Structural and Functional Analysis of Dentin Proteins

牙本质蛋白的结构和功能分析

• 批准号: 7569025
• 负责人: YASUO YAMAKOSHI
• 金额: $ 34.2万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-20 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7569025
• 关键词: Affect; American; Antibodies; Binding; Biological; Biological Assay; Cell-Matrix Junction; Cleaved cell; Collagen; Collagen Type I; DSPP gene; Defect; Dental; Dental crowns; Dentin; Dentin Dysplasia; Dentin Formation; Dentinogenesis Imperfecta; Development; Diagnosis; Digestion; Disease; Electron Microscopy; Elements; Environmental Risk Factor; Enzymes; Extracellular Matrix; Future; Gelatinase A; Gene Expression; Genes; Genetic; Goals; Health Benefit; Human; In Vitro; Injury; Intervention; Life; Light; Location; MMP-20; Maintenance; Molecular; N-terminal; Natural regeneration; Odontoblasts; Pathology; Peptide Hydrolases; Peptides; Procedures; Process; Property; Proteins; Proteolysis; Public Health; Recombinants; Research; Role; Site; Staging; Structural Protein; Testing; Tooth structure; base; biomineralization; improved; in vivo; repaired

DESCRIPTION (provided by applicant): Our long-term goal is to understand the molecular mechanisms of dentin biomineralization. The most abundant and important molecules in dentin are type I collagen and dentin sialophosphoprotein (DSPP). DSPP is a multifunctional protein that is cleaved into component parts by proteases. Proteases act by altering the properties of the proteins they cleave: by activating them, by allowing subunits to separate and function in different locations, and by degrading or inactivating them. Proteolysis is central to the biological activity of DSPP, and tailors its activities to serve the various requirements of intertubular and peritubular dentin formation and the maturation of dentin. Specific Aim 1: To identify the protease that catalyzes the initial cleavage of DSPP (generating DPP) and to determine if early cleavage products (DSP, DGP, and DPP) segregate preferentially into predentin, peritubular dentin, or intertubular dentin. The protease that releases DPP is assayed during purification procedures using a fluorescent peptide and confirmed by characterizing its cleavage of recombinant DSPP. Region specific DSPP antibodies are used in light and electron microscopy to immunolocalize each structural/functional domain. Special procedures are used to separate predentin and peritubular dentin from whole dentin, and DSPP-derived proteins in each extract are characterized. Specific Aim 2: To determine which DSPP-derived proteins are structural (long-lived) and which are transient (degraded) and characterize the enzymatic processes that determine this. Proteins are extracted at four successive stages of crown development and DSPP cleavage products in each stage are characterized. N-terminal sequencing identifies the specific cleavage sites catalyzed by enzymes in vivo. In vitro digestions identify the proteases that catalyze these cleavages. Specific Aim 3: To characterize the structural/functional properties of the major early DSPP- derived proteins and their long-lived cleavage products. DSPP proteins are tested for their ability to promote cell attachment and induce relevant changes in gene expression, and to bind collagen. The basis for DPP structural diversity is also determined. This research will advance our understanding of how proteases activate DSPP to promote predentin, peritubular and intertubular dentin formation, and degrade DSPP to facilitate dentin maturation. Understanding how dentin forms and hardens will help us induce the biological repair and regeneration of teeth and improve the diagnosis and treatment of dentin pathologies, which can arise from genetic or environmental factors, injury and disease. Defects in the dentin sialophosphoprotein (DSPP) gene cause dentinogenesis imperfecta and dentin dysplasia. We investigate how proteases cleave DSPP to support peritubular and intertubular dentin formation and degrade DSPP to promote hardening of formed dentin. A better understanding of dentin biomineralization is needed to develop treatments that induce the repair and regeneration of teeth.

描述（由申请人提供）：我们的长期目标是了解牙本质生物矿化的分子机制。牙本质中最丰富和最重要的分子是I型胶原蛋白和牙本质唾液磷蛋白（DSPP）。 DSPP是一种多功能蛋白，通过蛋白酶裂解成组件部分。蛋白酶通过改变它们切割的蛋白质的特性来起作用：通过激活它们，通过允许亚基分离并在不同位置分离和功能，并通过降解或灭活它们来分离和功能。蛋白水解对于DSPP的生物学活性至关重要，并为其活性定制，以满足细胞间和周围牙本质形成和牙本质成熟的各种要求。具体目的1：确定催化DSPP（生成DPP）初始切割的蛋白酶，并确定早期裂解产物（DSP，DGP和DPP）是否优先将其隔离到predentin，peperentin，周围牙本质牙本质牙本质或天间牙本质中。使用荧光肽在纯化过程中测定释放DPP的蛋白酶，并通过表征其重组DSPP的裂解来确认。区域特异性DSPP抗体用于光和电子显微镜，以使每个结构/功能域进行免疫定位。特殊的程序用于将预易蛋白和周围牙本质与整个牙本质分开，并且在每种提取物中的DSPP衍生蛋白质都具有表征。特定目的2：确定哪种DSPP衍生的蛋白质是结构性的（长寿），哪些是短暂的（降解），并表征了确定这一点的酶促过程。蛋白质是在冠状发育的连续四个阶段提取的，并且在每个阶段都表征了DSPP裂解产物。 N末端测序识别体内酶催化的特定裂解位点。体外消化确定催化这些切割的蛋白酶。特定目的3：表征主要早期DSPP蛋白质及其长寿命裂解产物的结构/功能特性。 DSPP蛋白的促进细胞附着并诱导基因表达和结合胶原蛋白的相关变化的能力进行了测试。还确定了DPP结构多样性的基础。这项研究将促进我们对蛋白酶如何激活DSPP的理解，以促进前蛋白，周围和细胞间牙本质形成，并降解DSPP以促进牙本质成熟。了解牙本质形成和硬化如何帮助我们诱导牙齿的生物修复和再生，并改善牙本质病理的诊断和治疗，这可能是由遗传或环境因素，损伤和疾病引起的。牙本质唾液磷酸蛋白（DSPP）基因的缺陷导致牙齿生成不完美和牙本质发育不良。我们研究了蛋白酶如何裂解DSPP以支持周围和细胞间牙本质形成并降解DSPP以促进形成的牙本质的硬化。需要更好地了解牙本质生物矿化，以开发诱导牙齿修复和再生的治疗方法。