DSP-PP Precursor Protein Processing

描述（申请人提供）：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，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编码序列来自单个DSP-PP基因，但在牙本质中存在1：6的DSP：PP比例，而不是预期的1：1比例。迄今为止，还不可能研究DSP-PP翻译后加工和切割，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，留下了诸如DSP-PP切割在何处发生（即，细胞内或细胞外）以及可能涉及的裂解酶。为了回答这些DSP-PP蛋白质加工的问题，我们利用杆状病毒表达系统从DSP-PP 240 cDNA中产生重组DSP-PP前体蛋白，DSP-PP 240 cDNA代表了几种内源性DSP-PP转录本之一，据信在牙本质矿化过程中发挥不同的作用。我们最近在J. Biol. Chemistry上发表的文章和我们的初步结果表明，DSP-PP 240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前体蛋白质加工。本研究的具体目的是利用各种不同的重组DSP-PP前体蛋白组合物来研究DSP-PP在各种表达系统中的加工（目的1）;利用定点诱变来确定DSP-PP的第一切割位点（目的2）;检查DSP-PP切割缺陷突变体对牙髓细胞矿化可能具有的功能效应（目的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结构域中的残基如何影响PP蛋白水解。我们预计，这一建议将可能导致一类新的蛋白酶的表征。我们还希望，这一建议将导致识别特定的单核苷酸多态性PP可能与牙齿相关的异常。 公共卫生相关性：牙齿发育需要一组复杂的蛋白质将预矿化牙本质转化为矿化牙本质。牙本质涎蛋白（DSP）和磷酸蛋白（PP）是牙齿发育过程中存在的两种非胶原蛋白，参与矿化过程。虽然这两种蛋白质在同一基因上编码，但很少有人知道它们是如何在牙齿矿化位点加工产生DSP和PP的。本研究的目的是确定DSP-PP加工事件，调节DSP-PP前体蛋白切割。这项研究将有助于更好地了解牙齿发育生物学。

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.