Vascular Extracellular Superoxide Dismutase Modulation

血管细胞外超氧化物歧化酶调节

• 批准号: 6855074
• 负责人: TOHRU FUKAI
• 金额: $ 26.6万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6855074
• 关键词: Golgi apparatus; angiotensin /renin /aldosterone hypertension; angiotensin II; cell line; copper; enzyme structure; free radical oxygen; laboratory mouse; membrane transport proteins; metal metabolism; molecular chaperones; oxidative stress; protein structure function; superoxide dismutase; vascular smooth muscle; yeasts

DESCRIPTION (provided by the applicant): Extracellular superoxide dismutase (ecSOD), a major form of SOD expressed in the vasculature, is a "secretory" copper-containing enzyme and plays an important role in regulating blood pressure and endothelial function by modulating the levels of O2 in the extracellular space. Particularly, in angiotensin H-induced hypertension model, the excessive 02 is observed in the vessel wall and the hypertension is ameliorated by treatment with membrane-targeted forms of SOD. Moreover, we have found that blood pressure and 02 production in the vessel were highly elevated in ecSOD-deficient mice infused with angiotensin II. Thus, ecS0D is a potentially important modulator of oxidative phenomena in the pathogenesis of hypertension. Recently, it has been shown that copper chaperones (CCS) are critical for copper transport and delivery to copper containing enzymes. Our preliminary data strongly suggests that CCS with signal peptide (CCS-SP) which targets to Golgi plays an important role in the transport of copper to ecSOD, which is required for full activity of the ecSOD. We will propose the following specific aim to address how ecSOD activity is controlled by copper transport system such as CCS and copper transporter in the yeast system, vascular cells and in vivo model of hypertension. In aim 1, we will characterize a role of copper transport system for full expression of ecSOD activity using the yeast system. First, by generating several CCS-SP cDNA constructs including the truncated form, we will determine which region is critical for copper loading-to ecSOD. Second, we will determine if copper loading to ecSOD requires MNK, a copper transporter in the trans-Golgi network, using the yeast strain deficient in MNK. In aim 2, we will identify endogenous copper chaperone for ecSOD in human aortic smooth muscle cells (HASM) that highly expresses ecSOD, by using the highly conserved region of CCS as a probe that have detected novel CCS-like transcript and protein in HASM. Next, we will determine if copper delivery to ecSOD requires MTNK in mammalian cells, by using the murine MNK-mutant fibroblast and aorta from MNK-mutant mice. In aim 3, we will examine the role of copper transport system for ecSOD in blood pressure, vascular O2 production and endothelial function in angiotensin II induced hypertension by using MNK-mutant mice. These studies will provide new insight into a copper transport system for ecSOD as a novel modulator of oxidative stress linked to the pathogenesis of hypertension and as essential to anti-oxidant therapy.

描述（由申请人提供）：细胞外超氧化物歧化酶 (ecSOD) 是在脉管系统中表达的 SOD 的主要形式，是一种“分泌” 含铜酶，在调节血液中起重要作用 通过调节血管中的 O2 水平来调节压力和内皮功能 细胞外空间。特别地，在血管紧张素H诱导的高血压模型中， 血管壁中观察到过量的02，则为高血压。 通过膜靶向形式的 SOD 治疗可以改善。此外，我们还有 发现血管中的血压和 02 产量显着升高 在注射了血管紧张素 II 的 ecSOD 缺陷小鼠中。因此，ecS0D 是 发病机制中氧化现象的潜在重要调节剂 高血压。最近，研究表明铜伴侣蛋白（CCS） 对于铜运输和递送至含铜酶至关重要。我们的 初步数据强烈表明带有信号肽（CCS-SP）的CCS 高尔基体的目标在铜向 ecSOD 的运输中发挥重要作用， 这是 ecSOD 充分发挥活性所必需的。我们将提出以下建议 具体目标是解决铜转运如何控制 ecSOD 活性 系统，例如酵母系统中的 CCS 和铜转运蛋白、血管细胞 和高血压体内模型。在目标 1 中，我们将扮演以下角色： 使用酵母充分表达 ecSOD 活性的铜转运系统 系统。首先，通过生成几个 CCS-SP cDNA 构建体，包括 截断形式，我们将确定哪个区域对铜至关重要 加载至 ecSOD。其次，我们将确定铜负载至 ecSOD 是否需要 MNK，跨高尔基体网络中的铜转运蛋白，使用酵母菌株 MNK 缺乏。在目标 2 中，我们将确定内源性铜伴侣 人主动脉平滑肌细胞 (HASM) 中高度表达 ecSOD， 通过使用 CCS 的高度保守区域作为探针，检测到了新的 HASM 中的 CCS 样转录物和蛋白质。接下来，我们将确定是否铜 递送至 ecSOD 需要哺乳动物细胞中的 MTNK，通过使用小鼠 来自 MNK 突变小鼠的 MNK 突变成纤维细胞和主动脉。在目标 3 中，我们将检查 ecSOD 铜转运系统在血压、血管 O2 中的作用 血管紧张素II诱导的高血压的产生和内皮功能 使用 MNK 突变小鼠。这些研究将为铜提供新的见解 ecSOD 转运系统作为氧化应激的新型调节剂 高血压的发病机制和抗氧化治疗至关重要。