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

• 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
• 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

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.

1型神经纤维瘤病（NF 1）是一种遗传性神经系统疾病，每3000人中就有1人患病。 NF 1基因的突变是这种疾病的基础，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--与 周围神经NF 1患者也经常发生恶性肿瘤，包括外周神经鞘 肿瘤的此外，NF 1基因已被鉴定为最常突变的肿瘤抑制基因之一， 包括胶质母细胞瘤和肺腺癌在内的许多其他癌症中的基因。NF 1基因编码 一种称为神经纤维蛋白的大蛋白质，其中心部分（称为差距域）负性 调节Ras，一种重要的细胞信号蛋白。NF 1突变通常会导致神经纤维蛋白的丢失或损害 其GAP结构域，导致Ras信号传导升高。然而，许多NF 1致病性错义突变， 已经鉴定了预测影响不同于差距域的神经纤维蛋白区域的蛋白。这 表明高度保守蛋白质的其他部分对其功能也是必不可少的。我们假设 差距域外的氨基酸取代可能会破坏重要的蛋白质相互作用， 神经纤维蛋白的亚细胞定位，这可能会干扰其活性，并可能导致各种 NF 1的临床症状这一假设将解决使用果蝇（果蝇）模型的NF 1， 研究NF 1错义突变的分子和细胞后果。具体目标1：我们将使用 果蝇快速评估含有相应突变的果蝇NF 1的剩余功能 患者我们将确定NF 1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，突变体将在功能测定中进行测试，包括它们拯救生长和行为的能力。 NF 1突变果蝇的缺陷。这将使我们能够将细胞和分子表型与特定的 突变破坏神经纤维蛋白的不同区域。具体目标2：我们最近进行了 果蝇的蛋白质组学研究，以确定与神经元中的神经纤维蛋白相关的蛋白质。这些研究 为神经纤维蛋白在神经元中的功能提供了可能的新线索。我们将确认这些推定的 在与神经纤维蛋白的蛋白质复合物中存在相互作用物，并使用遗传学方法探讨其功能意义 研究，以及生物化学和细胞分离实验。我们预计，这些研究涉及 在果蝇体内模型中进行NF 1的快速功能测试将使我们能够建立基因型-表型 一些患者源性NF 1突变的关系，以及进一步定义的功能作用， 通过研究新的蛋白质相互作用物在神经元中的神经纤维蛋白。这些知识将有助于优先考虑NF 1 在人类细胞中进一步分析突变，以发现新的生物标志物和治疗靶点， NF 1。