Regulation of Mast Cell Proteases

肥大细胞蛋白酶的调节

• 批准号: 7312452
• 负责人: Richard L Stevens
• 金额: $ 49.6万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7312452
• 关键词: cell biology; cell line; crystallization; enzyme activity; enzyme induction /repression; genetic regulation; genetic regulatory element; genetic transcription; genetically modified animals; immunocytochemistry; isozymes; laboratory mouse; mast cell; posttranscriptional RNA processing; protein purification; proteoglycan; serine proteinases; tryptase; yeast two hybrid system

Nearly the entire weight of a mouse mast cell's (MC's) secretory granules consists of 16 neutral proteases ionically bound to serglycin proteoglycans (SGPGs) that contain either heparin (HP) or highly sulfated chondroitin (ChS) chains such as ChS-E. We and others have shown that some of these protease/SGPG complexes play beneficial roles in innate and acquired immunity. For example, mouse MC protease (mMCP)-6/HP complexes play an anti-bacterial role in the lung. However, it is now clear that some of these complexes contribute to the pathology that occurs in MC-mediated clinical disorders. For example, exocytosed mMCP-5/HP complexes cause muscle rhabdomyolysis in a hindlimb ischemia-reperfusion model of hypoxia injury. The post-translational mechanism by which only properly folded proteases are stored in the granules and the mechanism by which MCs minimize autolysis of their associated proteases are unknown. Depending on the cytokine microenvironment, mouse and human MCs synthesize different types of glycosaminoglycans (GAGs) onto serglycin. The finding that HP dramatically alters the substrate specificity of the tryptase mMCP-6 raises the possibility that ChS-E plays a similar regulatory role for other MC tryptases and/or chymases. The overall objective of Project 1 is to use complementary biochemistry, immunology, cell biology, crystallographic, and molecular biology approaches to deduce how SGPGs and their unique GAGs control the expression, granule storage, enzymatic activity, and extracellular metabolism of the different MC granule proteases. In Specific Aim 1, we will screen our phage-display peptide libraries with recombinant MC proteases in the presence and absence of MC-derived GAGs to determine if the type of carbohydrate chain attached to a SGPG differentially alters the enzymatic activity of each investigated protease. The potential roles of SGPGs in the regulation of the extracellular metabolism of MC proteases, including their inactivation by serpins and other naturally occurring protease inhibitors will be investigated. We also will determine the crystal structure of human MC tryptase beta1 complexed to it's preferred GAG. In Specific Aim 2, we will evaluate the ability of our HP- and mMCP-5-null mice (as well as newly created mMCP-6- and ChS-E-null mice) to combat bacteria and helminth infections. These transgenic mice also will be use to ascertain the role of MC protease/SGPG complexes in baseline and antigen-induced airway reactivity, fibrosis, and remodeling.

小鼠肥大细胞（MC）分泌颗粒的几乎全部重量由16种中性蛋白酶组成，所述中性蛋白酶与含有肝素（HP）或高度硫酸化软骨素（ChS）链（如ChS-E）的丝甘肽蛋白聚糖（SGPs）离子结合。我们和其他人已经表明，这些蛋白酶/SGPG复合物中的一些在先天性和获得性免疫中发挥有益的作用。例如，小鼠MC蛋白酶（mMCP）-6/HP复合物在肺中发挥抗菌作用。 然而，现在清楚的是，这些复合物中的一些有助于MC介导的临床疾病中发生的病理学。例如，胞吐的mMCP-5/HP复合物在缺氧损伤的后肢缺血-再灌注模型中引起肌肉横纹肌溶解。翻译后的机制，只有正确折叠的蛋白酶储存在颗粒和机制，其中MC最大限度地减少其相关的蛋白酶的自溶是未知的。根据细胞因子微环境，小鼠和人MC在sergylycin上合成不同类型的糖胺聚糖（GAG）。HP显著改变类胰蛋白酶mMCP-6的底物特异性的发现提高了ChS-E对其他MC类胰蛋白酶和/或糜蛋白酶起类似调节作用的可能性。的总体目标 项目1是使用互补的生物化学，免疫学，细胞生物学，晶体学和分子生物学方法来推断SGPs及其独特的GAG如何控制不同MC颗粒蛋白酶的表达，颗粒储存，酶活性和细胞外代谢。在具体目标1中，我们将在存在和不存在MC衍生的GAG的情况下用重组MC蛋白酶筛选我们的噬菌体展示肽文库，以确定连接到SGPG的碳水化合物链的类型是否差异地改变每种研究的蛋白酶的酶活性。将研究SGPs在MC蛋白酶的细胞外代谢调节中的潜在作用，包括丝氨酸蛋白酶抑制剂和其他天然存在的蛋白酶抑制剂对它们的失活。我们还将确定与其首选GAG复合的人MC类胰蛋白酶β 1的晶体结构。在特定目标2中，我们将评估HP和mMCP-5缺失小鼠（以及新创建的mMCP-6和ChS-E缺失小鼠） 来对抗细菌和寄生虫感染。这些转基因小鼠也将用于确定MC蛋白酶/SGPG复合物在基线和抗原诱导的气道反应性、纤维化和重塑中的作用。