Targeting of Proteins into Peroxisomes

描述（由申请人提供）：我们的长期目标是了解过氧化物酶体（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）的稳态中涉及的生物发生和周转机制。过氧化物酶体生物合成障碍是许多致命的和使人衰弱的人类过氧化物酶体生物合成障碍（PBD）的根本原因。具有过氧化物酶体靶向信号的过氧化物酶体基质蛋白PTS 1或PTS 2分别被受体Pex 5和Pex 7识别。PTS 2途径也使用共受体。在以前的工作中，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，表明它们往返于过氧化物酶体。受体/共受体的再循环和降解依赖于单泛素化和多泛素化步骤，后一过程也涉及泛素蛋白酶体系统（UPS）。过氧化物酶体膜蛋白（PMP）的生物发生知之甚少，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）或可能两者被分选到过氧化物酶体膜是正在进行的争论。关于过氧化物酶体生物发生的生长和分裂与从头生物发生模型的相对贡献也有相当多的讨论。Pex 19被认为是许多PMPs翻译后插入过氧化物酶体膜的mPTS受体，但我们发现Pex 3和Pex 19在ER的前过氧化物酶体囊泡（PPV）的出芽中发挥新的作用。其他蛋白质，如Pex 3，Pex 16 L和Pex 25也在过氧化物酶体从头生物发生，分裂和遗传中发挥作用。本提案的目的1和2关注ER中过氧化物酶体的从头生物发生，研究四种蛋白质-Pex 3，Pex 16 L，Pex 19和Pex 25在此过程中的作用，包括两种过氧化物酶体膜裂变机制的组装，其以时空不同的方式发挥作用，导致ER中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。目标1询问Pex 19和Pex 3如何在PPV从ER出芽中发挥作用，重点关注PPV出芽中涉及的Pex 19结构域，测试关于Pex 3和Pex 19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。目标2测试特定的假设，Pex 16样（Pex 16 L）和Pex 25蛋白质的PPV出芽和/或过氧化物酶体分裂的作用，注意裂变机制的组成部分和它们的时间行动。Aim 3解决了PTS受体动力学及其通过代谢线索的调节，重点是Pex 7，其动力学迄今尚未研究。Aim 3还讨论了Pex 7和货物周转是否受UPS和代谢线索的调节。这些问题的答案不仅可以提供对这些过程的机械理解，还可以揭示人类疾病状态，因为我们关注的所有蛋白质在人类中都是保守的，在PBD中突变。

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.