Targeting of Proteins into Peroxisomes

过氧化物酶体是重要的亚细胞器，在脂肪酸的氧化中起关键作用， 谷胱甘肽的稳态，以及活性氧和氮物种（ROS和RNS，分别）。他们 在调节细胞内氧化还原状态和抗病毒信号传导以及细胞内 分化和代谢，以及他们的损害造成许多衰弱，往往是致命的，人类 过氧化物酶体生物发生障碍（PBD）。它们的生物发生由36个PEX基因协调，编码过氧化物酶， 参与过氧化物酶体膜和基质蛋白的生物发生，以及细胞器的控制 大小、数量和继承。它们的生物发生已经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种过氧化物及其作用方式是保守的。 已经了解到过氧化物酶体基质和膜蛋白的生物发生， 过氧化物酶体，远不知道这个细胞器是如何产生从头从其他内源性 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： 过氧化物酶体生物合成受损（例如人PBD），或在应激条件下（例如ROS）， 过氧化物酶体通过自噬（pexophagy）被翻转。事实上，任何与不平衡有关的疾病 原则上，可以通过操纵过氧化物酶体生物发生或其代谢产物来纠正过氧化物酶体的稳态。 正如我们所展示的那样，营业额。这种对过氧化物酶体机制的全面理解 自我平衡是我实验室的长期目标 在近30年的时间里，我们利用酵母，巴斯德毕赤酵母，提供了许多重要的见解， 过氧化物酶体生物发生和周转知识。这项建议的重点是收集更深入的了解 的蛋白质参与内质网内分选和出芽的过氧化物酶体膜蛋白（PMP）的前， ER上的过氧化物酶体出口位点（pER），至少有两种类型的前过氧化物酶体囊泡（PPV）从此处出芽， 最终产生过氧化物酶体，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 无托叶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 参与人类健康的同行。也有来自线粒体的ppV有助于 过氧化物酶体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，体外出芽反应和使用创新技术，以遵循 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如何发挥作用。目标是： 目的1 -分离和表征参与PPV出芽的ATPase/s和其他蛋白。 目的2 -Pex 25和Pex 36如何在刺激ER内分选和Pex 2和其他RING的出芽中起作用。 域过氧化物酶？ 目的3 -PPV出芽是否发生在酵母线粒体中，其生理相关性是什么？！

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?!