HUMAN MESOTRYPSIN S195A - COMPLEX WITH APPI - CRYSTAL SCREEN CONDITION #33

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 人胰酶同工酶有三种：阳离子胰酶、阴离子胰酶和中胰酶。所有这三种酶都是由胰腺作为消化酶原产生和分泌的，在其他组织中也有少量产生。胰蛋白酶通常是通过内源性小蛋白抑制剂的强烈抑制来调节的，但次要同工酶MesoTrypsin是唯一抵抗抑制的，它与小蛋白抑制剂结合并作为底物降解它们。通过清除保护性胰腺胰蛋白酶抑制剂SPINK1，MesoTrypsin的抑制剂降解活性被认为在胰腺炎的发生发展中发挥了作用。中胰蛋白酶在脑中也有高表达，并被认为在多发性硬化症的病理机制中参与髓鞘碱性蛋白的加工。此外，我们实验室的初步工作表明，中胰蛋白酶在几种癌症的恶变过程中发挥了作用。酶活性部位的单一氨基酸变化，即精氨酸取代保守的甘氨酸，似乎完全是导致中胰蛋白酶对蛋白质抑制剂产生抗性的原因。基于现有的几种胰酶/抑制剂复合体的X射线结构和未络合的MesoTrypsin的结构的模拟研究表明，这种精氨酸残基干扰了抑制剂在活性部位的对接，并且在MesoTrypsin能够与蛋白质抑制剂结合之前，必须在酶/抑制剂界面发生实质性的构象变化。中胰酶与各种胰酶抑制剂的相对结合亲和力与其他胰酶的偏好不同，这表明中胰酶/抑制物结合能可能受不同的相互作用所支配，而不是那些决定更多研究较好的胰酶特异性的相互作用。我们的目标是解决人中胰酶和人阳离子胰蛋白酶与几种蛋白抑制剂的复合体结构，包括牛胰腺胰蛋白酶抑制物(BPTI)、阿尔茨海默病前体蛋白抑制域(APPI)和大豆胰蛋白酶抑制物(SBTI)。我们希望这些结构的比较(1)将揭示允许中胰蛋白酶结合抑制剂的构象变化，(2)将提供线索来解释为什么中胰酶与阳离子胰蛋白酶相比能够切割结合的抑制剂，以及(3)将深入了解中胰蛋白酶和阳离子胰蛋白酶的不同特性。中胰蛋白酶参与了许多人类疾病，使其成为潜在的药物靶点，对该酶抑制剂耐药性的结构了解将有助于药物开发的努力。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. There are three human trypsin isozymes: cationic trypsin, anionic trypsin, and mesotrypsin. All three are produced and secreted by the pancreas as digestive proenzymes, and are also produced to a lesser extent in other tissues. Trypsins are typically regulated through strong inhibition by endogenous small protein inhibitors, but the minor isozyme mesotrypsin is uniquely resistant to inhibition, binding with reduced affinity to small protein inhibitors and degrading them as substrates. The inhibitor-degrading activity of mesotrypsin has been postulated to play a role in the development of pancreatitis, through clearance of the protective pancreatic trypsin inhibitor SPINK1. Mesotrypsin is also highly expressed in the brain, and has been postulated to play a role in the processing of myelin basic protein in the pathological mechanism of multiple sclerosis. In addition, preliminary work in our own laboratory suggests that mesotrypsin plays a role in progression to malignancy in several cancers. A single amino acid change in the enzyme active site, substitution of arginine for a conserved glycine, appears to be wholly responsible for the resistance of mesotrypsin to protein inhibitors. Modeling studies based on existing X-ray structures for several trypsin/inhibitor complexes and the structure of uncomplexed mesotrypsin indicate that this arginine residue interferes with inhibitor docking in the active site, and that substantial conformational changes must occur at the enzyme/inhibitor interface before mesotrypsin can bind to a protein inhibitor. The relative binding affinities of mesotrypsin to a variety of trypsin inhibitors do not parallel the preferences of other trypsins, indicating that mesotrypsin/inhibitor binding energetics may be dominated by different interactions than those that determine the specificities of more well-studied trypsins. It is our aim to solve the structures of human mesotrypsin and human cationic trypsin in complex with several protein inhibitors, including bovine pancreatic trypsin inhibitor (BPTI), Alzheimer precursor protein inhibitor domain (APPI), and soybean trypsin inhibitor (SBTI). We expect that comparison of these structures (1) will reveal the conformational changes that allow mesotrypsin to bind inhibitors, (2) will give clues to explain why mesotrypsin, in contrast to cationic trypsin, is then able to cleave bound inhibitors, and (3) will give insight into the different specificities of mesotrypsin and cationic trypsin. The involvement of mesotrypsin in a number of human diseases makes it a potential drug target, and a structural understanding of the inhibitor resistance of this enzyme will facilitate efforts towards drug development.