Targeting of Proteins into Peroxisomes

描述（由申请人提供）：我们的长期目标是了解与过氧化物酶体稳态相关的生物发生和周转机制，过氧化物酶体是一种重要的亚细胞细胞器，密切参与许多代谢途径，特别是脂质代谢。过氧化物酶体生物生成障碍是许多致命性和致残性人类过氧化物酶体生物生成障碍（PBDs）的根本原因。具有过氧化物酶体靶向信号PTS1或PTS2的过氧化物酶体基质蛋白分别被受体Pex5和Pex7识别。PTS2通路也使用共受体。在之前的工作中，我们和其他人证明了一些PTS受体/共受体在基质蛋白输入周期中的动态行为，表明它们穿梭于过氧化物酶体之间。受体/共受体的回收和降解依赖于单泛素化和多泛素化步骤，后者也参与泛素蛋白酶体系统（UPS）。过氧化物酶体膜蛋白（PMP）的生物发生机制尚不清楚，mpts作为过氧化物酶体膜靶向的信号。pmp是否仅通过其翻译后插入过氧化物酶体，或通过内质网（ER），或可能两者都被分类到过氧化物酶体膜上，目前仍在争论中。关于过氧化物酶体生物发生的生长和分裂与新生生物发生模型的相对贡献也有相当多的讨论。Pex19一直被认为是许多pmp在翻译后插入过氧化物酶体膜的mPTS受体，但我们发现了Pex3和Pex19在内质网的过氧化物酶体前囊泡（ppVs）出芽中的新作用。其他蛋白质，如Pex3、Pex16L和Pex25也在新生过氧化物酶体的生物发生、分裂和遗传中发挥作用。本提案的目的1和2聚焦于内质网过氧化物酶体的新生生物发生，研究了四种蛋白——Pex3、Pex16L、Pex19和Pex25在这一过程中的作用，包括两种过氧化物酶体膜裂变机制的组装，这两种机制以不同的时空方式起作用，导致内质网管状前过氧化物酶体的裂变，然后分裂成熟的过氧化物酶体。目的1探讨了Pex19和Pex3如何在内质网ppV的出芽过程中发挥作用，重点研究了参与ppV出芽的Pex19结构域，验证了关于Pex3和Pex19在两类ppV出芽和过氧化物酶体遗传中的功能的特定假设，以及所涉及的潜在机制。目的2测试了关于pex16样蛋白（Pex16L）和Pex25蛋白在ppV出芽和/或过氧化物酶体分裂中的作用的特定假设，并关注了裂变机制的组成部分及其时间作用。Aim3解决了PTS受体动力学及其代谢线索的调节，重点是Pex7，其动力学迄今尚未研究。Aim3还解决了Pex7和货物周转是否受到UPS和代谢线索的调节。这些问题的答案不仅将提供对这些过程的机制理解，而且还将揭示人类疾病状态，因为我们关注的所有蛋白质在人类中都是保守的，并且在pbd中发生突变。

DESCRIPTION (provided by applicant): Our long term goal is to understand both the biogenesis and turnover mechanisms involved in the homeostasis of peroxisomes, an essential subcellular organelle that is intimately involved in many metabolic pathways, particularly lipid metabolism. Impairment of peroxisome biogenesis is the underlying cause of many fatal and debilitating human peroxisome biogenesis disorders (PBDs). Peroxisomal matrix proteins with the peroxisomal targeting signals, PTS1 or PTS2, are recognized by receptors, Pex5 and Pex7, respectively. The PTS2 pathway also uses co-receptors. In previous work, we and others demonstrated the dynamic behavior of some PTS receptors/co-receptors during the matrix protein import cycle, showing that they shuttle to and from peroxisomes. Receptor/co-receptor recycling and degradation are dependent on mono- and poly-ubiquitination steps, with the latter process also engaging the ubiquitin proteasome system (UPS). Peroxisomal membrane protein (PMP) biogenesis is poorly understood, with mPTSs serving as signals for peroxisomal membrane targeting. Whether PMPs are sorted to peroxisome membranes solely by their post- translation insertion into peroxisomes, or via the endoplasmic reticulum (ER), or possibly both, are ongoing debates. There is also considerable discussion regarding the relative contributions of the growth and division versus de novo biogenesis models for peroxisome biogenesis. Pex19 has been viewed as the mPTS receptor for the post-translational insertion of many PMPs into peroxisome membranes, but we found new roles for Pex3 and Pex19 in the budding of pre-peroxisomal vesicles (ppVs) from the ER. Other proteins, such as Pex3, Pex16L and Pex25 also play a role in de novo peroxisome biogenesis, division and inheritance. Aims1 and 2 of this proposal focus on the de novo biogenesis of peroxisomes from the ER investigating the roles of four proteins - Pex3, Pex16L, Pex19 and Pex25 in this process, including the assembly of two peroxisome membrane fission machineries that function in a spatiotemporally distinct manner to cause fission of tubular pre-peroxisomes at the ER and later to divide mature peroxisomes. Aim1 asks how Pex19 and Pex3 perform a role in budding of ppVs from the ER, focusing on Pex19 domains involved in ppV budding, testing specific hypotheses regarding Pex3 and Pex19 functions in budding of two classes of ppVs and in peroxisome inheritance, as well as the underlying mechanisms involved. Aim2 tests specific hypotheses regarding the roles of Pex16-like (Pex16L) and Pex25 proteins in either ppV budding and/or peroxisome division, with attention on the components of the fission machineries and their temporal action. Aim3 addresses PTS receptor dynamics, and its regulation by metabolic cues, focusing on Pex7, whose dynamics has not been studied to date. Aim3 also addresses whether Pex7 and cargo turnover are regulated by the UPS and metabolic cues. Answers to these questions will not only provide a mechanistic understanding of these processes, but also shed light on human disease states because all the proteins we focus on are conserved in humans and mutated in PBDs.