RNF11, a novel E3 ubiquitin ligase associated with PD pathogenesis

RNF11，一种与 PD 发病机制相关的新型 E3 泛素连接酶

• 批准号: 8114974
• 负责人: Ranjita S Betarbet
• 金额: $ 33.07万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8114974
• 关键词: Animal Model; Autopsy; Biological Assay; Brain; Cell Culture System; Cell Death; Cell Survival; Corpus striatum structure; Cultured Cells; Cysteine; Cytoplasmic Inclusion; Degradation Pathway; Dopamine; Electron Microscope; Environmental Exposure; Environmental Risk Factor; Experimental Animal Model; Exposure to; Fingers; Fractionation; Genetic; Human; Hydrogen Peroxide; Lewy Bodies; Light Microscope; Membrane Proteins; Modeling; Modification; Mutation; N-terminal; Nerve Degeneration; Neurons; Neuroprotective Agents; Oxidants; Oxidative Stress; Paraquat; Parkinson Disease; Pathogenesis; Patients; Play; Predisposition; Property; Proteins; Protocols documentation; Rattus; Resistance; Role; Rotenone; Site; Solubility; Substantia nigra structure; Testing; Tissues; Toxic Environmental Substances; Transgenic Mice; Ubiquitin-Conjugating Enzymes; Ubiquitination; alpha synuclein; brain tissue; dopaminergic neuron; human RBX1 protein; immunocytochemistry; insight; mitochondrial dysfunction; mutant; myristoylation; novel; parkin gene/protein; synuclein; ubiquitin-protein ligase

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.

描述（由申请人提供）：为了描述导致夹杂物形成并诱导PD中多巴胺细胞死亡的机制，我们建议检查一种新型蛋白质，环形蛋白11（RNF11）及其与与PD病原体相关的环境毒素的相互作用。目的1：测试（a）RNF11是E3泛素连接酶，（b）RNF11是在多巴胺能神经元中表达的膜相关蛋白。理由：环域对于识别E2泛素偶联酶和泛素化酶是必不可少的。 RNF11在多巴胺能神经元中的表达将暗示其E3泛素连接酶活性及其在PD中的作用。方法：我们将检查RNF11的自夸脱特性，及其与其他蛋白质的相互作用，使用GST下拉测定法确定其在培养细胞中的底物。我们将通过（a）在光和电子显微镜水平上使用（a）免疫细胞化学，以及（b）亚细胞分级分级方案来确定其活性位点，通过（a）使用（a）使用（a）通过（a）使用（a）来检查正常大鼠和人脑的RNF11的区域，细胞和亚细胞分布。目的2：测试RNF11具有神经保护功能和RNF11的氧化修饰的假设，这会导致环境毒素暴露会损害其神经保护功能。理由：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清除并增加细胞活力。暴露于环境毒素和RNF11的氧化修饰可能会损害RNF11作为神经保护剂的作用。方法：在细胞培养系统中，我们将检查（a）RNF11对（i）暴露于氧化剂后的蛋白酶体活性和细胞存活的作用，以及（ii）遗传改变，包括突变体RNF11（b）RNF11亚细胞亚细胞分布和E3 Ligase分布和E3 ligase和细胞的可依性的作用。目的3：检验以下假设：RNF11的神经保护功能由于功能RNF11的耗竭而在PD中受到损害。基本原理：RNF11的修改或胞质夹杂物中RNF11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：我们将检查PD IE的（A）动物模型，大鼠和A-突触核蛋白转基因小鼠的RNF11的变化以及PD患者的尸检脑组织中的A-Synclein转基因小鼠。这项研究将确定RNF11的作用，以及它如何赋予帕金森氏病中多巴胺细胞死亡的保护。