RECOMBINANT SERPINS AND NEUTROPHILS IN ACUTE LUNG INJURY

急性肺损伤中的重组丝氨酸蛋白酶抑制剂和中性粒细胞

• 批准号: 2392552
• 负责人: MICHAEL I PLOTNICK
• 金额: $ 8.8万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-15 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2392552
• 关键词: enzyme activity; enzyme induction /repression; enzyme mechanism; free radical oxygen; human subject; inflammation; lung injury; metalloendopeptidases; neutrophil; protease inhibitor; protein structure function; recombinant proteins

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.

该建议概述了一项培训计划，将有助于我们 了解的生化特性和生物功能， 丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂），以及为我提供一个 作为分子生物学领域独立研究者的职业基础 以及调节肺部炎症的细胞生物学。 我们有兴趣研究丝氨酸蛋白酶抑制剂和中性粒细胞的相互作用 在维持或改变蛋白酶抗蛋白酶平衡的过程中， 炎症 中性丝氨酸蛋白酶，尤其是弹性蛋白酶（HNE）， 是急性肺损伤的主要介质。 的 丝氨酸蛋白酶抑制剂α-1-蛋白酶抑制剂（α 1 PI）和α-1- 抗糜蛋白酶（ACT）是中性粒细胞的主要抑制剂， 血浆和肺实质中的丝氨酸蛋白酶。 alpha 1 PI是 主要的HNE抑制剂 ACT和α 1 PI可抑制中性粒细胞 活性氧物质（ROS）通过NADPH氧化酶系统产生。 另一方面，嗜中性粒细胞可以形成微环境， α 1 PIs抑制活性被中性粒细胞ROS或裂解减弱 中性粒细胞金属蛋白酶（NMPs）和非靶丝氨酸蛋白酶。 我们在生产丝氨酸蛋白酶抑制剂的重组变体方面拥有丰富的经验 具有改变的抑制活性和其他改变的生化 特色 本提案中描述的研究使用体外 活化的中性粒细胞降解细胞外基质的模型， 设计用于抵抗ROS的α 1 PI重组变体 修饰和/或NMP裂解，以检验假设（1） 中性粒细胞衍生的ROS和（2）NMPs在炎症中起作用， 部分通过降低炎症部位的α 1 PI活性。 我们也 检验α 1 PI和ACT调节（1）中性粒细胞ROS的假设 产生和（2）NMP活化，从而限制中性粒细胞的 降低炎症部位丝氨酸蛋白酶抑制剂活性的能力。 的作用 HNE介导的ACT切割在改变ACT活性的位点 炎症也将被检查。 具体目标基于这些 假设包括：（1）评估野生型和变异体的作用 丝氨酸蛋白酶抑制剂对活性氧产生的影响;（2）设计抗氧化的α 1 PI 变体，并将其与野生型α 1 PI的功效进行比较 保护模型细胞外基质，并确定是否丝氨酸蛋白酶抑制剂 介导的ROS抑制增强了野生型α 1 PI的功效;（3） 评估野生型和变体丝氨酸蛋白酶抑制剂对NMP活化的影响; (4)设计抗NMP的alpha 1 PI变体，并将其与NMP进行比较- 在保护模型的功效方面敏感的α 1 PI变体 细胞外基质，并确定是否丝氨酸蛋白酶抑制剂介导的抑制NMP 激活增强了NMP敏感性P1 PI变体的功效;（5） 设计对HNE具有相对抗性的重组ACT变体 切割和NMP切割，并比较这些变体的有效性 在增强研究中使用野生型ACT。