LOCALIZED PROTEOLYSIS IN PULMONARY INFLAMMATION

炎性细胞降解细胞外蛋白质是一种免疫抑制剂。 有效炎症反应的基本要素。 然而当 炎症反应过度、不适当或延长， 蛋白水解性组织损伤成为多种疾病的特征， 共同影响身体的每个器官。 例如在 肺气肿，很明显，蛋白水解损伤细胞外 基质是一个关键的致病事件。抑制剂限制蛋白水解 炎性细胞对细胞周围的直接活性 微环境，但不能抑制蛋白质的降解， 与细胞直接接触。我们对细胞生物学的理解 这种有限的蛋白水解活性远远落后于我们对 酶和抑制剂本身的结构和生物化学。 在本供资期间开始的研究产生了重要的 新的概念和新的见解的机制，催化活性 酶可以局限于（或附近）细胞表面时， 炎症细胞被浸泡在含有高亲和力 酶的抑制剂。例如，我们在量子成像方面的成功 来自单个嗜天青颗粒的蛋白水解活性的爆发 嗜中性粒细胞的研究带来了新的见解和基于扩散的结构， 理解局部催化活性。我们还发现 中性粒细胞表面的催化活性丝氨酸蛋白酶 它们对天然存在的蛋白酶的抑制具有抗性 抑制剂的在拟议的工作中，我们将继续这些意见， 逻辑方向。具体而言，我们将实现以下目标：1） 研究细胞和受试者之间紧密接触区域的蛋白水解 a）检查蛋白水解底物的短暂爆发， 来自单个嗜天青颗粒释放的活性; B）模拟 酶在空间和时间上的分散和非各向同性抑制 在脱粒之后;和c）探索在脱颗粒中的组织损伤的机制。 α 1-抗胰蛋白酶缺乏通过实验和模拟。（二） 研究与中性粒细胞表面结合的丝氨酸蛋白酶， a）与细胞表面结合的机制; 和B）调节和焦点靶向。拟议的工作将提供 关于炎症细胞 正常降解细胞外蛋白质，对周围环境的损害很小 组织，以及它们的蛋白水解机制可能受到影响的方式 从失调。

Degradation of extracellular proteins by inflammatory cells is a fundamental element of effective inflammatory responses. However, when inflammatory responses are excessive, inappropriate, or prolonged, proteolytic tissue injury becomes a feature of a variety of diseases that together affect essentially every organ of the body. For example, in pulmonary emphysema, it is clear that proteolytic injury to extracellular matrix is a pivotal pathogenic event. Inhibitors confine the proteolytic activity of inflammatory cells to the immediate pericellular microenvironment, but cannot inhibit degradation of proteins that are in direct contact with the cells. Our understanding of the cell biology of this confined proteolytic activity has lagged far behind our knowledge of the structure and biochemistry of the enzymes and inhibitors themselves. Studies begun during the present funding period have produced important new concepts and new insights into mechanisms by which catalytic activity of enzymes can be confined to (or near) the cell surface when an inflammatory cell is bathed in a medium containing high-affinity inhibitors of the enzymes. For example, our success in imaging quantum bursts of proteolytic activity from single azurophil granules of neutrophils has led to new insights and a diffusion-based construct for understanding localized catalytic activity. Further we have identified catalytically active serine proteinases on the cell surface of neutrophils that are resistant to inhibition by naturally-occurring proteinase inhibitors. In the work proposed, we will pursue these observations in logical directions. Specifically, we will address the following aims: 1) Study proteolysis in zones of close contact between cells and subjacent substrates, through: a) Examining evanescent bursts of proteolytic activity from release of single azurophil granules; b) Simulating dispersion of enzymes, and non-isotropic inhibition, in space and time following degranulation; and c) Exploring mechanisms of tissue injury in alpha1-antitrypsin deficiency through experiments and simulations. 2) Study serine proteinases bound to the cell surface of neutrophils and monocytes, with regard to: a) Mechanism(s) of binding to the cell surface; and b) Modulation and focal targeting. The proposed work will provide important new information about mechanisms by which inflammatory cells normally degrade extracellular proteins with little damage to surrounding tissues, and also ways in which their proteolytic mechanisms may suffer from dysregulation.