Functional analysis of pathogenic mutations in Neurofibromatosis Type-1 (NF1)

1 型神经纤维瘤病 (NF1) 致病突变的功能分析

基本信息

批准号：
9245368
负责人：
James Anthony Walker
金额：
$ 26.32万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
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：
2016-09-01 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9245368
关键词：
Address Adult Affect Affinity Chromatography Amino Acid Substitution Amino Acids Behavior Behavioral Benign Biochemical Biological Assay Biological Markers Biology Cell Fractionation Cells Clinical Cognitive deficits Confocal Microscopy Defect Development Disease Drosophila genus GTPase-Activating Proteins Genes Genetic study Genotype Glioblastoma Growth Hereditary Disease Human Immunoprecipitation Inherited Investigation Knowledge Laboratories Lead Lung Adenocarcinoma Malignant Neoplasms Mammalian Cell Mass Spectrum Analysis Missense Mutation Modeling Molecular Mutate Mutation NF1 gene Neurofibromatosis 1 Neurofibromatosis Type 1 Protein Neurons Organelles Patients Peripheral Nerve Sheath Neoplasm Peripheral Nerves Phenotype Pigmentation physiologic function Proteins Proteomics Regulation Residual state Role Signal Transduction Signaling Protein Symptoms System Tertiary Protein Structure Testing Tissues Transgenes Transgenic Organisms Tumor Suppressor Genes Validation Walkers Work base fly in vivo man molecular phenotype mutant nervous system disorder neurofibroma novel protein complex research study skeletal abnormality therapeutic target tumor vascular abnormality

项目摘要

Neurofibromatosis type-1 (NF1) is an inherited neurological disorder affecting about 1 in 3000 people. Mutations in the NF1 gene underlie this disease, with patients’ symptoms including pigmentation defects, cognitive deficits, skeletal and vascular abnormalities, and neurofibromas - benign tumors associated with peripheral nerves. NF1 patients also frequently develop malignant tumors, including peripheral nerve sheath tumors. Further, the NF1 gene has been identified among the most frequently mutated tumor suppressor genes in a number of other cancers including glioblastoma and lung adenocarcinoma. The NF1 gene encodes a large protein, called neurofibromin, the central portion of which (termed the GAP domain) negatively regulates Ras, an important cell signaling protein. NF1 mutations often result in loss of neurofibromin or impair its GAP domain, resulting in elevated Ras signaling. However, many NF1 pathogenic missense mutations have been identified that are predicted to affect regions of neurofibromin distinct from the GAP domain. This suggests that other parts of the highly conserved protein are also essential for its function. We hypothesize that amino acid substitutions outside of the GAP domain may disrupt important protein interactions or subcellular localization of neurofibromin, which could perturb its activity and potentially contribute to the varied clinical symptoms of NF1. This hypothesis will be addressed using a fruit fly (Drosophila) model of NF1 to investigate the molecular and cellular consequences of NF1 missense mutations. Specific Aim 1: We will use Drosophila to rapidly assess the residual function of fly NF1 containing the corresponding mutations from NF1 patients. We will determine if NF1 mutations negatively affect cell signaling and localization within neurons. In addition, mutants will be tested in functional assays including their ability to rescue the growth and behavioral defects of NF1 mutant flies. This will enable us to correlate cellular and molecular phenotypes to specific mutations that disrupt different regions of neurofibromin. Specific Aim 2: We have recently conducted proteomic studies in Drosophila to identify proteins that associate with neurofibromin in neurons. These studies give possible new clues as to the function of neurofibromin in neurons. We will confirm that these putative interactors exist in protein complexes with neurofibromin and explore the functional significance using genetics studies, as well as biochemical and cell fractionation experiments. We anticipate that these studies involving rapid functional testing in an in vivo Drosophila model of NF1 will allow us to establish genotype-phenotype relationships for a number of patient-derived NF1 mutations, as well as further define the functional role of neurofibromin in neurons by investigating novel protein interactors. This knowledge will help prioritize NF1 mutations for further analysis in human cells in the discovery of new biomarkers and therapeutic targets for NF1.

神经纤维瘤病类型1（NF1）是一种影响3000人中约1人的遗传神经系统疾病。 NF1基因中的突变是该疾病的基础，患者的症状包括色素沉着缺陷，认知缺陷，骨骼和血管异常以及神经纤维瘤 - 良性肿瘤周围神经。 NF1患者也经常出现恶性肿瘤，包括周围神经鞘肿瘤。此外，在最常见的突变肿瘤抑制剂中已经鉴定出NF1基因许多其他癌症中的基因，包括胶质母细胞瘤和肺腺癌。 NF1基因编码一种称为神经纤维蛋白的大蛋白，其中心部分（称为间隙结构域）负调节RAS，这是一种重要的细胞信号蛋白。 NF1突变通常会导致神经纤维蛋白的丧失或损害它的间隙结构域，导致RAS信号升高。但是，许多NF1致病错义突变具有被鉴定出来会影响与间隙结构域不同的神经纤维蛋白区域。这表明，高度保守的蛋白质的其他部分对于其功能也是必不可少的。我们假设在间隙结构域之外的氨基酸取代可能会破坏重要的蛋白质相互作用或神经纤维蛋白的亚细胞定位，这可能会扰动其活性，并有可能导致多样化 NF1的临床症状。该假设将使用NF1的果蝇（果蝇）TO来解决研究NF1错义突变的分子和细胞后果。特定目标1：我们将使用果蝇快速评估含有NF1的相应突变的蝇NF1的残留功能患者。我们将确定NF1突变是否会对神经元内的细胞信号传导和定位产生负面影响。在此外，将在功能测定中测试突变体，包括挽救生长和行为的能力 NF1突变蝇的缺陷。这将使我们能够将细胞和分子表型与特定破坏神经纤维蛋白不同区域的突变。特定目标2：我们最近进行了果蝇中的蛋白质组学研究，以鉴定与神经元中神经纤维蛋白相关的蛋白质。这些研究提供有关神经纤维蛋白在神经元中功能的可能新线索。我们将确认这些推定的与神经纤维蛋白的蛋白质复合物中存在相互作用，并使用遗传学探索功能意义研究以及生化和细胞分馏实验。我们预计这些研究涉及 NF1体内果蝇模型中的快速功能测试将使我们能够建立基因型 - 表型许多患者衍生的NF1突变的关系，并进一步定义通过研究新型蛋白质相互作用者，神经纤维蛋白在神经元中。这些知识将有助于确定NF1的优先级在发现新的生物标志物和治疗靶点的人类细胞中进一步分析的突变 NF1。