Regulation of the Expression of G[alpha]i2 by Reactive Oxygen Species

活性氧对Gα 12 表达的调节

• 批准号: 8131830
• 负责人: Ifeanyi J. Arinze
• 金额: $ 32.63万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-02 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8131830
• 关键词: 2-tert-butylhydroquinone; Acids; Affinity; Aging; Alkanesulfonates; Antibodies; Antioxidants; Applications Grants; Atherosclerosis; Binding; Binding Sites; Biological Assay; CCAAT-Enhancer-Binding Proteins; Cardiovascular system; Cell Differentiation process; Cell Line; Cell Nucleus; Cells; Chemicals; Chimeric Proteins; Cloning; Co-Immunoprecipitations; Cytoplasm; Data; Development; Diabetes Mellitus; Differentiation Inducer; Disease; Electrophoretic Mobility Shift Assay; Event; Excision; Family; Fluorescence; Functional disorder; Funding; Future; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Gene Expression; Genes; Genetic Transcription; Half-Life; Heart failure; Hematopoietic; Hepatocyte; Heterotrimeric GTP-Binding Proteins; Hydrogen Peroxide; Hypertension; Imagery; Importins; Indium; Inflammation; Journals; K562 Cells; Kinetics; Leukemic Cell; Life; Link; Liver; Malignant Neoplasms; Measurement; Measures; Mediating; Messenger RNA; Metabolic Diseases; Metabolism; Methods; Molecular; Monitor; Mutate; Mutation; NF-kappa B; Neurodegenerative Disorders; Normal Cell; Nuclear; Nuclear Import; Nuclear Translocation; Outcome; Oxidants; Oxidation-Reduction; Oxidative Stress; Pathology; Pathway interactions; Peer Review; Pharmaceutical Preparations; Phosphoenolpyruvate Carboxylase; Photobleaching; Play; Process; Promoter Regions; Protein Family; Protein Isoforms; Proteins; Public Health; Publications; Reactive Oxygen Species; Regulation; Reporter Genes; Reporting; Research; Role; Signal Transduction; Site-Directed Mutagenesis; Solid; Sp1 Transcription Factor; Specificity; Sulfhydryl Compounds; System; Testing; Time; Up-Regulation; Western Blotting; Work; alpha Karyopherins; assault; base; beta Karyopherins; cell type; chromatin immunoprecipitation; fluorescence imaging; link protein; member; novel; oxidant stress; oxidation; promoter; protein protein interaction; receptor; research study; response; transcription factor

The overall objective of this project is to unravel molecular mechanisms by which reactive oxygen species (ROS) regulate expression of the signal transduction protein Ga{i2}. ROS are implicated in many disease states such as cancer, diabetes, cardiovascular problems, hypertension, aging, and inflammation. To extend preliminary studies showing that Ga{i2} is up-regulated by ROS through the Nrf2/Keap1 pathway, a pathway that underlies cells' response to oxidative stress, two specific aims are proposed. In specific aim 1, the objectives are to (a) test the hypothesis that protein-protein interaction between C/EBP1 and one or more of the other transcription factors (Sp1, Nrf2, and NF-kB) that bind at the ROS-responsive region of the Ga{i2} gene promoter constitute the mechanistic basis for the C/EBP1-induced suppression of Ga{i2} gene expression, and (b) explore the idea that Ga{i2} possesses redox-sensing capability. Protein-protein interaction will be monitored by co- immunoprecipitation, co-occupancy on, and/or recruitment (of the transcription factors) to the promoter. In specific aim 2, the objective is to assess mechanism(s) of the nuclear import of Nrf2, the key transcription factor in the Nrf2/Keap1 pathway. Its nuclear translocation will be monitored by fluorescence imaging of transfected green fluorescence protein (GFP)-tagged Nrf2, and by site-directed mutagenesis to assess the functionality of novel nuclear localization sequences in the protein. Affinity of such sequences to importin alphas that mediate this translocation will be measured by co-immunoprecipitation methods, and kinetics of nuclear import will be measured by fluorescence loss in photo bleaching (FLIP) assays. The work will be done primarily with K562 cells, a hematopoietic cell line, and in some experiments with HepG2 cells which are of liver origin. To elevate ROS levels, the cells will be exposed to the electrophile tert-butylhydroquinone or to H{2}O{2}. RELEVANCE. Oxygen radicals, also known as reactive oxygen species (ROS), are products of normal cell metabolism but they are toxic at high levels. They can be induced by drugs, environmental oxidants, and stress-inducing chemicals. ROS and the signal transduction proteins called G proteins are of enormous significance in public health because abnormalities in their function impact many diseases such as diabetes, heart failure, hypertension, atherosclerosis, and cancer. The outcome of the proposed research will contribute to further understanding of these pathologies.

该项目的总体目的是阐明分子机制，通过该机制，活性氧（ROS）调节信号转导蛋白Ga {i2}的表达。 ROS与许多疾病状态有关，例如癌症，糖尿病，心血管问题，高血压，衰老和炎症。为了扩展初步研究表明，ROS通过NRF2/KEAP1途径将GA {I2}上调，这是一种基于细胞对氧化应激的反应的途径，提出了两个特定的目标。在特定目的1中，目标是（a）检验以下假说：C/EBP1与一个或多个其他转录因子（SP1，NRF2和NF-KB）之间的蛋白质 - 蛋白质相互作用在GA {I2}基因启动子的ROS-响应性区域结合的c/eBP1-eBP1-诱导的机械基因（B）构成的GA {i2}基因构成的基础（B） - 均为B）的机械基础（B） - B） - inds in of cons of cons of cons in of-nf-kb（sp1，nrf2和nf-kb）。这个想法 GA {I2}具有氧化还原感应能力。蛋白质 - 蛋白质相互作用将通过共同沉淀，（转录因子）对启动子的共同沉淀，共募集和/或募集来监测。在特定目标2中，目的是评估NRF2的核进口机理，NRF2是NRF2/KEAP1途径中的关键转录因子。它的核转运将通过转染的绿色荧光蛋白（GFP）标记的NRF2的荧光成像进行监测，并通过定位定向的诱变来评估蛋白质中新型核定位序列的功能。这种序列与导入的介导该转运的亲和力将通过共免疫沉淀方法来衡量，核进口的动力学将通过光漂白（FLIP）测定中的荧光损失来衡量。这项工作将主要使用K562细胞，造血细胞系，以及在某些实验中对肝脏起源的HEPG2细胞进行的。为了升高ROS水平，细胞将暴露于亲电的叔丁基氢醌或H {2} o {2}。关联。氧自由基，也称为活性氧（ROS）是正常细胞代谢的产物，但它们在高水平上是有毒的。它们可以由药物，环境氧化剂和诱导压力的化学物质诱导。 ROS和称为G蛋白的信号转导蛋白在公共卫生中具有巨大的意义，因为其功能异常会影响许多疾病，例如糖尿病，心力衰竭，高血压，动脉粥样硬化和癌症。拟议研究的结果将有助于进一步理解这些病理。