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。在追求这一目标的过程中，我们分离出了一个18 kDa的 正常人红细胞中调节补体的蛋白质 激活。他把这种蛋白质称为膜反应抑制因子 裂解或MIRL。这种蛋白质的结构特征是 在功能上，并已被证明参与了一种 临床障碍，阵发性睡眠性血红蛋白尿。最新数据 提示MIRL的表达可能是对 补体激活导致了对潜在机制的研究 调节MIRL的表达。我们已经演示了MIRL的表达是 因接触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 级别也是如此。我们的直接目标是通过以下方式描述这一机制 其中MIRL的表达受到调控。 特定目的I：确定补体激活是否可诱发MIRL 表情。K562细胞将暴露于激活的补体和 检测MIRL RNA和蛋白表达。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表达的检测。具体目标三：解释 PMA诱导的MIRL RNA和蛋白质水平的差异。这个 增加的MIRL RNA的命运将由代谢决定 用抗体标记细胞和免疫沉淀MIRL。特定目标 IV：确定与PMA有关的顺式DNA序列 响应性。MIRL基因的5‘侧翼区将被克隆。 负责授予PMA响应性的特定序列将 由他们将PMA响应性授予 异源启动子和DNase 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(S) MIRL基因的元件。将构建一个cdna表达文库 来自K562核糖核酸。转录调节蛋白将被克隆 使用PMA响应元件作为探头。

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.