The role of MRP1 in Protection of Cardiac Injury

MRP1 在保护心脏损伤中的作用

• 批准号: 8115153
• 负责人: Mary Vore
• 金额: $ 26.19万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-25 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8115153
• 关键词: 4 hydroxynonenal; ABCC1 gene; ATP-Binding Cassette Transporters; Adriamycin PFS; Alkylation; Antibodies; Biochemical; Biological Assay; Cardiac; Cardiac Myocytes; Cardiotoxicity; Cell Line; Cell membrane; Cells; Collaborations; Coupled; Cysteine; Development; Dose; Esters; Fractionation; Genes; Genotype; Glutathione; Glutathione Disulfide; Goals; Heart; Histidine; Human; Immunohistochemistry; In Vitro; Injury; Knockout Mice; Lead; Lysine; Measures; Mediating; Messenger RNA; Mitochondria; Modality; Morphology; Mus; Normal tissue morphology; Oxidation-Reduction; Oxidative Stress; P-Glycoprotein; P-Glycoproteins; Production; Protein Isoforms; Proteins; Proteomics; Quantitative Microscopy; Regulation; Role; Sarcolemma; Schiff Bases; Site; Testing; Therapeutic; Time; Tissues; Toxic effect; Transgenic Mice; Transgenic Organisms; Tumor Necrosis Factor-alpha; Vesicle; Wild Type Mouse; adduct; base; buthionine; cancer therapy; chemotherapeutic agent; cytotoxic; functional group; in vivo; novel; overexpression; oxidant stress; oxidative damage; response; treatment effect; uptake

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.

空间 假如。 ' 本申请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。我们假设阿霉素 (ADR) 的癌症治疗领先 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 压力并导致正常组织损伤。 Mrp1 是一种 ATP 结合盒 (ABC) 转运蛋白，介导 谷胱甘肽 (GSH) 及其结合物（包括细胞毒性物质的 GSH 结合物）的 ATP 依赖性流出 氧化应激产物，4-羟基壬烯醛（HNE；GS-HNE）。我们将测试以下假设：1) 心脏 Mrp1 的表达通过介导 ADR 的流出来保护心脏免受 ADR 诱导的氧化应激和损伤 GS-HNE； 2) Mrp1表达增加并定位于质膜和线粒体作为响应 氧化应激和/或 TNF，以及 3) HNE 和 GS-HNE 的过量产生使 Mrp1 失活， 压倒其保护作用，并加剧氧化损伤。四个具体目标将测试这些 假设；目标 1 将利用 Mrp1 缺失小鼠评估其在保护心脏免受 ADR 诱导的作用中的作用 氧化应激和损伤、MnSOD 补偿 Mrp1 损失的能力以及 TNF 的功能 调节 Mrp1 的表达。目标 2 将描述 Mrp1 的亚细胞定位和功能 ADR 和 TNF 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 将通过以下方式表征 HNE 和 GS-HNE 灭活 Mrp1 的能力： 关键半胱氨酸、组氨酸或赖氨酸残基的烷基化。我们将利用各种基因型的小鼠（Mrp1 null 小鼠、MnSOD 转基因和杂合 (+/-) 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 心肌细胞中Mrp1表达定位分析，以及HEK293细胞中Mrp1表达的分析 表征其功能；蛋白质组分析将用于鉴定 Mrp1 的潜在结构亚型 以及Mrp1的HNE烷基化位点。了解 Mrp1、TNF 和 GSH 在保护细胞中的作用 ADR 引起的心脏组织损伤将导致辅助治疗方式的发展 防止这种伤害，从而允许使用更高剂量的这种高效 化疗剂。