A quantitative high resolution understanding of heat stress sensing and responses

对热应力传感和响应的定量高分辨率理解

• 批准号: 8771565
• 负责人: NICHOLAS J KAPLINSKY
• 金额: $ 30.88万
• 依托单位: SWARTHMORE COLLEGE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8771565
• 关键词: 26S proteasome; Alleles; Animals; Arabidopsis; Arabidopsis Proteins; Biochemical Genetics; Cell physiology; Cells; Cellular Stress; Characteristics; Custom; Data; Defect; Development; Disease; Ensure; Essential Genes; Eukaryotic Cell; Gene Expression; Genes; Genetic; Genetic Screening; Goals; Heat Stress Disorders; Heat shock proteins; Heat-Shock Response; Heating; High temperature of physical object; Human; Image Analysis; Individual; Kinetics; Link; Maps; Measures; Mediating; Microscopy; Molecular Chaperones; Monitor; Mutation; Organism; Pathway interactions; Phenotype; Population; Proteins; Quality Control; Reporter; Resolution; Signal Transduction; Stress; System; Temperature; Temperature Sense; Tertiary Protein Structure; human disease; insight; loss of function; multicatalytic endopeptidase complex; mutant; next generation sequencing; plant fungi; plant growth/development; prevent; programs; protein degradation; protein folding; protein misfolding; public health relevance; response; screening; tool

DESCRIPTION (provided by applicant): A wide range of important human diseases are characterized by protein misfolding. In addition to disease, protein misfolding is also caused by a range of cellular stresses. In the cell, protein chaperones maintain proteostasis by ensuring correct folding of newly synthesized proteins and the refolding of stress-denatured proteins. It follows that a full understanding of protein misfolding diseases depends on understanding both how chaperones function and how they are regulated in response to stress. High temperature stress is one of the stresses which causes protein misfolding. The two goals of this project are to understand 1) how cells sense high temperature stress, and 2) how the Arabidopsis protein chaperone BOBBER1 (BOB1) ensures normal cellular function under both non-stressful and stressful condition. The mechanisms which cells use to sense heat stress and misfolded proteins are only partially understood. Forward genetic approaches have been of limited use in understanding these mechanisms because of genetic redundancy. We plan on identifying genes involved in high temperature sensing and signaling, genes we call thermostat genes, using the RootScope, a custom automated microscopy system. This high-throughput system allows us to quantitatively monitor cellular responses to heat stress with high spatial and temporal resolution. It enables us to identify subtle changes in the kinetics of heat shock responses caused by mutations in redundant genes. The RootScope will allow us to identify and characterize genes involved in responding to protein misfolding which cannot be identified using conventional phenotyping approaches. BOB1 encodes a protein chaperone with functions in both development and temperature responses. BOB1 contains a conserved NudC protein domain. NudC genes are essential genes in animals, fungi, and plants suggesting that they have conserved, important, and unique functions. NudC genes are found in humans so understanding BOB1 function will contribute to our understanding of protein quality control in disease. We have taken advantage of the developmental defects in bob1 mutants to identify genetic interactions between a BOB1 partial loss of function allele and multiple genes encoding proteasome subunits. We plan on characterizing these interactions in order to understand how protein folding and degradation mechanisms are linked. We have also identified two additional genes which interact genetically with BOB1 which we will clone and characterize. Finally, we will continue an ongoing BOB1 genetic modifier screen to identify and characterize additional genes and cellular pathways which rely on BOB1 function. The identification and characterization of both heat shock response thermostat and BOB1 interacting genes will contribute to a mechanistic understanding of how eukaryotic cells sense and respond to disruptions to proteostasis caused by elevated temperatures and other stresses.

描述（由申请人提供）：多种重要的人类疾病的特征在于蛋白质错误折叠。除了疾病，蛋白质错误折叠也是由一系列细胞应激引起的。在细胞中，蛋白质伴侣通过确保新合成蛋白质的正确折叠和应激变性蛋白质的重折叠来维持蛋白质稳态。因此，对蛋白质错误折叠疾病的全面了解取决于对伴侣蛋白如何发挥作用以及它们如何在应激反应中受到调节的理解。高温胁迫是引起蛋白质错误折叠的胁迫之一。该项目的两个目标是了解1）细胞如何感知高温胁迫，以及2）拟南芥蛋白伴侣BOBBER 1（BOB1）如何确保非应激和应激条件下的正常细胞功能。细胞用来感知热应激和错误折叠蛋白质的机制只被部分理解。由于遗传冗余，正向遗传学方法在理解这些机制方面的作用有限。我们计划使用RootScope（一种定制的自动显微镜系统）来识别参与高温传感和信号传导的基因，我们称之为恒温基因。这种高通量系统使我们能够以高的空间和时间分辨率定量监测细胞对热应激的反应。 它使我们能够识别由冗余基因突变引起的热休克反应动力学的微妙变化。RootScope将使我们能够识别和表征参与响应蛋白质错误折叠的基因，这些基因无法使用传统的表型分析方法进行识别。BOB1编码一种蛋白伴侣，在发育和温度反应中具有功能。BOB1含有一个保守的NudC蛋白结构域。NudC基因是动物、真菌和植物的必需基因，具有保守、重要和独特的功能。NudC基因在人类中发现，因此了解BOB1功能将有助于我们了解疾病中的蛋白质质量控制。我们利用bob1突变体的发育缺陷来鉴定BOB1部分功能丧失等位基因和编码蛋白酶体亚基的多个基因之间的遗传相互作用。我们计划表征这些相互作用，以了解蛋白质折叠和降解机制是如何联系在一起的。我们还确定了两个额外的基因相互作用的基因与BOB1，我们将克隆和表征。最后，我们将继续进行BOB1遗传修饰剂筛选，以识别和表征依赖于BOB1功能的其他基因和细胞通路。热休克反应恒温器和BOB1相互作用基因的鉴定和表征将有助于真核细胞如何感知和响应由升高的温度和其他应激引起的蛋白质稳态破坏的机制理解。