Peroxisomal fatty acid metabolism in genetic and age-related disorders

项目摘要/摘要 过氧化物酶体是真核生物中普遍存在的细胞器，是多种代谢途径的焦点。脂类 新陈代谢，特别是与之相关的脂肪酸运输，在很大程度上依赖于过氧酶体膜。 专门为此目的而进化的蛋白质。三磷酸腺苷依赖盒(ABC)转运蛋白 D‘亚家族’驻留在过氧化物体膜上，负责将脂肪酸输入到 过氧酶体，其过程中的缺陷与各种代谢紊乱有关。ABCD1基因突变导致 X连锁肾上腺脑白质营养不良(X-ALD)，表现为轻度至重度中枢神经系统(CNS) 脱髓鞘。ABCD3和/或过氧化物酶体生物发生因子(PEXs)功能障碍可能导致Zellweger 综合征(ZS)，一组不同种类的过氧化物体组装障碍。除了遗传性疾病， 过氧化体功能降低与衰老和年龄相关获得性疾病的发病机制有关，如 糖尿病，神经退行性疾病。认识ABCDs在新陈代谢中的作用的关键差距 疾病是由于缺乏结构细节，特别是在底物过程中它们的构象可塑性 运输，以及它是如何在衰老和疾病期间受到损害的。我的目标是了解 衰老和与年龄相关的神经退行性疾病中过氧化脂肪酸代谢的研究 阿尔茨海默氏症，并最终作为这一领域的独立研究员领导一个研究小组。 我建议在没有的情况下，用低温电子显微镜在原子水平上阐明ABCDs功能的结构基础 底物的存在，以及ATP(目标1a和1b，指导)，ATPase和硫代酯酶之间的偶联 活性以及脂肪酸结合和运输(目标2a和2b，指导)。我将测定原子结构 ABCDs与PEXs(Pex3和Pex19)的复合体使用CryoEM(Aim 3a，指导)并评估 超低温电子断层扫描的过氧化物体形态；ABCD(1-3)的功能和膜组成 击倒细胞系、衰老细胞和阿尔茨海默病(AD)模型细胞系(目标3b、3c，3D指导/ 独立阶段)。这些目标促使我长期致力于研究过氧化物酶的功能及其与 细胞生理学和疾病，同时在结构的前沿工具中获得培训和经验 生物物理学。为了实现这些目标，我得到了一支非常强大的导师、顾问和 顾问。我的导师罗伯特·斯特劳德教授是膜蛋白结构和机制的先驱，我的合作伙伴 彼得·沃尔特教授是未折叠蛋白反应(UPR)的先驱，专门研究细胞和 细胞器生物学。我的合作导师亚当·弗罗斯特博士走在了CryoEM/CryoET的前沿。艾瑞克顾问教授 韦尔丁是衰老研究领域的领军人物。顾问教授马丁·坎普曼正在发现新的分子参与者 在公元后。科学顾问程一凡教授开创了冷冻EM，Charles Craik教授开发了 以抗体和Fab文库为工具。总之，这项提议将利用他们在各自领域的专业知识， 同时利用我的热情通过结构透镜研究细胞器生物学。

Project Summary/Abstract Peroxisomes are ubiquitous eukaryotic organelles that form a focal point for multiple metabolic pathways. Lipid metabolism, and in particular, fatty acid transport related to it, depend heavily on peroxisomal membrane proteins that have specifically evolved for such purposes. The ATP-dependent cassette (ABC) transporters of ‘D’ subfamily’ reside in the peroxisomal membrane and are responsible for fatty acid import into the peroxisomes, defects in which process are related to various metabolic disorders. Mutations in ABCD1 cause X-linked adrenoleukodystrophy (X-ALD) which manifests as mild to severe central nervous system (CNS) demyelination. Dysfunction of ABCD3 and/or peroxisome biogenesis factors (PEXs) may cause Zellweger syndrome (ZS), a heterogeneous group of peroxisome assembly disorders. In addition to inherited diseases, reduced peroxisomal function is associated with aging and pathogenesis of age-related acquired diseases like diabetes, neurodegenerative disorders. Key gaps in understanding the function of ABCDs in metabolism and disease are due to lack of structural details, particularly of their conformational plasticity during substrate transport, and how this is compromised during aging and disease. My goal is to understand the involvement of peroxisomal fatty acid metabolism in aging and age-related neurodegenerative diseases, specifically Alzheimer’s disease and ultimately to lead a research group as an independent researcher in this area. I propose to elucidate the structural basis for function of ABCDs at atomic level using CryoEM in the absence and presence of substrate, and ATP (Aim 1a and 1b, mentored), coupling between ATPase and thioesterase activity, and fatty acid binding and transport (Aim 2a and 2b, mentored). I will determine atomic structures of ABCDs in complex with PEXs (Pex3 and Pex19) using CryoEM (Aim 3a, mentored) and assess alterations in peroxisomal morphology using Cryo-Electron Tomography; function and membrane composition of ABCD(1-3) knockdown cell lines, aged cells and Alzheimer’s disease (AD) model cell lines (Aim 3b, 3c, 3d mentored/ independent phase). These aims foster my long-term aim of studying peroxisome function and its relation to cellular physiology and disease, while obtaining training and experience in forefront tools of structural biophysics. For executing these aims, I have support of a very strong team of mentors, consultants and advisors. My mentor Prof. Robert Stroud is a pioneer in membrane protein structure and mechanism, my co- mentor Prof. Peter Walter is a pioneer in the unfolded protein response (UPR) and specializes in cell and organelle biology. My co-mentor Dr. Adam Frost is at the forefront of CryoEM/CryoET. Consultant Prof. Eric Verdin is a leading scientist in aging. Consultant Prof. Martin Kampmann is discovering new molecular players in AD. Scientific advisors Prof. Yifan Cheng pioneered CryoEM and Prof. Charles Craik has developed antibody and Fab libraries as tools. Together, this proposal will utilize their expertise in the respective fields, while leveraging my passion to study organellar biology through a structural lens.