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

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。酿酒酵母中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表达基因的功能方面进行了大量的分析，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的信号转导途径知之甚少。这项提案中概述的研究试图识别和表征作为HSF1调节因子的蛋白质。进行了初步筛选，结果鉴定出11个潜在的阳性HSF1调节子。该提案的第一部分致力于研究这些基因中的每一个在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印迹分析)和系统级生物技术(如竞争性适应性分析和微阵列)的组合来实现。该提案的第二部分致力于开发一种通用的、定量的系统来识别转录因子的正负调控因子，该系统将用于进一步识别HSF1调控因子。除了荧光激活细胞分选(FACS)和使用下一代测序技术(Illumina Genome Analyzer II)的条形码DNA测序外，这将使用普通的重组DNA技术来实现。总体而言，这项工作将显著增加我们对热休克反应和真核细胞应激反应信号转导的了解。 公共卫生相关性：了解真核生物热休克反应的机制对许多应用具有潜在的深远意义。耐热性的操作可能会对使用酵母或其他真菌物种生产医药或工业化合物(例如，胰岛素、维生素和酒精由重组酵母生产)的过程产生直接影响。此外，许多不同的病理机制与热休克反应密切相关：热休克蛋白90信号是肿瘤形成和繁殖所必需的，蛋白质错误折叠(受热休克蛋白调控)是许多神经退行性疾病(阿尔茨海默病、亨廷顿病和普里恩病等)的标志，高温生长允许真菌病原体在人体内生存。同样，了解和调节热休克反应可能会导致这些类型的疾病的治疗。

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.