Peroxisomal fatty acid metabolism in genetic and age-related disorders

遗传和年龄相关疾病中的过氧化物酶体脂肪酸代谢

• 批准号: 10371815
• 负责人: Meghna Gupta
• 金额: $ 12.5万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10371815
• 关键词: 3-Dimensional; ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Acyl Coenzyme A; Adrenoleukodystrophy; Affect; Affinity; Aging; Alzheimer' s Disease; Alzheimer' s disease model; Antibodies; Area; Axon; Binding; Biochemical Reaction; Biogenesis; Biological Assay; Biology; Biophysics; Brain; Catalytic Domain; Cell Line; Cell membrane; Cell physiology; Cells; Central Nervous System Diseases; Complex; Confocal Microscopy; Coupled; Coupling; Cryo-electron tomography; Cryoelectron Microscopy; Cytoplasmic Receptors; Cytosol; Defect; Demyelinations; Diabetes Mellitus; Disease; Enzymes; Equilibrium; Ethers; Eukaryotic Cell; Event; Fatty Acids; Fostering; Functional disorder; Gene Deletion; Generations; Genetic; Goals; Heart; Hereditary Disease; Homeostasis; Human; Ions; Lead; Length; Libraries; Lipids; Liver; Mass Spectrum Analysis; Measures; Mediating; Medium chain fatty acid; Membrane; Membrane Proteins; Mentors; Metabolic Diseases; Metabolic Pathway; Metabolism; Mitochondria; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Structure; Morphology; Mutation; Myelin Sheath; NADH; Neuraxis; Neurodegenerative Disorders; Neuroglia; Neurons; Nucleotides; Organ; Organelles; Orthologous Gene; Oxidative Stress; Pancreas; Pathogenesis; Phase; Plants; Play; Process; Proteins; Protocols documentation; Reaction; Reactive Oxygen Species; Reporting; Research; Research Personnel; Resolution; Role; Scanning Electron Microscopy; Scientist; Side; Site; Specificity; Streptavidin; Structure; Sulfhydryl Compounds; Testing; Training; Zellweger Syndrome; age related; age related neurodegeneration; analog; base; bile salts; branched chain fatty acid; cell age; cryogenics; electron tomography; experience; fatty acid metabolism; fatty acid transport; gene repression; knock-down; lens; lipid metabolism; loss of function; member; mutant; oxidation; peroxisome; peroxisome lumen; peroxisome membrane; preservation; protein structure; response; tool; verdin photosensitizer

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.

项目总结/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。脂质 代谢，特别是与之相关的脂肪酸转运，在很大程度上依赖于过氧化物酶体膜 专门为此目的进化的蛋白质。ATP依赖性盒（ABC）转运蛋白 'D'亚家族'存在于过氧化物酶体膜中，负责脂肪酸输入到 过氧化物酶体，该过程中的缺陷与各种代谢紊乱有关。ABCD 1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ABCD 3和/或过氧化物酶体生物合成因子（PEXs）的功能障碍可能导致Zellweger 综合征（ZS），一组异质性过氧化物酶体组装障碍。除了遗传性疾病， 过氧化物酶体功能降低与衰老和年龄相关的获得性疾病的发病机制有关， 糖尿病神经退行性疾病在理解ABCD在代谢中的功能方面存在的主要差距， 疾病是由于缺乏结构细节，特别是在底物降解过程中它们的构象可塑性， 运输，以及在衰老和疾病期间这是如何受到损害的。我的目标是了解 过氧化物酶体脂肪酸代谢在衰老和与年龄相关的神经退行性疾病中的作用， 并最终以该领域独立研究员的身份领导一个研究小组。 我建议在缺乏的情况下，使用CryoEM在原子水平上阐明ABCD功能的结构基础。 底物和ATP（Aim 1a和1b，指导）的存在，ATP酶和硫酯酶之间的偶联 活性，脂肪酸结合和运输（目标2a和2b，指导）。我将确定 使用CryoEM（Aim 3a，指导）分析与PEX（Pex 3和Pex 19）复合的ABCD，并评估 过氧化物酶体形态学（冷冻电子断层扫描）; ABCD的功能和膜组成（1-3） 敲低细胞系、老化细胞和阿尔茨海默病（AD）模型细胞系（Aim 3b、3c、3d指导/ 独立阶段）。这些目标促进了我研究过氧化物酶体功能的长期目标， 细胞生理学和疾病，同时获得结构的前沿工具的培训和经验， 生物物理学为了实现这些目标，我得到了一个由导师、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， 导师Peter Walter教授是未折叠蛋白反应（UPR）的先驱，专门研究细胞和 细胞器生物学我的共同导师Adam Frost博士是CryoEM/CryoET的最前沿。顾问教授Eric Verdin是衰老领域的领先科学家。顾问教授马丁·坎普曼正在发现新的分子球员 在AD中。科学顾问Yifan Cheng教授开创了CryoEM，Charles Craik教授开发了 抗体和Fab文库作为工具。这项建议将利用他们在各自领域的专门知识， 同时利用我的热情通过结构透镜研究细胞器生物学。