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、醚类脂质和某些类固醇的氧化。与线粒体一起，这些细胞器从细胞的其余部分分离出有毒的活性氧，如过氧化氢和超氧阴离子，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，过氧化物酶体必须在过氧化物酶体膜上识别和运输一百多种不同的蛋白质。最近的研究已经证实，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低聚蛋白质。尽管有近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，转运通道的身份，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，我们建议对在这一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低聚物被靶向并通过过氧化物酶体膜运输的手段。

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.