Structural and Functional Analysis of Dentin Proteins

牙本质蛋白的结构和功能分析

• 批准号: 8197836
• 负责人: JAMES P SIMMER
• 金额: $ 33.52万
• 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 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8197836
• 关键词: Affect; American; Antibodies; Binding; Biological; Biological Assay; Cell-Matrix Junction; Cleaved cell; Collagen; Collagen Type I; Cytoskeleton; 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; 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; light microscopy; 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型胶原和牙本质涎磷蛋白（DSPP）。DSPP是一种多功能蛋白质，其被蛋白酶切割成组成部分。蛋白酶通过改变它们切割的蛋白质的性质来起作用：通过激活它们，通过允许亚基在不同位置分离和发挥功能，以及通过降解或灭活它们。蛋白水解是DSPP的生物活性的核心，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要求。具体目标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）是否优先分离至前牙本质、小管周围牙本质或小管间牙本质。在纯化过程中使用荧光肽测定释放DPP的蛋白酶，并通过表征其对重组DSPP的切割来确认。区域特异性DSPP抗体用于光学和电子显微镜以免疫定位每个结构/功能结构域。使用特殊的程序从整个牙本质中分离前牙本质和管周牙本质，并对每种提取物中的DSPP衍生蛋白进行表征。具体目标二：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。蛋白质提取在四个连续阶段的冠发展和DSPP裂解产物在每个阶段的特点。N-末端测序鉴定由酶在体内催化的特异性切割位点。体外酶切鉴定催化这些裂解的蛋白酶。具体目标3：表征主要早期DSPP衍生蛋白及其长寿命裂解产物的结构/功能特性。测试DSPP蛋白促进细胞附着和诱导基因表达的相关变化以及结合胶原蛋白的能力。还确定了DPP结构多样性的基础。本研究将进一步了解蛋白酶如何激活DSPP促进前牙本质、管周牙本质和管间牙本质的形成，以及降解DSPP促进牙本质成熟。了解牙本质如何形成和硬化将有助于我们诱导牙齿的生物修复和再生，并改善牙本质病变的诊断和治疗，这些病变可能由遗传或环境因素、损伤和疾病引起。牙本质涎磷蛋白（DSPP）基因缺陷可导致牙本质发育不良和牙本质发育不良。我们研究了蛋白酶如何切割DSPP以支持管周和管间牙本质的形成，以及如何降解DSPP以促进形成的牙本质的硬化。更好地了解牙本质生物矿化需要开发治疗，诱导牙齿的修复和再生。

项目成果

Porcine Amelogenin : Alternative Splicing, Proteolytic Processing, Protein - Protein Interactions, and Possible Functions.

• DOI: 10.1016/s1349-0079(11)80011-3
• 发表时间: 2011
• 期刊: Journal of oral biosciences
• 影响因子: 2.4
• 作者: Y. Yamakoshi

Structural features, processing mechanism and gene splice variants of dentin sialophosphoprotein.

牙本质唾液酸磷蛋白的结构特征、加工机制和基因剪接变异体。

• DOI: 10.1016/j.jdsr.2018.03.006
• 发表时间: 2018
• 期刊: The Japanese dental science review
• 作者: Yamakoshi,Yasuo;Simmer,JamesP
• 通讯作者: Simmer,JamesP

Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments.

• DOI: 10.1186/1471-2091-12-6
• 发表时间: 2011-02-03
• 期刊: BMC biochemistry
• 作者: Yamakoshi Y;Nagano T;Hu JC;Yamakoshi F;Simmer JP
• 通讯作者: Simmer JP

A Large Chondroitin Sulfate Proteoglycan, Versican, in Porcine Predentin.

猪 Predentin 中的一种大型硫酸软骨素蛋白多糖，Versacan。

• DOI: 10.2330/joralbiosci.53.72
• 发表时间: 2011
• 期刊: Journal of oral biosciences
• 影响因子: 2.4
• 作者: Okahata,Saori;Yamamoto,Ryuji;Yamakoshi,Yasuo;Fukae,Makoto
• 通讯作者: Fukae,Makoto

Astacin proteases cleave dentin sialophosphoprotein (Dspp) to generate dentin phosphoprotein (Dpp).

• DOI: 10.1002/jbmr.202
• 发表时间: 2011-01
• 期刊: JOURNAL OF BONE AND MINERAL RESEARCH
• 影响因子: 6.2
• 作者: Tsuchiya, Shuhei;Simmer, James P.;Hu, Jan C-C;Richardson, Amelia S.;Yamakoshi, Fumiko;Yamakoshi, Yasuo
• 通讯作者: Yamakoshi, Yasuo