Structure and function of polyamine transporters

项目总结 多胺是许多基本分子和细胞所必需的一类小的有机聚阳离子。 包括翻译、电信号、细胞增殖和自噬等过程。感染性和 增生性疾病以及许多自身免疫性、心血管和神经退行性疾病 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 摄取和分泌具有改善人类健康的巨大潜力。然而，尽管做了几十年的工作，这 主题继续令人费解。深入了解多胺运输的一个关键障碍是完整的 没有任何多胺转运体的原子结构。这个项目的目标是阐明 多胺转运的基本原理及其结构和功能结合的调节 接近了。ATP13A2是一种溶酶体P型三磷酸腺苷驱动泵，任务是进口精胺和 从溶酶体腔到胞浆的亚精胺。削弱ATP13A2功能的突变会导致谱系 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、神经元蜡样脂褐素沉着症和肌萎缩侧索硬化症。因此，ATP13A2是一种 潜在的毒品目标。我们在初步研究中取得了重大进展，以确定高分辨率 人ATP13A2的三维结构。结合功能分析，这些结构 揭示了ATP13A2‘S腔门控和多胺选择性的作用机制。根据这些初步结果， 我们将利用补充的电子冷冻显微镜，生物物理，生化，分析化学和 突变策略，以进一步使ATP13A2受到详细的机械性审查。具体来说，我们的目标是 研究：1)脂质如何调节ATP13A2活性；2)ATP13A2是否以及如何将其他阳离子泵入细胞内 以及3)ATP13A2如何将多胺穿梭于脂双层。通过解决这些问题， 这一研究项目将为ATP13A2的基本运行和调控机制提供新的见解， 这将广泛地促进我们对多胺运输和溶酶体生理学的理解。结构和 拟议工作中的机械性发现可能会为未来治疗靶向的合理设计提供参考 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.