Mechanism of PAI-1 Polarization to Myoendothelial Junctions

PAI-1 极化至肌内皮连接的机制

• 批准号: 8403973
• 负责人: Brant E Isakson
• 金额: $ 18.33万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-15 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8403973
• 关键词: American; Area; Arteries; Binding; Binding Proteins; Biological Assay; Biological Markers; Blood Circulation; Blood Vessels; Cardiovascular Diseases; Cartoons; Cells; Co-Immunoprecipitations; Coculture Techniques; Communication; Complex; Cytoskeleton; Data; Detection; Disease; Endothelium; Fatty acid glycerol esters; Figs - dietary; Genetic Translation; Goals; Grant; Hypertension; Immunoblotting; In Vitro; Inflammatory; Label; Laboratories; Lead; Left; Link; Location; Messenger RNA; Metabolic; Metabolic syndrome; Microtubules; Modeling; Movement; Mus; Non-Insulin-Dependent Diabetes Mellitus; Oranges; Peptides; Phosphorylation; Physiological; Plasminogen Activator Inhibitor 1; Play; Population; Production; Proteins; RNA-Binding Proteins; Regulation; Resistance; Ribosomes; Role; Smooth Muscle; Stimulus; Structure; TNF gene; Techniques; Testing; Therapeutic; Time; Translating; Translations; Work; abstracting; base; cell type; feeding; in vivo; mutant; new therapeutic target; nicotinamide phosphoribosyltransferase; novel; protein complex; protein transport; research study; response; tau Proteins

Abstract During the course of metabolic syndrome, there is a significant increase in plasminogen activator inhibitor-1 (PAI-1); however the pathological role that PAI-1 plays during the course of the disease is not clear. We have recently identified PAI-1 as being key to regulation of myoendothelial junctions (MEJs), which are critical heterocellular structures linking the endothelium and smooth muscle in the resistance vasculature, coordinating communication between the two cell types. In mice with metabolic syndrome, we have shown that PAI-1 accumulates in MEJs and increases the total number of these structures, indicating a possible role in altering heterocellular communication. In order to understand how PAI-1 may alter MEJ formation, we used TNF-a to mimic the effect of metabolic syndrome and demonstrate significant increases in PAI-1 mRNA throughout endothelium, MEJ, and smooth muscle. However, after application of TNF-a, there was only a significant increase in PAI-1 protein at the MEJ. This indicates that PAI-1 may be locally translated at the MEJ. Recently, the serpine binding protein 1 (SERBP1) has been described as a novel RNA binding protein (RBP) specifically for PAI-1 mRNA that promotes stabilization of the mRNA for translation. However, an RBP requires anchoring to the cytoskeleton to remain in a specific area of the cell (e.g., the MEJ). To that end, we discovered a microtubule binding domain in the novel protein nicotinamide phosphoribosyltransferase (NAMPT), demonstrated to increase concurrently with PAI-1 during the course of metabolic syndrome, which we found to be localized to the MEJ. Therefore, we hypothesize that SERBP1 and NAMPT act together as a PAI-1 RBP complex to localize PAI-1 mRNA to the MEJ for the rapid and specific dissemination of PAI-1 protein. To test this hypothesis, we have put forth two specific aims: 1) PAI-1 mRNA is locally translated at the MEJ and 2) NAMPT regulates localization of SERBP1 to the MEJ. This proposal will focus on how PAI-1 is capable of being polarized to the MEJ using such techniques as FlAsH/ReAsH to examine nascent PAI-1 protein production and how RBP proteins could be anchored to the cytoskeleton using peptides against the microtubule binding domain of NAMPT. The aggregate of the experiments will provide for the first time a pathological mechanism for the effect of increased PAI-1 seen in metabolic syndrome.

抽象的 在代谢综合征的病程中，纤溶酶原显着增加 激活剂抑制剂-1 (PAI-1)；然而，PAI-1 在该过程中发挥的病理作用 病情尚不清楚。我们最近发现 PAI-1 是监管的关键 肌内皮连接（MEJ），是连接肌内皮细胞的重要异质细胞结构 阻力脉管系统中的内皮细胞和平滑肌，协调沟通 两种细胞类型之间。在患有代谢综合征的小鼠中，我们发现 PAI-1 在 MEJ 中积累并增加这些结构的总数，表明可能存在 改变异细胞通讯的作用。为了了解 PAI-1 如何改变 MEJ 的形成，我们使用 TNF-a 来模拟代谢综合征的影响并证明 整个内皮、MEJ 和平滑肌中的 PAI-1 mRNA 显着增加。 然而，应用TNF-a后，PAI-1蛋白仅在 MEJ。这表明 PAI-1 可能在 MEJ 进行本地翻译。最近，蛇 结合蛋白 1 (SERBP1) 被描述为一种新型 RNA 结合蛋白 (RBP) 专门针对 PAI-1 mRNA，促进 mRNA 翻译的稳定性。然而， RBP 需要锚定到细胞骨架上以保留在细胞的特定区域（例如， MEJ）。为此，我们在新型蛋白质中发现了微管结合域 烟酰胺磷酸核糖转移酶 (NAMPT)，被证明与 代谢综合征过程中的 PAI-1，我们发现其定位于 MEJ。 因此，我们假设 SERBP1 和 NAMPT 一起作为 PAI-1 RBP 复合物发挥作用， 将 PAI-1 mRNA 定位到 MEJ，以实现 PAI-1 蛋白的快速和特异性传播。到 为了检验这一假设，我们提出了两个具体目标：1）PAI-1 mRNA 在以下位置进行本地翻译： MEJ 和 2) NAMPT 调节 SERBP1 在 MEJ 的定位。该提案将重点 关于 PAI-1 如何能够使用 Flash/ReAsH 等技术极化到 MEJ 检查新生 PAI-1 蛋白的产生以及 RBP 蛋白如何锚定到 使用针对 NAMPT 微管结合域的肽构建细胞骨架。总计 这些实验将首次提供影响的病理机制 代谢综合征中 PAI-1 升高。