Alpha4's regulation of PP2A activity: Role in tau hyperphosphorylation

描述（由申请人提供）：阿尔茨海默病是一种进行性神经退行性疾病，导致认知能力下降和记忆丧失，影响65岁以上的美国人中的1/8（1）。据估计，到2050年，1100万至1600万65岁及以上的美国人将受到影响（2）。目前，有一些策略可以暂时减轻阿尔茨海默氏症的症状，但没有治疗方法可以阻止神经退行性疾病的进展或治愈AD（3）。AD有两个主要的病理生理学特征，β淀粉样蛋白斑块和神经纤维缠结（4）。神经元缠结由过度磷酸化的tau蛋白组成（5，6）。正常tau是一种微管相关蛋白，可稳定微管（7）。tau的过度磷酸化降低其对微管的亲和力，并且过度磷酸化的tau螯合正常tau，防止其结合微管（8-10）。Tau被许多激酶磷酸化，但PP 2A被认为是参与Tau去磷酸化的主要磷酸酶（11-13）。已显示α 4调节PP 2A稳定性和表达（14，15），并由于其与Mid 1的关联而定位于微管（16）。具体而言，α 4通过抑制PP 2Ac的多聚泛素化来调节PP 2Ac的泛素化及其被蛋白酶体降解（15）。基于先前的研究和初步结构，工作假设是α 4的UIM结合到单泛素化的PP 2Ac，从而为泛素链加帽并阻止多聚泛素化，并且这种保护通过α 4的泛素化来调节。为了了解α 4调节PP 2Ac的机制，将进行双电子-电子共振（DEER）研究以观察α 4在与PP 2Ac和泛素相互作用中的柔性的作用。为了研究α 4调节PP 2A的机制，将用破坏蛋白质-蛋白质相互作用的α 4构建体转染神经元细胞系，并评估PP 2Ac泛素化和稳定性。PP 2A对tau的活性将通过使用可用的磷酸化tau抗体测量磷酸化tau的水平来评估。我们的数据表明，α 4可能通过泛素化调节。为了研究这一点，将用破坏蛋白质-蛋白质相互作用的α 4构建体转染293 FT细胞，并测量α 4泛素化。本项目的目的是阐明α 4调节PP 2A的机制，并研究α 4在调节PP 2A对tau的活性中所起的作用。

DESCRIPTION (provided by applicant): Alzheimer's disease is a progressive neurodegenerative disease causing cognitive decline and memory loss that effects 1 in 8 Americans over the age of 65 (1). It is estimated that by the year 2050 between 11 million and 16 million Americans age 65 and older will be affected (2). Currently, there are strategies to temporarily reduce the symptoms of Alzheimer's, but there are no therapies to stop the progression of neurodegeneration or cure AD (3). There are two main pathophysiological features of AD, beta amyloid plaques and neurofibrillary tangles (4). Neurofibrillary tangles are composed of hyperphosphorylated tau protein (5, 6). Normal tau is a microtubule-associated protein that stabilizes microtubules (7). Hyperphosphorylation of tau decreases its affinity for microtubules and hyperphosphorylated tau sequesters normal tau preventing it from binding to microtubules (8-10). Tau is phosphorylated by numerous kinases, but PP2A has been implicated as being the predominant phosphatase involved in tau dephosphorylation (11-13). Alpha4 has been shown to regulate PP2A stability and expression (14, 15) and localizes to microtubules due to its association with Mid1 (16). Specifically, alpha4 regulates ubiquitination of PP2Ac and its degradation by the proteasome by suppressing polyubiquitination of PP2Ac (15). Based on previous studies and preliminary structures, the working hypothesis is that the UIM of alpha4 binds to monoubiquitinated PP2Ac capping the ubiquitin chain and preventing polyubiquitination and that this protection is regulated through the ubiquitination of alpha4. In order to understand the mechanism by which alpha4 regulates PP2Ac, double electron-electron resonance (DEER) studies will be conducted to look at the role of flexibility of alpha4 in interacting with both PP2Ac and ubiquitin. To investigate the mechanism of alpha4 regulation of PP2A, a neuronal cell line will be transfected with alpha4 constructs that disrupt protein-protein interactions and PP2Ac ubiquitination and stability will be assessed. Activity of PP2A towards tau will be assessed using by measuring levels of phosho-tau using available phospho-tau antibodies. Our data indicate that alpha4 may be regulated via ubiqutination. To investigate this, 293FT cells will be transfected with alpha4 constructs that disrupt protein-protein interactions and alpha4 ubiquitination will be measured. The goal of this project is to elucidate the mechanism of alpha4 regulation of PP2A and investigate the role alpha4 plays in regulating PP2A activity towards tau.