UNIQUE MACROPHAGE METALLOELASTASE AND EMPHYSEMA

独特的巨噬细胞金属弹性蛋白酶和肺气肿

• 批准号: 2415603
• 负责人: STEVEN D SHAPIRO
• 金额: $ 23.71万
• 依托单位: BARNES-JEWISH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2415603
• 关键词: alpha 1 antitrypsin; catalyst; chemical binding; chemical kinetics; disease /disorder model; elastases; emphysema; enzyme biosynthesis; enzyme induction /repression; enzyme structure; extracellular matrix; genetic manipulation; genetic strain; human subject; inflammation; laboratory mouse; macrophage; metalloenzyme; molecular pathology; posttranslational modifications; protein structure function; recombinant proteins; respiratory toxin; smoking; tissue inhibitor of metalloproteinases

Metalloproteinases comprise a family of matrix degrading enzymes that are believed to play a role in normal development as well as in tissue remodeling and repair. Abnormal expression of metalloproteinases may also contribute to the pathogenesis of many destructive processes. We have recently cloned a murine macrophage elastase (MME), that is a distinct member of the metalloproteinase gene family with potent elastolytic activity. Subsequently, we cloned and expressed the human orthologue to MME that we call human macrophage metalloelastase (HME). Deduced amino acid sequence demonstrates that HME is a unique human metalloproteinase. HME mRNA and protein are expressed in human alveolar macrophages derived from several smokers. Similar to MME, both native and recombinant HME degrade elastin. The long-term goals of this grant are to test the central hypothesis that macrophage elastase contributes to the pathogenesis of destructive inflammatory lung diseases. Particularly, we will focus on the role of macrophage elastase in pulmonary emphysema related to cigarette smoking which is characterized by macrophage accumulation and elastin degradation. To achieve these goals, we will first define the elastolytic and other proteolytic properties of human and murine macrophage elastase. Recombinant macrophage elastase will be used to determine the binding affinity and kinetic parameters of catalysis for elastin. We will also delineate the capacity of macrophage elastase to degrade other relevant extracellular matrix molecules as well as alpha-1-antiproteinase. Cleavage sites will be determined on selected susceptible matrices to begin to characterize peptide bonds cleaved by HME/MME. Second, we will determine the structural basis of the unusual C-terminal processing of macrophage elastase and investigate the effect of C-terminal processing on elastin binding, catalysis, and TIMP inhibition. We will identify the C-terminal cleavage site(s), mutate it to prevent the processing event, and then compare the ability of the truncated vs mutant forms to degrade elastin. Third, to address the role of MME in emphysema and other destructive inflammatory diseases we will determine whether HME production by human alveolar macrophages correlates with smoking and emphysema. We will identify mediators of inflammation that regulate HME biosynthesis in macrophages with a future goal being to define the molecular mechanisms responsible for regulation. Fourth, to more directly determine the role of macrophage elastase in emphysema, we will create a strain of mice with a targeted mutation in the macrophage elastase gene and examine the capacity of these MME-deficient mice and normal littermates to develop emphysema and other inflammatory lung diseases.

金属蛋白酶包括一个基质降解酶的家族 相信在正常发育以及组织中发挥作用 重塑和维修。 金属蛋白酶的异常表达也可能 有助于许多破坏性过程的发病机理。我们有 最近克隆了一种鼠巨噬细胞弹性酶（MME），这是一个独特的 具有有效催化性的金属蛋白酶基因家族的成员 活动。随后，我们克隆并表达了人类直系同源 MME称为人类巨噬细胞金属弹性酶（HME）。推导的氨基 酸序列表明HME是一种独特的人类金属蛋白酶。 HME mRNA和蛋白质在人类肺泡巨噬细胞中表达 来自几个吸烟者。类似于MME，无论是天然和重组HME 降解弹性蛋白。 这笔赠款的长期目标是检验中心假设 巨噬细胞弹性蛋白酶有助于破坏性的发病机理 炎症性肺部疾病。特别是，我们将专注于 与吸烟有关的肺肺气肿中的巨噬细胞弹性蛋白酶 其特征是巨噬细胞的积累和弹性蛋白降解。 为了实现这些目标，我们将首先定义弹性和其他 人和鼠巨噬细胞弹性酶的蛋白水解特性。 重组巨噬细胞弹性酶将用于确定结合 弹性蛋白催化的亲和力和动力学参数。我们也会 描述巨噬细胞弹性酶降解其他相关的能力 细胞外基质分子以及α-1-抗蛋白酶。乳沟 将在选定的易感矩阵上确定站点以开始 表征由HME/MME裂解的肽键。第二，我们将确定 巨噬细胞不寻常的C末端处理的结构基础 弹性酶并研究C末端加工对弹性蛋白的影响 结合，催化和TIMP抑制。我们将确定C端 切割站点，将其突变以防止处理事件，然后 比较截短与突变体形式降解弹性蛋白的能力。 第三，解决MME在肺气肿和其他破坏性中的作用 炎症性疾病我们将确定人类是否生产HME 肺泡巨噬细胞与吸烟和肺气肿相关。我们将 确定调节HME生物合成的炎症介体 巨噬细胞的未来目标是定义分子机制 负责监管。第四，更直接地确定 巨噬细胞弹性蛋白酶在肺气肿中，我们将与A产生一小鼠的菌株 巨噬细胞弹性酶基因的靶向突变并检查能力 这些缺乏MME的小鼠和正常同窝材料会发展出肺气肿 和其他炎症性肺部疾病。