Role of FAM20C in the Phosphorylation of SIBLING Protein

FAM20C 在 SIBLING 蛋白磷酸化中的作用

• 批准号: 8622956
• 负责人: XIAOFANG WANG
• 金额: $ 10.91万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8622956
• 关键词: Affect; Amino Acid Sequence; Biological Assay; C-terminal; Calvaria; Categories; Cell Culture System; Cells; Collagen Type I; Data; Defect; Dental Cementum; Dental Enamel; Dentin; Dentinogenesis; Disease; Enzymes; Event; Extracellular Matrix; Failure; Foundations; Future; Glycoproteins; Hypophosphatemia; In Vitro; Incubated; Individual; Integrin Binding; Knowledge; Lead; Ligands; Link; Mass Spectrum Analysis; Minerals; Modality; Mus; Nodule; Odontoblasts; One-Step dentin bonding system; Osteoblasts; Osteogenesis; Peptide Hydrolases; Peptides; Phosphoproteins; Phosphorylation; Phosphoserine; Phosphotransferases; Physiologic calcification; Post-Translational Protein Processing; Protein Kinase; Proteins; Recombinants; Research Proposals; Rickets; Role; Serine; Site; Skeleton; Solid; Testing; Tissues; Tooth structure; Wild Type Mouse; Work; biomineralization; bone; bone sialoprotein; craniofacial complex; dentin matrix protein 1; mineralization; novel; osteopontin; protein aminoacid sequence; public health relevance; therapy development

DESCRIPTION (provided by applicant): This research proposal focuses on the role of FAM20C (also known as "dentin matrix protein 4" or "DMP4") in the phosphorylation of SIBLING proteins. Our recent data have established that FAM20C is essential to the formation and mineralization of bone and tooth, and that this protein kinase may be the primary enzyme responsible for catalyzing the phosphorylation of SIBLING proteins. We discovered: 1) FAM20C is highly expressed in the cells forming bone, dentin, cementum and enamel; 2) the inactivation of Fam20C in mice leads to rickets with extensive hypomineralization in murine bone; 3) Fam20C-KO mice have significant defects in the formation and mineralization of dentin, cementum and enamel; 4) the total phosphorylation level of non-collagenous proteins in the bone or dentin of Fam20C-KO mice is significantly lower than that of wild type mice. Furthermore, we and others revealed that recombinant FAM20C effectively phosphorylates some of the SIBLING proteins in vitro. Our preliminary data lead us to form the central hypothesis that FAM20C is the primary kinase catalyzing the phosphorylation of SIBLING proteins and the defects in the hard tissues of the Fam20C-KO mice may result from a significant loss of phosphorylation in the SIBLING proteins. To test this novel hypothesis, we propose the following three specific aims: (1) To determine the effects of FAM20C deficiency on the phosphorylation of SIBLING proteins. We will isolate OPN, BSP, the C-terminal fragment of DMP1, and DPP from the bone and dentin of Fam20C-KO mice and compare the phosphorylation level of these proteins with that in tissues from WT mice. We will also examine the effects of FAM20C on the phosphorylation of SIBLING proteins via 32P incorporation assay in the cultures of calvarial osteoblasts derived from Fam20C-KO and WT mice. (2) To examine phosphoserines in the SIBLING proteins isolated from the bone and dentin of Fam20C-KO and WT mice by digesting OPN, BSP, DMP1 C-terminal fragment, and DPP with proteases, separating the proteolytic peptides, and sequencing the isolated peptides with mass spectrometry. (3) To examine the effects of SIBLING proteins isolated from the Fam20C-KO mice on the mineralized nodule formation by osteoblasts and odontoblasts. The multipronged approaches proposed in this application will advance our understanding of the role of FAM20C in the phosphorylation of SIBLING proteins, and serve as the foundation for further elucidating the mechanism of biomineralization associated with the phosphorylation of SIBLINGs.

描述（由申请人提供）：本研究计划重点关注 FAM20C（也称为“牙本质基质蛋白 4”或“DMP4”）在 SIBLING 蛋白磷酸化中的作用。我们最近的数据表明，FAM20C 对于骨骼和牙齿的形成和矿化至关重要，并且这种蛋白激酶可能是负责催化 SIBLING 蛋白磷酸化的主要酶。我们发现：1）FAM20C在形成骨、牙本质、牙骨质和牙釉质的细胞中高表达； 2) 小鼠体内 Fam20C 失活导致佝偻病，小鼠骨骼广泛矿化不足； 3）Fam20C-KO小鼠牙本质、牙骨质和牙釉质的形成和矿化存在明显缺陷； 4)Fam20C-KO小鼠骨骼或牙本质中非胶原蛋白的总磷酸化水平显着低于野生型小鼠。此外，我们和其他人揭示了重组 FAM20C 在体外有效磷酸化一些 SIBLING 蛋白。我们的初步数据使我们形成了中心假设，即 FAM20C 是催化 SIBLING 蛋白磷酸化的主要激酶，Fam20C-KO 小鼠的硬组织缺陷可能是由于 SIBLING 蛋白磷酸化的显着丧失造成的。为了检验这一新假设，我们提出以下三个具体目标：（1）确定 FAM20C 缺陷对 SIBLING 蛋白磷酸化的影响。我们将从 Fam20C-KO 小鼠的骨骼和牙本质中分离 OPN、BSP、DMP1 的 C 端片段和 DPP，并将这些蛋白质的磷酸化水平与 WT 小鼠组织中的磷酸化水平进行比较。我们还将在源自 Fam20C-KO 和 WT 小鼠的颅骨成骨细胞培养物中通过 32P 掺入测定检查 FAM20C 对 SIBLING 蛋白磷酸化的影响。 (2) 通过用蛋白酶消化 OPN、BSP、DMP1 C 端片段和 DPP，分离蛋白水解肽并对分离的肽进行测序，检查从 Fam20C-KO 和 WT 小鼠的骨骼和牙本质中分离的 SIBLING 蛋白中的磷酸丝氨酸与质谱分析。 (3)研究从Fam20C-KO小鼠中分离的SIBLING蛋白对成骨细胞和成牙本质细胞形成矿化结节的影响。本申请提出的多管齐下的方法将增进我们对 FAM20C 在 SIBLING 蛋白磷酸化中的作用的理解，并为进一步阐明与 SIBLING 磷酸化相关的生物矿化机制奠定基础。