RNF11, a novel E3 ubiquitin ligase associated with PD pathogenesis

描述（由申请人提供）：为了描述在PD中导致包涵体形成和诱导多巴胺细胞死亡的机制，我们建议检查一种新的蛋白质，环指蛋白11（RNF 11）及其与PD发病机制相关的环境毒素的相互作用。目的1：验证RNF 11是一种E3泛素连接酶，RNF 11是多巴胺能神经元表达的膜相关蛋白。RING结构域对于E2泛素结合酶的识别和特异性底物的泛素化是必需的。RNF 11在多巴胺能神经元中的表达将提示其E3泛素连接酶活性的位点及其在PD中的作用。方法：我们将研究RNF 11的autoubiquitination特性，以及它与其他蛋白质的相互作用，使用GST下拉测定法来确定其在培养细胞中的底物。我们将通过使用（a）光镜和电镜水平的免疫细胞化学和（B）亚细胞分级方案来确定其活性位点，来检查正常大鼠和人脑中RNF 11的区域、细胞和亚细胞分布。目标二：为了检验RNF 11具有神经保护功能和暴露于环境毒素导致的RNF 11的氧化修饰将损害其神经保护功能的假设。原理：作为蛋白酶体降解途径的一个组成部分，RNF 11将促进或促进蛋白质的清除并增加细胞活力。暴露于环境毒素和RNF 11的氧化修饰可能会损害RNF 11作为神经保护剂的作用。方法：在细胞培养系统中，我们将研究（A）RNF 11对蛋白酶体活性和细胞存活的作用，（i）暴露于氧化剂和（ii）遗传改变，包括突变型RNF 11（B）RNF 11的亚细胞分布和E3连接酶活性的结构修饰对细胞活力的影响。目的3：验证在PD中由于功能性RNF 11的耗尽而损害RNF 11的神经保护功能的假设。基本原理：RNF 11的修饰或RNF 11在细胞质内含物中的隔离将降低功能性RNF 11的水平并损害其神经保护功能。方法：我们将检查（a）PD动物模型中RNF 11的变化，在大鼠和α-突触核蛋白转基因小鼠中的鱼藤酮模型和（B）来自PD患者的尸检脑组织中。这项研究将确定RNF 11的作用，以及它如何保护帕金森病中的多巴胺细胞死亡。

DESCRIPTION (provided by applicant): In an effort to delineate the mechanisms that cause the formation of inclusions and induce dopamine cell death in PD, we propose to examine a novel protein, RING-Finger Protein 11 (RNF11) and its interactions with environmental toxins associated with PD pathogenesis. Aim 1: To test that (a) RNF11 is an E3 ubiquitin ligase and (b) that RNF11 is a membrane-associated protein expressed in dopaminergic neurons. Rationale: The RING-domain is essential for recognition of E2 ubiquitin-conjugating enzyme and ubiquitination of spec- ific substrates. Expression of RNF11 in dopaminergic neurons will be suggestive of its site of E3 ubiquitin ligase activity and its role in PD. Approach: We will examine the autoubiquitination properties of RNF11, and its interaction with other proteins using GST pull down assays to determine its substrate/s in cultured cells. We will examine regional, cellular and subcellular distribution of RNF11 in normal rat and human brain by using (a) immunocytochemistry at light and electron microscope levels and (b) sub-cellular fractionation protocols to determine its site of activity. Aim 2: To test the hypothesis that RNF11 has a neuroprotective function and oxidative modification(s) of RNF11 as a result of exposure to environmental toxins will compromise its neuroprotective function. Rationale: As a component of the proteasomal degradation pathway, RNF11 will facilitate or promote clearance of proteins and increase cell viability. Exposure to environmental toxins and oxidative modifications of RNF11 could impair RNF11's role as a neuroprotectant. Approach: In cell culture systems we will examine (A) the role of RNF11 on proteasomal activity and cell survival following (i) exposure to oxidizing agents and (ii) genetic alterations including mutant RNF11 (B) Effects of structural modifications of RNF11 's subcellular distribution and E3 ligase activity and on cell viability. Aim 3: To test the hypothesis that neuroprotective function of RNF11 is compromised in PD due to depletion of functional RNF11. Rationale: Modifications of RNF11 or sequestration of RNF11 in cytoplasmic inclusions will reduce the levels of functional RNF11 and compromise its neuroprotective function. Approach: We will examine changes in RNF11 in (a) animal models of PD ie., rotenone model in rats and a-synuclein transgenic mice and (b) in autopsy brain tissue from PD patients. This study will determine a role for RNF11 and how it confers protection against dopamine cell death seen in Parkinson's disease.