Kapβ2 recognition of cargos involved in neurodegenerative/neurodevelopmental diseases

Kapβ2 识别涉及神经退行性/神经发育疾病的货物

• 批准号: 10269917
• 负责人: Abner Gonzalez-Castro
• 金额: $ 3.96万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-29 至 2022-09-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10269917
• 关键词: ALS patients; Affect; Affinity; Amyotrophic Lateral Sclerosis; Binding; Binding Sites; Biological Assay; C-terminal; Calorimetry; Cell Nucleus; Cell physiology; Cells; Cellular Structures; Complex; Cryoelectron Microscopy; Crystallization; Cytoplasm; Data; Defect; Disease; Gene Expression Regulation; Goals; Human; Human Cell Line; Immobilization; Importins; Karyopherins; Knowledge; Lead; Link; Maps; Measures; Mediating; Mental Retardation; Motor Neurons; Mutate; Mutation; N-terminal; Nerve Degeneration; Neurodegenerative Disorders; Neurodevelopmental Disorder; Nuclear; Nuclear Import; Nuclear Localization Signal; Nuclear Pore Complex; Patients; Permeability; Phase; Primary Lateral Sclerosis; Proline; Proteins; RNA-Binding Proteins; RRM2 gene; Resolution; Ribonucleoproteins; Severity of illness; Signal Transduction; Structure; Syndrome; Testing; Titrations; Tyrosine; Variant; Work; X-Ray Crystallography; early onset; experimental study; familial amyotrophic lateral sclerosis; frontotemporal lobar dementia-amyotrophic lateral sclerosis; fused in sarcoma; interest; macromolecule; mutant; nervous system disorder; novel; nucleocytoplasmic transport; receptor; stress granule; therapy development

Karyopherin β2 (Kapβ2) is a nuclear import factor that transports many heterogeneous ribonucleoproteins (hnRNPs) into the nucleus by recognizing the nuclear localization signal (NLS) known as PY-NLS. Many mutations in Kapβ2 cargoes are linked to neurological diseases by causing protein mislocalization to the cytoplasm, followed by aggregate formation and fibrillization. In this proposal, I seek to understand the Kapβ2- mediated nuclear import mechanism of cargoes Fused in Sarcoma (FUS) and hnRNP H2, which are involved in neurodegenerative and neurodevelopmental disease, respectively. Aim 1. Many mutations in the FUS proteins are associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. One mutation results in the mutant FUS495X protein, which is truncated at the C-terminus and missing its PY-NLS and causes a severe type of familial ALS. When FUS495X is expressed in cells, it is localized to both the cytoplasm and the nucleus, suggesting a fraction is still imported into the nucleus despite missing its PY-NLS. Preliminary studies suggest that Kapβ2 uses its PY-NLS binding site to bind FUS495X. Our goaI here is to 1) map the region(s) of FUS495X that binds Kapβ2, 2) use isothermal titration calorimetry (ITC) to measure binding affinities of different FUS495X regions for Kapβ2, 3) test if these FUS region(s) can be transported into the nucleus in cells, and 3) solve the crystal structure of Kapβ2 bound to FUS495X. Using qualitative pull-down binding assays and quantitative ITC experiments, I have mapped Kapβ2-binding to the C-terminal 130 residues of FUS495X, which spans its RGG2, ZnFinger and RGG3 domains/regions and binds Kapβ2 with a KD of 200 nM. I will test cellular localization of this region of FUS495X to assess if it can be imported into the nucleus by Kapβ2. I will express FUS 371-495 in a few human cell lines to determine if it can localize to the nucleus. I will also attempt to solve the crystal structure of Kapβ2 complexed this FUS fragment to understand how it binds the importin. Aim 2. Three mutations in hnRNP H2 have been found in patients with MRXSB (mental retardation, X-linked, syndromic, Bain type) diseases. The goal here is to understand how these mutations affect Kapβ2-binding. I have generated various hnRNP H2 constructs and used both qualitative pull- down binding assays and ITC to map Kapβ2-binding to a fragment containing RRM2 and a PY-NLS. ITC using this hnRNP H2 fragment showed that notable losses of affinity for Kapβ2-binding of the MRXSB mutants and these results correlate well with hnRNP H2 mutants localization to stress granules and with disease severity. The interactions between hnRNP H2 and Kapβ2 is highly unusual in that it involves a folded RRM domain in addition to a PY-NLS. I will determine the structure of Kapβ2 complexed with hnRNP H2 RRM-PY-NLS to understand this novel mode of interaction and also to understand how MRXSB disease mutations result in loss of Kapβ2-binding.

核仁组成蛋白2(Karyopherinβ2，KAPβ2)是一种核输入因子，可运输多种异质核糖核蛋白 (HnRNPs)通过识别被称为PY-NLS的核定位信号(NLS)进入细胞核。许多 KAPβ2货物的突变与神经系统疾病有关，因为它导致蛋白质错误定位到 细胞质，随后是聚集体形成和纤化。在这项建议中，我试图理解KAPβ2- 参与肉瘤融合(FUS)和hnRNP H2融合的货物的核进口机制 分别在神经退行性疾病和神经发育疾病中。目标1.FU中的许多突变 蛋白质与家族性肌萎缩侧索硬化症(ALS)和额颞部痴呆有关。一 突变导致突变的FUS495X蛋白，该蛋白在C末端被截断并丢失其PY-NLS 导致一种严重的家族性肌萎缩侧索硬化症。当FUS495X在细胞中表达时，它被定位到两个 细胞质和细胞核，这表明尽管缺少PY-NLS，但仍有一部分被输入到细胞核中。 初步研究表明，KAPβ2利用其PY-NLS结合位点与FUS495X结合。我们的目标是1) 将FUS495X与KAPβ结合的区域(S)作图，2)用等温滴定量热法测定 不同FUS495X区域对KAPβ的结合亲和力2，3)测试这些FUS区域(S)是否能被转运到 3)解决了与FUS495X结合的KAPβ2的晶体结构。使用定性下拉菜单 结合分析和国际贸易中心定量实验，我已经将KAPβ2-结合映射到C-末端130 FUS495X的残基，它横跨其RgG2、锌指和RgG3结构域/区域，并与KAPβ2结合 Kd为200 nm。我将测试FUS495X的这个区域的细胞定位，以评估它是否可以导入到 我将在几个人细胞系中表达FUS371-495，以确定它是否能定位于β。 原子核。我还将尝试解开KAPβ2的晶体结构复杂的这个FUS片段来理解 它是如何绑定进口的。目的2.在MRXSB患者中发现了hnRNP H2的三个突变 (精神发育迟缓，X连锁，综合征，贝恩型)疾病。这里的目标是了解这些 突变会影响KAPβ2的结合。我已经生成了各种hnRNP H2结构，并使用了定性的拉动- 下结合试验和ITC将KAPβ2结合映射到包含RRM2和PY-NLS的片段。ITC使用 HnRNPH2片段表明，mrxsb突变体和mrxsb突变体与KAPβ2结合的亲和力显著降低。 这些结果与hnRNP H2突变体对应激颗粒的定位以及疾病严重程度有很好的相关性。 HnRNPH2和KAPβ2之间的相互作用非常不寻常，因为它涉及一个折叠的RRM域 添加到PY-NLS。我将确定KAPβ2与hnRNP H2 RRM-PY-NLS络合的结构，以 了解这种新的相互作用模式，并了解MRXSB疾病突变如何导致丢失 KAPβ2结合。