Structural and Functional Analysis of Dentin Proteins

描述（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是一种多功能蛋白，可被蛋白酶裂解成组成部分。蛋白酶通过改变它们所切割的蛋白质的性质来起作用：激活它们，允许亚基在不同的位置分离和起作用，以及降解或使它们失活。蛋白水解是DSPP生物活性的核心，并调整其活性以满足管间和管周牙本质形成和牙本质成熟的各种需求。特异性目的1：鉴定催化DSPP初始裂解（生成DPP）的蛋白酶，并确定早期裂解产物（DSP、DGP和DPP）是否优先分离为前牙本质、管周牙本质或管间牙本质。释放DPP的蛋白酶在纯化过程中使用荧光肽进行分析，并通过表征其对重组DSPP的裂解来确认。区域特异性DSPP抗体在光镜和电子显微镜下用于免疫定位每个结构/功能域。使用特殊程序从整个牙本质中分离牙本质前质和小管周围牙本质，并对每种提取物中的dspp衍生蛋白进行表征。具体目标2：确定哪些dspp衍生蛋白是结构性的（长寿命的），哪些是瞬时的（降解的），并表征决定这一点的酶促过程。在树冠发育的四个连续阶段提取蛋白质，并对每个阶段的DSPP裂解产物进行表征。n端测序在体内鉴定酶催化的特定裂解位点。在体外消化鉴定催化这些裂解的蛋白酶。具体目标3：表征主要的早期DSPP衍生蛋白及其长寿命裂解产物的结构/功能特性。DSPP蛋白能够促进细胞附着，诱导相关基因表达变化，并结合胶原蛋白。确定了DPP结构多样性的基础。本研究将有助于我们进一步了解蛋白酶如何激活DSPP促进牙本质前、小管周围和管间形成，并降解DSPP促进牙本质成熟。了解牙本质是如何形成和硬化的，将有助于我们诱导牙齿的生物修复和再生，并提高牙本质病理的诊断和治疗，这些病理可能是由遗传或环境因素、损伤和疾病引起的。牙本质唾液磷蛋白（DSPP）基因缺陷导致牙本质发育不全和牙本质发育不良。我们研究了蛋白酶如何裂解DSPP以支持小管周围和管间牙本质的形成，并降解DSPP以促进形成的牙本质硬化。为了更好地了解牙本质生物矿化，需要开发诱导牙齿修复和再生的治疗方法。

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.