Investigating the Import Mechanism of Folded Proteins into Peroxisomes

过氧化物酶体是真核细胞中普遍存在的细胞器，负责脂肪酸、醚脂和某些类固醇的氧化。这些细胞器与线粒体一起将有毒的活性氧（例如过氧化氢和超氧阴离子）与细胞的其他部分隔离开来，以防止与许多疾病（包括癌症）相关的蛋白质和核酸受到损害。为了支持这些功能，过氧化物酶体必须识别并运输一百多种不同的蛋白质穿过过氧化物酶体膜。最近的研究表明，与其他几种以未折叠状态输入蛋白质的细胞器不同，过氧化物酶体能够输入折叠甚至寡聚的蛋白质。尽管近二十种蛋白质参与了这一过程，但其输入机制的细节仍不清楚。此外，有关该途径的许多基本方面尚未确定，包括对接时受体与过氧化物酶体膜的相互作用、易位通道的身份、泛素化对受体循环的调节以及过氧化物酶体的受体输出。因此，我们建议对该过程中鉴定的三种关键蛋白质复合物进行生化分析，以阐明折叠的货物蛋白和寡聚物靶向并跨过氧化物酶体膜运输的方式。 该提案的具体目标是：（1）表征过氧化物酶体输入对接复合物及其与货物蛋白和受体的相互作用； (2) 分离假定的泛素连接酶复合物并研究泛素化在受体再循环中的作用； (3) 阐明参与过氧化物酶体受体输出的 AAA ATP 酶的膜募集机制。 这项研究解决了细胞生物学的一个核心问题，具有重要的医学意义，因为这一输入途径的几乎每一步的缺陷都与过氧化物酶体生物发生障碍有关，包括齐薇格综合征和婴儿雷夫苏姆病。这项工作的长期目标是开发一种过氧化物酶体蛋白质输入的体外系统，该系统将允许精确测定过氧化物酶体在蛋白质低聚物输入过程中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活性氧。

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.