RECOMBINANT SERPINS AND NEUTROPHILS IN ACUTE LUNG INJURY

这项建议概述了一项培训计划，这将有助于我们的 对其生化特性和生物学功能的认识 丝氨酸蛋白酶抑制剂(Serpins)以及为我提供 作为分子领域的独立研究员的职业生涯的基础 以及调节肺部炎症的细胞生物学。 我们对研究蛇类和中性粒细胞之间的相互作用很感兴趣。 在维持或改变蛋白水解酶抗蛋白酶平衡的过程中 发炎。中性粒细胞丝氨酸蛋白酶，特别是弹性蛋白酶(HNE)， 被认为是主要的介质或急性肺损伤。这个 丝氨酸α-1-蛋白酶抑制物(α1PI)和α-1- 抗糜蛋白酶(ACT)是中性粒细胞的主要抑制物 血浆和肺实质中的丝氨酸蛋白酶。Alpha1PI是 主要的HNE抑制剂。ACT和α1PI可能抑制中性粒细胞 活性氧(由NADPH氧化酶系统产生的ROS0。 另一方面，中性粒细胞可能形成微环境， 中性粒细胞ROS或裂解减弱α1PI抑制活性 中性粒细胞金属蛋白酶(NMPs)和非靶向丝氨酸蛋白酶。 我们有丰富的生产蛇类重组变种的经验 具有改变的抑制活性和其他改变的生化 特点。这项建议中描述的研究使用的是体外 激活的中性粒细胞降解细胞外基质的模型 专为抵抗ROS而设计的Alpha1PI的重组变体 修饰和/或NMP裂解以检验假说(1) 中性粒细胞来源的ROS和(2)NMP在炎症中发挥作用，在 部分，通过降低炎症部位的α1PI活性。我们也 检验α1PI和ACT调节(1)中性粒细胞ROS的假说 产生和(2)NMP激活，从而限制中性粒细胞的 能够减少炎症部位的蛇毒活性。角色 HNE介导的ACT裂解对ACT活性的影响 还将检查炎症的情况。以此为基础的具体目标 假设包括：(1)评估野生型和变异型的效果 ROS生产中的蛇形；(2)设计抗氧化Alpha1PI 并在疗效方面与野生型alpha1PI进行比较 在保护模型细胞外基质中，并确定是否有丝氨酸 介导的ROS抑制增强野生型alpha1PI的疗效；(3) 评价野生型和变异型蛇毒对NMP激活的影响； (4)设计抗NMP的Alpha1PI变体，并将其与NMP- 敏感的Alpha1PI变体在保护模式中的有效性 细胞外基质，并确定丝氨酸是否介导对NMP的抑制 激活增强NMP敏感的alph1PI变异体的疗效；(5) 设计对HNE具有相对抗性的重组ACT变异体 裂解和NMP裂解并比较这些变体的效果 与野生型ACT在扩增研究中的应用。

This proposal outlines a training program which will contribute to our understanding of the biochemical properties and biological functions of serine protease inhibitor (serpins) as well as provide me with a foundation for a career as an independent investigator in the molecular and cellular biology of the regulation of lung inflammation. We are interested in studying the interactions of serpins and neutrophils in maintaining or altering the protease antiprotease balance during inflammation. Neutrophil serine proteases, especially elastase (HNE), have been implicated as major mediators or acute lung injury. The serpins alpha-1-protease inhibitor (alpha1PI) and alpha-1- antichymotrypsin (ACT) are the predominant inhibitors of neutrophil serine proteases in the plasma and lung parenchyma. alpha1PI is the predominant HNE inhibitor. ACT and alpha1PI may inhibit neutrophil reactive oxygen species (ROS0 production by the NADPH oxidase system. The neutrophil, on the other hand, may form microenvironments where alpha1PIs inhibitory activity is attenuated by neutrophil ROS or cleavage by neutrophil metalloproteinases (NMPs) and non-target serine proteases. We have extensive experience producing recombinant variants of serpins with altered inhibitory activity and other altered biochemical characteristics. The studies described in this proposal use in vitro models of extracellular matrix degradation by activated neutrophils and recombinant variants of alpha1PI that are designed to resist ROS modification and/or NMP cleavage in order to examine the hypotheses (1) neutrophil derived ROS and (2) NMPs play a role in inflammation, in part, by decreasing alpha1PI activity at sites of inflammation. We also examine the hypotheses that alpha1PI and ACT regulate (1) neutrophil ROS production and (2) NMP activation thereby limiting the neutrophil's ability to decrease serpin activity at sites of inflammation. The role of HNE mediated cleavage of ACT on altering the activity of ACT at sites of inflammation will also be examined. The Specific Aims based on these hypotheses include: (1) evaluate the effect of wild type and variant serpins on ROS production; (2) design oxidation resistant alpha1PI variants and compare them to wild type alpha1PI with regard to efficacy in protecting model extracellular matrix, and determine if serpin mediated ROS inhibition augments the efficacy of wild type alpha1PI; (3) evaluate the effect of wild type and variant serpins on NMP activation; (4) design NMP-resistant alpha1PI variants and compare them to NMP- sensitive alpha1PI variants with regard to efficacy in protecting model extracellular matrix, and determine if serpin mediated inhibition of NMP activation augments the efficacy of NMP-sensitive alph1PI variants; (5) design recombinant ACT variants that are relatively resistant to HNE cleavage and NMP cleavage and compare the effectiveness of these variants with wild type ACT in the augmentation studies.