HUMAN CATIONIC TRYPSIN S195A/R117H - COMPLEX WITH BPTI - CRYSTAL SCREEN CONDITIO

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 有三种人胰蛋白酶同工酶：阳离子胰蛋白酶、阴离子胰蛋白酶和中胰蛋白酶。 所有这三种都是由胰腺作为消化酶原产生和分泌的，并且在其他组织中也以较小的程度产生。 胰蛋白酶通常通过内源性小蛋白抑制剂的强烈抑制来调节，但次要同工酶中胰蛋白酶独特地对抑制具有抗性，以降低的亲和力与小蛋白抑制剂结合并将其降解为底物。 中胰蛋白酶的降解活性被认为通过清除保护性胰蛋白酶抑制剂SPINK 1在胰腺炎的发展中发挥作用。 中胰蛋白酶也在脑中高度表达，并且已经假定在多发性硬化的病理机制中在髓鞘碱性蛋白的加工中发挥作用。 此外，我们实验室的初步研究表明，中胰蛋白酶在几种癌症的恶性进展中发挥作用。 酶活性位点中的单个氨基酸变化，精氨酸取代保守的甘氨酸，似乎是中胰蛋白酶对蛋白质抑制剂的抗性的全部原因。 基于现有的几种胰蛋白酶/抑制剂复合物的X-射线结构和未复合的中胰蛋白酶的结构的建模研究表明，该精氨酸残基干扰抑制剂在活性位点的对接，并且在中胰蛋白酶可以结合到蛋白质抑制剂之前，必须在酶/抑制剂界面发生实质性的构象变化。 中胰蛋白酶对各种胰蛋白酶抑制剂的相对结合亲和力与其他胰蛋白酶的偏好不平行，表明中胰蛋白酶/抑制剂结合能可能受不同相互作用的支配，而不是那些确定更充分研究的胰蛋白酶特异性的相互作用。 我们的目标是解决人中胰蛋白酶和人阳离子胰蛋白酶与几种蛋白质抑制剂复合物的结构，包括牛胰蛋白酶抑制剂（BPTI），阿尔茨海默病前体蛋白抑制剂结构域（APPI）和大豆胰蛋白酶抑制剂（SBTI）。 我们期望这些结构的比较（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蛋白酶与阳离子胰蛋白酶相反，然后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异性。 中胰蛋白酶在许多人类疾病中的参与使其成为潜在的药物靶标，并且对这种酶的抑制剂抗性的结构理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.