Comparative Biology Elucidation of Environmental Pathways and Susceptibility

描述(由申请人提供)： 许多人类疾病，如亨廷顿氏症、帕金森氏症和阿尔茨海默氏症，以及正常的生理状态，如衰老，都受到环境压力的强烈影响。然而，关于环境应激源在动物身上被检测到并相互区分的机制，人们知之甚少。这项工作的长期目标是利用线虫的功能基因组方法来确定基本的环境胁迫感知和信号机制。这类研究将更准确地确定环境在人类健康和疾病中的作用，并有助于确定许多有毒物质的作用模式。在这项建议中要检验的具体假设是，隔室特异性蛋白质损伤构成了区分不同形式的环境应激的反馈抑制机制。这一假说基于以下观察结果：1)正常情况下阻止某些形式的蛋白质损伤积累的蛋白质稳态(PH)基因的敲除会激活渗透敏感基因的表达；2)激活渗透敏感基因表达的PH基因的敲除会加速蛋白质的聚集；3)高温和氧化应激诱导的蛋白质损伤不会激活渗透敏感基因的表达。这项提议的具体目的是：1)定义受环境应激源转录调控的PH基因。我们将使用I)比较全基因组微阵列分析野生型和转录因子突变动物的热、氧化和渗透诱导的基因表达；II)GFP标记的PH基因在暴露于环境应激期间的体内定位；III)过度表达PH靶标的转基因动物的应激存活分析，来研究PH基因在环境应激反应中的时空和功能参与。2)测试蛋白质错误折叠作为应激诱导基因表达的激活剂的作用。蛋白质损伤将通过RNAi敲除特定的PH复合体来遗传诱导。每个基因敲除对热、氧化和渗透应激信号通路的激活的影响将通过i)表达应激特异性GFP报告的动物的在体高通量荧光测量，ii)使用压力感应转录因子突变体的上位性分析，iii)在PH敲除动物中的全动物存活率分析来量化。

DESCRIPTION (provided by applicant): Many human diseases, such as Huntington's, Parkinson's, and Alzheimer's, and normal physiological states, such as aging, are strongly influenced by exposure to environmental stress. However, little is known about the mechanisms by which environmental stressors are detected and discriminated from one another in animals. The long term objective of this work is to utilize functional genomic approaches in the nematode C. elegans to define fundamental environmental stress sensing and signaling mechanisms. Such studies will more precisely define the role of the environment in human health and disease and help to define the modes of action of numerous toxic substances. The specific hypothesis to be tested in this proposal is that compartment specific protein damage constitutes a feedback inhibition mechanism for discrimination between different forms of environmental stress. This hypothesis is based on the observations that 1) knockdown of protein homeostasis (PH) genes that normally prevent the accumulation of some forms of protein damage activates osmosensitive gene expression 2) Knockdown of PH genes that activate osmosensitive gene expression accelerates protein aggregation 3) Protein damage induced by heat and oxidative stress does not activate osmosensitive gene expression. The specific aims of this proposal are: 1) Define the PH genes that are transcriptionally regulated by environmental stressors. We will examine the spatial and temporal, and functional involvement of PH genes in response to environmental stress using i) comparative whole genome microarray analysis of heat, oxidative, and osmotically induced gene expression in wild type and transcription factor mutant animals ii) in vivo localization of GFP tagged PH genes during exposure to environmental stress iii) stress survival assays in transgenic animals overexpressing PH targets. 2) Test the role of protein misfolding as an activator of stress induced gene expression. Protein damage will be genetically induced using RNAi knockdown of specific PH complexes. The effects of each gene knockdown on the activation of heat, oxidative, and osmotic stress signaling pathways will be quantified by i) in vivo high throughput fluorescence measurements of animals expressing stress-specific GFP reporters ii) epistasis analysis using stress sensing transcription factor mutants iii) whole animal survival assays in PH knockdown animals.