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

1型神经纤维瘤病(NF1)是一种遗传性神经疾病，每3000人中就有1人患病。 NF1基因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--与以下疾病相关的良性肿瘤 周围神经。NF1患者还经常发展为恶性肿瘤，包括周围神经鞘 肿瘤。此外，nf1基因已被鉴定为最频繁突变的肿瘤抑制因子。 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。NF1基因编码 一种大的蛋白质，称为神经纤维素，其中央部分(称为GAP结构域)为负 调节RAS，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丢失或受损 它的GAP结构域，导致RAS信号升高。然而，许多nf1致病错义突变具有 据预测，影响神经纤维蛋白的区域不同于GAP结构域。这 这表明高度保守的蛋白质的其他部分对其功能也是必不可少的。我们假设 GAP结构域外的氨基酸替换可能会扰乱重要的蛋白质相互作用或 神经纤维蛋白的亚细胞定位，这可能扰乱其活性并可能导致不同的 NF1的临床症状。这一假说将使用NF1的果蝇(果蝇)模型来解决 研究NF1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NF1突变的果蝇NF1的残留功能 病人。我们将确定NF1突变是否会对细胞信号和神经元内的定位产生负面影响。在……里面 此外，还将在功能分析中测试突变体，包括它们挽救生长和行为的能力 NF1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 破坏神经纤维蛋白不同区域的突变。具体目标2：我们最近进行了 果蝇蛋白质组学研究以确定神经元中与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可能的新线索。我们将确认这些推定的 蛋白质与神经纤维蛋白复合体中的相互作用及其功能意义的遗传学探讨 研究，以及生化和细胞分级实验。我们预计这些研究涉及 NF1体内果蝇模型的快速功能测试将使我们能够建立基因-表型 一系列患者来源的NF1突变的关系，以及进一步定义NF1的功能作用 通过研究新的蛋白质相互作用研究神经元中的神经纤维蛋白。这一知识将有助于确定NF1的优先顺序 在人类细胞中进一步分析突变，以发现新的生物标记物和治疗靶点 NF1。

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.