Mechanisms Underlying the Dominant Negative Phenotype in Hereditary Angioedema

遗传性血管性水肿病是由SERPING1基因突变引起的一种常染色体显性遗传病。差不多了 所有的HAE患者都是杂合子，有一个正常的和一个突变的SERPING1拷贝 突变型和野生型C1INH蛋白在同一细胞中的同时表达。这个 由此导致的单倍体功能不全预计会导致患者的C1INH水平是正常水平的50% 然而，HAE患者的血浆功能性C1INH水平通常在10-10之间。 是正常的20%。导致这种出乎意料的低水平功能性C1INH的机制从未 已被理解，并且是本应用程序的重点。我们已经证明突变的C1INH蛋白干扰 野生型(WT)C1INH蛋白的分泌。这一应用的总体假设是突变体C1INH 对野生型C1INH产生显性负效应，使功能性C1INH水平降至 肿胀所需的阈值，因此是HAE的显性阴性表型的原因。 这一显性负性表型的机制将通过两种转基因细胞进行研究。 野生型和突变型C1INH以及HAE患者单核细胞。野生型和突变型的特异性标记 C1INH蛋白将被用来特异性地跟踪C1INH蛋白在体内的运输和分泌 转基因细胞。HAE和对照单核细胞也将被研究。目标1将描述细胞内的 野生型C1INH在表达野生型和突变型C1INH蛋白的细胞中的运输和处置。 我们将使用共聚焦和免疫电子技术来确定这些蛋白质在细胞内的保留位置。 显微镜。然后我们将确定野生型C1INH是否与突变型C1INH使用天然凝胶形成低聚物 标记蛋白的免疫印迹和下拉实验。接下来，我们将评估激活的证据 表达WT+突变体C1INH的细胞中的自噬通量，以及自噬与WT抑制的相关性 C1INH分泌。我们还将分析影响内质网应激的途径，包括未折叠的蛋白质反应 与内质网相关的降解，对显性负效应。然后目标2将阐明生物物理 C1INH在突变体中表达时对细胞内滞留易感性的特性 丝氨酸蛋白。我们将通过交换同源结构来创建嵌合的C1INH和A1-AT蛋白 并确定显性负性表型所需的关键序列。我们还将确定 在细胞内与C1INH相互作用的蛋白质。最后，我们将使用多变量分析来了解 这些参数中的每一个都可能有助于HAE单核细胞分泌功能性的C1INH。 到本项目结束时，预计对野生型C1INH分泌的主要负面影响 海尔将会被清楚地理解。这将为试图开发的后续研究奠定基础 治疗方法可以消除这种显性的负面影响，增加野生型C1INH的分泌， 并将C1INH水平恢复到接近正常水平的50%，在这一水平上，患者将没有症状。

Hereditary angioedema (HAE) is an autosomal dominant disease caused by mutations in SERPING1. Almost all HAE patients are heterozygotes, having one normal and one mutated copy of SERPING1 with the concurrent expression of both mutant and wild-type C1 inhibitor (C1INH) proteins in the same cell. The resulting haploinsufficiency would be expected to result in patients having 50% of the normal level of C1INH in their plasma; however, HAE patients typically have plasma levels of functional C1INH that are between 10- 20% of normal. The mechanism responsible for this unexpectedly low level of functional C1INH has never been understood and is the focus of this application. We have shown that mutant C1INH proteins interfere with the secretion of wild-type (WT) C1INH protein. The overall hypothesis of this application is that mutant C1INH exerts a dominant negative effect on wild-type C1INH, reducing the level of functional C1INH below the threshold required for swelling and thus is responsible for the dominant negative phenotype of HAE. The mechanisms of this dominant negative phenotype will be studied using both transfected cells expressing wild-type and mutant C1INH as well as in HAE patient monocytes. Specific tagging of wild-type and mutant C1INH proteins will be utilized to specifically follow trafficking and secretion of both C1INH proteins in transfected cells. HAE and control monocytes will also be studied. Aim 1 will characterize the intracellular trafficking and disposition of wild-type C1INH in cells expressing both wild-type and mutant C1INH proteins. We will determine where these proteins are retained within the cell using confocal and immunoelectron microscopy. We will then determine if wild-type C1INH forms oligomers with mutant C1INH using native gel immunoblots and pull-down experiments with tagged proteins. Next we will assess evidence for activation of autophagic flux in cells expressing WT plus mutant C1INH, and correlate autophagy with inhibition of WT C1INH secretion. We will also analyze the impact ER stress pathways, including the unfolded protein response and ER associated degradation, on the dominant negative effect. Aim 2 will then elucidate the biophysical properties of C1INH that contribute to its susceptibility to intracellular retention when expressed with mutant serpin proteins. We will create chimeric C1INH and a1-AT proteins though swapping of homologous structures and define critical sequences required to manifest the dominant negative phenotype. We will also identify proteins that interact with C1INH within the cell. Finally, we will use multivariate analyses to understand how each of these parameters may contribute to the secretion of functional C1INH in HAE monocytes. By the end of this project, it is anticipated that the dominant negative effect on wild-type C1INH secretion in HAE will be clearly understood. This would set the stage for subsequent studies attempting to develop therapeutic approaches that could abrogate this dominant negative effect, increase wild-type C1INH secretion, and restore C1INH levels to close to 50% of normal at which level patients would be asymptomatic.