The mechanism of action of a NudC domain small heat shock protein

描述(申请人提供)：一系列重要的人类疾病都以蛋白质错误折叠为特征。在细胞中，蛋白质伴侣确保新合成蛋白质的正确折叠以及应激变性蛋白质的重新折叠。因此，对蛋白质错误折叠疾病的充分理解依赖于对伴侣机制的理解。本项目的目的是了解BOBBER1(BOB1)的生物学功能，BOB1是最近发现的一种拟南芥蛋白伴侣。Bob1是第一个在发育和温度反应中都发挥作用的伴侣，这表明它具有重要和独特的功能。Bob1含有一个进化上保守的NudC蛋白结构域。由于像Bob1这样的NudC结构域伴侣存在于包括人类在内的许多生物体中，了解Bob1的功能将有助于从根本上理解蛋白质质量控制的细胞机制。这项建议包括研究1)Bob1蛋白结构的变化如何影响其功能，以及2)确定与Bob1相互作用的其他蛋白质和基因。了解Bob1功能的方法包括使用一系列突变体来系统地确定每个突变对生物耐热性、伴侣活性和BOB1s亚细胞定位的影响。在高温下，Bob1被结合到热休克颗粒(HSGs)中，这是一种应激诱导的亚细胞结构，其功能和行为的特征尚不清楚。在活细胞中观察Bob1蛋白将有助于更多地了解HSGs的功能和组成。为了确定与Bob1相互作用的蛋白质和基因，将使用生物化学和遗传学方法。Bob1将被亲和纯化，相互作用的蛋白质将用质谱学鉴定。相互作用的蛋白质预计包括伴侣底物和HSG蛋白，这些相互作用的蛋白质将被表征，以了解Bob1发挥作用的细胞环境。为了确定与Bob1相互作用的基因和遗传途径，将在Bob1-3突变背景下进行敏化筛选。使用这些方法确定的蛋白质和基因将有助于从机制上理解Bob1的功能。这将加强对nudC伴侣功能的理解，更广泛地说，将为蛋白质折叠的细胞机制提供新的见解。 与公共健康相关：该项目涉及确定BOBBER1蛋白如何使植物在高温下存活，以及确保植物正常生长和发育。BOBBER1是一种分子伴侣，可以帮助其他蛋白质正确折叠和发挥功能，在包括人类在内的大多数生物体中都发现了类似BOBBER1的蛋白质。蛋白质错误折叠是许多人类疾病的特征，因此了解BOBBER1的功能将为这一重要的生物过程提供有价值的见解。

DESCRIPTION (provided by applicant): A wide range of important human diseases are characterized by protein misfolding. In the cell, protein chaperones ensure correct folding of newly synthesized proteins as well as the refolding of stress-denatured proteins. It follows that a full understanding of protein misfolding diseases depends on understanding chaperone mechanisms. The goal of this project is to understand the biological functions of BOBBER1 (BOB1), a recently identified Arabidopsis protein chaperone. BOB1 is the first chaperone of its kind with roles in both development and in temperature responses suggesting that it has important and unique functions. BOB1 contains an evolutionarily conserved NudC protein domain. Because NudC domain chaperones like BOB1 are found in many organisms including humans, understanding how BOB1 functions will contribute to a fundamental understanding of cellular mechanisms of protein quality control. This proposal involves investigating 1) how changes in BOB1 protein structure affect its function, and 2) identifying other proteins and genes which interact with BOB1. The approach for understanding BOB1 function involves using a series of mutants to systematically determine the effects of each mutation on organismal thermotolerance, on chaperone activity, and on BOB1s sub-cellular localization. At high temperatures BOB1 is incorporated into heat shock granules (HSGs), stress-induced sub-cellular structures whose function and behavior are poorly characterized. Observation of BOB1 protein in living cells will be used to learn more about the function and composition of HSGs. In order to identify proteins and genes which interact with BOB1 biochemical and genetic approaches will be used. BOB1 will be affinity purified and interacting proteins will be identified using mass spectrometry. Interacting proteins are predicted to include chaperone substrates and HSG proteins, and these interactors will be characterized in order to understand the cellular context in which BOB1 functions. To identify genes and genetic pathways that interact with BOB1 a sensitized screen in a bob1-3 mutant background will be performed. The proteins and genes identified using these approaches will contribute to a mechanistic understanding of BOB1 function. This will enhance understanding of NudC chaperone functions, and more broadly will provide novel insights into cellular mechanisms of protein folding. PUBLIC HEALTH RELEVANCE: This project involves determining how the BOBBER1 protein enables plants to survive at high temperatures as well as ensuring that plants grow and develop normally. BOBBER1 is a molecular chaperone which helps other proteins fold and function correctly and BOBBER1-like proteins are found in most organisms including humans. Protein misfolding is a characteristic feature of many human diseases, so understanding how BOBBER1 functions will provide valuable insights into this important biological process.