Biochemical Characterization of Parkin

描述（由申请人提供）：编码帕金森氏症的基因突变与常染色体隐性遗传性少年帕金森氏症（AR-JP）有关。Parkin是一种多结构域蛋白，编码泛素蛋白（E3）连接酶，泛素化底物用于蛋白酶体介导的降解，据报道在未折叠蛋白的降解中起特定作用。编码α -突触核蛋白（a- syn）和UCH-Li的基因突变也有助于帕金森病（PD），并为该疾病的遗传基础提供了令人信服的证据。值得注意的是，泛素途径降解a- syn，而UCH-Li是一种切割泛素前体的神经元蛋白酶。因此，在这些蛋白（Parkin， a-Syn和UCH-LI）和泛素/蛋白酶体途径之间存在有趣的联系。

DESCRIPTION (provided by applicant): Mutations in the gene encoding Parkin have been implicated in autosomal recessive juvenile parkinsonism (AR-JP). Parkin is a multi-domain protein that encodes a ubiquitin protein (E3) ligase, which ubiquitinates substrates for proteasome-mediated degradation, and was reported to play a specific role in the degradation of unfolded proteins. Mutations in the genes encoding alpha-synuclein (a-Syn) and UCH-Li also contribute to Parkinson's disease (PD), and provide compelling evidence for a genetic basis for this disease. Remarkably, the ubiquitin pathway degrades a-Syn, while UCH-Li is a neuronal protease that cleaves ubiquitin precursors. Consequently, there is an intriguing link between these proteins (Parkin, a-Syn, and UCH-LI) and the ubiquitin/proteasome pathway. Parkin contains RiNG domains that bind ubiquitin-conjugating (E2) enzymes. However, it is not known why Parkin contains two such sequences. Parkin also contains an amino-terminal ubiquitin-like (UbL) domain that might interact with the 26S proteasome. If UbL domains provide a general mechanism for interacting with the 26S proteasome, it would predict novel proteolytic functions for many UbL-containing proteins, including Parkin. Alpha-synuclein displays a propensity for self-association and forms insoluble aggregates in neuronal cells. These precipitates may contribute to PD, and similar phenomena could underlie the biochemical defects in other neurodegenerative diseases. In this regard, it is significant that a-Syn is ubiquitinated by Parkin and degraded by the proteasome, suggesting a link between the formation of intracellular aggregates and proteasome-mediated degradation. We will use our expertise in the ubiquitin field to define the biological functions of Parkin, by addressing the following critical questions. i). A potential interaction between Parkin and the proteasome will be examined in vitro, and in human cells. ii). We will determine if the two RING domains in Parkin are redundant, or play unique roles. iii). We will determine if the E2 enzymes UbcH7 and UbcH8 bind Parkin simultaneously to regulate the assembly of substrate-linked multi-Ub chains. iv). Our long-term objective is to characterize the ubiquitination of a-Syn and other physiological substrates by Parkin.