Application of ABC transporters toward enhanced arsenic detoxification

ABC 转运蛋白在增强砷解毒中的应用

• 批准号: 7325759
• 负责人: Melissa Sue LeBlanc
• 金额: $ 5.13万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7325759
• 关键词: ABCC1 gene; ATP-Binding Cassette Transporters; Address; Amino Acids; Animals; Arabidopsis; Arsenic; Arsenites; Biochemical; Cadmium; Carcinogens; Cell Line; Cell Survival; Cells; Chinese Hamster; Chinese Hamster Ovary Cell; Complement; Cytoplasm; DNA; DNA Sequence; Drug Metabolic Detoxication; Engineering; Exposure to; Genes; Glutathione; Goals; Homologous Gene; Human; Individual; Lead; Libraries; Maps; Measures; Mediating; Melissa; Mercury; Modeling; Multidrug Resistance Gene; Mutagenesis; Mutate; Mutation; Ovary; P-Glycoproteins; Pharmaceutical Preparations; Phenotype; Plants; Play; Polymerase Chain Reaction; Property; Proteins; Pump; Range; Relative (related person); Resistance; Role; Screening procedure; Site; Site-Directed Mutagenesis; Specificity; Stress; Structure; Substrate Specificity; Survival Rate; System; Tertiary Protein Structure; Testing; Toxic Environmental Substances; Toxic effect; Transcript; Transgenic Organisms; Vacuole; Work; World Health Organization; Yeasts; cell type; expression vector; homologous recombination; human disease; interest; member; metal poisoning; mutant; promoter; protein transport; repaired; research study; toxicant; uptake

The World Health Organization recognizes inorganic arsenic as a carcinogen, and a serious threat to millions of people. One mechanism cells use to deal with exposure to toxicants such as arsenic, is to pump them out of the cytoplasm using a special type of ABC transporter called a multidrug resistance protein (MRP). The yeast MRP, YCF1, actively pumps glutathione:arsenite conjugates into the vacuole, resulting in arsenic resistance. A YCF1 deletion strain lacks pump activity and is hypersensitive to arsenic. We propose that enhanced versions of YCF1 will confer increased arsenic uptake, accumulation, and resistance upon transgenic cells, and the Aycfl deletion strain will provide a convenient experimental system to test this hypothesis. The goal of this work is to identify conserved residues and domains within MRPs responsible for regulating arsenic toxicity, and will be addressed with the following specific aims: (1) Enhance the arsenic- transporting pump activity of YCF1. We will use PCR to randomly mutagenize the YCF1 gene, homologous recombination with a yeast expression vector to generate a library of mutants in a Aycfl yeast background, and phenotypic screening to identify mutations conferring enhanced arsenic resistance on Aycfl cells. (2) Explore the effects of corresponding mutations on human and plant YCF1 homologs. Human (MRP1, 2) and plant (AtMRPI, 3) multidrug resistance genes will be introduced into Aycfl yeast, and tested for their ability to rescue the Aycfl arsenic-sensitivity. Promising mutations identified in YCF1 will be introduced into the corresponding sites of these human and plant MRPs by site-directed mutagenesis, and the ability of these mutant MRPs to complement Aycfl yeast with enhanced arsenic pump activity will be examined. (3) Demonstrate an MRP-mediated reduction in arsenic-induced toxicity. Independent plant and CHO (Chinese hamster ovary) cell lines carrying the most promising mutant MRP versions identified will be generated and examined for acquisition of enhanced arsenic pump activity, measured by cell survival rates, arsenic accumulation, and induction of stress-related transcripts. Our coordinated work with yeast, plant, and human MRPs, in three different biochemical backgrounds, will define evolutionarily conserved regions that are important for arsenic pump activity and/or substrate specificity. RELEVANCE: This information will contribute toward dissecting the functions of the entire MRP subfamily of ABC transporters, several members of which have been implicated in human diseases, resistance to chemotherapeutic drugs, or metal toxicity. In addition, because YCF1 activity contributes not only to arsenic, but also to cadmium, mercury, and lead resistance in yeast, our results will likely be applicable toward several environmental toxicants.

世界卫生组织将无机砷视为致癌，对 数百万人。一种机制细胞用于处理暴露于毒素（例如砷）的方法是抽水 它们使用称为多药耐药性蛋白的特殊类型的ABC转运蛋白从细胞质中脱出 （MRP）。酵母MRP，YCF1，积极泵送谷胱甘肽：阿森特缀合物进入液泡，导致 砷耐药性。 YCF1缺失菌株缺乏泵活性，对砷过敏。我们建议 增强的YCF1版本将赋予增加砷的吸收，积累和抵抗力 转基因细胞和AYCFL缺失应变将提供一个方便的实验系统来测试这一点 假设。这项工作的目的是确定MRP内保守的残留物和域 调节砷毒性，并将以以下特定目的解决：（1）增强砷 - 运输YCF1的泵活动。我们将使用PCR随机诱变YCF1基因，同源 用酵母表达载体重组以在AYCFL酵母背景中生成突变体库， 和表型筛查以鉴定突变，赋予AYCFL细胞上砷耐药性增强。 （2） 探索相应突变对人和植物YCF1同源物的影响。人（MRP1，2）和 植物（ATMRPI，3）将将多药抗性基因引入AYCFL酵母菌，并测试其能力 营救Aycfl砷敏感性。在YCF1中确定的有希望的突变将引入 这些人类和植物MRP的相应位置通过位置定向的诱变以及这些的能力 将检查突变的MRP，以补充AYCFL酵母具有增强的砷泵活性。 （3） 证明了MRP介导的砷诱导的毒性降低。独立的植物和Cho（中文 将生成带有最有前途的突变体MRP版本的仓鼠卵巢），将产生，并产生 检查了通过细胞存活率测量的砷泵活性增强的砷泵活性 积累和诱导应力相关转录本。我们与酵母，植物和人类的协调合作 MRP在三种不同的生化背景中，将定义进化保守的区域 对于砷泵活性和/或底物特异性重要。相关性：此信息将 有助于剖析ABC转运蛋白的整个MRP亚科的功能，有几个 其成员与人类疾病有关，对化学治疗药物的抗性 毒性。此外，由于YCF1活性不仅有助于砷，而且对镉，汞， 和酵母中的铅耐药性，我们的结果可能适用于几种环境有毒物质。