Study of mTOR Regulation by Rheb Using Deuterium Exchange Mass Spectrometry

描述(申请人提供)：雷帕霉素的哺乳动物靶点(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；该通路中的基因改变导致良性肿瘤和恶性肿瘤的发生。Rheb是Ras家族小分子GTP酶家族中的一员，它直接与mTOR催化结构域相互作用，不依赖于Rheb的核苷酸负载状态。当Rheb从不活跃的GDP结合状态改变为活跃的GTP结合状态时，其Switch I区发生构象变化，即结合mTOR的区域。我们假设Rheb GTP负载诱导了mTOR的构象变化，从而导致了激酶的激活。MTOR的催化活性也可能受到C末端催化结构域两侧多个特征不佳的结构域之间的相互作用的调节。氢交换质谱学(DXMS)包括蛋白质主链酰胺氢的氢/氢交换，然后快速和灵敏地定位氢化残基。这是一种研究蛋白质结构域结构和动力学的有效方法，因为蛋白质的酰胺质子的交换率与它们的溶剂可及性成正比，交换率的变化反映了蛋白质构象的动态变化和/或蛋白质结合作用的变化。我们建议使用DXMS来：(I)定位Rheb和mTOR之间的相互作用界面，并探测Rheb-GTP诱导的mTOR的构象变化；以及(Ii)确定mTOR C-末端区域之间的边界，并研究它们之间的相互作用和在调节激酶活性中的功能。DXMS鉴定的Rheb/mTOR界面和结构域间接触中涉及的特定残基将通过定点突变得到确认；突变对mTOR构象、Rheb相互作用和激酶活性的影响将被确定。这些研究将有助于更好地理解mTOR活性的调控机制，并为未来mTOR的结晶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.