Human Cathepsin G: Expression, C-Terminal Processing and Dual Specificity

描述（由申请人提供）：人组织蛋白酶G （CatG）和人中性粒细胞弹性酶（HNE）通过中性粒细胞杀死细菌发挥重要的先天免疫作用。这两种丝氨酸蛋白酶都作为活性酶储存在细胞质颗粒中。它们还经历了一个不寻常的处理步骤，去除10和19个氨基酸的羧基末端肽。在HNE的情况下，不能去除这种肽导致转运到膜和中性粒细胞减少。在Aim#1中，这些蛋白酶将通过甲醇酵母菌毕赤酵母（Pichia pastoris）的分泌表达为活性重组酶，这是表达类似酶的一种经过验证的方法。将制作全长和c端截短格式。在目标2中，这些重组酶将与从中性粒细胞中分离的天然蛋白酶进行比较。同样，全长和c端截断的酶将在动力学、与抑制剂的反应以及与膜和脂质体的结合方面进行比较，因为膜结合是c端延伸的可能功能。将比较全长和截断形式的CatG的细胞运输，使用类似形式的HNE作为对照。为了进一步研究异常的c端加工事件的机制，还需要酶的全长形式。虽然HNE上的C-末端延伸已被证明在转运和与膜结合中发挥作用，但CatG上较短的C-末端延伸是否具有相似或不同的功能尚不清楚。在Aim#3中，将研究双特异性CatG。虽然CatG是凝乳胰蛋白酶家族的成员，但它也被证明可以切割胰蛋白酶样底物。这种双重特异性被认为是由于S1底物结合袋中的Glu226，该残基将突变为其他氨基酸，以观察双重特异性是否受到影响。预期的结果将为进一步研究CatG在中性粒细胞和肥大细胞中的功能打开大门，同时提供目前无法获得的重组酶，这将有助于其他研究人员。重组HNE将允许产生由单核苷酸多态性引起的突变体，这可能导致其他疾病，如肺气肿和关节炎。这个区域项目为学生提供了DNA克隆、微生物技术、细胞生物学、发酵方法、蛋白质纯化、结构/功能研究、酶/抑制剂反应和酶动力学方面的经验。本实验室在培养本科生、医学生和研究生方面有着良好的记录。通过这个项目获得的知识将提高我们对中性粒细胞功能和疾病的理解，包括对细菌感染、炎症和中性粒细胞减少症的易感性。这个学术研究增强奖将允许本科生和研究生在医学院环境中获得重要的研究经验，为他们从事科学研究做好准备。将在酵母中产生两种人类蛋白质降解酶，并对其进行研究，以获得有关不寻常加工事件的额外知识。众所周知，这些酶和储存它们的身体细胞在我们抵御细菌感染和包括过敏在内的疾病方面起着至关重要的作用。因此，所获得的知识和经验将有助于我们改善健康的努力。

DESCRIPTION (provided by applicant): Human Cathepsin G (CatG) and human neutrophil elastase (HNE) play significant innate immunity roles via neutrophil killing of bacteria. Both serine proteases are stored as active enzymes in cytoplasmic granules. They also undergo an unusual processing step that removes carboxyl terminal peptides of 10 and 19 amino acids. In the case of HNE, failure to remove this peptide results in trafficking to the membrane and neutropenia. In Aim#1 these proteases will be expressed as active recombinant enzymes via secretion from the methanolic yeast Pichia pastoris, a proven approach for the expression of similar enzymes. Both full-length and C-terminally truncated forms will be produced. In Aim#2 these recombinant enzymes will be compared with native proteases isolated from neutrophils. Likewise, full-length and C-terminally truncated enzymes will be compared with respect to kinetics, reaction with inhibitors and binding to membranes and liposomes, because membrane binding is a possible function of the C-terminal extension. Cellular trafficking of full-length and truncated forms of CatG will be compared, using similar forms of HNE as controls. Full-length forms of the enzymes are also needed for future studies to investigate the mechanism of the unusual C-terminal processing event. Although the C- terminal extension on HNE has been shown to play a role in trafficking and binding to membranes, it is unknown whether the shorter C-terminal extension on CatG has similar or different function. In Aim#3 the dual specificity CatG will be studied. Although CatG is a member of the chymotrypsin family, it has been shown to also cleave trypsin-like substrates. This dual specificity is thought to be due to Glu226 in the S1 substrate binding pocket and this residue will be mutated to other amino acids to see if the dual specificity is affected. Expected results will open the door to further studies on the function of CatG in neutrophils and mast cells, while providing presently unavailable recombinant enzymes that will aid other researchers. Recombinant HNE will allow the generation of mutants resulting from single nucleotide polymorphisms, which might contribute to other diseases such as emphysema and arthritis. This AREA project provides students with experience in DNA cloning, microbiological techniques, cell biology, fermentation methods, protein purification, structure/function studies, enzyme/inhibitor reactions and enzyme kinetics. This laboratory has a solid record in the training of undergraduates, medical students and graduate students. Knowledge to be gained through this project will improve our understanding of neutrophil function and diseases, including susceptibility to bacterial infections, inflammation and neutropenia. This Academic Research Enhancement Award will allow undergraduate and graduate students to gain important research experience in a medical school environment that will prepare them for careers in scientific research. Two human protein degrading enzymes will be produced in yeast and studied to gain additional knowledge concerning an unusual processing event. These enzymes and the cells of the body that store them are known to play critical roles in our defenses to bacterial infection and in diseases, including allergies. Consequently, the knowledge and experiences to be gained will aid in our efforts to improve health.