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?!

过氧化物酶体是重要的亚细胞细胞器，在脂肪酸的氧化和 谷胱甘肽以及活性氧和氮（分别为 ROS 和 RNS）的稳态。他们 在细胞内氧化还原状态和抗病毒信号传导以及细胞 分化和新陈代谢及其损伤会导致人类许多衰弱甚至致命的疾病 过氧化物酶体生物发生障碍（PBD）。它们的生物起源由 36 个 PEX 基因精心策划，编码过氧化物酶， 参与过氧化物酶体膜和基质蛋白的生物合成，以及细胞器的控制 大小、数量和继承。它们的生物发生已在从酵母到植物的许多生物体中进行了研究 从酵母到人类，超过 15 种过氧化物酶及其作用方式是保守的。虽然很多 已经了解了过氧化物酶体基质和膜蛋白与预先存在的生物发生 过氧化物酶体，人们对这种细胞器如何从其他内源性从头产生的知之甚少 膜。这种从头产生新过氧化物酶体的能力显然与以下条件相关： 过氧化物酶体生物发生受损（例如人类PBD），或在应激条件下（例如ROS） 过氧化物酶体通过自噬（pexophagy）进行翻转。事实上，任何与失衡有关的疾病 原则上，过氧化物酶体稳态可以通过操纵过氧化物酶体生物发生或其 正如我们所展示的，营业额。对过氧化物酶体相关机制的全面了解 体内平衡是我实验室的长期兴趣。 近 3 年来，我们利用毕赤酵母为我们的酵母提供了许多重要的见解。 过氧化物酶体生物发生和周转的知识。该提案的重点是收集更深入的理解 参与内质网内分选和过氧化物酶体膜蛋白 (PMP) 出芽的蛋白质到预- 内质网 (pER) 上的过氧化物酶体出口位点，至少有两种类型的前过氧化物酶体囊泡 (ppV) 在此萌芽 最终生成过氧化物酶体，或者通过与预先存在的过氧化物酶体融合，或者当过氧化物酶体被重新生成时重新生成。 缺席的。 ppV 的出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具有 涉及人类健康的同行。还有报道称，源自线粒体的 ppV 有助于 过氧化物酶体生物发生。我们的方法基于使用新颖的遗传和生化策略，包括 ppV 纯化和表征、体外出芽反应以及后续创新技术的使用 这些新颖的蛋白质有什么作用、在哪里发挥作用、与谁相互作用以及它们如何发挥作用。目标是： 目标 1 - ATP 酶和其他参与 ppV 出芽的蛋白质的分离和表征。 目标 2 – Pex25 和 Pex36 如何作用于刺激 Pex2 和其他 RING 的内质网内排序和出芽 域过氧化物酶？ 目标 3 – ppV 出芽是否发生在酵母线粒体中？其生理相关性是什么？！