Functions of Family with Sequence Similarity 20 - Member C (FAM20C) andMember A (FAM20A) in Amelogenesis and Dentinogenesis

序列相似性家族20-成员C (FAM20C)和成员A (FAM20A)在釉质发生和牙本质发生中的功能

• 批准号: 10263267
• 负责人: Yongbo Lu
• 金额: $ 41.14万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-14 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10263267
• 关键词: Affect; Ameloblasts; Amelogenesis; Biological Process; Catalysis; Cell Extracts; Cells; Clinical Management; Complex; Consensus; Cultured Cells; Cyclic AMP-Dependent Protein Kinases; DSPP gene; Defect; Dental Enamel; Dental Pulp; Dentin; Dentin Formation; Dentinogenesis; Development; Disease Management; Electrophoresis; Enamel Formation; Enamel Organ; Family; Genes; Goals; Golgi Apparatus; Human; Hydrophobicity; In Vitro; Incisor; Lead; Molecular; Molecular Sieve Chromatography; Mus; Mutation; Names; Nature; Odontoblasts; Organelles; Pathologic; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Proteins; Research; Research Project Grants; Role; Serine; Source; Stretching; Testing; Therapeutic; Tooth structure; Transmembrane Domain; Two-Dimensional Gel Electrophoresis; Work; ameloblastin; amelogenin; base; dentin matrix protein 1; design; disease-causing mutation; enamel matrix proteins; enamelin; in vivo; member; novel; osteopontin; paralogous gene; secretory protein; therapeutic development

PROJECT SUMMARY Phosphorylation is essential for the phosphorylated proteins to exert their functions in regulating the relevant biological processes. Recent studies have demonstrated that a protein kinase known as “FAM20C” (Family with sequence similarity 20 – member C), together with its paralog named “FAM20A”, plays key and non-redundant functions in phosphorylating serine residues within the Ser-x-Glu/pSer motifs of the secretory proteins, including enamel and dentin matrix proteins that are essential to amelogenesis and dentinogenesis. Although studies have confirmed that FAM20C resides in the Golgi apparatus, it is unclear as to how FAM20C, which does not possess a transmembrane domain, is retained in this organelle to exert its kinase function. While in vitro studies suggest that FAM20A may enhance the kinase activity of FAM20C, whether such functional interaction occurs in ameloblasts and odontoblasts in vivo remains to be determined; FAM20A mutations in humans or its inactivation in mice only affect enamel formation but have no pathological effect on dentin formation, whereas loss of FAM20C function leads to both enamel and dentin defects. The goals of this project are to determine 1) the molecular mechanisms that govern the Golgi-retention of FAM20C, and 2) how FAM20C interacts with FAM20A during amelogenesis and dentinogenesis. Preliminary studies have shown that 1) a FAM20C-related supramolecular complex was present in the cell extract but not in the culture medium when FAM20C was expressed in the transfected cells; 2) a similar large FAM20C-related protein band was also found in the total proteins extracted from the enamel organ (containing ameloblasts) and dental pulp (containing odontoblasts) of mouse teeth; 3) Fam20a-deficient mice developed enamel defects, but had no dentin abnormalities, whereas Fam20c-deficient mice had both enamel and dentin defects; 4) Fam20a-deficient mice had a dramatic reduction in the expression levels of genes encoding the enamel matrix proteins, but showed no expression changes in the genes encoding the major dentin matrix proteins; and 5) Fam20a deletion reduced the level of FAM20C protein in ameloblasts and odontoblasts. These findings lead to the hypothesis that FAM20C forms a supramolecular complex, through which this kinase is retained within the Golgi apparatus, and that FAM20A broadens the substrate spectrum of FAM20C and is required for the proper phosphorylation of the hydrophobic enamel matrix proteins. Two Aims are proposed to test this novel hypothesis: Aim 1 – to determine the mechanisms that govern the Golgi-retention of FAM20C in ameloblasts and odontoblasts. Aim 2 – to determine the functions of FAM20A and FAM20C in ameloblasts and odontoblasts. Successful completion of the proposed work will not only elucidate the molecular mechanisms by which FAM20C and FAM20A function in the phosphorylation of enamel and dentin matrix proteins, but may also provide clues for the development of therapeutic strategies for the clinical management of diseases caused by the FAM20C and FAM20A mutations.

项目摘要 磷酸化对于磷酸化蛋白质发挥其调节相关蛋白质的功能是必需的。 生物过程。最近的研究表明，一种称为“FAM 20 C”（FAM 20 C家族）的蛋白激酶， 序列相似性（20个成员C）及其命名为“FAM 20 A”的同源性是关键的，非冗余的 在磷酸化分泌蛋白的Ser-x-Glu/pSer基序内的丝氨酸残基中起作用，包括 牙釉质和牙本质基质蛋白质，它们是牙釉质形成和牙本质形成所必需的。虽然研究 证实了FAM 20 C存在于高尔基体中，但不清楚FAM 20 C是如何存在的，它不具有 跨膜结构域保留在该细胞器中以发挥其激酶功能。虽然体外研究表明 FAM 20 A可以增强FAM 20 C的激酶活性，无论这种功能性相互作用是否发生在 成釉细胞和成牙本质细胞在体内仍有待确定;人类FAM 20 A突变或其失活 在小鼠中仅影响釉质形成，但对牙本质形成没有病理影响，而 FAM 20 C功能导致牙釉质和牙本质缺陷。该项目的目标是确定1） 控制FAM 20 C高尔基体保留的分子机制，以及2）FAM 20 C如何与FAM 20 A相互作用 在牙釉质形成和牙本质形成过程中。初步研究表明，1）FAM 20 C相关 当FAM 20 C时，超分子复合物存在于细胞提取物中，但不存在于培养基中 在转染细胞中表达; 2）在总的转染细胞中也发现了类似的大的FAM 20 C相关蛋白条带。 从成釉器官（含有成釉细胞）和牙髓（含有成牙本质细胞）中提取的蛋白质， 小鼠牙齿; 3）Fam 20 a缺陷小鼠出现釉质缺陷，但没有牙本质异常，而 Fam 20 c缺陷小鼠存在牙釉质和牙本质缺陷; 4）Fam 20 a缺陷小鼠的牙釉质和牙本质缺陷显著减少， 在编码釉基质蛋白的基因的表达水平中， Fam 20 a缺失降低了牙本质基质蛋白FAM 20 C的表达水平 成釉细胞和成牙本质细胞中的蛋白质。这些发现导致了FAM 20 C形成一种 超分子复合物，通过该复合物，该激酶被保留在高尔基体内， FAM 20 A拓宽了FAM 20 C的底物谱，并且是FAM 20 C的适当磷酸化所必需的。 疏水性釉质基质蛋白。提出了两个目的来检验这一新的假设：目的1 - 确定成釉细胞和成牙本质细胞中FAM 20 C高尔基体滞留的机制。目的2 - 研究FAM 20 A和FAM 20 C在成釉细胞和成牙本质细胞中的功能。成功完成 这项工作不仅将阐明FAM 20 C和FAM 20 A发挥功能的分子机制， 在牙釉质和牙本质基质蛋白磷酸化中起重要作用，但也可能为牙釉质和牙本质基质蛋白的发展提供线索。 FAM 20 C和FAM 20 A突变引起的疾病的临床管理的治疗策略。