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: 7532765
• 负责人: Melissa Sue LeBlanc
• 金额: $ 5.34万
• 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/7532765
• 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; 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.

世界卫生组织确认无机砷为致癌物，对人类健康构成严重威胁 数百万人。细胞用来处理暴露于砷等有毒物质的一种机制是泵送 使用一种特殊类型的 ABC 转运蛋白（称为多药耐药蛋白）将它们从细胞质中排出 （物料需求计划）。酵母 MRP，YCF1，主动将谷胱甘肽：亚砷酸盐结​​合物泵入液泡，从而产生 抗砷性。 YCF1 缺失菌株缺乏泵活性并且对砷过敏。我们建议 YCF1 的增强版本将增加砷的吸收、积累和抵抗力 转基因细胞和Aycfl缺失菌株将提供一个方便的实验系统来测试这一点 假设。这项工作的目标是识别 MRP 中负责的保守残基和结构域 监管砷毒性，并将通过以下具体目标来解决：（1）加强砷 YCF1 的运输泵活性。我们将使用PCR随机诱变YCF1基因，同源 与酵母表达载体重组以在 Aycfl 酵母背景中生成突变体文库， 和表型筛选，以确定赋予 Aycfl 细胞增强砷抗性的突变。 (2) 探索相应突变对人类和植物 YCF1 同源物的影响。人类（MRP1、2）和 植物（AtMRPI，3）多药抗性基因将被引入Aycfl酵母中，并测试其能力 拯救 Aycfl 砷敏感性。 YCF1 中鉴定出的有希望的突变将被引入到 通过定点突变确定这些人类和植物 MRP 的相应位点，以及这些的能力 将检查突变型 MRP 以增强砷泵活性来补充 Aycfl 酵母。 (3) 证明 MRP 介导的砷诱导毒性降低。独立工厂及CHO（中文 将生成携带最有希望的突变 MRP 版本的仓鼠卵巢）细胞系，并 检查是否获得增强的砷泵活性，通过细胞存活率、砷来测量 应激相关转录本的积累和诱导。我们与酵母、植物和人类的协调工作 MRP，在三种不同的生化背景下，将定义进化上保守的区域 对于砷泵活性和/或底物特异性很重要。相关性：此信息将 有助于剖析 ABC 转运蛋白的整个 MRP 亚家族的功能， 其中的成员与人类疾病、化疗药物或金属耐药性有关 毒性。此外，由于YCF1活性不仅有助于砷，还有助于镉、汞、 和酵母的铅抗性，我们的结果可能适用于几种环境毒物。

项目成果

Enhanced arsenic tolerance of transgenic eastern cottonwood plants expressing gamma-glutamylcysteine synthetase.

表达γ-谷氨酰半胱氨酸合成酶的转基因东部三叶杨植物的砷耐受性增强。

• 发表时间: 2011-08
• 影响因子: 3.7
• 作者: LeBlanc, Melissa S;Lima, Amparo;Montello, Paul;Kim, Tehryoung;Meagher, Richard B;Merkle, Scott
• 通讯作者: Merkle, Scott

Hijacking membrane transporters for arsenic phytoextraction.

劫持膜转运蛋白用于砷植物提取。

• DOI: 10.1016/j.jbiotec.2012.10.013
• 发表时间: 2013-01-10
• 期刊: Journal of biotechnology
• 影响因子: 4.1
• 作者: LeBlanc, Melissa S.;McKinney, Elizabeth C.;Meagher, Richard B.;Smith, Aaron P.
• 通讯作者: Smith, Aaron P.