Structural Insights into Leucine Transport for mTORC1 Activation

mTORC1 激活亮氨酸转运的结构见解

• 批准号: 10607075
• 负责人: Ampon Sae Her
• 金额: $ 6.95万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607075
• 关键词: Affect; Amino Acid Transporter; Antineoplastic Agents; Apoptosis; Binding; Biochemical; Biochemistry; Biological Assay; Career Choice; Carrier Proteins; Catabolic Process; Cell Membrane Proteins; Cell Proliferation; Cells; Ceramides; Complex; Cryoelectron Microscopy; Data; Disease; Drug Design; Environment; Essential Amino Acids; FRAP1 gene; Foundations; Gatekeeping; Goals; In Vitro; Integral Membrane Protein; Kinetics; Lead; Leucine; Link; Lipids; Liposomes; Lysosomes; Malignant Neoplasms; Measures; Membrane; Membrane Proteins; Molecular; Molecular Conformation; Molecular Probes; Mutation; Neoplasm Metastasis; Oncoproteins; Outcome; Pathway interactions; Persons; Phosphorylation; Process; Prognosis; Protein Isoforms; Proteins; Public Health; Regulation; Research; Research Personnel; Resolution; Resources; Signal Transduction; Structure; Structure-Activity Relationship; Surface; Techniques; Therapeutic; Training; Up-Regulation; Work; analog; cancer cell; cancer drug resistance; career; design; detection of nutrient; drug development; electron diffraction; experimental study; inhibitor; insight; interest; neoplastic cell; overexpression; particle; prevent; protein complex; protein protein interaction; protein structure; proteoliposomes; reconstitution; recruit; structural biology; tool; training opportunity; uncontrolled cell growth; uptake

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.

项目概要/摘要 雷帕霉素复合物 1 (mTORC1) 通路的机制靶标是平衡合成代谢和代谢的看门人。 通过感知营养物质的分解代谢过程会导致诸如此类的衰弱性疾病。 作为癌症，癌细胞中最丰富的必需氨基酸之一是亮氨酸。 亮氨酸传感和转运激活mTORC1途径的相关性尚不清楚。 跨膜蛋白 (LAPTM4b) 是一种癌蛋白，参与将亮氨酸转运蛋白定位到 溶酶体表面激活 mTORC1 通路，也起到神经酰胺转运蛋白的作用。 分隔神经酰胺以帮助癌细胞逃避细胞凋亡目前还没有高分辨率的方法。 该蛋白质的结构，以阐明其神经酰胺转运和与亮氨酸形成复合物的机制 详细探索 LAPTM4b 的结构和功能至关重要，因为超过 ~70% 的癌症存在 LAPTM4b 上调，并且 LAPTM4b 与不良预后相关。 该提案的重点是了解 LAPTM4b 及其蛋白质复合物的结构和功能，以 为通过亮氨酸激活 mTORC1 奠定基础 这些结构将使用最先进的技术来解析。 低温电子显微镜技术，包括微晶电子衍射 (MicroED) 和单次电子衍射 在目标 1 中，将通过高通量阐明 LAPTM4b 中神经酰胺结合的机制。 使用 MicroED 解析 LAPTM4b 与神经酰胺复合物的结构。 将为 LAPTM4b 的全球确认提供关于神经酰胺影响的见解，并提供基础 LAPTM4b 与亮氨酸转运蛋白复合物的结构引导药物开发。 目标 2 将研究脂质体测定。将在体外评估亮氨酸转运蛋白的动力学，以提供 深入了解 LAPTM4b、神经酰胺和神经酰胺类似物如何影响亮氨酸转运 在目标 3 中，结构。 将解析 LAPTM4b 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该复合物以实现 mTORC1 失活。 申请人的职业目标是利用结构生物学的工具结合生物化学实验来回答 关于疾病相关蛋白质的科学问题由于缓解癌症是公共卫生的迫切需要， 申请人旨在研究 mTORC1 通路中具有挑战性的膜蛋白。 这些蛋白质的功能关系将指导未来设计有效癌症疗法的研究 所提议的研究是申请人职业道路上不可或缺的一步。 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。