Mechanism of VCP interaction and processing of neurodegenerative tau fibrils

项目摘要 本提案的目的是研究AAA+ ATP酶相互作用的结构和动力学， 含有缬沙素的蛋白（VCP）与疾病相关的tau原纤维，以确定VCP介导的疾病 进展和治疗潜力。阿尔茨海默病（AD）和皮质基底节变性（CBD）是两种常见的 超过20种以不同形式的纤维状tau聚集体为特征的“tau蛋白病”。tau的程度 尸检病理学与认知能力下降相关，但神经毒性、疾病 发展和疾病进展仍有待阐明。聚集性疾病 表明蛋白质稳定功能障碍-蛋白质组的调节和平衡。VCP扮演着重要的角色， 通过从复合物、膜和聚集体（如形成的那些）中去除蛋白质，在蛋白质稳定中起作用 因此，VCP是蛋白质抑制功能障碍的重要参与者，也是一种潜在的药物 靶向tau蛋白病。 VCP具有多种辅助因子，使其能够执行一系列重要的细胞功能。Ufd 1和Npl 4 辅助因子（UN）一起特异性地使VCP能够识别聚泛素化底物。AD tau纤维具有 高速率的泛素化，可能是因为细胞已经标记了这些纤维，以便被VCP等蛋白质去除。 此外，VCP内的突变与神经退行性疾病有关。首先是一个低活性的VCP突变 爱德华·李实验室描述的临床表现为痴呆症，并已显示出促进tau纤维 病理低活性VCP突变被认为降低了VCP功能，导致tau蛋白的积累。 纤维，导致神经退行性疾病。然而，VCP不充分清除tau蛋白的类似机制， 在散发性Tau蛋白病的情况下可能存在原纤维。VCP/UN，突变型或野生型，也可能卡在 tau原纤维（可能由于原纤维的高稳定性）并且不能分解原纤维或被释放， 有效的VCP功能通过捕获它的原纤维。这在26 S中的类似AAA+ ATP酶中已经观察到。 蛋白酶体和神经变性聚集体。 我建议，对于生产性解聚酶活性VCP/UN结合非结构化泛素在任何一端的 然后tau原纤维一次拉下一个tau单体。我还建议行动机制不会改变， 由于原纤维结构的差异，化学计量和反应速率将在tau原纤维之间变化， 泛素化为了测试这个模型，在目标1中，我将从结构上描述VCP/UN如何与大脑相互作用- 衍生的AD和CBD tau原纤维。从结构上理解VCP/UN如何与tau纤维相互作用将有助于 阐明对VCP/UN功能至关重要的蛋白质区域，并了解这种相互作用如何变化 与原纤维类型的关系将有助于理解VCP/UN原纤维相互作用的普遍性。在目标2中，我将 表征VCP/UN-tau相互作用的动力学。了解自愿协商进程/联合国互动的动态 将进一步阐明VCP/UN与疾病相关的tau原纤维相关的机制。

Project Summary The goal of this proposal is to study the structure and dynamics of the interaction of the AAA+ ATPase, valosin-containing protein (VCP) with disease-relevant tau fibrils to determine VCP-mediated disease progression and therapeutic potential. Alzheimer's Disease (AD) and Corticobasal degeneration (CBD) are 2 of more than 20 “tauopathies” characterized by different forms of fibrillar tau aggregates. The degree of tau pathology on autopsy correlates with cognitive decline, yet the mechanism of neurotoxicity, disease development, and disease progression in tauopathies remains to be elucidated. Diseases of aggregation indicate a dysfunction in proteostasis – the regulation and balance of the proteome. VCP plays an essential role in proteostasis by removing proteins from complexes, membranes, and aggregates such as those formed by fibrillar tau, thus pointing to VCP as an important player in proteostatic dysfunction and as a potential drug target for tauopathies. VCP has a variety of cofactors that enable it to perform an array of vital cellular. Ufd1 and Npl4 cofactors (UN) together specifically enable VCP to recognize poly-ubiquitinated substrates. AD tau fibrils have high rates of ubiquitination, likely because cells have marked these fibrils for removal by proteins such as VCP. Additionally, mutations within VCP are linked to neurodegenerative disease. A hypoactive VCP mutation first described by the Edward Lee lab clinically presents as dementia and has been shown to promote tau fibril pathology. The hypoactive VCP mutation is thought to reduce VCP function causing the accumulation of tau fibrils, leading to neurodegenerative disease. However, a similar mechanism of VCP inadequately clearing tau fibrils may be present in cases of sporadic tauopathies. VCP/UN, mutant or wild-type, may also get stuck on tau fibrils (possibly due to fibrils' high stability) and be unable to break down the fibril or be released, reducing effective VCP function by trapping it on the fibril. This has been seen with a similar AAA+ ATPase in the 26S proteasome and neurodegenerative aggregates. I propose that for productive disaggregase activity VCP/UN binds unstructured ubiquitin at either end of tau fibrils then pulls off one tau monomer at a time. I also propose the mechanism of action will not change but the stoichiometry and rate of reaction will change between tau fibrils due to differences in fibril structure and ubiquitination. To test this model, in Aim 1 I will structurally characterize how VCP/UN interacts with brain- derived AD and CBD tau fibrils. Structurally understanding how VCP/UN interacts with tau fibrils will help elucidate the protein regions that are crucial to VCP/UN function, and knowing how this interaction changes with fibril type will help understand the generalizability of the VCP/UN fibril interaction. In Aim 2, I will characterize the dynamics of the VCP/UN-tau interaction. Understanding the dynamics of VCP/UN interaction with these fibrils will further elucidate the mechanism of VCP/UN in relation to disease-relevant tau fibrils.