Investigating the Import Mechanism of Folded Proteins into Peroxisomes

过氧化体是真核细胞中普遍存在的细胞器，负责脂肪酸、乙醚类脂和某些类固醇的氧化。与线粒体一起，这些细胞器将有毒的活性氧物种，如过氧化氢和超氧阴离子，与细胞的其余部分隔开，以防止与包括癌症在内的许多疾病相关的蛋白质和核酸受到损害。为了支持这些功能，过氧体必须识别和运输100多种不同的蛋白质通过过氧体膜。最近的研究表明，与其他几个以未折叠状态输入蛋白质的细胞器不同，过氧化物体能够输入折叠的蛋白质，甚至是寡聚蛋白质。尽管已有近20种蛋白质参与了这一过程，但其机制细节仍不清楚。此外，关于这一途径的许多基本方面还没有确定，包括受体与过氧化物体膜对接时的相互作用，易位通道的识别，泛素化对受体循环的调节，以及过氧化体的受体输出。因此，我们建议对这一过程中确定的三个关键蛋白质复合体进行生化分析，以阐明折叠的货物蛋白和寡聚体靶向并通过过氧化体膜运输的方式。 这项建议的具体目的是：(1)表征过氧化体输入对接复合体及其与货物蛋白和受体的相互作用；(2)分离可能的泛素连接酶复合体，并研究泛素化在受体循环中的作用；(3)阐明参与过氧体受体输出的AAA ATPase的膜募集机制。 这项研究解决了细胞生物学中的一个中心问题，并具有关键的医学意义，因为这一输入途径几乎每一步的缺陷都与包括Zellweger综合征和婴儿Refsum病在内的过氧化体生物发生障碍有关。这项工作的长期目标是开发一种过氧体蛋白质进口的体外系统，该系统将能够精确地确定在蛋白质低聚体进口过程中，过氧酶体如何保持其膜的完整性，而不会泄漏可能导致细胞损伤和人类疾病的危险的活性氧物种。

Peroxisomes are ubiquitous organelles in eukaryotic cells and are responsible for the oxidation of fatty acids, ether lipids, and certain steroids. Along with mitochondria, these organelles segregate toxic reactive oxygen species, such as hydrogen peroxide and superoxide anions, from the rest of the cell to prevent damage to proteins and nucleic acids associated with many diseases, including cancer. To support these functions, the peroxisome must recognize and transport over one hundred distinct proteins across the peroxisomal membrane. Recent studies have established that in contrast to several other cellular organelles, which import proteins in an unfolded state, peroxisomes are capable of importing folded and even oligomeric proteins. Although nearly twenty proteins have been implicated in this process, the mechanistic details of import are still unclear. In addition, many fundamental aspects regarding this pathway have yet to be defined, including the interactions of the receptor with the peroxisomal membrane upon docking, the identity of the translocation channel, the regulation of receptor cycling by ubiquitination, and receptor export from the peroxisome. Therefore, we propose to undertake a biochemical analysis of three critical protein complexes identified in this process in order to elucidate the means by which folded cargo proteins and oligomers are targeted to and transported across the peroxisomal membrane. The specific aims of this proposal are: (1) to characterize the peroxisomal import docking complex and its interactions with the cargo protein and receptor; (2) to isolate the putative ubiquitin ligase complex and investigate the role of ubiquitination in receptor recycling; (3) to elucidate the mechanism of membrane recruitment of the AAA ATPases implicated in receptor export from the peroxisome. This research addresses a central question in cellular biology and is of critical medical relevance, as defects in nearly every step of this import pathway have been linked to the peroxisomal biogenesis disorders including Zellweger syndrome and infantile Refsum's disease. The long-term goal of this work is to develop an in vitro system of peroxisomal protein import, which will allow for the precise determination of how the peroxisome maintains the integrity of its membrane during the import of protein oligomers without leaking dangerous reactive oxygen species that can lead to cellular damage and human disease.