PROTEASES ESSENTIAL IN MACROPHAGE ACTIVATION

巨噬细胞激活所必需的蛋白酶

• 批准号: 2899117
• 负责人: MICHAEL J PABST
• 金额: $ 24.44万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2899117
• 关键词: G protein; biological signal transduction; enzyme activity; enzyme biosynthesis; enzyme inhibitors; enzyme substrate; gel electrophoresis; human tissue; leukocyte activation /transformation; lipopolysaccharides; macrophage; mass spectrometry; monocyte; nuclear factor kappa beta; phosphatase inhibitor; phospholipase inhibitor; phosphorylation; polymerase chain reaction; protein kinase C; protein sequence; protein tyrosine kinase; radiotracer; serine proteinases; tissue /cell culture; western blottings

The aggressiveness of macrophages in fighting infection or killing cancer cells is regulated by macrophage activation. Activation in vitro is called "priming". Primed macrophages release more microbicidal oxygen radicals. Macrophages are primed by bacterial factors like lipopolysaccharide (LPS) and muramyl peptides, by cytokines like interferon-gamma, and by lipid mediators like platelet-activating factor. Earlier, we showed in mice that the activator muramyl dipeptide enhanced non-specific resistance to lethal infections. We recently found that priming of monocytes was inhibited by the serine protease inhibitor AEBSF. This suggests that, in addition to kinases and other signal transduction elements, proteases are essential in macrophage activation. Objective: Elucidate the role of proteases in macrophage activation. Specific Aim 1: Identify the negative regulator of priming. Hypothesis: Priming is negatively regulated by a protein, whose stability, or continuous synthesis, is required to keep monocytes unprimed. This regulator protein can be identified in two-dimensional gels, comparing monocytes q LPS q AEBSF. Specific Aim 2: Identify the protease responsible for inactivating the negative regulator of priming. Hypothesis: Priming is prevented by a monocyte protein that acts as a negative regulator; removal of the regulator by an endogenous protease causes priming. The protease is inactivated by AEBSF, which blocks priming. The protease can be identified by tagging it with radiolabeled AEBSF. Specific Aim 3: Investigate the mechanism by which LPS inactivates or blocks the synthesis of the negative regulator of priming. Hypothesis: LPS and other primers activate the protease, or induce synthesis of the protease, that cleaves the negative regulator of priming. Covalent modification or synthesis of the protease, or removal of an inhibitor, can be observed in response to LPS. Specific Aim 4: Determine how the protease and its substrate interact with other signal elements like kinases and phospholipases. Hypothesis: The substrate of the protease (the negative regulator) is an inhibitor of a phospholipase or kinase that is essential for macrophage activation. Significance: Knowledge of mechanisms of macrophage activation will help us to fight infection, which will increase due to aging of the population, more immuno-compromised patients, and antibiotic-resistant microbes.

巨噬细胞在抗击感染或杀死癌细胞中的侵略性受巨噬细胞的激活调节。体外激活称为“启动”。 底漆巨噬细胞释放更多的微生物氧自由基。巨噬细胞由细菌因子（例如脂多糖（LPS）和穆拉米基肽），干扰素 - 伽马等细胞因子以及脂质介质等血小板激活因子等细胞因子。 早些时候，我们在小鼠中表明，激活剂穆拉米尔二肽增强了对致命感染的非特异性抗性。我们最近发现，丝氨酸蛋白酶抑制剂AEBSF抑制了单核细胞的启动。 这表明，除了激酶和其他信号转导元件外，蛋白酶在巨噬细胞激活中至关重要。 目的：阐明蛋白酶在巨噬细胞激活中的作用。特定目的1：确定启动的负调节剂。假设：启动受到蛋白质的负调节，蛋白质的稳定性或连续合成需要保持单核细胞未刺激。 可以在二维凝胶中鉴定该调节蛋白，并比较单核细胞Q LPS Q AEBSF。特定目的2：确定负责灭活启动的负调节剂的蛋白酶。 假设：单核细胞蛋白可以阻止启动，该蛋白充当负调节剂。通过内源性蛋白酶去除调节剂会导致启动。 蛋白酶被阻断启动的AEBSF灭活。 可以通过用放射性标记的AEBSF对其进行标记来识别蛋白酶。 特定目标3：研究LP灭活或阻断启动负调节剂的合成的机制。 假设：LP和其他引物激活蛋白酶的蛋白酶，或诱导蛋白酶的合成，从而切割了启动的负调节剂。 可以响应LPS观察到蛋白酶的共价修饰或合成或去除抑制剂。 特定目标4：确定蛋白酶及其底物如何与其他信号元素（如激酶和磷脂酶）相互作用。 假设：蛋白酶（阴性调节剂）的底物是磷脂酶或激酶的抑制剂，对于巨噬细胞激活至关重要。 意义：巨噬细胞激活机制的知识将有助于我们与感染作斗争，由于人群的老龄化，更多的免疫功能低下的患者和抗抗生素耐药的微生物将增加感染。