Study of mTOR Regulation by Rheb Using Deuterium Exchange Mass Spectrometry

描述（由申请人提供）：哺乳动物雷帕霉素靶点（mTOR）是一种高度保守的丝氨酸/苏氨酸蛋白激酶，控制细胞生长，是癌症治疗的重要药物靶点。mTOR是磷脂酰肌醇3-激酶(PI3K)/Akt/Rheb信号通路的一部分；这一途径的遗传改变导致良性肿瘤并导致恶性肿瘤。Rheb是小gtpase Ras家族的一员，直接与mTOR催化结构域相互作用，独立于Rheb的核苷酸负载状态。当Rheb从不活跃的gtp结合状态转变为活跃的gtp结合状态时，其开关I区（与mTOR结合的区域）发生构象变化。我们假设Rheb GTP负载诱导mTOR的构象变化导致激酶激活。mTOR的催化活性也可能受到c端催化结构域两侧多个特征不明确的结构域之间相互作用的调节。氘交换质谱法（DXMS）涉及蛋白质骨架酰胺氢的氢/氘交换，随后是氘化残基的快速和敏感定位。这是研究蛋白质结构域结构和动力学的有力方法，因为蛋白质酰胺质子的交换速率与其溶剂可及性成正比，交换速率的变化反映了蛋白质构象的动态变化和/或蛋白质结合相互作用引起的变化。我们建议使用DXMS:(i)绘制Rheb与mTOR之间的相互作用界面，并探测Rheb- gtp引起的mTOR构象变化；（ii）确定mTOR c端区域结构域之间的边界，研究它们在调节激酶活性中的相互作用和功能。参与Rheb/mTOR界面和DXMS鉴定的域间接触的特定残基将通过位点定向诱变确定；突变体对mTOR构象、Rheb相互作用和激酶活性的影响将被确定。这些研究将有助于进一步了解mTOR活性的调控机制，并为今后mTOR的晶体学和详细结构分析奠定基础。在这项工作中，首席调查员将得到几名高级调查员的协助，该奖项将使他能够过渡到独立的调查生涯。

DESCRIPTION (provided by applicant): The mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine protein kinase which controls cell growth, and is an important drug target for cancer therapy. mTOR is part of the phosphatidylinositol 3-kinase (PI3K)/Akt/Rheb signaling pathway; genetic alterations in this pathway lead to benign tumors and contribute to malignancy. Rheb, a member of the Ras family of small GTPases, directly interacts with the mTOR catalytic domain, independently of Rheb's nucleotide-loaded state. When Rheb changes from an inactive GDP-bound, to an active GTP-bound state, a conformational change occurs in its switch I region, the region which binds mTOR. We hypothesize that Rheb GTP loading induces a conformational change in mTOR leading to kinase activation. mTOR's catalytic activity may also be regulated by interactions between multiple, poorly characterized domains flanking the catalytic domain in the C-terminus. Deuterium exchange mass spectrometry (DXMS) involves hydrogen/deuterium exchange of protein backbone amide hydrogens, followed by rapid and sensitive localization of deuterated residues. It is a powerful method to study protein domain structure and dynamics, because exchange rates of a protein's amide protons are proportional to their solvent accessibility, and changes in exchange rates reflect dynamic changes in protein conformation and/or changes resulting from protein binding interactions. We propose using DXMS to: (i) map the interaction interface between Rheb and mTOR, and probe for Rheb-GTP-induced conformational changes in mTOR; and (ii) to determine the boundaries between domains in mTOR's C-terminal region, and study their interaction and function in regulating kinase activity. Specific residues involved in the Rheb/mTOR interface and in inter-domain contacts identified by DXMS will be confirmed by site-directed mutagenesis; the mutants' effects on mTOR conformation, Rheb interaction, and kinase activity will be determined. These studies will provide a better understanding of the mechanisms regulating mTOR activity, and lay the foundation for future crystallographic and detailed structural analyses of mTOR. During this work, the principal investigator will be assisted by several senior investigators, and the award will allow him to transition to an independent investigative career.