Molecular Pathomechanism of Hereditary Pancreatitis

描述（申请人的摘要）：本申请的广泛的、长期的目标 应用程序是为了了解人类突变的机制， 阳离子胰蛋白酶原基因（PRSS 1）导致遗传性胰腺炎（HP）。HP是 一种常染色体显性遗传病，特征是早发性复发 急性胰腺炎发作，经常进展为慢性胰腺炎 偶尔也会导致胰腺癌HP属于遗传形式， 特发性慢性胰腺炎是一种遗传异质性疾病， 其中不仅在PRSS 1中发现突变，而且在囊性 纤维化跨膜传导调节因子（CFTR）基因和胰腺 分泌型胰蛋白酶抑制剂基因（SPINK 1）。惠普一直被广泛认为是 所有形式的人类胰腺炎的高度相关的模型系统。在 大多数情况下，三个突变，Arg 117-His，Asn 2l-，Ile和Ala 8-瓦尔， 在PRSSI中识别。由HP突变引起的分子缺陷将 在当前发病机制模型的背景下进行研究，这表明 HP是由胰腺内胰蛋白酶活性过度引起的， 机制：（i）增加胰蛋白酶原激活，（ii）减少胰蛋白酶 降解;或（iii）胰腺分泌胰蛋白酶的抑制作用受损 抑制剂（PSTI）。实验设计的主要目的是 研究体外HP突变的影响，使用重组人 胰蛋白酶原野生型和突变型胰蛋白酶原将在大肠杆菌中表达。 大肠杆菌，并纯化至具有ecotin亲和性的均一性 层析催化性质和自催化降解（自溶） 胰蛋白酶的自激活和自催化降解（酶原分解） 胰蛋白酶原将被表征。此外，野生型之间的相互作用 以及阳离子胰蛋白酶（ogen）与阴离子胰蛋白酶（ogen）的突变形式， 将检查中胰蛋白酶（ogen）。最后，抑制人胰蛋白酶 野生型和突变型PSTI蛋白的同种型和HP突变型胰蛋白酶将被 研究了

DESCRIPTION (Applicant's abstract): The broad, long-term objectives of this application are to understand the mechanism by which mutations in the human cationic trypsinogen gene (PRSS1) lead to hereditary pancreatitis (HP). HP is an autosomal dominant genetic disorder characterized by early-onset recurrent attacks of acute pancreatitis with frequent progression to chronic pancreatitis and occasionally to pancreatic cancer. HP belongs to the inherited forms of idiopathic chronic pancreatitis, a genetically heterogeneous disease group, where mutations have been found not only in PRSS1, but also in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and in the pancreatic secretory trypsin inhibitor gene (SPINK1). HP has been widely recognized as a highly relevant model system for all forms of human pancreatitis. In the majority of cases, three mutations, Arg117-His, Asn2l-,lle, and Ala8-Val, have been identified in PRSSI. The molecular defects caused by the HP mutations will be studied within the context of a current pathogenesis model, which suggests that HP is caused by excessive intrapancreatic trypsin activity via one of 3 mechanisms: (i) increased trypsinogen activation, (ii) decreased trypsin degradation; or (iii) impaired inhibition by pancreatic secretory trypsin inhibitor (PSTI). The principal objective of the experimental design is to study the effects of the HP-mutations in vitro, using recombinant human trypsinogens. Wild-type and mutant trypsinogens will be expressed in Escherichia coli, and purified to homogeneity with ecotin affinity chromatography. Catalytic properties and autocatalytic degradation (autolysis) of trypsins and autoactivation and autocatalytic degradation (zymogenolysis) of trypsinogens will be characterized. In addition, interactions between wild-type and mutant forms of cationic trypsin(ogen) with anionic trypsin(ogen) and mesotrypsin(ogen) will be examined. Finally, inhibition of human trypsin isoforms and HP mutant trypsins by wild-type and mutant PSTI proteins will be studied.