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: 7628888
• 负责人: Ifeanyi J. Arinze
• 金额: $ 32.66万
• 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/7628888
• 关键词: 2-tert-butylhydroquinone; Abbreviations; Acetates; 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; Guanine Nucleotides; Half-Life; Heart failure; Hematopoietic; Hepatocyte; Heterotrimeric GTP-Binding Proteins; Histone Deacetylase; Hydrogen Peroxide; Hypertension; Imagery; Indium; Inflammation; Journals; K562 Cells; Karyopherins; Kinetics; Leukemic Cell; Life; Link; Liver; MAP Kinase Gene; Malignant Neoplasms; Measurement; Measures; Mediating; Messenger RNA; Metabolic Diseases; Metabolism; Methods; Mitogen-Activated Protein Kinases; Molecular; Monitor; Mutate; Mutation; NF-E2-related factor 2; NF-kappa B; Neurodegenerative Disorders; Normal Cell; Nuclear; Nuclear Import; Nuclear Localization Signal; Nuclear Translocation; Outcome; Oxidants; Oxidation-Reduction; Oxidative Stress; Pathology; Pathway interactions; Peer Review; Pharmaceutical Preparations; Phosphotransferases; Photobleaching; Play; Process; Promoter Regions; Protein Family; Protein Isoforms; Protein Kinase C; Proteins; Public Health; Publications; Reactive Oxygen Species; Regulation; Reporter Genes; Reporting; Research; Response Elements; 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; cell type; chromatin immunoprecipitation; enhancer binding protein; extracellular; factor C; fibrosarcoma; fluorescence imaging; genetic regulatory protein; link protein; member; novel; oxidant stress; oxidation; phorbol-12-myristate; promoter; protein protein interaction; public health relevance; receptor; research study; response; transcription factor

DESCRIPTION (provided by applicant): The overall objective of this project is to unravel molecular mechanisms by which reactive oxygen species (ROS) regulate expression of the signal transduction protein G1i2. ROS are implicated in many disease states such as cancer, diabetes, cardiovascular problems, hypertension, aging, and inflammation. To extend preliminary studies showing that G1i2 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 G1i2 gene promoter constitute the mechanistic basis for the C/EBP1-induced suppression of G1i2 gene expression, and (b) explore the idea that G1i2 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 H2O2. 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. Public Health Relevance: The overall objective of this project is to unravel molecular mechanisms by which reactive oxygen species (ROS) regulate expression levels of G1i2, a member of the heterotrimeric G-protein family of signal transduction proteins, and to explore mechanisms of the nuclear import of the transcription factor Nrf2, an upstream event in the ROS-induced upregulation of G1i2. ROS and the signal transduction proteins called G proteins are of enormous significance in public health because abnormalities in their function are implicated in a plethora of diseases and metabolic derangements such as cancer, diabetes, cardiovascular problems vis-`- vis heart failure, hypertension, neurodegenerative disorders, inflammation leading to atherosclerosis, and aging.

描述（由申请人提供）：该项目的总体目标是揭示活性氧（ROS）调节信号转导蛋白G1i2表达的分子机制。 ROS 与许多疾病状态有关，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 G1i2 通过 Nrf2/Keap1 途径（细胞对氧化应激反应的基础途径）被 ROS 上调，提出了两个具体目标。在具体目标 1 中，目标是 (a) 检验以下假设：C/EBP1 与在 ROS 响应位点结合的一个或多个其他转录因子（Sp1、Nrf2 和 NF-kB）之间存在蛋白质-蛋白质相互作用。 G1i2 基因启动子区域构成了 C/EBP1 诱导的 G1i2 基因表达抑制的机制基础，并且 (b) 探索了 G1i2 具有氧化还原感应的观点 能力。蛋白质-蛋白质相互作用将通过共免疫沉淀、启动子上的共占据和/或（转录因子的）募集来监测。具体目标 2 的目标是评估 Nrf2（Nrf2/Keap1 通路中的关键转录因子）的核输入机制。其核易位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，并通过定点诱变来评估蛋白质中新型核定位序列的功能。这些序列与介导这种易位的输入α的亲和力将通过免疫共沉淀方法来测量，并且核输入的动力学将通过光漂白（FLIP）测定中的荧光损失来测量。这项工作将主要使用 K562 细胞（一种造血细胞系）进行，并且在一些实验中使用肝脏来源的 HepG2 细胞。为了提高 ROS 水平，细胞将暴露于亲电子试剂叔丁基氢醌或 H2O2。关联。氧自由基，也称为活性氧 (ROS)，是正常细胞代谢的产物，但高浓度时具有毒性。它们可以由药物、环境氧化剂和诱发压力的化学物质诱发。 ROS 和称为 G 蛋白的信号转导蛋白对公共健康具有重要意义，因为它们的功能异常会影响许多疾病，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了解这些病理学。 公共卫生相关性：该项目的总体目标是揭示活性氧 (ROS) 调节 G1i2（信号转导蛋白异源三聚体 G 蛋白家族成员）表达水平的分子机制，并探索细胞核的机制。转录因子 Nrf2 的导入，是 ROS 诱导的 G1i2 上调的上游事件。 ROS 和称为 G 蛋白的信号转导蛋白对公共健康具有重要意义，因为它们的功能异常与多种疾病和代谢紊乱有关，例如癌症、糖尿病、心力衰竭、高血压、神经退行性疾病等心血管问题疾病、导致动脉粥样硬化的炎症和衰老。