HYPERTHERMIA AND THE FUNCTIONS OF HSP70

热疗和 HSP70 的功能

• 批准号: 2733035
• 负责人: GLORIA C LI
• 金额: $ 28.17万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2733035
• 关键词: DNA binding protein; DNA damage; DNA footprinting; DNA repair; X ray; chemical association; embryonic stem cell; gene deletion mutation; gene expression; genetic promoter element; genetically modified animals; human genetic material tag; hyperthermia; laboratory mouse; messenger RNA; molecular site; mutant; protein structure function; reporter genes; site directed mutagenesis; southern blotting; stress proteins; tissue /cell culture; transcription factor; transfection /expression vector

Ku autoantigen is a DNA binding protein consisting of 7O-kDa (Ku70) and 86-kDa (Ku80) subunits. Recent data have implicated Ku80 in the repair of DNA double strand breaks, and Ku70 in the suppression of heat-induced hsp70 expression. The long term goal of this project is to understand the molecular bases of the cellular response to stress. Work in the next granting period will focus on the mechanistic and functional aspects of Ku protein, in modulating cellular response to heat shock and to ioizing radiation. There are three specific aims: Specific Aim I focuses on the molecular mechanism(s) by which Ku suppresses heat-induction of hsp70. We will test a hypothesis inferred from our preliminary studies that Ku regulates the heat shock response by modulating the binding of transcription factors to their respective regulatory elements in the promoter region of the hsp70 gene. By in vivo genomic footprinting, the effects of Ku on the pattern of protein binding to the hsp70 promoter, especially its effects on the binding of the heat shock transcription activator HSF1 and other transcription factors to their respective binding sites will be determined. The possibility that Ku binds to a specific element in the regulatory region of the hsp70 gene will be tested. In addition to in vivo genomic footprinting, mutations will be introduced into the promoter of hsp70, linked to a reporter gene construct, to test for the presence of a cis-element(s), which when mutated abolishes the Ku-mediated suppression of heat-induction of reporter gene expression. In Specific Aim II we will study the structural and functional domains of Ku that are involved in the modulation of hsp70 gene expression. Rodent cell lines stably and constitutively expressing various mutant Ku genes will be established. The heat shock response of these cells and the biochemical properties of wild type and mutant Ku subunits will be examined. In Specific Aim III we propose to establish homozygous rodent cell lines in which both alleles of Ku70 or Ku80 are inactivated through gene-targeting mutagenesis. These "double knockout" Ku7O-/- and Ku8- /- cell lines will enable us to study various physiological roles of the individual subunits of Ku and to dissect the functional domains involved in the cellular response to heat shock and ionizing radiation. Because of the biological importance and the multiple cellular roles of Ku, the significance of constructing Ku70-/- and Ku80-/- lines extends beyond the immediate goal of elucidating the role of Ku in hsp70 gene regulation, and in the sensitivity of cells to x-rays. These cell lines and their derivatives expressing various mutant Ku subunits can be used directly in the analysis of any functional aspect of Ku. The choice of embryonic stem cells for this work further assures that such studies can be readily extended to animal models, through the construction of knockout mice strains.

KU自动抗原是由7o-KDA（KU70）组成的DNA结合蛋白 和86 kDa（ku80）亚基。最近的数据已牵涉到Ku80 DNA双链断裂的修复和抑制作用的KU70 热诱导的Hsp70表达。这个项目的长期目标是 了解细胞对应激反应的分子碱基。 在下一个授予期内的工作将重点放在机械上， KU蛋白的功能方面，调节细胞对 热量冲击和辐射。有三个特定的目标： 具体目的我重点介绍了ku的分子机制 抑制HSP70的热诱导。我们将检验一个假设 根据我们调节热量的初步研究来推断 通过调节转录因子与 它们在启动子区域的各自调节元素 HSP70基因。 通过体内基因组足迹，KU的影响对 蛋白质与HSP70启动子结合，尤其是其对 热休克转录激活剂HSF1和其他的结合 转录因子到其各自的结合位点将是 决定。 KU与特定元素结合的可能性 HSP70基因的调节区域将进行测试。此外 为了体内基因组足迹，将引入突变 HSP70的启动子与报告基因构建体相关，以测试 为了存在顺式元素，当突变时废弃时 KU介导的报告基因的热诱导抑制 表达。 在特定目标II中，我们将研究结构和功能 与HSP70基因调制有关的KU域 表达。啮齿动物细胞系稳定且组成型表达 将建立各种突变的KU基因。热冲击响应 这些细胞以及野生型和突变体的生化特性 将检查KU亚基。 在特定的目标III中，我们建议建立纯合啮齿动物细胞 ku70或ku80等位基因均被灭活的线 基因靶向诱变。这些“双重淘汰” ku7o - / - 和ku8-- / - 细胞系将​​使我们能够研究 KU的各个亚基并剖析功能域 参与热冲和电离的细胞反应 辐射。由于生物学的重要性和多重 ku的细胞作用，构建ku70 - / - 和 ku80 - / - 线扩展到阐明该目标的直接目标 KU在HSP70基因调控和细胞敏感性中的作用 进行X射线。这些细胞系及其衍生物表达各种 突变的KU亚基可直接用于分析任何 KU的功能方面。为此选择胚胎干细胞 工作进一步确保，这些研究很容易扩展到 动物模型，通过敲除小鼠菌株的结构。