Molecular basis of fatty acid transport by peroxisomal ABC transporters

项目摘要 ABCD1、ABCD2和ABCD3等ABCD1、ABCD2和ABCD3等过氧体ABC转运体运送超长链脂肪酸(VLCFAs)， 支链脂肪酸(BCFAs)和胆汁酸前体形成过氧化体。他们的功能障碍导致 到由磷脂和脂肪酸紊乱引起的严重神经和代谢病理 代谢，包括X连锁肾上腺脑白质营养不良(X-ALD)，既是最常见的脑白质营养不良，也是 常见的由ABCD1基因突变引起的无法治愈的过氧素体疾病，以及胆汁酸 合成缺陷和由ABCD3功能受损引起的肝病。而其他角色则为abu zai 更广泛的疾病途径中的转运蛋白继续被发现，潜在的机制 管理它们的底物识别、运输和运输调节仍然知之甚少。长的- 该项目的学期目标是深入了解过氧化物酶体ABCD转运蛋白的功能和调控。 分子细节。我们将结合使用生化和细胞生物学工具，高分辨率结构 冷冻电子显微镜和连续波电子顺磁共振(CW-EPR)分析 揭示ABCD使用的功能相关结构特征和构象状态的光谱 脂肪酸易位中的转运体，X-ALD中ABCD1突变可能如何改变它们，以及ABCD1如何改变 与ABCD2和ABCD3在机制上是不同的，尽管它们的功能重叠。特定目标1涉及的是 测定底物专一性分布和转运的体外分析方法的发展和应用 ABCD1、ABCD2和ABCD3的属性。具体目标2涉及获得高分辨率结构 ABCD1、ABCD2和ABCD3在生理脂质环境中功能相关状态的信息 通过低温电子显微镜分析。具体目标3处理获取有关ABCD1结构动力学的信息 通过CW-EPR研究。我们的结果将为研究过氧素体ABC转运蛋白提供基本的见解 可用于ABCD1靶向诊断和治疗工具的功能，以改善X-ALD患者 结果，为设计和开发研究ABCD家族的化学探针提供了一个框架 功能，并在体外和硅胶中产生可靠的工具，以加速药物开发/发现工作 瞄准他们。

Project Summary Peroxisomal ABC transporters like ABCD1, ABCD2, and ABCD3 shuttle very long chain fatty acids (VLCFAs), branched chain fatty acids (BCFAs), and bile acid precursors into peroxisomes. Their functional impairment leads to severe neurological and metabolic pathologies stemming from disrupted phospholipid and fatty acid metabolism, including X-linked Adrenoleukodystrophy (X-ALD), both the most common leukodystrophy and most common peroxisomal disorder that is caused by mutations in ABCD1 and for which no cure exists, and bile acid synthesis defects and liver disease stemming from impaired ABCD3 function. While additional roles for ABCD transporters in a wider array of disease pathways continue to be uncovered, the underlying mechanisms governing their substrate recognition, transport, and transport regulation remain poorly understood. The long- term objectives of this project are to gain insight into peroxisomal ABCD transporter function and regulation in molecular detail. We will use a combination of biochemical and cell biological tools, high resolution structural analysis by cryo-electron microscopy, and continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy to reveal the functionally relevant structural features and conformational states used by ABCD transporters in fatty acid translocation, how they may be altered by ABCD1 mutation in X-ALD, and how ABCD1 is mechanistically distinct from ABCD2 and ABCD3 despite their functional overlap. Specific Aim 1 deals with the development and utilization of in vitro assays for determining substrate specificity profiles and transport properties of ABCD1, ABCD2, and ABCD3. Specific Aim 2 deals with obtaining high resolution structural information of ABCD1, ABCD2, and ABCD3 in functionally relevant states in a physiological lipid environment through cryo-EM analysis. Specific Aim 3 deals obtaining information on the structural dynamics of ABCD1 through CW-EPR studies. Our results will provide fundamental insights into peroxisomal ABC transporter functioning that can be exploited for ABCD1 targeted diagnostic and therapeutic tools to improve X-ALD patient outcomes, provide a framework for the design and development of chemical probes to study ABCD family function, and generate reliable in vitro and in silico tools to accelerate drug development/discovery efforts targeting them.