MONOCYTE PATHOGENESIS--RESPIRATORY BURST OXIDASE

单核细胞发病机制--呼吸爆发氧化酶

• 批准号: 2766775
• 负责人: Martha K Cathcart
• 金额: $ 24.73万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2766775
• 关键词: active sites; antisense nucleic acid; enzyme mechanism; human subject; isozymes; leukocyte activation /transformation; leukocyte oxidative burst; monocyte; phlebotomy; phospholipase A2; phosphorylation; protein kinase C; respiratory burst oxidase; superoxides

Monocytes are critical for normal immune function, both for innate and specific immunity. They are also important components of inflammatory responses. Monocytes can be activated by a variety of stimuli to produce superoxide anion (O2-). Superoxide anion production appears to be critical for normal and pathologic contributions of this leukocyte and is generated by a multi- component enzyme complex, the respiratory burst oxidase (RB0). In this application, we propose to study in detail the regulation of the RB0 in activated human monocytes. Little is known about the regulation of this complex in monocytes, other than the regulation appears to be substantially different from that observed in neutrophils, the common model used for studies of this enzyme complex. We have recently shown that O2-production by activated monocytes is essential for monocytes to oxidize LDL, a process believed to have important pathologic roles in inflammation and particularly in atherogenesis. We have also shown that activation-induced PKC and cPLA2 activities are required for O2-production. Here we propose to rigorously evaluate the mechanisms for the regulation of this critical RBO complex by these important signal-transducing enzymes. In Aim 1 we will test the hypothesis that cPLA2 (through arachidonic acid) regulates phosphorylation, translocation and/or interaction of RBO components. In Aim 2 we will identify the PKCalpha-dependent phosphorylation site on cPLA2 that regulates cPLA2 activity and ultimately controls the activity of the RBO. Finally, in Aim 3 we will test the hypothesis that an isoform of PKC, other than PKCalpha or PKCbeta, is involved in regulating RBO activity. These studies will elucidate important regulatory pathways that control this critical enzyme complex. The novelty of these studies is derived from our unique opportunity to specifically dissect the roles of these pathways in intact cells by virtue of the fact that monocytes are very conducive to antisense ODN manipulation of specific protein expression. These studies will substantially influence our appreciation of the modes of regulation of the RB0 complex and will likely suggest specific and selective means for modulating its activity.

单核细胞对于正常的免疫功能是至关重要的，无论是对于先天免疫还是特异性免疫。 它们也是炎症反应的重要组成部分。 单核细胞可被多种刺激物激活产生超氧阴离子（O2-）。 超氧阴离子的产生似乎对这种白细胞的正常和病理贡献至关重要，并且由多组分酶复合物呼吸爆发氧化酶（RB 0）产生。在本申请中，我们建议详细研究活化的人单核细胞中RB 0的调节。 关于单核细胞中这种复合物的调节知之甚少，除了调节似乎与中性粒细胞中观察到的调节有很大不同之外，中性粒细胞是用于研究这种酶复合物的常见模型。 我们最近发现，由活化的单核细胞产生的O2-是单核细胞氧化LDL所必需的，这一过程被认为在炎症，特别是在动脉粥样硬化形成中具有重要的病理作用。 我们还表明，激活诱导的PKC和cPLA 2活动所需的O2生产。 在这里，我们建议严格评估这些重要的信号转导酶调节这一关键RBO复合物的机制。 在目标1中，我们将测试假设cPLA 2（通过花生四烯酸）调节磷酸化，易位和/或RBO组件的相互作用。 在目标2中，我们将鉴定cPLA 2上的PKCa依赖性磷酸化位点，其调节cPLA 2活性并最终控制RBO的活性。 最后，在目标3中，我们将检验以下假设：PKC的亚型，而不是PKC α或PKC β，参与调节RBO活性。这些研究将阐明控制这种关键酶复合物的重要调控途径。 这些研究的新奇来自于我们独特的机会，特别是解剖这些途径在完整细胞中的作用，凭借单核细胞是非常有益的反义ODN操纵特定的蛋白质表达的事实。 这些研究将极大地影响我们对RB 0复合物调节模式的认识，并可能提出调节其活性的特异性和选择性方法。