Structure and function of the protein chaperone Tti2

蛋白伴侣 Tti2 的结构和功能

• 批准号: RGPIN-2015-04394
• 负责人: Brandl, Christopher
• 金额: $ 2.48万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=612939
• 关键词: Structure; function; protein; chaperone; Tti2

The folding of many proteins requires chaperones. Chaperones also untangle misfolded proteins or target them for degradation. Chaperones are particularly critical in the response to stress; thus global warming is making mechanisms to modulate their activity genetically or chemically increasingly valuable. Our studies focus on the TTT chaperone complex. The components of this complex, Tel2, Tti1 and Tti2, are essential in eukaryotes. Using genetic selections in S. cerevisiae we identified mutations in TTI2 that enhance the folding of a member of the PIKK (Phosphatidylinositol 3-kinase-related kinase) protein family. This effect results from partial loss of Tti2 function, leading us to hypothesize that Tti2 targets slowly folding proteins for degradation. Similar mutations are not found in TEL2 or TTI1, suggesting that Tti2's function may be unique. The goal of our research program is to determine the function of the components of the TTT-complex. An initial emphasis will be placed on Tti2, because of its potentially unique activities and since we have many of the key tools for its analysis. Our studies use yeast because the combination of experimental approaches yeast allow is ideal for solving fundamental biological problems. Specific objectives for this five-year period are: 1. Identify the protein interaction networks of the components of the TTT-complex. The functions of Tel2, Tti1 and Tti2 will be more clearly defined when their interacting partners are determined. We will determine the interaction partners of the three proteins using biochemical approaches. The significance of novel interactions will be examined by mapping the interaction surface through mutagenesis, then introducing the mutagenized alleles into cells. 2. Characterize the structure/function relationships of Tti2. Using unigenic evolution we have identified clusters of invariant residues in Tti2. These residues will be mutated and their phenotypic consequences determined by plasmid shuffling in a tti2 knockout strain we constructed. The effects of the mutations on Tti2 expression, localization, folding of the PIKK proteins, and protein-protein interactions will be examined. Alleles that reduce cell growth will be valuable tools for suppressor genetic studies that will guide experiments to determine the function of Tti2. 3. Identify the “client” proteins for Tti2. The importance of the TTT proteins in the folding of members of the PIKK family has been demonstrated. Whether other clients exist is unknown. We will search for proteins whose expression depends on Tti2 and Tel2 using conditional alleles and protein identification through stable isotope labeling with amino acids in cell culture. A second approach to identify both targets and interacting proteins will be through the BioID strategy. This method was designed for mammalian cells, so we will start by identifying targets of mammalian Tti2 in cell culture.

许多蛋白质的折叠需要分子伴侣。分子伴侣还可以解开错误折叠的蛋白质或将其降解。伴侣在应对压力方面尤其重要;因此，全球变暖使得调节其遗传或化学活性的机制越来越有价值。我们的研究集中在TTT伴侣复合物。该复合物的组分Tel 2、Tti 1和Tti 2在真核生物中是必需的。利用S.在酿酒酵母中，我们鉴定了TTI 2中增强PIKK（磷脂酰肌醇3-激酶相关激酶）蛋白家族成员折叠的突变。这种效应是由Tti 2功能的部分丧失引起的，这使我们假设Tti 2靶向缓慢折叠的蛋白质进行降解。在TEL 2或TTI 1中没有发现类似的突变，这表明Tti 2的功能可能是独特的。我们研究计划的目标是确定TTT复合物组分的功能。由于Tti 2具有潜在的独特活动，并且我们拥有许多用于分析的关键工具，因此最初的重点将放在Tti 2上。我们的研究使用酵母，因为酵母允许的实验方法的组合是解决基本生物学问题的理想选择。这五年期间的具体目标是： 1.确定TTT复合物组分的蛋白质相互作用网络。Tti 2、Tti 1和Tti 2的功能将在它们的相互作用伙伴被确定后被更清楚地定义。我们将使用生物化学方法确定这三种蛋白质的相互作用伙伴。通过诱变绘制相互作用表面，然后将诱变的等位基因引入细胞，来检查新相互作用的意义。 2.描述Tti 2的结构/功能关系。使用单基因进化，我们已经确定了Tti 2不变的残基簇。这些残基将被突变，其表型结果通过我们构建的tti 2敲除菌株中的质粒改组来确定。将检查突变对Tti 2表达、定位、PIKK蛋白折叠和蛋白-蛋白相互作用的影响。降低细胞生长的等位基因将是抑制基因研究的有价值的工具，这将指导实验以确定Tti 2的功能。 3.鉴定Tti 2的“客户”蛋白。TTT蛋白在PIKK家族成员折叠中的重要性已得到证实。是否有其他客户端是未知的。我们将寻找其表达依赖于Tti 2和Tel 2的蛋白质，使用条件等位基因和蛋白质鉴定，通过稳定的同位素标记与细胞培养中的氨基酸。第二种方法来确定目标和相互作用的蛋白质将通过BioID策略。该方法是针对哺乳动物细胞设计的，因此我们将从鉴定细胞培养物中哺乳动物Tti 2的靶标开始。