Biochemical Characterization of Parkin

描述(由申请人提供)：Parkin编码基因的突变被认为与常染色体隐性遗传性青少年帕金森综合征(AR-JP)有关。Parkin是一种多结构域蛋白，编码泛素蛋白(E3)连接酶，泛素化蛋白酶体介导的降解底物，据报道在未折叠蛋白的降解中发挥特殊作用。编码α-突触核蛋白(a-Syn)和UCH-LI的基因突变也与帕金森病(PD)有关，并为这种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α-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。 Parkin含有结合泛素结合酶(E2)的环状结构域。然而，目前尚不清楚为什么帕金包含两个这样的序列。Parkin还含有一个氨基末端泛素样结构域，可能与26S蛋白酶体相互作用。如果UBL域提供了与26S蛋白酶体相互作用的一般机制，它将预测包括Parkin在内的许多包含UBL域的蛋白质的新的蛋白分解功能。 α-突触核蛋白表现出自结合的倾向，并在神经细胞中形成不可溶的聚集体。这些沉淀物可能导致帕金森病，类似的现象可能是其他神经退行性疾病的生化缺陷的基础。在这方面，重要的是α-Syn被Parkin泛素化并被蛋白酶体降解，这表明细胞内聚集体的形成与蛋白酶体介导的降解之间存在联系。 我们将利用我们在泛素领域的专业知识，通过解决以下关键问题来定义Parkin的生物学功能。i)。帕金和蛋白酶体之间的潜在相互作用将在体外和人类细胞中进行检测。Ii)。我们将确定Parkin中的两个环域是冗余的，还是起着独特的作用。三)。我们将确定E2酶UbcH7和UbcH8是否同时与Parkin结合，以调节底物连接的多Ub链的组装。四)。我们的长期目标是研究Parkin对α-Syn和其他生理底物的泛素化作用。

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.