Selective Aggregation and Sorting of Salivary Proteins

描述：这项研究的长期目标是确定唾液腺中受调节的分泌蛋白的分类和存储机制。这个问题的答案对于我们理解唾液腺蛋白质分泌的调节以及唾液的组成是很重要的。在第一个赠款期间，在实现这一目标方面取得了重大进展。结果表明，钙和pH介导的聚集对腮腺分泌颗粒中分泌蛋白的储存没有显著贡献，这表明该腺体的分选不同于其他内分泌腺和外分泌腺。事实上，已经证明了硫酸蛋白多糖是有效储存大鼠腮腺腺泡细胞分泌颗粒中的分泌蛋白所必需的。硫酸蛋白多聚糖似乎缓冲了分泌颗粒的内部pH。这些结果导致了一种假设，即硫酸化蛋白多糖作为缓冲剂，调节腮腺腺泡细胞中蛋白质的分类和储存。特殊目标1将测试硫酸化蛋白多糖是否在腮腺分泌颗粒中起到缓冲剂的作用。目的2将测试蛋白多糖核心蛋白的表达和翻译后修饰是否调节其他腮腺分泌蛋白的分类和储存。这些目标将利用异丙肾上腺素治疗在大鼠腮腺中过表达酸性和碱性PRPS，并结合体外组织培养方法。作为替代，我们将使用在唾液腺中过度表达PRPS的转基因小鼠。在目标3中，将测试硫酸化蛋白多糖是否在PSP的蛋白或膜结合中发挥作用。特定目标4将测试硫酸化蛋白多糖或其他酸性蛋白是否参与颌下腺分泌蛋白的分类和存储，颌下腺是目前基因治疗方案的目标。这项拟议的研究将在唾液细胞和完整动物的蛋白质表达实验中使用细胞生物学和分子方法。这项研究的结果将为深入了解唾液腺中发挥作用的独特分类机制提供依据。这将有助于更好地了解唾液的形成，并为有效地从唾液腺输送治疗性蛋白质提供基础。

DESCRIPTION: The long-term goal of this research is to determine the mechanisms for sorting and storage of regulated secretory proteins in salivary glands. The answer to this question is important for our understanding of the regulation of protein secretion from salivary glands and, hence, the composition of saliva. Significant progress towards this goal was made in the first grant period. It was shown that calcium- and pH-mediated aggregation do not contribute significantly to the storage of secretory proteins in parotid secretory granules, suggesting that sorting in this gland differs from that of other endocrine and exocrine glands. Indeed, it was demonstrated that sulfated proteoglycans are necessary for efficient storage of secretory proteins in secretory granules of rat parotid acinar cells. The sulfated proteoglycan appears to buffer the internal pH of secretory granules. These results led to the hypothesis that sulfated proteoglycans act as buffering agents that regulate protein sorting and storage in parotid acinar cells. Specific Aim 1 will test if sulfated proteoglycans act as buffering agents in parotid secretory granules. Aim 2 will test if expression and) post-translational modification of the proteoglycan core proteins regulates the sorting and storage of other) parotid secretory proteins. These Aims will employ isoproterenol treatment to overexpress acidic and basic PRPs in rat parotid glands combined with in vitro tissue culture approaches. As an alternative, we will use transgenic mice that overexpress PRPs in salivary glands. In Aim 3, it will be tested if sulfated proteoglycans play a role in protein or membrane binding of PSP. Specific Aim 4 will test if sulfated proteoglycans or other acidic proteins are involved in sorting and storage of secretory proteins in the submandibular gland, a current target for gene therapy protocols. The proposed research will use cell biological and molecular methods in salivary cells and protein expression experiments in intact animals. The results of this research will provide insight into the unique sorting mechanisms that function in salivary glands. This will lead to a better understanding of the formation of saliva and provide the basis for efficient delivery of therapeutic proteins from salivary glands.