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在调节相关方面发挥作用至关重要 生物过程。最近的研究表明，一种称为“ FAM20C”的蛋白激酶（家庭 序列相似性20 - 成员c）及其名称为“ FAM20A”，扮演钥匙和非冗余 磷酸化丝氨酸的功能保留在秘密蛋白的SER-X-GLU/PSER基序中，包括 牙釉质和牙本质基质蛋白对阿米他酮和牙本质生成至关重要。虽然研究有 确认FAM20C位于高尔基体中 跨膜结构域保留在此细胞器中以执行其激酶功能。而体外研究表明 FAM20A可能会增强FAM20C的激酶活性，是否发生在 体内的成成细胞和牙胶细胞仍有待确定。人类中的FAM20A突变或其失活 在小鼠中仅影响牙釉质形成，但对牙本质的形成没有病理作用，而失去 FAM20C功能会导致牙釉质和牙本质缺陷。该项目的目标是确定1） 控制FAM20C的Golgi的分子机制，以及2）FAM20C如何与FAM20A相互作用 在没有降解和牙本质发生期间。初步研究表明1）与FAM20C相关 超分子复合物存在于细胞提取物中，但在FAM20C为时不存在于培养基中 在翻译的细胞中表达； 2）在总计中也发现了类似的大型FAM20C相关蛋白带 从搪瓷器官（包含成成细胞）和牙髓（包含牙糖细胞）中提取的蛋白质 小鼠牙齿； 3）FAM20A缺陷小鼠患有牙釉质缺陷，但没有牙本质异常，而 FAM20C缺陷小鼠既有牙釉质和牙本质缺陷; 4）FAM20A缺陷小鼠的减少幅度很大 在编码搪瓷基质蛋白的基因的表达水平中，但没有显示出表达的变化 编码主要牙本质基质蛋白的基因； 5）FAM20A删除降低了FAM20C的水平 蛋白质细胞和odontoblasts中的蛋白质。这些发现导致了以下假设：FAM20C形成A 超分子复合物，通过该复合物将这种激酶保留在高尔基体中，并且 FAM20A扩大了FAM20C的底物光谱，是正确磷酸化所必需的 疏水搪瓷基质蛋白。提出了两个目标来检验这个新的假设：目标1 - 确定在木质细胞和odontoblasts中控制FAM20C的高尔基抗性的机制。目标2 - 确定FAM20A和FAM20C在成成组细胞和Odontoblasts中的功能。成功完成 拟议的工作不仅将阐明FAM20C和FAM20A功能的分子机制 在搪瓷和牙本质基质蛋白的磷酸化中，但也可能为发展提供线索 由FAM20C和FAM20A突变引起的疾病临床管理的治疗策略。