Targeting of Proteins into Peroxisomes

将蛋白质靶向过氧化物酶体

• 批准号: 10246358
• 负责人: Suresh Subramani
• 金额: $ 51.2万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-20 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10246358
• 关键词: ATP phosphohydrolase; Antiviral Agents; Autophagocytosis; Biochemical; Biogenesis; Cellular Membrane; Cytosol; Disease; Docking; Endoplasmic Reticulum; Firefly Luciferases; Funding; Future; Genes; Genetic; Glean; Glutathione; Grant; Growth; Health; Homeostasis; Human; Human Development; Impairment; In Vitro; Intracellular Membranes; Investigation; Knowledge; Mammalian Cell; Mammals; Membrane; Membrane Proteins; Metabolism; Mitochondria; Nitrogen; Organelles; Organism; Oxidation-Reduction; Oxygen; Peripheral; Physiological; Pichia; Plants; Play; Population; Protein Import; Proteins; Quality Control; Reaction; Receptor Signaling; Recycling; Regulation; Reporting; Role; Saccharomyces cerevisiae; Signal Transduction; Site; Sorting - Cell Movement; Stress; Suggestion; System; Techniques; Tertiary Protein Structure; Ubiquitin; Vesicle; Work; Yeast Model System; Yeasts; base; fatty acid oxidation; genetic approach; innovation; insight; interest; man; novel; peroxisome; receptor

Peroxisomes are essential subcellular organelles that play crucial roles in the oxidation of fatty-acids and homeostasis of glutathione, as well as reactive oxygen and nitrogen species (ROS and RNS, respectively). They play critical roles in the regulation of intracellular redox states and antiviral signaling, as well as cellular differentiation and metabolism, and their impairment causes many debilitating, and often fatal, human peroxisome biogenesis disorders (PBDs). Their biogenesis is orchestrated by 36 PEX genes, encoding peroxins, involved in the biogenesis of peroxisomal membrane and matrix proteins, as well as in the control of organelle size, number and inheritance. Their biogenesis has been studied in many organisms from yeast to plants and mammals, and more than 15 peroxins and their modes of action are conserved from yeast to man. While much has been learned about the biogenesis of peroxisomal matrix and membrane proteins to pre-existing peroxisomes, far less is known about how this organelle is generated de novo from other endogenous membranes. Such an ability to generate new peroxisomes de novo is obviously relevant under conditions where peroxisome biogenesis is impaired (e.g. human PBDs), or under conditions of stress (e.g. ROS) when peroxisomes are turned over by autophagy (pexophagy). Indeed, any disorders associated with imbalanced peroxisome homeostasis can be corrected, in principle, by manipulating either peroxisome biogenesis or its turnover, as we have shown. Such a global understanding of the mechanisms involved in peroxisome homeostasis is the long-term interest of my lab. Over almost 3 decades, we exploited the yeast, Pichia pastoris, to provide many major insights into our knowledge of peroxisome biogenesis and turnover. This proposal focuses on gleaning a deeper understanding of the proteins involved in the intra-ER sorting and budding of peroxisomal membrane proteins (PMPs) to a pre- peroxisomal exit site on the ER (pER) from where at least two type of pre-peroxisomal vesicles (ppVs) bud to ultimately generate peroxisomes, either by fusion with pre-existing peroxisomes or anew when peroxisomes are absent. Budding of ppVs is conserved between yeast and mammals and several proteins we will study have counterparts involved in human health. There are also reports of ppVs derived from mitochondria contributing to peroxisome biogenesis. Our approach is based on the use of novel genetic and biochemical strategies, including ppV purification and characterization, in vitro budding reactions and the use of innovative techniques to follow what these novel proteins do, where they act, who they interact with and how they function. The Aims are: Aim 1 - Isolation and characterization of the ATPase/s and other proteins involved in ppV budding. Aim 2 – How do Pex25 and Pex36 act in stimulating intra-ER sorting and budding of Pex2 and other RING- domain peroxins? Aim 3 – Does ppV budding occur from yeast mitochondria and what is its physiological relevance?!

过氧酶体是必需的亚细胞细胞器，在脂肪酸和 谷胱甘肽以及活性氧和氮物种(分别为ROS和RNS)的动态平衡。他们 在细胞内氧化还原状态的调节和抗病毒信号的调节中起关键作用 分化和新陈代谢，以及它们的损害，导致许多衰弱的，往往是致命的人类 过氧化物酶体生物发生障碍(PBDS)。它们的生物发生是由36个PEX基因协调的，编码过氧化物素， 参与过氧化物膜和基质蛋白的生物发生，以及细胞器的控制 大小、数量和遗传。它们的生物发生已经在许多生物中进行了研究，从酵母到植物和 从酵母到人类，15种以上的过氧化物素及其作用方式都是保守的。虽然有很多 已了解到过氧化物体基质和膜蛋白的生物发生 过氧酶体，关于这种细胞器是如何从其他内源 膜。这种从头产生新的过氧化物酶体的能力显然与以下条件有关 过氧化物酶体生物发生受损(例如，人的PBD)，或在应激条件下(例如，ROS) 过氧化物体通过自噬(吞噬)来翻转。事实上，任何与失衡相关的疾病 原则上，可以通过控制过氧酶体的生物发生或其自身的 营业额，正如我们已经展示的那样。这样一种对过氧化酶体所涉及的机制的全球理解 动态平衡是我实验室的长期利益所在。 在近30年的时间里，我们利用毕赤酵母为我们提供了许多重要的见解 关于过氧化物体生物发生和周转的知识。这项建议的重点是收集更深层次的理解 过氧化物体膜蛋白(PMPs)在内质网分选和萌发过程中所涉及的蛋白质 内质网(PER)上的过氧体出口部位(PER)，至少两种类型的过氧体前囊泡(PPV)从哪里萌芽到 最终产生过氧酶体，要么通过与先前存在的过氧酶体融合，要么在过氧酶体 缺席。PPV的发芽在酵母和哺乳动物之间是保守的，我们将研究的几种蛋白质 与人类健康有关的同行。也有报道称，来自线粒体的PPV有助于 过氧化物酶体的生物发生。我们的方法是基于使用新的遗传和生化策略，包括 PPV的纯化和表征、体外萌发反应和创新技术的使用 这些新的蛋白质做什么，它们在哪里起作用，它们与谁相互作用，以及它们是如何发挥作用的。目标是： 目的1-腺苷三磷酸酶/S及其他与细小病毒萌发相关的蛋白的分离与鉴定。 目的2-Pex25和Pex36如何在刺激Pex2和其他环的内质网内分选和萌发中起作用- 域过氧化物素？ 目标3-PPV发芽是否发生在酵母线粒体中，其生理相关性是什么？！