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的蛋白质动态平衡的长期目标，目前的提案重点放在CHIP上，这是一种重要的泛素连接酶，最近被证明可以抑制由各种错误折叠的疾病蛋白质引起的神经退化。目的1探讨CHIP调控泛素链形成的机制。体外泛素化分析将系统地测试一系列候选芯片交互作用因子调节由芯片形成的泛素链连接的能力；测试候选对象将包括所有已知的泛素结合酶(E2s)以及一组选定的去泛素化酶(DUB)和泛素相互作用基序包含蛋白(UIMs)。这些研究将确定哪些E2与CHIP起作用，每个E2-CHIP对形成什么类型的泛素链，以及DUBS和含UIM的蛋白质在限制CHIP形成特定类型的泛素链连接方面可能发挥什么作用。目标2是对目标1的补充，因为它寻求识别直接或间接与芯片相互作用的蛋白质，以维持大脑中适当的蛋白质稳态。将采用两种平行的方法：1)酵母双杂交分析与验证性免疫共沉淀分析相结合；以及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蛋白复合体。识别正常和神经退行性疾病大脑中功能芯片复合体的成分是了解芯片在提供神经保护方面可能扮演的不同角色的第一步。总而言之，AIMS 1和AIMS 2中的研究将包括对什么构成全功能芯片泛素连接酶的首次全面分析。AIM 3将建立并进行高通量筛选，以识别刺激芯片活性的化合物，因为预计芯片活性增强剂将增强其神经保护作用。这将通过建立基于生化FRET的猝灭试验来实现，以高通量筛选减少荧光素排放的化合物。刺激芯片活性的化合物可能会发展成为一类新的治疗药物。这一提议将为芯片在各种神经退行性疾病中维持神经元蛋白质稳态的机制提供洞察力。ChIP是大脑中一种重要的泛素连接酶，在人类疾病的各种动物模型中被认为是大脑退行性变的关键调节因子。目前的项目既寻求了解CHIP如何调节神经保护，也寻求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剂。

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.