Single-Molecule Dissection of mTOR Complexes

描述（由申请人提供）：哺乳动物雷帕霉素靶蛋白（mTOR）信号传导网络已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、细胞分化和代谢。雷帕霉素的多次FDA批准的临床应用证明了mTOR在医学中的核心重要性。最近的证据也揭示了mTOR信号除了影响健康寿命外，还在调节寿命方面发挥着关键作用。mTOR调控网络的分子解剖将促进针对衰老和衰老相关疾病（包括心血管疾病、代谢疾病和癌症）的治疗剂的开发。 mTOR组装两种生物化学和功能不同的蛋白质复合物-mTORC 1和mTORC 2，其成核不同的通路，但广泛地串扰。目前mTOR复合物组装的知识主要来源于常规的生物化学表征，其通常需要可能破坏天然复合物的冗长且费力的纯化程序，并且对复合物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，该方法能够直接从全细胞裂解物中以单复合物分辨率快速灵敏地分析蛋白复合物。在这里，我们建议解剖的组装mTORC 1和mTORC 2采用SiMPul的方法。将确定复合物中各组分的化学计量。还将检查上游信号对复合物的调节以及两个复合物之间的潜在物理串扰。此外，我们将开发一个单分子激酶测定与SiMPull平台，并应用这种新的方法来询问各种mTOR复合物的活性。 这项研究有望在单复合体水平上破译mTORC 1和mTORC 2的组装，并揭示mTOR组装体中的结构-功能关系。从这项研究中获得的知识将是非常宝贵的，在指导未来的mTOR调控网络的调查，并在促进潜在的新的治疗策略的设计。

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.