DSP-PP Precursor Protein Processing

DSP-PP 前体蛋白质加工

• 批准号: 7583614
• 负责人: HELENA H Ritchie
• 金额: $ 33.14万
• 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: 2008-12-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7583614
• 关键词: Affect; Amino Acid Sequence; Amino Acids; Applications Grants; Area; Baculovirus Expression System; Baculoviruses; Binding; C-terminal; Canis familiaris; Caseins; Cells; Chemistry; Cleaved cell; Code; Complementary DNA; Complex; Culture Media; Defect; Dental; Dental Pulp; Dentin; Dentin Dysplasia; Dentin Formation; Dentinogenesis Imperfecta; Development; Developmental Biology; Diagnostic; Didelphidae; Enzymes; Escherichia coli; Event; Exhibits; Extracellular Space; Gel; Gelatin; Gelatin Zymography; Genes; Genetic Processes; Human; Hydroxyapatites; Immunohistochemistry; In Situ; In Vitro; Insecta; Kidney; Kinetics; Knockout Mice; Lead; Left; Length; Link; Macaca mulatta; Methods; Molecular; Molecular Genetics; Monitor; Mus; Mutagenesis; Mutate; Mutation; Organogenesis; Pan Genus; Paper; Peptide Hydrolases; Peptides; Phosphorylation; Play; Positioning Attribute; Procedures; Process; Protease Inhibitor; Protein Isoforms; Protein Precursors; Proteins; Proteolysis; Publications; Publishing; Rattus; Recombinant Proteins; Recombinants; Research Proposals; Role; Salivary Glands; Series; Set protein; Single Nucleotide Polymorphism; Site; Site-Directed Mutagenesis; Structure of thyroid parafollicular cell; System; Testing; Tissue Differentiation; Tissue Sample; Tissues; Tooth Diseases; Tooth structure; Transcript; base; bone; dentin sialoprotein; expression vector; gel electrophoresis; glycosylation; hearing impairment; human DLC1 protein; in vivo; mRNA Expression; mineralization; mutant; phosphophoryn; polyanion; programs; protein expression; public health relevance; rat Dspp protein; research study; tool; vector

DESCRIPTION (provided by applicant): Dentin sialoprotein (DSP) and phosphophoryn (PP) are the two most abundant noncollagenous proteins in dentin, and have more recently been found in bone, kidney and salivary glands, suggesting that the DSP-PP gene may participate in a variety of processes during organogenesis. DSP and PP coding sequences are derived from a single DSP-PP gene, yet in dentin there exists a 1:6 ratio of DSP:PP instead of the expected 1:1 ratio. To date it has not been possible to study DSP-PP post-translational processing and cleavage because no DSP- PP precursor protein has been identified in any cell or tissue sample, leaving unanswered such questions as where DSP-PP cleavage occurs (i.e., intracellularly or extracellularly) and what cleavage enzyme(s) may be involved. To answer these DSP-PP protein-processing questions, we utilized a baculovirus expression system to produce recombinant DSP-PP precursor proteins from a DSP-PP240 cDNA, which represents one of several endogenous DSP-PP transcripts believed to play different roles during dentin mineralization. Our recent publication in the J. Biol. Chemistry, and our Preliminary Results, demonstrate that DSP-PP240 precursor proteins are produced by this system, and are capable of self-processing to yield both DSP and PP proteins. This application proposes a series of studies to better define and characterize DSP-PP precursor protein processing. The Specific Aims of this proposal are to utilize a variety of different recombinant DSP-PP precursor protein compositions to investigate DSP-PP processing in various expression systems (Aim 1); to determine the first DSP-PP cleavage site using site mutagenesis (Aim 2); to examine functional effects that DSP-PP cleavage defective mutants may have on dental pulp cell mineralization (Aim 3); and in Aim 4 we will investigate proteolytic activity of DSP-PP and PP using kinetic studies and protease inhibitors, and determine how residues within specific PP domains may affect PP proteolysis. We expect that this proposal will potentially lead to the characterization of a new class of protease. We also expect that this proposal will lead to the identification of specific single nucleotide polymorphisms in PP that may be associated with dental related abnormalities. PUBLIC HEALTH RELEVANCE: Tooth development requires a complex set of proteins to convert pre-mineralized dentin to mineralized dentin. Two non-collagenous proteins present in developing teeth, dentin sialoprotein (DSP) and phosphophoryn (PP), participate in the mineralization process. While these two proteins are encoded on the same gene, little is know about how they are processed to produce DSP and PP at tooth mineralization sites. The aim of this study is to define the DSP-PP processing events that regulate DSP-PP precursor protein cleavage. This study should lead to a greater understanding of tooth developmental biology.

描述（申请人提供）：牙本质唾液蛋白（DSP）和磷酸蛋白（PP）是牙本质中最丰富的两种非胶原蛋白，最近在骨、肾和唾液腺中发现，表明DSP-PP基因可能参与器官发生过程中的多种过程。 DSP 和 PP 编码序列源自单个 DSP-PP 基因，但在牙本质中，DSP:PP 的比例为 1:6，而不是预期的 1:1 比例。迄今为止，还不可能研究 DSP-PP 翻译后加工和裂解，因为在任何细胞或组织样品中都没有鉴定出 DSP-PP 前体蛋白，从而留下了诸如 DSP-PP 裂解发生在何处（即细胞内或细胞外）以及可能涉及哪些裂解酶等问题。为了回答这些 DSP-PP 蛋白质加工问题，我们利用杆状病毒表达系统从 DSP-PP240 cDNA 产生重组 DSP-PP 前体蛋白，它代表了几种内源 DSP-PP 转录物之一，据信在牙本质矿化过程中发挥不同的作用。我们最近在《生物杂志》上发表的文章。化学和我们的初步结果表明，DSP-PP240 前体蛋白是由该系统产生的，并且能够自我加工以产生 DSP 和 PP 蛋白。本申请提出了一系列研究，以更好地定义和表征 DSP-PP 前体蛋白加工。该提案的具体目标是利用各种不同的重组 DSP-PP 前体蛋白组合物来研究各种表达系统中的 DSP-PP 加工（目标 1）；使用位点诱变确定第一个 DSP-PP 切割位点（目标 2）；检查 DSP-PP 裂解缺陷突变体可能对牙髓细胞矿化产生的功能影响（目标 3）；在目标 4 中，我们将使用动力学研究和蛋白酶抑制剂研究 DSP-PP 和 PP 的蛋白水解活性，并确定特定 PP 结构域内的残基如何影响 PP 蛋白水解。我们预计该提案将有可能导致一类新蛋白酶的表征。我们还期望该提案将导致 PP 中可能与牙齿相关异常相关的特定单核苷酸多态性的鉴定。 公众健康相关性：牙齿发育需要一组复杂的蛋白质才能将预矿化牙本质转化为矿化牙本质。发育中的牙齿中存在两种非胶原蛋白：牙本质唾液酸蛋白 (DSP) 和磷酸蛋白 (PP)，参与矿化过程。虽然这两种蛋白质由同一基因编码，但人们对它们如何在牙齿矿化位点加工产生 DSP 和 PP 知之甚少。本研究的目的是确定调节 DSP-PP 前体蛋白裂解的 DSP-PP 加工事件。这项研究应该可以加深对牙齿发育生物学的了解。