DSP-PP Precursor Protein Processing

DSP-PP 前体蛋白质加工

• 批准号: 7725834
• 负责人: HELENA H Ritchie
• 金额: $ 34.41万
• 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/7725834
• 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（由申请人提供）：牙本质唾液蛋白（DSP）和磷phophoryn（PP）是牙本质中两种最丰富的非胶原蛋白，最近在骨，肾脏和唾液腺中发现了骨骼，肾脏和唾液腺，这表明DSP-PP PPP基因可能参与了在机器发生过程中的各种过程中。 DSP和PP编码序列源自单个DSP-PP基因，但在Dentin中存在DSP：PP的1：6比例，而不是预期的1：1比率。迄今为止，由于在任何细胞或组织样品中都没有发现DSP-PP的翻译后处理和裂解，因此无法研究DSP-PP，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，因此在诸如dsp-pp-ppp裂解的地方（即细胞内或外部或外部外部或外部外部）以及哪些cleaeavage and cleavage（可能涉及dsp-pp）。为了回答这些DSP-PP蛋白质处理问题，我们利用了杆状病毒表达系统从DSP-PP240 cDNA中产生重组DSP-PP-PP PREP蛋白，这代表了在牙本质矿化过程中据信被认为起着不同作用的几种内源性DSP-PP-PPP转录本之一。我们最近在J. Biol的出版物。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，并且能够自我处理以产生DSP和PP蛋白。该应用提出了一系列研究，以更好地定义和表征DSP-PP PRECROR蛋白处理。该建议的具体目的是利用各种不同的重组DSP-PP PPP前体蛋白组成来研究各种表达系统中的DSP-PP处理（AIM 1）；使用位点诱变确定第一个DSP-PP裂解位点（AIM 2）；为了检查DSP-PP裂解有缺陷突变体可能对牙髓细胞矿化有缺陷的功能效应（AIM 3）；在AIM 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结构域中的残基如何影响PP蛋白水解。我们预计该建议将有可能导致新的蛋白酶的表征。我们还希望该建议将导致PP中特定的单核苷酸多态性的鉴定，这可能与牙齿相关的异常有关。 公共卫生相关性：牙齿发育需要一组复杂的蛋白质，将矿物化牙本质转换为矿物化牙本质。发育中的两种非胶原蛋白存在于发育中的牙本质唾液蛋白（DSP）和磷phophophoryn（PP）中，参与了矿化过程。尽管这两种蛋白质是在同一基因上编码的，但几乎不知道如何处理它们在牙齿矿化位点产生DSP和PP。这项研究的目的是定义调节DSP-PP PPP前体蛋白裂解的DSP-PP处理事件。这项研究应导致对牙齿发育生物学的更多了解。