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 对于各种致命神经退行性疾病，没有有效的治疗方法，包括肌萎缩症 硬化症（ALS），额颞叶变性（FTLD）或多系统蛋白质病（MSP），其中 特定的RNA结合蛋白（RBP），具有prion样域的域错误定位和聚集在细胞质中 退化神经元。例如，野生型FUS，TAF15和EWSR1积聚在细胞质中 聚集体并以某些形式的FTLD中的神经元中的核中耗尽，而野生 类型或突变的HNRNPA1和HNRNPA2在退化的神经元和其他时机中表现出这种表型 MSP。对于所有这些RBP，都带有核核定位信号（NLS）以及TDP-43，它们 带有独特的规范NLS，一个关键的病理事件是将其错误定位到细胞质聚集体中。 实际上，从这个角度来看，ALS，FTD和MSP可以从根本上看作是核传播疾病。 我们假设代理可以逆转RBP错误定位和聚合，从而恢复 RBP到天然形式，功能和核定位将通过简单地减轻毒性 消除：（1）错误折叠形式功能的任何有毒毒性获得； （2）由于 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Karyopherin-β2（KAPβ2，也称为Transperin）就是这样的药物。那， KAPβ2可以防止并逆转带有PY-NLS的各种RBP的聚集，然后 将它们运回细胞核。 KAP2作为核进口因素的作用已得到充分确立。然而， 我们发现KAP2具有分解酶活性的活性是前所未有的。 FUS PY-NL中的突变是 与ALS有关，这些突变直接削弱了KAP2与FUS之间的相互作用。在这里，我们 提案一系列使用纯蛋白质生物化学，酵母，哺乳动物神经元的多学科研究 培养和果蝇模型的RBP播种模型以满足两个目的：（1）定义KAP2在预防和 在体外和体内逆转聚集，错误定位和特定疾病连接的RBP的毒性； （2） 工程师增强了KAP2变体，以识别和分解与ALS相关的FUS变体轴承突变 在他们的py-nls中。这，我们将把KAP2作为双功能分解酶和核进口因素 战斗发病机理与FUS，TAF15，EWSR1， HNRNPA1和HNRNPA2。我们提出的研究将阐明核进口因子KAP2如何如何 利用和设计以防止和扭转这些已删除的RBP错误定位和错误折叠 事件，这将增强针对ALS，FTD和MSP的特定形式的疗法的开发。