Structural characterization of the protein kinase "target of rapamycin" and its interactions with regulators and substrates

雷帕霉素的靶标(TOR)是一种蛋白激酶，通过拦截不同的信号级联来调节细胞生长。严格控制细胞生长是细胞、器官和生物体达到特定大小所必需的。细胞生长的失调会导致细胞、器官和生物体大小的病理变化，并导致代谢障碍、癌症和神经退行性疾病，如阿尔茨海默氏症。TOR是一个约280 kDa的模块化蛋白质。除催化结构域外，TOR还具有其他功能区域，可调节与其他细胞成分的相互作用。该项目的长期目标是对TOR的所有结构域及其与调节剂和底物的相互作用进行结构表征。该项目中的一个重要问题是TOR-脂质和蛋白质相互作用的结构特征，这解释了TOR在不同细胞膜(内质网、高尔基体、线粒体、溶酶体、质膜)和细胞核中的定位。TOR的准确定位决定了哪些特定的细胞过程受到调控。在过去的一段时间里，我们表征了氧化还原敏感的C末端FATC结构域的膜结合特性，并已经发表了部分数据。FKBP12-雷帕霉素结合(FRB)结构域可介导脂质磷脂酸(PA)对TOR的调节。对此，我们可以表明，在膜模拟存在的情况下，FRB结构域在有和没有调节剂PA的情况下经历了强烈的构象变化。为了更好地了解FRB结构域作为条件膜锚的作用，必须确定其在膜模拟物存在下的结构。此外，我们还为几个结合伙伴(Rheb、LST8、FKBP38)和TOR上各自的结合区建立了纯化协议，并为各种结构建立了表达系统，用于表征激酶结构域的结构、结构域之间的接触以及与进一步已知的复杂伙伴和调节子的相互作用。所有这些工作都是拟议继续进行该项目的基础。

The "target of rapamycin" (TOR) is a protein kinase that regulates cellular growth by intercepting different signalling cascades. A stringent control of cell growth is mandatory for cells, organs, and organisms to achieve a characteristic size. Misregulation of cell growth can result in pathological changes of cell, organ, and organism size and in metabolic disorders, cancer and neurodegenerative diseases such Alzheimer's.TOR is a modular, about 280 kDa big protein. Besides a catalytic domain, TOR has additional functional regions that mediate interactions with other cellular components. The long-term goal of this project is the structural characterization of all domains of TOR and their interactions with regulators and substrates. An important issue within this project is the structural characterization of TOR-lipid and -protein interactions that account for the localization of TOR at different cellular membranes (ER, Golgi, mitochondria, lysosomes, plasma membrane) and in the nucleus. The exact localization of TOR determines which specific cellular processes are regulated. In the past time we characterized the membrane-binding properties of the redox-sensitive, C-terminal FATC domain and published already part of the data. The FKBP12-rapamycin binding (FRB) domain was suggested to mediate the regulation of TOR by the lipid phosphatidic acid (PA). Regarding this, we could show that the FRB domain undergoes strong conformational changes in the presence of membrane-mimetics with and without the regulator PA. To better understand the role of the FRB domain as conditional membrane anchor, its structure in the presence of membrane-mimetics shall be determined. In addition, we established purification protocols for several binding partners (Rheb, LST8, FKBP38) and the respective binding regions on TOR as well as expression systems for various constructs for the structural characterization of the kinase domain, interdomain contacts and interactions with further known complex partners and regulators. All of this work is the foundation for the proposed continuation of the project.