Structural and functional analyses of the FAM46 proteins.

FAM46 蛋白的结构和功能分析。

• 批准号: 10087901
• 负责人: Xuewu Zhang
• 金额: $ 37.06万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-08 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10087901
• 关键词: Aneuploidy; BRCA2 gene; Binding; Biochemical; Biological Assay; Biophysics; CDKN1A gene; Cell Cycle; Cell Cycle Stage; Cell Nucleus; Cell physiology; Cells; Centrosome; Chromosome Segregation; Cilia; Clinic; Complex; Crystallization; Cytoplasm; DNA Damage; DNA Repair; Data; Development; Disease; Ensure; Family; Family member; Fluorescence; Fluorescence Microscopy; Genes; Goals; Homeostasis; Hyperactivity; Interphase Cell; Knowledge; Link; Malignant Neoplasms; Mediating; Microtubules; Mitotic spindle; Molecular; Multiple Myeloma; Mutate; Mutation; Mutation Analysis; Nuclear; Organelles; Organogenesis; PLK1 gene; Pathologic; Patients; Physiological; Play; Process; Protein Analysis; Proteins; Regulation; Resolution; Roentgen Rays; Role; Signal Pathway; Signal Transduction; Structure; Testing; Therapeutic; Tissues; Work; base; biophysical techniques; cilium biogenesis; human disease; insight; kinetosome; new therapeutic target; novel; nucleotidyltransferase; protein protein interaction; small molecular inhibitor

FAM46C (Family with sequence similarity 46, C) is one of the most frequently mutated genes in multiple myeloma (found in over 20% of the patients). Mutations of other FAM46 family members (A, B and D) are also associated with various human diseases. Despite the strong connections with diseases, the functions of FAM46 in either physiological or pathological settings are unknown. The goal of the project is to fill the knowledge gap on the functions of the FAM46 proteins and the underlying mechanisms, paving the way for developing therapeutic strategies for the associated diseases. The project is based on our preliminary work showing the direct physical interactions of FAM46 with polo-like kinase 4 (Plk4) and BRCA2 and CDKN1A(p21) interacting protein (BCCIP). Plk4 is the master regulator of centrosome duplication and ciliogenesis. BCCIP has been shown to be involved in regulating DNA repair and centrosome duplication. These together suggest that the FAM46 proteins regulate centrosome duplication, ciliogenesis and DNA repair. Centrosomes organize the bi-polar spindle formation and proper chromosome segregation in the cell cycle. In non-dividing cell, centrosomes mediate the formation of primary cilia, specialized signaling organelles critical for organogenesis and tissue homeostasis. A role in regulating these processes are consistent with the strong connection between FAM46 mutations and cancer. These hypotheses will be tested in three aims by combining X-ray crystallographic, biophysical, biochemical and cell-based functional approaches. Aim 1 will be focused on biophysical and structural analyses of the FAM46/Plk4 interaction. These studies will reveal the binding mode between FAM46 and Plk4, and identify key residues that can be tested by mutations in functional assays. In addition, potential mutual regulation of the enzymatic activities between FAM46 and Plk4 will be analyzed. Aim 2 will be focused on biophysical and structural analyses of the FAM46/BCCIP interaction. The crystal structure of the FAM46 and BCCIP complex will be determined to elucidate their interaction in atomic detail. Structural analyses will also be directed at the potential FAM46, Plk4 and BCCIPα tripartite complex. Mutational analyses will follow to test the binding mode and interface residues. The goal of Aim 3 is to analyze the roles of the interactions among FAM46, Plk4 and BCCIPα in regulating centrosome duplication, ciliogenesis and DNA repair. Fluorescence microscopy will be used to probe the localization of FAM46, Plk4 and BCCIPα, and their colocalization in and out of centrosomes in various stages of the cell cycle. The regulation of centrosome duplication, ciliogenesis and DNA repair by FAM46 through the interactions with Plk4 and BCCIP will also be analyzed by fluorescence microscopy.

FAM46C（具有序列相似性46，c）是多个中最常见的突变基因之一 骨髓瘤（在20％以上的患者中发现）。其他FAM46家庭成员的突变（A，B和D）也是 与各种人类疾病有关。尽管与疾病有密切的联系，但 FAM46在物理或病理环境中尚不清楚。该项目的目的是填补 知识差距关于FAM46蛋白质和基本机制的功能差距，为 为相关疾病制定治疗策略。该项目基于我们的初步工作 显示FAM46与polo样激酶4（PLK4）和BRCA2和CDKN1A的直接物理相互作用（P21） 相互作用的蛋白质（BCCIP）。 PLK4是中心体重复和纤毛发生的主要调节剂。 BCCIP 已显示与调节DNA修复和中心体重复有关。这些共同暗示 FAM46蛋白调节中心体重复，纤毛生成和DNA修复。中心体组织 在细胞周期中，双极纺锤体形成和适当的染色体分离。在非分裂的细胞中， 中心体介导原代纤毛的形成，专门的信号细胞器对器官发生至关重要 和组织稳态。在控制这些过程中的作用与牢固的联系一致 在FAM46突变和癌症之间。这些假设将通过组合X射线来以三个目标进行测试 晶体学，生物物理，生化和基于细胞的功能方法。 AIM 1将重点放在 FAM46/PLK4相互作用的生物物理和结构分析。这些研究将揭示结合模式 在FAM46和PLK4之间，并确定可以通过功能测定突变测试的密钥保留。在 此外，将分析FAM46和PLK4之间酶活性的潜在相互调节。目的 2将重点放在FAM46/BCCIP相互作用的生物物理和结构分析上。晶体结构 FAM46和BCCIP综合体将确定以原子细节阐明其相互作用。结构 分析也将针对潜在的FAM46，PLK4和BCCIPα三方复合物。突变分析 将遵循测试绑定模式和接口残差。目标3的目的是分析 FAM46，PLK4和BCCIPα之间的相互作用在调节中心体重复，纤毛生成和DNA中的相互作用 维修。荧光显微镜将用于探测FAM46，PLK4和BCCIPα的定位，且它们的定位 在细胞周期的各个阶段，共定位在中心体中进出。中心体的调节 FAM46通过与PLK4和BCCIP的相互作用的重复，纤毛生成和DNA修复也将是 通过荧光显微镜分析。