Analysis of CHIP, a ubiquitin ligase implicated in neurodegeneration

描述（由申请人提供）：为了了解大脑中泛素依赖蛋白的稳态，目前的提案侧重于CHIP，一种重要的泛素连接酶，最近被证明可以抑制由多种错误折叠的疾病蛋白引起的神经退行性变。目的1探讨CHIP调控泛素链形成的机制。体外泛素化试验将系统地测试一系列候选CHIP相互作用物调节由CHIP形成的泛素链连接类型的能力；测试的候选物将包括所有已知的泛素偶联酶（E2s）以及一组选定的去泛素化酶（DUBs）和泛素相互作用含基元蛋白（UIMs）。这些研究将确定哪些E2s与CHIP一起起作用，每个E2-CHIP对形成什么类型的泛素链键，以及dub和含um的蛋白在限制CHIP形成特定类型的泛素链键方面可能发挥什么作用。Aim 2是对Aim 1的补充，因为它寻求识别与CHIP直接或间接相互作用的蛋白质，以维持大脑中适当的蛋白质稳态。将采用两种平行方法：1)酵母双杂交分析与验证性共免疫沉淀分析相结合；2)从野生型小鼠和多谷氨酰胺神经退行性疾病小鼠模型中分离和分离CHIP蛋白复合物。确定正常和神经退行性疾病大脑中功能性CHIP复合物的成分是了解CHIP在提供神经保护方面可能发挥的多种作用的第一步。总之，目标1和目标2中的研究将首次对全功能CHIP泛素连接酶的构成进行全面分析。Aim 3将建立并执行高通量筛选，以识别刺激CHIP活性的化合物，因为预计CHIP活性增强剂可以增强其神经保护作用。这将通过建立一个基于FRET的生化猝灭实验来完成，对减少荧光素发射的化合物进行高通量筛选。刺激CHIP活性的化合物可能会发展成为一类新的治疗药物。这一建议将有助于深入了解CHIP在各种神经退行性疾病中维持神经元蛋白稳态的机制。CHIP是一种重要的脑泛素连接酶，在多种人类疾病动物模型中被认为是脑退化的关键调节因子。目前的项目旨在了解CHIP如何介导神经保护并识别增强这种活性的化合物，因为它们可能代表各种神经退行性疾病的新治疗药物。

DESCRIPTION (provided by applicant): With the long term objective of understanding ubiquitin-dependent protein homeostasis in the brain, the current proposal focuses on CHIP, an important ubiquitin ligase recently shown to suppress neurodegeneration caused by a variety of misfolded disease proteins. Aim 1 explores the mechanisms regulating ubiquitin chain formation by CHIP. In vitro ubiquitination assays will systematically test a range of candidate CHIP interactors for their ability to regulate the types of ubiquitin chain linkages formed by CHIP; tested candidates will include all known ubiquitin conjugating enzymes (E2s) as well as a panel of selected de ubiquitinating enzymes (DUBs) and Ubiquitin Interacting Motif-containing proteins (UIMs). These studies will determine which E2s function with CHIP, what types of linkages of ubiquitin chains are made by each E2-CHIP pair, and what role DUBs and UIM-containing proteins may play in restricting CHIP to the formation of specific types of ubiquitin chain linkages. Aim 2 is complementary to Aim 1 in that it seeks to identify proteins that interact, directly or indirectly, with CHIP in maintaining proper protein homeostasis in the brain. Two parallel approaches will be employed: 1) yeast two-hybrid analysis coupled with confirmatory co-immunoprecipitation assays; and 2 )chromatographic separation and isolation of CHIP protein complexes from wild type mice and from a polyglutamine neurodegenerative disease mouse model. Identifying the components of functional CHIP complexes in normal and neurodegenerative disease brain is a first step towards understanding the diverse roles CHIP may play in providing neuroprotection. Together, the studies in aims 1 and 2 will comprise the first comprehensive analysis of what constitutes a fully functional CHIP ubiquitin ligase. Aim 3 will establish and perform a high-throughput screen to identify compounds that stimulate CHIP activity, as enhancers of CHIP activity are predicted to boost its neuroprotective action. This will be accomplished by establishing a biochemical FRET based quenching assay to do high- throughput screening for compounds that decrease fluorescein emission. Compounds that stimulate CHIP activity may be developed into a new class of therapeutics. This proposal will provide insight into the mechanism by which CHIP maintains neuronal protein homeostasis in various neurodegenerative diseases. CHIP, an important ubiquitin ligase in the brain, has been implicated as a key regulator of brain degeneration in various animal models of human disease. The current project seeks both to understand how CHIP mediates neuroprotection and to identify compounds that enhance this activity, as they could represent new therapeutic agents for various neurodegenerative diseases.