Structural Insights into Leucine Transport for mTORC1 Activation

项目总结/摘要 雷帕霉素复合物1（mTORC 1）途径的机制靶点是平衡合成代谢和代谢的看门人。 通过感知营养物质来分解代谢过程。这一途径的失调导致衰弱性疾病， 癌症癌细胞中最丰富的必需氨基酸之一是亮氨酸。然而， 亮氨酸传感和转运激活mTORC 1途径尚不清楚。溶酶体相关的 跨膜蛋白（LAPTM 4 b）是一种癌蛋白，参与亮氨酸转运蛋白定位于 溶酶体表面激活mTORC 1通路。LAPTM 4 b也作为神经酰胺转运蛋白发挥作用， 将神经酰胺区室化以帮助癌细胞逃避凋亡。目前还没有高分辨率的 这种蛋白质的结构，以阐明其机制的神经酰胺运输和复杂的形成与亮氨酸 传送器详细探索LAPTM 4 b的结构和功能是至关重要的，因为超过70%的LAPTM 4 b蛋白是由LAPTM 4 b基因编码的。 的癌症具有LAPTM 4 b上调，并且LAPTM 4 b与不良预后有关。 该提案的重点是了解LAPTM 4 b及其蛋白质复合物的结构和功能， 为通过亮氨酸激活mTORC 1奠定基础。这些结构将使用最先进的 低温电子显微镜技术，包括微晶电子衍射（MicroED）和单 颗粒分析（SPA）。在目的1中，将通过高表达来阐明LAPTM 4 b中神经酰胺结合的机制。 使用MicroED解析与神经酰胺复合的LAPTM 4 b的结构。在此目的中解决的结构 将为LAPTM 4 b的全球确认提供关于神经酰胺作用的见解，并为 结构导向药物开发。LAPTM 4 b与亮氨酸转运蛋白复合物的功能研究 脂质体测定将在目标2中研究。将在体外评价亮氨酸转运蛋白的动力学，以提供 深入了解LAPTM 4 b、神经酰胺和神经酰胺类似物如何影响亮氨酸转运。目标3：结构 将解决LAPTM 4 b和亮氨酸转运蛋白的蛋白质复合物，以了解它们的相互作用 并指导药物开发以破坏这种复合物使mTORC 1失活。 申请人的职业目标是利用结构生物学中的工具结合生物化学实验来回答 疾病相关蛋白质的科学问题。由于缓解癌症是一项紧迫的公共卫生需求， 本申请人旨在研究涉及mTORC 1途径的挑战性膜蛋白。结构- 这些蛋白质的功能关系将指导未来设计有效的癌症治疗方法的研究， 破坏这条通道。所提议的研究是申请人职业道路上不可或缺的一步。申请人将 在结构生物学领域的领先研究人员的指导下接受强化培训和建设性指导。

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.