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

“雷帕霉素靶标”（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。严格控制细胞生长对于细胞、器官和生物体来说是强制性的，以实现特征尺寸。细胞生长的失调可导致细胞、器官和生物体大小的病理变化，以及代谢紊乱、癌症和神经退行性疾病如阿尔茨海默病。TOR是一种模块化的约280 kDa的大蛋白。除了催化结构域之外，TOR还具有介导与其他细胞组分相互作用的其他功能区域。该项目的长期目标是TOR所有结构域的结构表征及其与调节剂和底物的相互作用。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表征，其解释了TOR在不同细胞膜（ER、高尔基体、线粒体、溶酶体、质膜）和细胞核中的定位。TOR的精确定位决定了哪些特定的细胞过程受到调节。在过去的时间里，我们的特点是氧化还原敏感，C-末端FATC结构域的膜结合特性，并已公布的部分数据。FKBP 12-雷帕霉素结合域（FRB）被认为介导脂质磷脂酸（PA）对TOR的调节。关于这一点，我们可以表明，FRB结构域经历强烈的构象变化，在存在和不存在调节PA的膜模拟物。为了更好地理解FRB结构域作为条件性膜锚的作用，应确定其在膜模拟物存在下的结构。此外，我们建立了几个结合伙伴（Rheb，LST 8，FKBP 38）和TOR上的相应结合区域的纯化方案，以及用于激酶结构域的结构表征，结构域间接触和与其他已知的复合物伙伴和调节剂的相互作用的各种构建体的表达系统。所有这些工作都是拟议继续执行该项目的基础。

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.