Large Scale Identification and Characterization of Hsf1-mediated Heat Shock Respo

Hsf1 介导的热休克反应的大规模鉴定和表征

• 批准号: 8122807
• 负责人: Patrick Allen Gibney
• 金额: $ 5.13万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8122807
• 关键词: Address; Alcohols; Alzheimer' s Disease; Binding; Biochemical; Biological; Biological Assay; Biological Models; Cell Separation; Cellular Stress; Collection; DNA; DNA Sequence; Defect; Disease; Drug resistance; Elements; Eukaryota; Fluorescence-Activated Cell Sorting; Gene Deletion; Gene Expression; General Population; Genes; Genetic; Genetic Engineering; Genetic Transcription; Genome; Growth; Heat shock proteins; Heat-Shock Proteins 90; Heat-Shock Response; Heating; High temperature of physical object; Human; Human body; Huntington Disease; Insulin; Knowledge; Lead; Malignant Neoplasms; Mediating; Messenger RNA; Microarray Analysis; Molecular; Neurodegenerative Disorders; Orthologous Gene; Pathology; Phenotype; Phosphorylation; Phosphotransferases; Physiologic pulse; Post-Translational Protein Processing; Prion Diseases; Process; Production; Proteins; Recombinants; Regulation; Research; Role; Saccharomyces cerevisiae; Signal Transduction; Signal Transduction Pathway; Stress; System; Systems Analysis; Techniques; Technology; Testing; Translation Process; Vitamins; Western Blotting; Work; Yeasts; base; biological adaptation to stress; biological systems; cell type; chromatin immunoprecipitation; deletion analysis; environmental change; fitness; heat shock transcription factor; interest; mutant; next generation; pathogen; promoter; protein folding; protein misfolding; research study; response; sensor; thermal stress; transcription factor; tumor

DESCRIPTION (provided by applicant): The ability to sense and respond to changing environmental conditions is a fundamental aspect of all biological systems. The conserved heat shock response in Saccharomyces cerevisiae has proven to be a paradigm for general eukaryotic stress responses. Despite much analysis at the level of gene expression and the functions of those expressed genes, very little is known about the cellular sensor or potential signal transduction pathway that activates the conserved heat shock transcription factor, the Hsf1 protein (referred to as Hsf1). The research outlined in this proposal seeks to identify and characterize proteins that act as regulators of Hsf1. A preliminary screen was performed and resulted in identification of 11 potential positive Hsf1 regulators. The first part of this proposal is dedicated to examining each of these genes for their functional and conserved role in the Hsf1-mediated heat shock response. This will be accomplished using a combination of classic genetic and biochemical techniques (such as deletion analysis and Western blot analysis) and systems-level biological techniques (such as competitive fitness assays and microarrays). The second part of this proposal is dedicated to developing a versatile, quantitative system for identification of both positive and negative regulators of transcription factors, which will be used to further identify Hsf1 regulators. This will be accomplished using common recombinant DNA technology in addition to fluorescent activated cell sorting (FACS) and barcode DNA sequencing using next generation sequencing technology (Illumina Genome Analyzer II). Overall, this work will significantly increase our knowledge of the heat shock response, and of eukaryotic stress- responsive signal transduction, in general. PUBLIC HEALTH RELEVANCE: Understanding the mechanism of the eukaryotic heat shock response has potential far-reaching significance for a number of applications. Manipulation of thermo tolerance could have a direct impact on processes that use yeast or other fungal species for production of medicinal or industrial compounds (for example, insulin, vitamins, and alcohol are produced by recombinant yeast). In addition, the mechanism of many different pathologies are intimately connected to the heat shock response: heat shock protein 90 signaling is required for tumor formation and propagation, protein misfolding (regulated by heat shock proteins) is a hallmark of many neurodegenerative diseases (Alzheimer's, Huntington's, and prion diseases, among others), and high temperature growth allows fungal pathogens to survive in the human body. Again, understanding and modulating the heat shock response could lead to therapies for these types of diseases.

描述（由申请人提供）：感知和响应不断变化的环境条件的能力是所有生物系统的基本方面。酿酒酵母菌保守的热休克反应已被证明是一般真核生物应激反应的范例。尽管在基因表达水平和这些表达基因的功能上进行了大量的分析，但对激活保守的热休克转录因子Hsf1蛋白（简称Hsf1）的细胞传感器或潜在信号转导途径知之甚少。本提案中概述的研究旨在识别和表征作为Hsf1调节因子的蛋白质。进行了初步筛选，结果鉴定出11个潜在的Hsf1阳性调节因子。本提案的第一部分致力于研究这些基因在hsf1介导的热休克反应中的功能和保守作用。这将使用经典的遗传和生化技术（如缺失分析和Western blot分析）和系统级生物技术（如竞争适应度分析和微阵列）的组合来完成。本提案的第二部分致力于开发一种多功能的定量系统，用于鉴定转录因子的正调控因子和负调控因子，这将用于进一步鉴定Hsf1调控因子。除了荧光活化细胞分选（FACS）和使用下一代测序技术（Illumina Genome Analyzer II）的条形码DNA测序外，这将使用常见的重组DNA技术来完成。总的来说，这项工作将显著增加我们对热休克反应和真核生物应激反应信号转导的认识。