Genetic and Molecular Analyses of Protein Biogenesis in the Neuroretina

描述(申请人提供)：泛素-蛋白酶体系统(UPS)的失控是许多神经退行性疾病的一个显著特征，包括视网膜退行性疾病，这涉及到光感受器蛋白质的错误折叠和失控。我们的长期目标是了解UPS-伴侣机制的调节复合体在控制底物降解和折叠方面的作用，并利用这些信息确定新的治疗策略的靶点，目前这些策略对UPS的选择性生物学和疾病过程缺乏疗效。这一提议的目的是了解维持蛋白质周转和生物发生之间平衡的分子和细胞内机制，这两个紧密耦合的过程对视网膜神经元的功能和生存至关重要，并与人类各种神经视觉障碍的发病机制有关。这一目标的实现将使我们和其他人能够确定新的药理靶点并开发治疗策略，以操纵UPS-伴侣机制，防止在各种视网膜疾病状态下正常或突变蛋白表达的毒性放松调控，以及导致UPS-伴侣机制和神经视觉功能受损的衰老表现。这项拟议的研究建立在我们之前的研究结果的基础上，该研究表明，底物在合成时被呈递给蛋白酶体，并且蛋白酶体的活性在细胞中受到调节和划分。Ran结合蛋白2(RanBP2)通过两个不同的结构域，即亲环素(CLD)和亲环素(CY)结构域，是这一过程的关键控制者。这一建议验证了我们的总体假设，即RanBP2及其CLD和CY结构域控制着两个紧密耦合的过程，即选择性蛋白的UPS依赖的周转和生物发生，这对选择性视网膜神经元和视网膜色素上皮(RPE)细胞在正常和病理生理条件下的功能和生存至关重要。我们将通过实现以下特定目标来验证这一假说，这些目标集中在RanBP2及其CLD和CY结构域在调节蛋白质稳态和正常和疾病应激条件下选择性视网膜神经元存活方面的机制作用。目的1.验证RanBP2功能丧失在视网膜神经元中具有不同的生物学和病理效应的假说，其中一些可能对这些神经元的存活或蛋白质稳态是必不可少的。目的2.验证以下假设：选择性丧失RanBP2的CLD活性会上调UPS的选择性功能，而选择性功能对选择性视网膜蛋白的稳态、视网膜神经元和RPE细胞的功能和存活至关重要。目的3.验证RanBP2 CY结构域顺-反式丙氨酰异构酶(PPIase)活性控制选择性蛋白质的生物发生的假说，这些蛋白质对视网膜神经元和RPE细胞的功能或生存至关重要。 与公共健康相关：视网膜中蛋白质降解和生物发生的调节对其功能至关重要。这些过程的损害被认为是影响视网膜神经元的几种退行性和老年性疾病的基础。该提案旨在阐明Ran结合蛋白2在视网膜蛋白质动态平衡和相关病理生理状态中的作用。

DESCRIPTION (provided by applicant): The deregulation of the ubiquitin-proteasome system (UPS) is a hallmark feature of many neurodegenerative disorders, including retinal degenerations, which involve misfolding and deregulation of protein turnover in photoreceptors. Our long-range objective is to understand the role of a regulatory complex of the UPS- chaperone machinery in controlling substrate degradation and folding and use this information to identify targets for novel therapeutic strategies, which presently lack efficacy toward selective biological and disease processes of the UPS. The goal of this proposal is to understand the molecular and intracellular mechanisms that sustain the balance between turnover and biogenesis of proteins, two tightly coupled processes that are vital for retinal-neuron function and survival and that are implicated in the pathogenesis of a wide variety of neuro-visual disorders of the human. Achievement of this goal will allow us and others to identify novel pharmacological targets and develop therapeutic strategies to manipulate the UPS-chaperone machinery, prevent the toxic deregulation of normal or expression of mutated proteins under various retinal disease states and aging manifestations causing the impairment of the UPS-chaperone machinery and neuro-visual function. The proposed research builds upon our prior research findings which demonstrated that substrates are presented to the proteasome upon synthesis and that the activity of the proteasome is regulated and compartmentalized in the cell. Ran-binding protein 2 (RanBP2) via two distinct domains, the cyclophilin-like (CLD) and cyclophilin (CY) domains, is a key controller of such processes. This proposal tests our overall hypothesis that RanBP2 and its CLD and CY domains control two tightly coupled processes, the UPS-dependent turnover and biogenesis of selective proteins that are critical to the function and survival of selective retinal neurons and retinal pigment epithelium (RPE) cells under normal and pathophysiological conditions. We will test this hypothesis by accomplishing the following specific aims, which focus on the mechanistic roles of RanBP2, and its CLD and CY domains, in the regulation of protein homeostasis and survival of selective retinal neurons under normal and disease stress conditions. Aim 1. Test the hypothesis that loss-of-function of RanBP2 has differential biological and pathological effects among retinal neurons, some of which may be essential to the viability or protein homeostasis of such neurons. Aim 2. Test the hypothesis that selective loss-of-function of the CLD activity of RanBP2 up-regulates selective functions of the UPS that are critical to homeostasis of selective retinal proteins, function and survival of retinal neurons and RPE cells. Aim 3. Test the hypothesis that the cis-trans prolyl isomerase (PPIase) activity in the CY domain of RanBP2 controls the biogenesis of selective proteins that are critical to the function or survival of retinal neurons and RPE cells. PUBLIC HEALTH RELEVANCE: The modulation of protein degradation and biogenesis in the retina is of vital importance to its function. Impairment of these processes is known to underlie the pathogenesis of several degenerative and aging disorders affecting retinal neurons. The proposal aims at elucidating the role of Ran-binding protein 2 in protein homeostasis and allied pathohysiological states in the retina.