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

描述(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，65岁以上的美国人中有1/8(1)。据估计，到2050年，将有1100万至1600万65岁及以上的美国人受到影响[2]。目前，有暂时减轻阿尔茨海默氏症症状的策略，但没有阻止神经退化或治愈AD的疗法(3)。阿尔茨海默病有两个主要的病理生理特征：β淀粉样斑块和神经原纤维缠结(4)。神经原纤维缠结由过度磷酸化的tau蛋白(5，6)组成。正常tau是一种微管相关蛋白，稳定微管(7)。Tau的过度磷酸化降低了它与微管的亲和力，过度磷酸化的tau隔离了正常的tau，阻止了它与微管的结合(8-10)。Tau被许多激酶磷酸化，但PP2A被认为是参与tau去磷酸化的主要磷酸酶(11-13)。Alpha4已被证明调节PP2A的稳定性和表达(14，15)，并由于其与MID1(16)的关联而定位于微管。具体地说，Alpha4通过抑制PP2Ac的泛素化来调节PP2Ac的泛素化及其被蛋白酶体的降解(15)。根据以前的研究和初步结构，工作假说是Alpha4的UIM与覆盖泛素链的单素化PP2Ac结合，并防止多泛素化，这种保护是通过Alpha4的泛素化来调节的。为了了解Alpha4调控PP2Ac的机制，将进行双重电子-电子共振(DeER)研究，以观察Alpha4在与PP2Ac和泛素相互作用中的灵活性所起的作用。为了研究PP2A的α4调控机制，将用α4构建的干扰蛋白质-蛋白质相互作用的α4构建体转染神经细胞系，并评估PP2Ac泛素化和稳定性。PP2A对tau的活性将通过使用可用的磷酸tau抗体测量phsho-tau的水平来评估。我们的数据表明，α4可能通过泛量化来调节。为了研究这一点，293FT细胞将被导入破坏蛋白质-蛋白质相互作用的Alpha4结构，并将测量Alpha4泛素化。本项目的目的是阐明α4对PP2A的调节机制，并研究α4在调节PP2A对tau的活性中所起的作用。 与公共卫生相关：tau过度磷酸化是阿尔茨海默病(AD)的一个标志，由蛋白磷酸酶2A(PP2A)调节。本研究探讨了α4‘S对PP2A的调节机制及其对tau磷酸化的影响。这项研究的结果将进一步加深我们对tau过度磷酸化的分子基础的理解，并导致可能的新的治疗靶点。

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. PUBLIC HEALTH RELEVANCE: Tau hyperphosphorylation, a hallmark of Alzheimer's disease (AD), is regulated by protein phosphatase 2A (PP2A). This research addresses the mechanism of alpha4's regulation of PP2A and attendant effects on tau phosphorylation. The results of this research will further our understanding of the molecular basis of tau hyperphosphorylation and lead to possible new therapeutic targets.