并随后调节基因表达和/或酶功能。本研究利用抑制性消减杂交技术分离Ap 4A差异表达的cDNA克隆，并将其序列与GenBank数据库进行比较。这种比较将为我们提供Ap 4A上调哪些代谢过程的信息。我们还将表征Ap 4A被内化的机制，并确定内化的Ap 4A的细胞内定位。我们将通过确定内分泌抑制剂是否抑制Ap 4A内化以及Ap 4A是否与小窝或网格蛋白包被的囊泡相关来实现这一点。此外，我们将确定Ap 4A摄取到各种亚细胞组分或细胞外空间的时间依赖性。我们还将确定Ap 4A是否诱导钙从内质网上的钙释放通道释放。本提案中获得的信息将使我们了解如何制定未来的提案，生物化学和分子实验，以确定Ap 4A作为报警物的机制。

DESCRIPTION (provided by applicant): Adenylated dinucleotides(ApxA)s have been termed alarmones because they are released from blood platelets following stress and modulate gene or enzyme activities. In support of this hypothesis our laboratory has demonstratedAp4A (the most abundant and best characterized Ap4A) is a signaling device that triggers the synthesis of protein(s)required to deliver L-Arg to bovine aortic endothelial cell (BAEC) nitric oxide synthase to generate nitric oxide. Our laboratory has also demonstrated that Ap4A is internalized by BAEC via endocytosis and this internalized Ap4A is not degraded. These data support the hypothesis that the internalizedAp4A may have a physiological role in vascular biology. We have also demonstrated that Ap4A acts as a partial agonist for ATP. These data suggest that Ap4Amay be a modulator of the physiological responses elicited by ATP. Thus, Ap4A truly fits the definition of an alarmone alerting the cells to the onset of metabolic stress and subsequently regulating gene expression and/or enzyme function. In this proposal we will use suppression subtractive hybridization to isolateAp4A differentially expressed cDNA clones and compare their sequences to Gen-Bank database. This comparision will give us information on what metabolic processes that Ap4A up regulates. We will also characterize the mechanism by which Ap4A is internalized and also determine the intracellular localization of the internalizedAp4A. We will accomplish this by determining whether inhibitors of endoctosis inhibit Ap4A internalization and whetherAp4A can be found associated with caveolae or clathrin-coated vesicles. In addition we will determine the time dependency of Ap4A uptake into various subcellular fractions or into the extracellular spaces. We will also determine whether Ap4A induces the release of calcium from calcium release channels on the endoplasmic reticulum. The information obtained in this proposal will give us insight on how to develop future proposals, biochemical and molecular experiments to determine the mechanism(s)by which Ap4A acts as an alarmone.