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

Alpha4 对 PP2A 活性的调节：在 tau 过度磷酸化中的作用

• 批准号: 8366203
• 负责人: Michele Laura LeNoue-Newton
• 金额: $ 2.69万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-12-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8366203
• 关键词: Address; Affect; Affinity; Age; Alzheimer' s Disease; American; Antibodies; Binding; Brain; Caspase; Catalytic Domain; Cell Line; Cells; Complement; Complex; Data; Elderly; Electrons; Elements; Engineering; Event; Exhibits; Face; Goals; Impaired cognition; Lead; Measures; Memory Loss; Microtubule-Associated Proteins; Microtubules; Modeling; Molecular; Molecular Structure; Mono-S; Nerve Degeneration; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; Patients; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Polyubiquitination; Post-Translational Protein Processing; Protein Dephosphorylation; Protein Phosphatase 2A Regulatory Subunit PR53; Protein phosphatase; Proteins; Regulation; Research; Role; Senile Plaques; Spectrum Analysis; Structure; Symptoms; Ubiquitin; Ubiquitination; Work; base; flexibility; genetic regulatory protein; hyperphosphorylated tau; insight; interest; multicatalytic endopeptidase complex; mutant; new therapeutic target; prevent; protein protein interaction; scaffold; tau Proteins; tau phosphorylation; ubiquitin-protein ligase

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.

描述（由申请人提供）：阿尔茨海默氏病是一种进行性神经退行性疾病，会导致认知能力下降和记忆丧失，影响 8 分之一的 65 岁以上美国人 (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，但没有任何疗法可以阻止神经退行性病变的进展或治愈 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 的过度磷酸化会降低其与微管的亲和力，并且过度磷酸化的 tau 会隔离正常的 tau，从而阻止其与微管结合 (8-10)。 Tau 被多种激酶磷酸化，但 PP2A 被认为是参与 tau 去磷酸化的主要磷酸酶 (11-13)。 Alpha4 已被证明可以调节 PP2A 稳定性和表达 (14, 15)，并且由于与 Mid1 相关而定位于微管 (16)。具体而言，α4 通过抑制 PP2Ac 的多泛素化来调节 PP2Ac 的泛素化及其被蛋白酶体的降解 (15)。根据之前的研究和初步结构，工作假设是α4的UIM与单泛素化的PP2Ac结合，封盖泛素链并防止多泛素化，并且这种保护是通过α4的泛素化来调节的。为了了解 alpha4 调节 PP2Ac 的机制，将进行双电子-电子共振 (DEER) 研究，以观察 alpha4 的灵活性在与 PP2Ac 和泛素相互作用中的作用。为了研究 PP2A 的 α4 调节机制，将用破坏蛋白质-蛋白质相互作用的 α4 构建体转染神经元细胞系，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 抗体测量磷酸 tau 水平来评估。我们的数据表明 alpha4 可能通过泛素化进行调节。为了研究这一点，将用破坏蛋白质-蛋白质相互作用的 α4 构建体转染 293FT 细胞，并测量 α4 泛素化。该项目的目标是阐明 α4 调节 PP2A 的机制，并研究 α4 在调节 PP2A 对 tau 活性中所起的作用。