RNF11, a novel E3 ubiquitin ligase associated with PD pathogenesis

描述(申请人提供)：为了阐明导致帕金森病内含物形成和诱导多巴胺细胞死亡的机制，我们建议研究一种新的蛋白质，环指蛋白11(RNF11)及其与与帕金森病发病相关的环境毒素的相互作用。目的：检测(A)RNF11是否为E3泛素连接酶，(B)RNF11是否是多巴胺能神经元表达的膜相关蛋白。原理：环状结构域对于识别E2泛素结合酶和特定底物的泛素化是必不可少的。RNF11在多巴胺能神经元中的表达将提示其E3泛素连接酶的活性部位及其在帕金森病中的作用。方法：我们将检测RNF11的自素化特性，以及它与其他蛋白质的相互作用，使用谷胱甘肽转移酶下拉试验来确定其底物/培养细胞中的S。我们将研究RNF11在正常大鼠和人脑中的区域、细胞和亚细胞分布，方法是(A)在光镜和电子显微镜水平上进行免疫细胞化学，以及(B)亚细胞分离方法来确定其活性部位。目的：验证RNF11具有神经保护功能的假说，环境毒素对RNF11的氧化修饰(S)将损害其神经保护功能。理论基础：作为蛋白酶体降解途径的一个组成部分，RNF11将促进或促进蛋白质的清除，并提高细胞活力。暴露于环境毒素和RNF11的氧化修饰可能会损害RNF11的神经保护剂S的作用。方法：在细胞培养系统中，我们将研究(A)RNF11对蛋白酶体活性和细胞存活的作用，(I)暴露于氧化剂和(II)包括突变型RNF11在内的遗传变化(B)RNF11‘S亚细胞分布和E3连接酶活性的结构改变以及对细胞存活率的影响。目的：验证RNF11在帕金森病患者中的神经保护功能因RNF11功能缺失而受损的假说。理论基础：RNF11的修饰或RNF11在胞浆内的隔离将降低功能性RNF11的水平，并损害其神经保护功能。方法：我们将检测(A)帕金森病动物模型，即大鼠鱼藤酮模型和a-突触核蛋白转基因小鼠的RNF11的变化，以及(B)帕金森病患者尸检脑组织中RNF11的变化。这项研究将确定RNF11的作用以及它如何提供保护，防止帕金森病中出现的多巴胺细胞死亡。

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.