Exploring and enhancing Karyopherin beta-2 disaggregate activity

探索和增强核传递蛋白 beta-2 解聚活性

• 批准号: 9182306
• 负责人: James Shorter
• 金额: $ 19.6万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-15 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9182306
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Amyotrophic Lateral Sclerosis; Back; Binding; Biological Models; C9ORF72; Cell Nucleus; Cells; Couples; Cytoplasm; DNA Shuffling; Data; Defect; Development; Directed Molecular Evolution; Disease; Drosophila genus; EWSR1 gene; Engineering; Event; Exhibits; Frontotemporal Lobar Degenerations; Heat shock proteins; Homeostasis; In Vitro; Karyopherins; Libraries; Link; Messenger RNA; Modeling; Molecular Chaperones; Mutation; Neurodegenerative Disorders; Neurons; Nuclear; Nuclear Import; Nuclear Localization Signal; Nuclear Protein; Pathogenesis; Peptide Signal Sequences; Pharmaceutical Preparations; Phenotype; Precision therapeutics; Protein Biochemistry; Proteins; RNA-Binding Proteins; Rattus; Recovery; Role; Series; Signaling Protein; Spinal Cord; TAF15 gene; Therapeutic; Tissues; Toxic effect; Translating; Ursidae Family; Variant; Yeasts; beta Karyopherins; combat; effective therapy; empowered; gain of function; in vivo; innovation; loss of function; meetings; multidisciplinary; mutant; nucleocytoplasmic transport; prevent; prion-like; protein TDP-43; protein aggregation; small molecule; therapy development

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.

项目摘要 对于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）或多系统蛋白质病（MSP），其中 具有朊病毒样结构域的特异性RNA结合蛋白（RBP）错误定位并聚集在细胞质中， 退化的神经元例如，野生型FUS、TAF 15和EWSR 1在细胞质中积累， 在某些形式的FTLD中，在变性神经元中聚集并从细胞核中耗尽，而野生型- 型或突变型hnRNPA 1和hnRNPA 2在退化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RBP，以及携带核定位信号（NLS）的TDP-43， 具有独特的典型NLS，一个关键的病理事件是它们错误定位到细胞质聚集体。 事实上，从这个角度来看，ALS，FTD和MSP基本上可以被视为核转运障碍。 我们假设，能够逆转RBP错误定位和聚集，从而恢复 将RBP转化为天然形式、功能和核定位， 消除：（1）错误折叠形式的任何毒性功能获得;和（2）由于以下原因导致的任何功能丧失： 螯合在细胞质聚集体中。值得注意的是，我们的初步研究结果表明， 核输入因子Karyopherin-β2（Kapβ2，也称为转运蛋白）就是这样一种因子。因此，在本发明中， Kapβ2可以阻止和逆转携带PY-NLS的各种RBP的聚集， 把它们送回原子核Kap 102作为核输入因子的作用已经确立。然而，在这方面， 我们发现Kap β 2具有解聚酶活性是前所未有的。FUS的PY-NLS中的突变是 与ALS相关，这些突变直接削弱了Kap β 2和FUS之间的相互作用。这里我们 提出了一系列多学科的研究，采用纯蛋白质生物化学，酵母，哺乳动物神经元， 培养和果蝇模型的RBP-病，以满足两个目标：（1）定义Kap β 2活性， 逆转体外和体内特定疾病相关RBP的聚集、错误定位和毒性;（2） 工程师增强了Kap β 2变体，以识别和解聚ALS相关的携带突变的FUS变体 在他们的PY-NLS中。因此，我们将利用Kap β 2作为双功能解聚酶和核输入因子， 对抗与FUS、TAF 15、EWSR 1的细胞质错误定位和聚集相关的发病机制， hnRNPA 1和hnRNPA 2。我们提出的研究将阐明核输入因子Kap β 2是如何被 利用和设计来防止和逆转这些有害的RBP错误定位和错误折叠 活动，这将有助于开发特定形式的ALS，FTD和MSP的疗法。