Regulation of Mast Cell Proteases

肥大细胞蛋白酶的调节

基本信息

批准号：
7422404
负责人：
Richard L Stevens
金额：
$ 49.06万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-06-01 至 2010-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7422404
关键词：
Allergens Animals Anti-Bacterial Agents Antibodies Antigens Autolysis Bacteria Basophilic leukemia Binding Biochemistry Bone Marrow Carbohydrates Carboxypeptidase Cellular biology Chondroitin Sulfate C Chondroitin Sulfates Chymase Clinical Communities Complex Cutaneous Cytoplasmic Granules Data Deacetylase Disease Disruption Embryo Endopeptidases Failure Family Fibrosis Figs - dietary Generations Genes Glycosaminoglycans Helminths Heparin High Pressure Liquid Chromatography Hindlimb Human Hypoxia Immunoglobulins Immunology In Vitro Infection Injury Interleukins Ischemia KITLG gene Knockout Mice Lung Mass Spectrum Analysis Mediating Metabolism Modeling Molecular Molecular Biology Mus Muscle Numbers Oligosaccharides Ovalbumin PAR-2 Receptor Pathology Pattern recognition receptor Peptide Hydrolases Peptide Phage Display Library Peptides Phosphate Buffer Physiological reperfusion Plasmin Plasminogen Play Polyacrylamide Gel Electrophoresis Population Preparation Process Protease Inhibitor Proteinase-Activated Receptors Proteins Proteoglycan RNA Rattus Reagent Recombinants Regulation Reperfusion Therapy Reverse Transcriptase Polymerase Chain Reaction Rhabdomyolysis Role Saline Screening procedure Secretory Vesicles Serine Protease Serpins Small Interfering RNA Sodium Dodecyl Sulfate-PAGE Stem Cell Factor Structure Substrate Specificity Testing Thinking Transgenic Animals Transgenic Mice Trypsin Tryptase Tumor Necrosis Factor-alpha Tumor Necrosis Factors Urokinase Weight acquired immunity airway hyperresponsiveness airway remodeling cytokine extracellular granule cell heparin proteoglycan homologous recombination human TNF protein human mast cell tryptase in vivo jejunum mast cell mast cell protease 6 progesterone 11-hemisuccinate-(2-iodohistamine)programs research study serglycin size stem sulfotransferase

项目摘要

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）分泌颗粒的整个重量几乎由16个中性蛋白酶与塞糖蛋白蛋白聚糖（SGPGS）结合，其中包含肝素（HP）或高度硫化软骨蛋白（CHS）链，例如CHS-E，例如CHS-E。我们和其他人表明，其中一些蛋白酶/SGPG复合物在先天和获得的免疫力中起有益的作用。例如，小鼠MC蛋白酶（MMCP）-6/HP复合物在肺中起抗细菌作用。但是，现在很明显，其中一些复合物有助于MC介导的临床疾病中发生的病理。例如，在后肢缺血 - 重新灌注的低氧损伤模型中，胞外MCP-5/HP复合物引起肌肉横纹肌溶解。翻译后的机制仅存储在颗粒中，而MCS最大程度地减少其相关蛋白酶的自溶的机制未知。根据细胞因子微环境，小鼠和人MC将不同类型的糖胺聚糖（GAGS）合成到Serglycin上。 HP显着改变了胰蛋白酶MMCP-6的底物特异性的发现增加了CHS-E对其他MC胰酶和/或Chymase扮演类似调节作用的可能性。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SGPG及其独特的插入如何控制不同MC颗粒蛋白酶的表达，颗粒存储，酶活性，酶促活性和细胞外代谢。在特定的目标1中，我们将在存在和不存在MC衍生的插孔的情况下用重组MC蛋白酶筛选我们的噬菌体 - 转化肽库，以确定与SGPG相连的碳水化合物链的类型是否差异地改变了每个研究过的蛋白酶的酶活性。 SGPG在调节MC蛋白酶细胞外代谢的调节中的潜在作用，包括它们被SERPINS灭活和其他天然存在的蛋白酶抑制剂。我们还将确定人MC胰酶beta1的晶体结构，使其首选的GAG复合。在特定目标2中，我们将评估HP和MMCP-5-NULL小鼠的能力（以及新创建的MMCP-6-和CHS-E-NULL小鼠）打击细菌和蠕虫感染。这些转基因小鼠还将用于确定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。