DSP-PP Precursor Protein Processing

DSP-PP 前体蛋白质加工

• 批准号: 8197913
• 负责人: HELENA H Ritchie
• 金额: --
• 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 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8197913
• 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; Fluorescein-5-isothiocyanate; 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; System; Testing; Tissue Differentiation; Tissue Sample; Tissues; Tooth Diseases; Tooth structure; Transcript; abstracting; 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; rat Dspp protein; research study; tool; vector

Abstract: 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.

摘要： 牙本质涎蛋白(DSP)和磷蛋白(PP)是两种含量最丰富的非胶原蛋白 牙本质中的蛋白质，以及最近在骨骼、肾脏和唾液腺中发现的蛋白质， 提示DSP-PP基因可能参与了多种过程。 器官发生。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。迄今为止，它还没有 之所以能够研究DSP-PP的翻译后加工和切割，是因为没有DSP- PP前体蛋白已经在任何细胞或组织样本中被确定，留下了这样的未回答 问题如DSP-PP在哪里发生切割(即细胞内还是细胞外)以及什么 可能与裂解酶(S)有关。为了回答这些DSP-PP蛋白质处理问题， 我们利用杆状病毒表达系统表达了重组的DSP-PP前体蛋白 来自一个DSP-PP240 cDNA，它代表几个内源DSP-PP转录本之一 据信在牙本质矿化过程中起着不同的作用。我们最近在《生物周刊》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，并且能够自我处理以产生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。这项提议的具体目的是利用各种 不同重组DSP-PP前体蛋白组成对DSP-PP的研究 在各种表达系统中的处理(目标1)；确定第一个DSP-PP裂解位点 利用定点突变(目标2)；检测DSP-PP裂解缺陷的功能效应 突变体可能对牙髓细胞矿化有影响(目标3)；在目标4中，我们将研究 用动力学研究和酶抑制剂研究DSP-PP和PP的蛋白水解性 确定特定PP结构域中的残基如何影响PP蛋白分解。我们期待着 这一提议可能会导致一类新的蛋白酶的特性。我们也 预计这项提议将导致特定的单核苷酸的鉴定 PP基因的多态性可能与牙齿相关的异常有关。