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

描述（由申请人提供）：许多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力变性蛋白质的重新折叠。因此，对蛋白质错误折叠疾病的全面理解取决于对伴侣蛋白机制的理解。本项目的目的是了解BOBBER1 （BOB1）的生物学功能，BOB1是最近发现的拟南芥蛋白伴侣。BOB1是第一个在发育和温度反应中都有作用的伴侣，这表明它具有重要而独特的功能。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中，了解BOB1的功能将有助于对蛋白质质量控制的细胞机制有一个基本的了解。本课题包括研究1)BOB1蛋白结构的变化如何影响其功能，2)鉴定与BOB1相互作用的其他蛋白和基因。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种突变对机体耐热性、伴侣活性和BOB1亚细胞定位的影响。在高温下，BOB1被纳入热休克颗粒（hsg），应力诱导的亚细胞结构，其功能和行为的特征很差。在活细胞中观察BOB1蛋白将有助于进一步了解hsg的功能和组成。为了鉴定与BOB1相互作用的蛋白质和基因，将使用生化和遗传方法。BOB1将被亲和纯化，相互作用的蛋白将用质谱法鉴定。预计相互作用蛋白包括伴侣底物和HSG蛋白，这些相互作用蛋白将被表征，以了解BOB1功能的细胞环境。为了鉴定与BOB1相互作用的基因和遗传途径，将在BOB1 -3突变背景下进行增敏筛选。使用这些方法鉴定的蛋白质和基因将有助于对BOB1功能的机制理解。这将增强对NudC伴侣功能的理解，并更广泛地为蛋白质折叠的细胞机制提供新的见解。

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.