Structural and functional analyses of the FAM46 proteins.

FAM 46 C（Family with sequence similarity 46，C）是多个基因组中最常突变的基因之一， 骨髓瘤（发现于超过20%的患者中）。其他FAM 46家族成员（A、B和D）的突变也是 与各种人类疾病有关。尽管与疾病有很强的联系， FAM 46在生理或病理环境中是未知的。该项目的目标是填补 关于FAM 46蛋白质功能和潜在机制的知识差距，为 为相关疾病制定治疗策略。该项目是基于我们的前期工作 显示FAM 46与polo样激酶4（Plk 4）和BRCA 2和CDKN 1A（p21）的直接物理相互作用 相互作用蛋白（BCCIP）。plk 4是中心体复制和纤毛发生的主要调节因子。BCCIP 已被证明参与调节DNA修复和中心体复制。这些共同表明 FAM 46蛋白调节中心体复制、纤毛发生和DNA修复。中心体组织 双极纺锤体的形成和细胞周期中染色体的适当分离。在非分裂细胞中， 中心体介导初级纤毛的形成，初级纤毛是对器官发生至关重要的专门的信号细胞器 和组织内环境稳定。在调节这些过程的作用是一致的强连接 FAM 46突变与癌症之间的联系这些假设将在三个目标进行测试，结合X射线 晶体学、生物物理学、生物化学和基于细胞的功能方法。目标1将侧重于 FAM 46/Plk 4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FAM 46和Plk 4之间，并确定可以通过功能测定中的突变进行测试的关键残基。在 此外，还将分析FAM 46和Plk 4之间酶活性的潜在相互调节。目的 2将侧重于FAM 46/BCCIP相互作用的生物物理和结构分析。晶体结构 的FAM 46和BCCIP复合物将被确定，以阐明它们的原子细节的相互作用。结构 还将分析潜在的FAM 46、Plk 4和BCCIPα三方复合物。突变分析 接下来将测试结合模式和界面残留物。目标3的目标是分析 FAM 46、Plk 4和BCCIPα在调控中心体复制、纤毛发生和DNA中的相互作用 修复.荧光显微镜将用于探测FAM 46、Plk 4和BCCIPα的定位，以及它们的表达。 在细胞周期的各个阶段，中心体内外的共定位。中心体的调控 FAM 46通过与Plk 4和BCCIP的相互作用进行复制、纤毛发生和DNA修复也将被研究。 通过荧光显微镜分析。

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.