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.

项目摘要/摘要 雷帕霉素复合物1（MTORC1）途径的机械靶标是平衡合成代谢和 通过感知营养的分解代谢过程。这种途径的失调导致令人衰弱的疾病 作为癌症。癌细胞中最丰富的必需氨基酸之一是亮氨酸。但是， 亮氨酸敏感性和激活MTORC1途径的运输尚不清楚。溶酶体相关 跨膜蛋白（LAPTM4B）是一种癌蛋白，涉及将亮氨酸转运蛋白定位于 溶酶体表面激活MTORC1途径。 LAPTM4B还用作神经酰胺转运蛋白和 分裂化神经酰胺以帮助癌细胞逃避凋亡。目前，没有高分辨率 该蛋白质的结构以阐明其神经酰胺的转运机理和与亮氨酸的复合形成 转运蛋白。详细探讨LAPTM4B的结构和功能至关重要，因为超过70％ 癌症中有LAPTM4B上调，LAPTM4B与预后不良有关。 该建议重点是理解LAPTM4B及其蛋白质复合物的结构和功能 通过亮氨酸为MTORC1激活奠定基础。结构将使用最新的 低温电子显微镜技术，包括微晶电子衍射（微晶）和单个 粒子分析（SPA）。在AIM 1中，LAPTM4B中神经酰胺结合的机理将被高 LAPTM4B的分辨率与神经酰胺在复合物中使用微型。在这个目标中解决的结构 将提供有关神经酰胺对LAPTM4B全球确认的影响的见解，并为 结构指导药物开发。 LAPTM4B的功能研究与亮氨酸转运蛋白在复合物中 脂质体测定将在AIM 2中研究。亮氨酸转运蛋白的动力学将在体外评估以提供 深入了解LAPTM4B，神经酰胺和神经酰胺类似物如何影响亮氨酸的运输。在AIM 3中，结构 LAPTM4B和亮氨酸转运蛋白的蛋白质复合物将被求解以了解其相互作用 并指导药物开发破坏了MTORC1失活的这种复合物。 申请人的职业目标是在结构生物学中使用工具与生物化学实验相结合来回答 有关疾病相关蛋白质的科学问题。由于紧急公共卫生需要减轻癌症，因此 适当的旨在研究MTORC1途径中涉及的膜蛋白的挑战。结构 - 这些蛋白质的功能关系将指导对设计有效癌症治疗的未来研究 破坏这一道路。拟议的研究是申请人职业道路不可或缺的一步。申请人将 在结构生物学领域的主要研究人员的领导下，接受密集的培训和建设性指导。