Redox Regulation of Intracellular Calcium Signaling

 描述(由申请人提供)：细胞内游离钙浓度的升高是细胞对激素、生长因子和神经递质做出反应的机制的一个组成部分。D-肌醇-1，4，5-三磷酸(IP3)是一种细胞内信使，通过与作为配体门控钙通道的无所不在的受体(IP3R)相互作用，介导细胞内钙离子的动员。IP3R是氧化还原敏感通道，可被氧化应激敏化。然而，这一调控的分子基础却知之甚少。从IP3Rs释放的钙离子被局部传递到线粒体，并可以刺激新陈代谢，在较高的量下，也可以启动细胞死亡。本研究的主要假设是，IP3Rs的氧化还原调节是细胞死亡途径中钙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]衡量和绘制IP3R的氧化还原变化情况。我们已经开发了一些方法来确定IP3Rs在体内的氧化还原状态，这将被用来定量外源性和内源性因素引起的氧化应激的影响。使用质谱学的初步研究确定了在IP3R-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。氧化还原敏感的硫醇将发生突变，并对氧化应激的功能敏感性进行评估。2]测定内质网/线粒体连接处的IP3R氧化还原状态。我们将测试这一假设，即位于ER/MITO结处的IP3R池特别容易发生ROS修饰。我们将使用亚细胞分离和成像方法，利用靶向IP3R、ROS敏感荧光蛋白、产生ROS的光敏探针和靶向过氧化氢酶。3]探讨IP3R氧化还原改变在内质网应激/细胞凋亡模型中的作用。我们将检验这一假设，即内质网驻留的NADPH氧化酶在IP3R氧化还原调节中发挥重要作用。将肝脏作为诱导内质网应激的实验模型。将研究IP3R氧化还原调节在果糖激活的内质网应激途径中的作用。该提案的长期目标是详细了解氧化应激在正常和疾病条件下如何影响细胞内钙信号。

 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.