DSP-PP Precursor Protein Processing

摘要： 牙本质涎蛋白（DSP）和磷酸蛋白（PP）是牙本质中最丰富的两种非胶原蛋白， 牙本质中的蛋白质，并且最近在骨、肾和唾液腺中发现， 这表明DSP-PP基因可能参与多种过程， 器官发生DSP和PP编码序列来源于单个DSP-PP基因，但在 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， 研究DSP-PP翻译后加工和切割是可能的，因为没有DSP- PP前体蛋白已在任何细胞或组织样品中鉴定，留下未回答的此类问题。 如DSP-PP裂解发生在何处的问题（即，细胞内或细胞外）， 可能涉及切割酶。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生产重组DSP-PP前体蛋白 来自DSP-PP 240 cDNA，其代表几种内源性DSP-PP转录物之一 在牙本质矿化过程中发挥着不同的作用。我们最近在J. Biol. 化学和我们的初步结果表明，DSP-PP 240前体蛋白是 由该系统产生，并且能够自处理以产生DSP和PP蛋白。 本申请提出了一系列的研究，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，以研究DSP-PP 在各种表达系统中进行加工（目的1）;确定第一个DSP-PP切割位点 使用位点诱变（目的2）;检查DSP-PP切割缺陷的功能效应 突变体可能对牙髓细胞矿化的影响（目的3）;在目的4中，我们将研究 使用动力学研究和蛋白酶抑制剂的DSP-PP和PP的蛋白水解活性，和 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 该建议将潜在地导致一类新的蛋白酶的表征。我们也 我希望这一建议将导致识别特定的单核苷酸 可能与牙齿相关异常相关的PP多态性。

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.