Exploring and enhancing Karyopherin beta-2 disaggregate activity

探索和增强核传递蛋白 beta-2 解聚活性

基本信息

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
项目类别：
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

项目摘要

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.

项目概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，没有有效的治疗方法硬化症 (ALS)、额颞叶变性 (FTLD) 或多系统蛋白病 (MSP) 具有朊病毒样结构域的特异性 RNA 结合蛋白 (RBP) 在细胞质中错误定位和聚集例如，野生型 FUS、TAF15 和 EWSR1 在细胞质中积累。在某些形式的 FTLD 中，退化神经元中聚集并从细胞核中耗尽，而野生- 类型或突变型 hnRNPA1 和 hnRNPA2 在退化神经元和其他组织中表现出这种表型对于所有这些带有 PY 核定位信号 (NLS) 的 RBP，以及 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 从根本上可以被视为核运输障碍。我们寻求能够逆转 RBP 错误定位和聚集的代理，从而恢复 RBP 的天然形式、功能和核定位将通过同时减轻毒性消除：(1) 错误折叠形式的任何有毒功能获得；以及 (2) 由于错误折叠而导致的任何功能丧失；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）就是这样一种试剂。 Kapβ2可以阻止和逆转带有PY-NLS的各种RBP的聚集，随后 Kap2 作为核输入因子的作用已得到证实。我们发现 Kap2 具有解聚酶活性，FUS 的 PY-NLS 突变是前所未有的。与 ALS 相关，这些突变直接削弱了 Kap2 和 FUS 之间的相互作用。提出一系列采用纯蛋白质生物化学、酵母、哺乳动物神经元的多学科研究 RBP 疾病的文化和果蝇模型，以满足两个目标：（1）定义 Kap2 在预防和治疗中的活性在体外和体内逆转特定疾病相关 RBP 的聚集、错误定位和毒性 (2) 设计增强的 Kap2 变体来识别和分解带有突变的 ALS 相关 FUS 变体因此，我们将利用 Kap2 作为双功能解聚酶和核输入因子来对抗与 FUS、TAF15、EWSR1 的细胞质错误定位和聚集相关的发病机制，我们提出的研究将阐明核输入因子 Kap2 是如何被表达的。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，这将有助于开发针对特定形式的 ALS、FTD 和 MSP 的疗法。