Ceramide Signaling in Coronary Endothelial Dysfunction

描述（由申请人提供）：在上一个资助期内完成的工作表明，神经酰胺的生产和膜筏（MR）（以前的脂筏）的聚集驱动NADPH氧化酶（Nox）亚基与内皮细胞（ECs）膜中的其他调节分子聚集，形成MR氧化还原信号平台。这个信号平台产生超氧化物来调节冠状动脉内皮功能。这种竞争性的更新现在试图阐明介导这些MR信号平台的形成和功能的分子机制。正在验证的假设是，通过以snare为中心的胞外机制，膜近端溶酶体与质膜的快速融合介导了ASMase的易位、局部神经酰胺的产生以及随后冠状动脉内皮细胞中MR氧化还原信号平台的形成。这种溶酶体- mr氧化还原信号体在响应死亡受体和其他危险信号的激活时有助于内皮功能障碍。特异性目的1将确定MR氧化还原信号平台的形成是否由冠状动脉内皮细胞中膜近端溶酶体通过SNARE中心胞外机制与质膜的快速融合介导。特异性目的2将利用扫描和免疫金电镜鉴定冠状动脉内皮细胞中溶酶体- mr信号体的地形特征，并确定不同分子之间可能的相互作用。特异性Aim 3将确定冠状动脉内皮细胞中溶酶体MR信号体的功能，重点是ASMase活性和Nox激活的相互作用。特异性Aim 4将确定通过以snare为中心的胞外机制形成的溶酶体MR信号体是否有助于完整冠状动脉内皮的内皮功能障碍。这些建议的研究将阐明介导胞内细胞器和分子转运到细胞质膜进行跨膜信号传导的分子机制。这些结果将揭示在内皮细胞中作为信号细胞器的一种新的溶酶体功能，它可能重要地促进MR氧化还原信号平台的形成，从而在生理和病理条件下调节内皮功能。

DESCRIPTION (provided by applicant): Work done over the last funding period has demonstrated that ceramide production and clustering of membrane rafts (MR) (formerly lipid rafts) drive aggregation of NADPH oxidase (Nox) subunits together with other regulatory molecules in the membrane of endothelial cells (ECs) forming a MR redox signaling platform. This signaling platform produces superoxide to regulate coronary endothelial function. This competitive renewal now seeks to elucidate the molecular mechanisms mediating the formation and function of such MR signaling platforms. The hypothesis being tested is that a rapid fusion of membrane proximal lysosomes to plasma membrane via the SNARE-centered exocytic machinery mediates translocation of ASMase, local ceramide production, and consequent formation of MR redox signaling platforms in coronary arterial endothelial cells. This lysosome-MR redox signalosome contributes to endothelial dysfunction in response to activation of death receptors and other danger signals. Specific Aim 1 will determine whether the formation of MR redox signaling platforms are mediated by a rapid fusion of membrane proximal lysosomes to plasma membrane via a SNARE- centered exocytic machinery in coronary ECs. Specific Aim 2 will identify the topographic characteristics of lysosome-MR signalosomes in coronary ECs using scanning and immunogold electron microscopy and determine possible interactions of different molecules. Specific Aim 3 will determine the functionality of the lysosome MR signalosomes in coronary ECs with a focus on interactions of ASMase activity and Nox activation. Specific Aim 4 will determine whether the formation of such lysosome MR signalosomes via the SNARE-centered exocytic machinery contributes to endothelial dysfunction in the intact coronary arterial endothelium. These proposed studies will elucidate the molecular mechanisms mediating the trafficking of intracellular organelles and molecules to cell plasma membrane for transmembrane signaling. The results will reveal a novel lysosomal function serving as a signaling organelle in ECs, which may importantly contribute to the formation of MR redox signaling platform and thereby to the regulation of endothelial function under physiological and pathological conditions.