Vascular Extracellular Superoxide Dismutase Modulation

血管细胞外超氧化物歧化酶调节

• 批准号: 6721148
• 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/6721148
• 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.

性状（申请方提供）：细胞外超氧化物歧化酶 内皮超氧化物歧化酶（ecSOD）是在血管系统中表达的SOD的主要形式， 含铜酶，在调节血液中起重要作用 压力和内皮功能通过调节O2的水平， 细胞外间隙特别地，在血管紧张素H诱导的高血压模型中， 在血管壁中观察到过量的O2， 通过用膜靶向形式的SOD治疗而改善。而且我们 发现血管中的血压和O2产生高度升高， 在输注血管紧张素II的ecSOD缺陷小鼠中。因此，ecS 0D是一个 氧化现象的发病机制中的潜在重要调节剂 高血压最近，已经表明铜分子伴侣（CCS）是 对于铜转运和递送至含铜酶至关重要。我们 初步数据强烈表明，CCS与信号肽（CCS-SP）， 靶向高尔基体在铜向ecSOD的运输中起重要作用， 这是ecSOD完全活性所必需的。我们将提出以下建议 具体目标是解决ecSOD活性如何受铜转运控制 系统，如CCS和酵母系统中的铜转运蛋白，血管细胞 和高血压的体内模型。在目标1中，我们将描述 利用酵母表达ecSOD活性的铜转运系统 系统首先，通过产生几种CCS-SP cDNA构建体，包括 截断形式，我们将确定哪个区域是关键的铜 加载到ecSOD。其次，我们将确定铜加载到ecSOD是否需要 MNK，一种在trans-Golgi网络中的铜转运蛋白，使用酵母菌株 缺乏MNK。在目标2中，我们将鉴定内源性铜分子伴侣， 高表达ecSOD的人主动脉平滑肌细胞（HASM）中的ecSOD， 通过使用CCS的高度保守区域作为探针， HASM中的CCS样转录物和蛋白。接下来，我们将确定铜是否 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，我们将研究 铜转运系统对ecSOD在血压、血管氧合中作用 血管紧张素II诱导的高血压中血管紧张素II的产生和内皮功能 使用MNK突变小鼠。这些研究将提供新的见解铜 ecSOD转运系统作为一种新的氧化应激调节剂， 高血压的发病机制和抗氧化治疗的必要性。