Mechanisms Underlying the Dominant Negative Phenotype in Hereditary Angioedema

遗传性血管性水肿（HAE）是一种由SERPING 1突变引起的常染色体显性遗传疾病。几乎 所有HAE患者都是杂合子，具有一个正常的SERPING 1和一个突变的SERPING 1拷贝， 在同一细胞中同时表达突变型和野生型C1抑制剂（C1 INH）蛋白。的 预期所产生的单倍不足将导致患者在24小时内具有50%的正常C1 INH水平。 然而，HAE患者的功能性C1 INH的血浆水平通常在10- 100 μ g/ml之间， 正常的20%。导致功能性C1 INH水平意外降低的机制从未被证实。 这一点已经被理解，并且是本申请的重点。我们已经证明，突变的C1 INH蛋白干扰 野生型（WT）C1 INH蛋白的分泌。本申请的总体假设是突变型C1 INH 对野生型C1 INH发挥显性负效应，将功能性C1 INH水平降低至低于 这是肿胀所需的阈值，因此是HAE显性阴性表型的原因。 这种显性阴性表型的机制将使用两种转染的细胞进行研究， 野生型和突变型C1 INH以及HAE患者单核细胞。野生型和突变体的特异性标记 C1 INH蛋白将被用于特异性地跟踪两种C1 INH蛋白在细胞中的运输和分泌。 转染细胞还将研究HAE和对照单核细胞。目的1将表征细胞内 野生型C1 INH在表达野生型和突变型C1 INH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显微镜来确定这些蛋白质在细胞内的保留位置。 显微镜然后，我们将使用天然凝胶电泳确定野生型C1 INH是否与突变型C1 INH形成寡聚体 免疫印迹和用标记的蛋白质的下拉实验。接下来，我们将评估激活 表达WT加突变型C1 INH的细胞中的自噬通量，以及自噬与WT抑制的相关性 C1 INH分泌。我们还将分析影响内质网应激途径，包括未折叠蛋白反应 和ER相关的降解，对显性负效应。目标2将阐明生物物理 C1 INH的特性，当用突变体表达时，有助于其对细胞内滞留的敏感性 丝氨酸蛋白酶抑制剂蛋白。我们将通过同源结构的交换来创建嵌合C1 INH和a1-AT蛋白 并定义了表现显性阴性表型所需的关键序列。我们还将确定 在细胞内与C1 INH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单核细胞中功能性C1 INH的分泌。 到本项目结束时，预期在小鼠中对野生型C1 INH分泌的显性负效应在小鼠中是显著的。 HAE将被清楚地理解。这将为随后的研究奠定基础， 可以消除这种显性负效应，增加野生型C1 INH分泌， 并将C1 INH水平恢复到接近正常水平的50%，在该水平患者将无症状。

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.