Redox Regulation of Intracellular Calcium Signaling

 描述（由申请方提供）：细胞溶质游离钙浓度升高是细胞对激素、生长因子和神经递质应答机制的组成部分。D-肌肌醇1，4，5-三磷酸盐（IP 3）是一种细胞内信使，通过与一种普遍存在的受体（IP 3R）相互作用，介导细胞内钙库中钙的动员，IP 3R是一种配体门控钙通道。IP 3R是氧化还原敏感性通道，并被氧化应激敏化。然而，这种调节的分子基础知之甚少。从IP 3R释放的Ca 2+局部传递到线粒体，可以刺激新陈代谢，并且在更高的量下，也可以引发细胞死亡。本研究的首要假设是，IP 3Rs的氧化还原调节是细胞死亡途径中Ca 2+信号调节的重要组成部分。该提案包括以下三个具体目标：1]测量和绘制IP 3R中的氧化还原变化。我们已经开发了方法来确定在体内的氧化还原状态的IP 3Rs，这将被用来定量的外源性和内源性剂引起的氧化应激的影响。使用质谱的初步研究鉴定了在IP 3R-1中被氧化的11个半胱氨酸的子集。将确定发生的氧化修饰的类型。将突变氧化还原敏感性硫醇，并评估对氧化应激的功能敏感性。2]测量ER/线粒体连接处的IP 3R氧化还原状态。我们将检验位于ER/mito连接处的IP 3R库特别容易发生ROS修饰的假设。我们将采用亚细胞分级和成像方法，利用靶向IP 3Rs，ROS敏感的荧光蛋白，ROS产生的光敏探针和靶向过氧化氢酶。3]研究IP 3R氧化还原变化在内质网应激/凋亡模型中的作用。我们将测试的假设，ER居民NADPH氧化酶发挥重要作用，在IP 3R氧化还原调节。肝脏将被用作诱导ER应激的实验模型。将研究IP 3R氧化还原调节在果糖激活的ER应激途径中的作用。该提案的长期目标是详细了解氧化应激如何影响正常和疾病条件下的细胞内Ca 2+信号传导。

 DESCRIPTION (provided by applicant): An elevation of cytosolic free calcium concentration is an integral component of the mechanism by which cells respond to hormones, growth factors and neurotransmitters. D-myo-inositol 1,4,5-trisphosphate ( IP3 ) is an intracellular messenger mediating the mobilization of Ca2+ from intracellular stores by interaction with an ubiquitous receptor ( IP3R ) that acts as a ligand-gated Ca2+ channel. IP3Rs are redox sensitive channels and are sensitized by oxidative stress. However, the molecular basis of this regulation is poorly understood. Ca2+ released from IP3Rs is locally transmitted to the mitochondria and can stimulate metabolism, and in higher amounts, can also initiate cell death. The overarching hypothesis of this study is that redox modulation of IP3Rs is an important component of the regulation of Ca2+ signals in cell death pathways. The proposal encompasses the following three specific aims: 1] To measure and map redox changes in IP3Rs. We have developed methods to determine the redox state of IP3Rs in vivo which will be used to quantitate the effects of exogenous and endogenous agents causing oxidative stress. Preliminary studies using mass-spectroscopy identify a subset of 11 cysteines that become oxidized in IP3R-1. The type of oxidative modifications occurring will be identified. Redox-sensitive thiols will be mutate and the functional sensitivity to oxidative stress will be assessed. 2] To measure IP3R redox state at the ER/mitochondrial junction. We will test the hypothesis that the pool of IP3Rs located at the ER/mito junction is particularly prone to ROS modifications. We will employ subcellular fractionation and imaging methods utilizing targeted IP3Rs, ROS-sensitive fluorescent proteins, ROS-producing photosensitive probes and targeted catalases. 3] To investigate the role of IP3R redox changes in models of ER stress/apoptosis. We will test the hypothesis that ER-resident NADPH oxidases play an important role in IP3R redox regulation. Liver will be used as an experimental model to induce ER stress. The role of IP3R redox regulation in ER stress pathways activated by fructose will be examined. The long-term goal of the proposal is to obtain a detailed understanding of how oxidative stress impacts intracellular Ca2+ signaling under normal and disease conditions.