Exploring and enhancing Karyopherin beta-2 disaggregate activity

项目总结 目前还没有有效的治疗方法来治疗各种致命的神经退行性疾病，包括肌萎缩侧索硬化症 硬化症(ALS)、额颞叶变性(FTLD)或多系统蛋白病(MSP) 具有蛋白样结构域的特异性RNA结合蛋白(RBPs)错位聚集在细胞质中 退化的神经元。例如，野生型FUS、TAF15和EWSR1在细胞质中积累 在某些形式的FTLD的变性神经元中，聚集并从细胞核中耗尽，而野生的- 类型或突变的hnRNPA1和hnRNPA2在变性的神经元和其他组织中表现出这种表型 MSP。对于所有这些携带PY核定位信号(NLS)的限制性商业惯例，以及TDP-43，它 具有明显的典型性NLS，一个关键的病理事件是它们错误地定位于细胞质聚集体。 事实上，从这个角度来看，ALS、FTD和MSP从根本上可以被视为核运输障碍。 我们假设，能够逆转RBP错误定位和聚集的代理从而恢复 天然形式、功能和核定位的限制性商业惯例将通过同时减轻毒性 消除：(1)错误折叠形式的功能的任何有毒收益；和(2)由于以下原因而导致的任何功能损失 在细胞质聚集体中的隔离。值得注意的是，我们的初步发现表明 核进口因子KAP-β2(KAPβ2，又称转运素)就是这样的制剂。因此， KAPβ2可以防止和逆转携带PY-NLS的各种限制性商业惯例的聚集，并随后 把它们运回原子核。KAP2作为核进口因子的作用已经确立。然而， 我们发现KAP2具有解聚酶活性，这是史无前例的。FUS的PY-NLS突变是 这些突变直接削弱了KAP2和FUS之间的相互作用。在这里，我们 提出一系列使用纯蛋白质生物化学、酵母、哺乳动物神经细胞的多学科研究 培养和果蝇RBP病模型以达到两个目的：(1)确定KAP2活性在预防和治疗中的作用 逆转体内和体外特定疾病相关限制性商业惯例的聚集、错位和毒性；(2) 工程师增强了KAP2变体，以识别和分解携带突变的肌萎缩侧索硬化症连锁FUS变体 在他们的PY-NLS里。因此，我们将利用KAP2作为双功能解聚酶和核进口因子来 FUS、TAF15、EWSR1、FUS、TAF15、EWSR1、 HnRNPA1和hnRNPA2。我们建议的研究将阐明核进口因子Kap2如何能够 利用和设计，以防止和扭转这些有害的RBP错误定位和错误折叠 活动，这将使针对特定形式的ALS、FTD和MSP的治疗方法的开发成为可能。

Project summary There are no effective treatments for various fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), or multisystem proteinopathy (MSP) in which specific RNA-binding proteins (RBPs) with prion-like domains mislocalize and aggregate in the cytoplasm of degenerating neurons. For example, wild-type FUS, TAF15, and EWSR1 accumulate in cytoplasmic aggregates and are depleted from the nucleus in degenerating neurons in some forms of FTLD, whereas wild- type or mutant hnRNPA1 and hnRNPA2 exhibit this phenotype in degenerating neurons and other tissues in MSP. For all of these RBPs, which bear a PY-nuclear localization signal (NLS), as well as TDP-43, which bears a distinct canonical NLS, a key pathological event is their mislocalization to cytoplasmic aggregates. Indeed, from this perspective ALS, FTD, and MSP can be viewed fundamentally as nuclear-transport disorders. We hypothesize that agents able to reverse RBP mislocalization and aggregation and thereby restore the RBPs to native form, function, and nuclear localization would mitigate toxicity by simultaneously eliminating: (1) any toxic gain of function of the misfolded form; and (2) any loss of function due to sequestration in cytoplasmic aggregates. Remarkably, our preliminary findings suggest that the nuclear import factor, Karyopherin-β2 (Kapβ2, also known as transportin), is such an agent. Thus, Kapβ2 can prevent and reverse the aggregation of various RBPs bearing a PY-NLS, and subsequently transport them back to the nucleus. A role for Kap2 as a nuclear import factor is well established. However, our discovery that Kap2 has disaggregase activity is unprecedented. Mutations in the PY-NLS of FUS are linked with ALS, and these mutations directly weaken the interaction between Kap2 and FUS. Here, we propose a series of multidisciplinary studies that employ pure protein biochemistry, yeast, mammalian neuronal culture, and Drosophila models of RBP-opathies to meet two aims: (1) Define Kap2 activity in preventing and reversing aggregation, mislocalization, and toxicity of specific disease-linked RBPs in vitro and in vivo; (2) Engineer enhanced Kap2 variants to recognize and disaggregate ALS-linked FUS variants bearing mutations in their PY-NLS. Thus, we will exploit Kap2 as a bifunctional disaggregase and nuclear import factor to combat pathogenesis associated with cytoplasmic mislocalization and aggregation of FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2. Our proposed studies will elucidate how a nuclear import factor, Kap2, can be harnessed and engineered to prevent and reverse these deleterious RBP mislocalization and misfolding events, which will empower the development of therapies for specific forms of ALS, FTD, and MSP.