The role of MRP1 in Protection of Cardiac Injury

空间 但前提是。‘ 本应用的目的是表征多药耐药蛋白1(MRP1)在 保护心脏免受氧化应激的伤害。我们假设用阿霉素(ADR)治疗癌症 氧化应激，进而导致肿瘤坏死因子-a(TNF)的产生，从而放大氧化 应激并导致正常组织损伤。MRP1是一种三磷酸腺苷结合盒(ABC)转运体，它介导 依赖于ATP的谷胱甘肽(GSH)外流及其结合物，包括细胞毒的GSH结合物 氧化应激产物4-羟基壬烯醛(HNE；GS-HNE)。我们将检验以下假设：1)心脏 MRP1的表达通过介导心肌细胞外排保护ADR所致的心肌氧化应激和损伤 GS-HNE；2)作为响应，mrp1表达增加并定位于质膜和线粒体 对氧化应激和/或肿瘤坏死因子，以及3)过量产生HNE和GS-HNE使MRP1失活， 压倒了它的保护作用，加剧了氧化损伤。四个具体的目标将检验这些 假设：目标1将利用mrp1基因缺失的小鼠来评估其在保护心脏免受ADR诱导的心脏损伤中的作用 氧化应激与损伤、MnSOD对MRp1缺失的补偿能力及肿瘤坏死因子的作用 调节mrp1的表达。目标2将描述mrp1的亚细胞定位和功能 ADR和肿瘤坏死因子治疗。目标3将评估氧化应激在调节mrp1表达中的作用 和本地化。最后，目标4将描述HNE和GS-HNE通过以下方式使MRP1失活的能力 关键的半胱氨酸、组氨酸或赖氨酸残基的烷基化。我们将利用不同基因类型(mrp1为空)的小鼠 转基因小鼠和杂合(/-)小鼠)，以评估这些基因在保护中的作用 抗心脏损伤、共聚焦免疫荧光免疫组织化学和定量免疫金 MRp1在心肌细胞和HEK293细胞中表达的定位分析 为了确定其功能，将使用蛋白质组学分析来确定mrp1的潜在结构异构体。 和Mr p1的HNE烷基化位点。了解MRp1、肿瘤坏死因子和谷胱甘肽在保护血管内皮细胞中的作用 心脏因ADR引起的组织损伤将导致辅助治疗手段的发展 防止这种伤害，从而允许使用更高剂量的高度有效的 化疗药物。

SPACE PROVIDED. ' The goal of the present application is to characterize the role of multidrug resistance protein 1 (Mrp1) in protecting the heart from oxidative stress. We postulate that cancer treatment with Adriamycin (ADR)leads to oxidative stress, which in turn leads to production of tumor necrosis factor-a (TNF) that amplifies oxidative stress and causes normal tissue injury. Mrp1 is an ATP-binding cassette (ABC) transporter that mediates the ATP-dependent efflux of glutathione (GSH) and its conjugates, including the GSH conjugate of the cytotoxic product of oxidative stress, 4-hydroxynonenal (HNE; GS-HNE). We will test the hypotheses that 1) Cardiac expression of Mrp1 protects the heart from oxidative stress and injury induced by ADR by mediating efflux of GS-HNE; 2) Mrp1 expression increases and is localized in plasma membrane and mitochondria in response to oxidative stress and/or TNF, and 3) excessive production of HNE and GS-HNE inactivates Mrp1, overwhelming its protective role, and exacerbating oxidative injury. Four Specific Aims will test these hypotheses; Aim 1will utilize Mrp1 null mice to assess its role in protecting the heart from ADR-induced oxidative stress and injury, the ability of MnSODto compensate for loss of Mrp1, and the function of TNF in regulating Mrp1 expression. Aim 2 will characterize the subcellular localization and function of Mrp1 following ADR and TNF treatment. Aim 3 will assessthe role of oxidative stress in the regulation of Mrp1 expression and localization. Finally, Aim 4 will characterize the ability of HNEand GS-HNE to inactivate Mrp1 by alkylation of key cysteine, histidine or lysine residues. We will utilize mice of various genotypes (Mrp1 null mice, MnSOD transgenic and heterozygous (+/-) mice) to assessthe roles of these genes in protection against cardiac injury, confocal immunofluorescent immunohistochemistry and quantitative immunogold analysis for localization of Mrp1 expression in the cardiomyocyte, and HEK293 cells for expression of Mrp1 to characterize its function; proteomic analyses will be used to identify potential structural isoforms of Mrp1 and the sites of HNE alkylation of Mrp1. Understanding the roles of Mrp1, TNF and GSH in protecting the heart from ADR-induced tissue injury will lead to the development of ancillary therapeutic modalities to protect against such injury, and thus permit utilization of higher doses of this highly effective chemotherapeutic agent.