DSP-PP Precursor Protein Processing

描述(申请人提供)：牙本质唾液蛋白(DSP)和磷蛋白(PP)是牙本质中含量最丰富的两种非胶原蛋白，最近在骨、肾脏和唾液腺中发现，提示DSP-PP基因可能参与器官发生的各种过程。DSP和PP的编码序列来源于单个的DSP-PP基因，但在牙本质中，DSP：PP的比例是1：6，而不是预期的1：1。到目前为止，由于在任何细胞或组织样本中都没有发现dsp-PP前体蛋白，因此还不可能对dsp-PP的翻译后加工和切割进行研究，这就留下了一些悬而未决的问题，如dsp-PP在哪里发生切割(即细胞内还是细胞外)以及什么切割酶(S)可能参与其中。为了回答这些DSP-PP蛋白加工问题，我们利用杆状病毒表达系统，从DSP-PP240 cDNA中产生重组的DSP-PP前体蛋白，这是几个被认为在牙本质矿化过程中发挥不同作用的内源性DSP-PP转录本之一。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的，并且能够自我加工得到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。这项建议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(目标1)；利用定点突变来确定第一个DSP-PP裂解位点(目标2)；检测DSP-PP裂解缺陷突变体对牙髓细胞矿化的功能影响(目标3)；以及在目标4中，我们将使用动力学研究和蛋白酶抑制剂来研究DSP-PP和PP的蛋白分解活性，并确定特定PP结构域中的残基如何影响PP蛋白分解。我们预计这项提议可能会导致一类新的蛋白酶的特征。我们还期望这一建议将导致识别PP中可能与牙科相关异常相关的特定单核苷酸多态。 与公共健康相关：牙齿发育需要一组复杂的蛋白质来将预矿化的牙本质转化为矿化的牙本质。发育中牙齿中存在两种非胶原蛋白，即牙本质涎蛋白(DSP)和磷蛋白(PP)，它们参与矿化过程。虽然这两种蛋白质是在同一个基因上编码的，但人们对它们是如何在牙齿矿化部位产生DSP和PP的知之甚少。本研究的目的是定义调节DSP-PP前体蛋白切割的DSP-PP加工事件。这项研究应该会对牙齿发育生物学有更好的理解。

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.