Structure and function of polyamine transporters

项目摘要 多胺是许多基本分子和细胞所必需的一类小分子有机聚阳离子 这些过程包括翻译、电信号、细胞增殖和自噬。传染病和 过度增殖性疾病以及许多自身免疫、心血管和神经变性疾病， 与多胺丰度的扰动密切相关。多胺转运在细胞内起着重要作用， 健康和异常细胞中的多胺稳态。了解多胺的分子基础 吸收和分泌具有改善人类健康的巨大潜力。然而，尽管经过几十年的努力， 主题继续神秘化。深入了解多胺转运的一个关键障碍是完整的 缺乏任何多胺转运蛋白的原子结构。这个项目的目标是阐明基本的 多胺转运及其调节的基本原理， 接近。ATP 13 A2是一种溶酶体P型ATP驱动的泵，其任务是输入精胺， 亚精胺从溶酶体腔到胞质溶胶。削弱ATP 13 A2功能的突变导致一系列 神经退行性疾病，包括Kufor-Rakeb综合征，早发性帕金森病，遗传性 痉挛性截瘫、神经元蜡样质脂褐质沉积症和肌萎缩侧索硬化症。ATP 13 A2是一种 潜在的药物靶点我们在初步研究中取得了重大进展， 人ATP 13 A2的三维结构。结合功能分析，这些结构 揭示了ATP 13 A2的腔门控和多胺选择性机制。在这些初步结果的基础上， 我们将利用互补的电子冷冻显微镜，生物物理，生物化学，分析化学和 诱变策略，以进一步使ATP 13 A2经受详细的机制审查。具体而言，我们的目标是 研究：1）脂质如何调节ATP 13 A2活性; 2）ATP 13 A2是否以及如何将其他阳离子泵入 溶酶体;和3）ATP 13 A2如何穿梭多胺通过脂质双层。通过解决这些问题， 这项研究项目将为ATP 13 A2的基本操作和调节机制提供新的见解， 这将广泛地推进我们对多胺转运和溶酶体生理学的理解。结构和 从拟议的工作中发现的机制可能会为未来合理设计治疗靶向药物提供信息。 ATP13A2。

PROJECT SUMMARY Polyamines are a class of small organic polycations indispensable for many basic molecular and cellular processes including translation, electrical signaling, cell proliferation, and autophagy. Infectious and hyperproliferative diseases as well as many autoimmune, cardiovascular and neurodegenerative disorders are deeply connected to perturbations in polyamine abundance. Polyamine transport plays a major role in cellular polyamine homeostasis in both healthy and abnormal cells. Understanding the molecular basis of polyamine uptake and secretion has enormous potential to improve human health. However, despite decades of work, this subject continues to mystify. A critical barrier to deeper knowledge in polyamine transport is the complete absence of atomic structures of any polyamine transporter. The goal of this project is to elucidate the fundamental principles underlying polyamine transport and its regulation using a combination of structural and functional approaches. ATP13A2 is a lysosomal P-type ATP-driven pump tasked with the import of spermine and spermidine from the lysosome lumen to the cytosol. Mutations that cripple ATP13A2 function causes a spectrum of neurodegenerative diseases including Kufor-Rakeb syndrome, early-onset Parkinson’s disease, hereditary spastic paraplegia, neuronal ceroid lipofuscinosis and amyotrophic lateral sclerosis. ATP13A2 is, thus, a potential drug target. We have made significant inroads in our preliminary studies to determine high-resolution three-dimensional structures of human ATP13A2. In combination with functional analysis, these structures revealed ATP13A2’s luminal gating and polyamine selectivity mechanisms. Building on these preliminary results, we will leverage complementary electron cryo-microscopy, biophysical, biochemical, analytical chemical and mutagenesis strategies to further subject ATP13A2 to detailed mechanistic scrutiny. Specifically, we aim to investigate: 1) how lipids regulate ATP13A2 activity; 2) whether and how ATP13A2 pumps other cations into the lysosome; and 3) how ATP13A2 shuttles polyamines through the lipid bilayer. By addressing these questions, this research project will provide new insights into the basic operating and regulatory mechanisms of ATP13A2, which will broadly advance our understanding of polyamine transport and lysosome physiology. Structural and mechanistic discoveries from the proposed work may inform future rational design of therapeutics targeting ATP13A2.