LOCALIZED PROTEOLYSIS IN PULMONARY INFLAMMATION

炎症细胞降解细胞外蛋白是一种 有效炎症反应的基本要素。但是，当 炎症反应过度、不适当或持续时间长， 蛋白水解性组织损伤成为各种疾病的特征 两者共同影响着身体的每一个器官。例如，在 肺气肿，显然是蛋白水解性细胞外损伤 基质是一种关键的致病事件。抑制剂限制了蛋白质的降解。 炎症细胞对即刻细胞周围的活性 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 这种有限的蛋白质分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 对催化活性的机理的新概念和新见解 的酶可以被限制在(或接近)细胞表面 炎性细胞沐浴在含有高亲和力的介质中 酶的抑制剂。例如，我们在量子成像方面的成功 单粒天青颗粒的蛋白水解性爆发 中性粒细胞带来了新的见解和基于扩散的结构 了解局部催化活性。此外，我们还确认了 中性粒细胞表面具有催化活性的丝氨酸蛋白酶 它们能抵抗自然产生的蛋白酶的抑制 抑制剂。在拟议的工作中，我们将在以下方面进行这些观察 合乎逻辑的方向。具体来说，我们将解决以下目标：1) 细胞与细胞间紧密接触区蛋白质分解的研究 底物，通过：a)检查蛋白水解物的消逝爆发 单粒天青的释放活性；b)模拟 酶在空间和时间上的分散和非各向同性抑制 C)探讨组织损伤的机制。 通过实验和模拟发现α1-抗胰蛋白酶缺乏症。2) 研究中性粒细胞表面结合的丝氨酸蛋白酶和 单核细胞，关于：A)与细胞表面结合的机制(S)； 以及b)调制和焦点定位。拟议的工作将提供 关于炎症细胞机制的重要新信息 正常降解胞外蛋白质，对周围环境几乎没有损害 组织，以及它们的蛋白分解机制可能受到影响的方式 不受监管失调的影响。

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.