TRANSCRIPTIONAL CONTROL--COMPLEMENT REGULATORY PROTEIN

我们的长期目标是了解细胞对补体的调节 表面蛋白 为了实现这一目标，我们分离出了一个18 kDa的 来自正常人红细胞的调节补体的蛋白质 activation. 他把这种蛋白质称为反应性细胞膜抑制剂 裂解或MIRL。 这种蛋白质的结构特征和 在功能上，并已被证明是参与的发病机制， 临床病症，阵发性睡眠性血红蛋白尿。 最近的数据 这表明MIRL表达可能是对以下因素的反应而增加的： 补体激活导致了对潜在机制的研究 调节MIRL表达。 我们已经证明MIRL表达是 通过暴露于PMA而增强。 此外，这种增强发生在 转录水平，并可能在翻译后调节 水平也是。 我们的近期目标是将该机制的特征描述为： MIRL的表达受到调控。 特定目的I：确定补体激活是否可诱导MIRL 表情 K562细胞将暴露于活化的补体， 测定MIRL RNA和蛋白质表达。具体目标二： 确定蛋白激酶C是否介导PMA诱导的MIRL表达。 K562 细胞将在PMA或PMA与蛋白激酶的存在下孵育 C抑制剂和MIRL表达测定。目的三：解释 PMA诱导的MIRL RNA和蛋白水平之间的差异。 的 增加的MIRL RNA的命运将通过代谢来确定。 标记细胞和用抗体免疫沉淀MIRL。具体目标 IV：确定负责赋予PMA的顺式DNA序列 响应能力。 将克隆MIRL基因的5'侧翼区。 负责赋予PMA响应性的特定序列将 由其赋予PMA对 异源启动子和DNA酶I足迹研究。具体目标五： 分离与PMA反应性结合的反式作用蛋白 MIRL基因。 构建cDNA表达文库 K562 RNA 转录调节蛋白将被克隆 以PMA响应元件为探针。

Our long term goal is to understand the regulation of complement by cell surface proteins. In pursuing this goal we have isolated an 18 kDa protein from normal human erythrocytes that regulates complement activation. He refer to this protein as membrane inhibitor of reactive lysis or MIRL. This protein has been characterized structurally and functionally and has been shown to be involved in the pathogenesis of a clinical disorder, paroxysmal nocturnal hemoglobinuria. Recent data suggesting that MIRL expression may be increased in response to complement activation has led to investigation of potential mechanisms regulating MIRL expression. We have demonstrated MIRL expression is enhanced by exposure to PMA. Furthermore, this enhancement occurs at the transcriptional level and may be modulated at the post-translational level as well. Our immediate goal is to characterize the mechanism by which MIRL expression is regulated. Specific Aim I: To determine if complement activation can induce MIRL expression. K562 cells will be exposed to activated complement and assayed for MIRL RNA and protein expression. Specific Aim II: To determine if protein kinase C mediates PMA induced MIRL expression. K562 cells will be incubated in the presence of PMA or PMA with protein kinase C inhibitors and MIRL expression assayed. Specific Aim III: To explain the discrepancy between PMA induced MIRL RNA and protein levels. The fate of the increased MIRL RNA will be determined by metabolically labeling cells and immunoprecipitating MIRL with antibody. Specific Aim IV: To determine the cis-DNA sequence responsible for conferring PMA responsiveness. The 5' flanking region of the MIRL gene will be cloned. The specific sequences responsible for conferring PMA responsiveness will be determined by their ability to confer PMA responsiveness to a heterologous promoter and DNase I footprinting studies. Specific Aim V: To isolate the trans-acting protein(s) which bind to PMA responsive element of the MIRL gene. A cDNA expression library will be constructed from K562 RNA. The transcriptional regulatory protein will be cloned using the PMA responsive element as probe.