Investigating the role of AAA+-ATPases in peroxisome biology

研究 AAA -ATP 酶在过氧化物酶体生物学中的作用

• 批准号: 10001560
• 负责人: Brooke Meghan Gardner
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10001560
• 关键词: ATP phosphohydrolase; Adaptor Signaling Protein; Architecture; Autophagocytosis; Bile Acids; Biochemical; Biochemical Genetics; Biochemistry; Biogenesis; Biological Assay; Biology; Brain; Cells; Complex; Cryoelectron Microscopy; Crystallization; Cytosol; Defect; Development; Disease; Dissection; Electron Microscopy; Encapsulated; Endoplasmic Reticulum; Environment; Enzymes; Eukaryota; Fluorescence Microscopy; Functional disorder; Genes; Goals; Growth; Health; Homeostasis; Human; Hydrogen Peroxide; In Vitro; Knowledge; Lipids; Liver Dysfunction; Longevity; Maintenance; Mass Spectrum Analysis; Membrane; Membrane Biology; Membrane Protein Traffic; Membrane Proteins; Mentors; Mentorship; Metabolic; Metabolism; Microscopy; Mitochondria; Modeling; Molecular; Motor; Mutate; Mutation; Myelin Sheath; Nerve Degeneration; Organelles; PHEX protein; Phenotype; Play; Process; Protein Import; Proteins; Quality Control; Reaction; Regulation; Research; Resources; Role; SNAP receptor; Structure; Syndrome; System; Techniques; Testing; Therapeutic Intervention; Training; Translating; Very Long Chain Fatty Acid; developmental disease; disease-causing mutation; effective therapy; fascinate; high throughput screening; improved; novel; novel therapeutic intervention; novel therapeutics; peroxisome; peroxisome membrane; programs; receptor; reconstitution; response; skills; trafficking

Project Summary: Peroxisomes are ubiquitous, membrane-bound organelles that encapsulate specialized metabolic reactions, typically including those that produce hydrogen peroxide as a byproduct. In humans, where peroxisomes breakdown very long chain fatty acids and synthesize precursors of bile acids and myelin sheath lipids, defects in peroxisomes cause Peroxisome Biogenesis Disorders (PBDs), characterized by neuronal degeneration, liver dysfunction, and decreased lifespan. There are currently no treatments for PBDs, and our understanding of peroxisomes in human health is hindered by our limited understanding of the biochemical mechanisms of peroxisome molecular membrane biology. De novo biogenesis of peroxisomes requires approximately 30 dedicated Pex proteins. During this process, the peroxisome membrane and membrane proteins traffic through the endoplasmic reticulum, while the matrix proteins are imported fully folded from the cytosol. The majority of PBDs are caused by mutations in Pex1 and Pex6, two AAA+-ATPase motor proteins that perform an uncharacterized task crucial for peroxisome matrix protein import. A complete understanding of Pex1 and Pex6 architecture, substrates, processing mechanism, and function at the peroxisome would reveal new mechanistic details of peroxisome formation, novel therapeutic strategies, and expand our understanding of the role of peroxisomes in disease. As a Miller Fellow in Dr. Andreas Martin's lab, I used in vitro biochemistry and electron microscopy to determine the architecture of the active Pex1/Pex6 complex and its interaction and regulation by its membrane tether protein Pex15. This represents the first structural and biochemical characterization of Pex1/Pex6 and a unique molecular handle to dissect the energy-dependent steps of peroxisome assembly. In this proposal I will expand my research to understand the function of Pex1/Pex6 in the context of the cell and peroxisome dysfunction in the context of cellular homeostasis. In Aim 1, with the mentorship of Dr. Martin, an expert on AAA+-ATPase mechanisms, I propose to identify the substrates of Pex1/Pex6 and determine their processing mechanism. In Aim 2, with mentorship from Dr. Schekman, an expert in biochemical dissections of membrane trafficking, I will determine Pex1/Pex6 function in peroxisome matrix protein import using a novel cell-free reconstitution. Finally, I propose to determine the cellular response to induced peroxisome dysfunction and identify novel proteins required for peroxisome maintenance. With the support of my mentors and the greater research environment at UC Berkeley, I will receive training in mass spectrometry, cryo-electron microscopy, cell-free reconstitutions, fluorescence microscopy, and high throughput screening. These skills will help me bridge my background in biochemistry with my fascination with organelle biology to build a successful independent research program investigating the biochemical mechanisms of peroxisome biology.

项目概述：过氧化物酶体是普遍存在的，膜结合的细胞器， 代谢反应，通常包括产生过氧化氢作为副产物的那些。在人类中， 在那里过氧化物酶体分解长链脂肪酸并合成胆汁酸和髓磷脂的前体 鞘脂质，过氧化物酶体的缺陷导致过氧化物酶体生物发生障碍（PBD），其特征在于 神经元变性、肝功能障碍和寿命缩短。目前还没有治疗PBD的方法， 我们对过氧化物酶体在人类健康中的理解受到我们对 过氧化物酶体分子膜生物学的生化机制。 过氧化物酶体的从头生物发生需要大约30种专用的Pex蛋白。在此 在这一过程中，过氧化物酶体膜和膜蛋白通过内质网运输， 基质蛋白从胞质溶胶中完全折叠输入。大多数PBD是由以下基因突变引起的： Pex 1和Pex 6，两种AAA+-ATP酶马达蛋白，执行对过氧化物酶体至关重要的未知任务 基质蛋白输入。全面了解Pex 1和Pex 6架构、基板和工艺 机制，和功能的过氧化物酶体将揭示新的机制的细节过氧化物酶体形成， 新的治疗策略，并扩大我们对过氧化物酶体在疾病中的作用的理解。 作为安德烈亚斯·马丁博士实验室的米勒研究员，我使用体外生物化学和电子显微镜， 确定活性Pex 1/Pex 6复合物的结构及其膜的相互作用和调节 系链蛋白Pex 15。这代表了Pex 1/Pex 6的第一个结构和生化特征， 独特的分子手柄来剖析过氧化物酶体组装的能量依赖步骤。在这份提案中，我将 扩展我的研究，以了解Pex 1/Pex 6在细胞和过氧化物酶体中的功能 在细胞内稳态的背景下的功能障碍。在目标1中，在马丁博士的指导下， AAA+-ATP酶机制，我建议确定Pex 1/Pex 6的底物，并确定其加工 机制在目标2中，在膜生化解剖专家Schekman博士的指导下 贩运，我将确定Pex 1/Pex 6功能过氧化物酶体基质蛋白进口使用新的无细胞 重组最后，我建议确定诱导过氧化物酶体功能障碍的细胞反应， 鉴定过氧化物酶体维持所需的新蛋白质。 在我的导师和加州大学伯克利分校更大的研究环境的支持下，我将获得 质谱、冷冻电子显微镜、无细胞重组、荧光显微镜、 和高通量筛选。这些技能将帮助我把我的生物化学背景和我的 迷恋细胞器生物学，建立一个成功的独立研究计划，调查 过氧化物酶体生物学的生化机制。