Single-Molecule Dissection of mTOR Complexes

描述(申请人提供)：哺乳动物的雷帕霉素靶标(MTOR)信号网络已经成为细胞和发育生物学中基本过程的主要调节因子，包括细胞生长、增殖、细胞分化和新陈代谢。FDA批准的雷帕霉素的多个临床应用证明了mTOR在医学上的核心重要性。最近的证据还显示，除了影响健康寿命外，mTOR信号在调节寿命方面也发挥了关键作用。MTOR调控网络的分子解剖将促进针对衰老和衰老相关疾病的治疗药物的开发，包括心血管疾病、代谢性疾病和癌症。MTOR组装了两个生化和功能不同的蛋白质复合体-mTORC1和mTORC2，它们形成了不同的途径，但却存在广泛的串扰。目前关于mTOR复合体组装的知识很大程度上来自于传统的生化表征，这通常需要漫长而繁琐的纯化过程，这可能会扰乱天然的复合体，并对复合体形成的化学计量和异质性提供有限的见解。我们(Ha实验室)最近开发了一种单分子下拉(SiMPull)方法，可以直接从全细胞裂解液中以单一络合物的分辨率快速而灵敏地分析蛋白质复合体。在这里，我们建议使用SiMPul方法剖析mTORC1和mTORC2的组装。将确定络合物中每个组分的化学计量比。还将研究上游信号对复合体的调节，以及两个复合体之间的潜在物理串扰。此外，我们将开发一种基于SiMPull平台的单分子激酶分析方法，并将这种新方法应用于询问各种mTOR复合体的活性。这项拟议的研究有望在单复合体水平上破译mTORC1和mTORC2的组装，并揭示mTOR组装中的结构-功能关系。从这项研究中获得的知识对于指导未来对mTOR调节网络的研究以及促进潜在的新的治疗策略的设计将是非常宝贵的。

DESCRIPTION (provided by applicant): The mammalian target of rapamycin (mTOR) signaling network has emerged as a master regulator of essential processes in cell and developmental biology, including cell growth, proliferation, cellular differentiation, and metabolism. The multiple FDA-approved clinical applications of rapamycin attest to the central importance of mTOR in medicine. Recent evidence has also revealed a key role for mTOR signaling in modulating lifespan in addition to impacting health span. Molecular dissection of the mTOR regulatory network will facilitate development of therapeutics against aging and aging-related diseases, including cardiovascular diseases, metabolic diseases, and cancer. mTOR assembles two biochemically and functionally distinct protein complexes - mTORC1 and mTORC2, which nucleate distinct pathways and yet crosstalk extensively. The current knowledge of mTOR complex assembly is largely derived from conventional biochemical characterizations, which typically require lengthy and strenuous purification procedures that may disrupt native complexes, and offer limited insights into the stoichiometry and heterogeneity of complex formation. We (the Ha laboratory) have recently developed a single-molecule pull-down (SiMPull) method that enables rapid and sensitive analysis of protein complexes at single-complex resolution, directly from whole cell lysates. Here we propose to dissect the assembly of mTORC1 and mTORC2 employing the SiMPul approach. Stoichiometry of each component in the complexes will be determined. Regulation of the complexes by upstream signals, and potential physical crosstalk between the two complexes, will also be examined. Furthermore, we will develop a single-molecule kinase assay with the SiMPull platform, and apply this novel approach to interrogating the activities of various mTOR complexes. The proposed study is expected to decipher the assembly of both mTORC1 and mTORC2 at the single-complex level and reveal structure-function relationships in the mTOR assemblies. Knowledge to be gained from this study will be invaluable in guiding future investigation of the mTOR regulatory network and in facilitating potentially novel design of therapeutic strategies.