Structural Insights into Leucine Transport for mTORC1 Activation

点击翻译按钮获取中文摘要

Project Summary/Abstract The mechanistic target of rapamycin complex 1 (mTORC1) pathway is a gatekeeper that balances anabolic and catabolic processes through sensing nutrients. Dysregulations of this pathway lead to debilitating diseases such as cancer. One of the most abundant essential amino acids in cancer cells is leucine. However, the process of leucine sensing and transport to activate mTORC1 pathway is not clear. The lysosomal associated transmembrane protein (LAPTM4b) is an oncoprotein that is involved in localizing leucine transporter to the lysosomal surface to activate mTORC1 pathway. LAPTM4b also functions as a ceramide transporter and compartmentalizes ceramide to help cancer cells evade apoptosis. Currently, there is no high-resolution structure of this protein to elucidate its mechanism of ceramide transport and complex formation with the leucine transporter. It is paramount to explore the structure and functions of LAPTM4b in detail since more than ~70% of cancers have LAPTM4b upregulation and LAPTM4b has been linked to poor prognosis. This proposal focuses on understanding the structure and functions of LAPTM4b and its protein complexes to lay the foundation on mTORC1 activation through leucine. The structures will be solved using state-of-the-art cryogenic electron microscopy techniques including Microcrystal Electron Diffraction (MicroED) and Single Particle Analysis (SPA). In Aim 1, the mechanism of ceramide binding in LAPTM4b will be elucidated by high resolution structures of LAPTM4b in complex with ceramide using MicroED. The structures solved in this aim will provide insights on the effect of ceramide to the global confirmation of LAPTM4b and provide basis on structure guided drug development. Functional studies of LAPTM4b in complex with the leucine transporter in liposome assays will be investigated in Aim 2. Kinetics of leucine transporter will be evaluated in vitro to provide insights into how LAPTM4b, ceramide, and ceramide analogues affect leucine transport. In Aim 3, the structure of the protein complex of LAPTM4b and the leucine transporter will be solved to understand their interactions and guide drug development to disrupt this complex for mTORC1 inactivation. The applicant’s career goal is to use tools in structural biology combined with biochemistry experiments to answer scientific questions on disease-related proteins. Since there is an urgent public health need to alleviate cancer, the applicant aims to study challenging membrane proteins involved in the mTORC1 pathway. The structure- function relationship of these proteins will guide future research on designing effective cancer therapeutics to disrupt this pathway. The proposed research is an integral step of the applicant’s career path. The applicant will receive intensive training and constructive guidance under a leading researcher in the structural biology field.