Mechanisms Underlying the Dominant Negative Phenotype in Hereditary Angioedema

遗传性血管性水肿显性阴性表型的机制

• 批准号: 10516092
• 负责人: Bruce L. Zuraw
• 金额: --
• 依托单位: VA SAN DIEGO HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-10-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10516092
• 关键词: Address; Alzheimer' s Disease; Angioneurotic Edema; Autophagocytosis; Biochemical; Biological Assay; Cells; Chimeric Proteins; Clinical; Confocal Microscopy; Data; Degradation Pathway; Disease; Dominant-Negative Mutation; Endoplasmic Reticulum Degradation Pathway; Gel; Genetic Techniques; Heterozygote; Human; Image; Immunoelectron Microscopy; Infectious Agent; Laboratories; Mass Spectrum Analysis; Mediating; Morbidity - disease rate; Multivariate Analysis; Mutate; Mutation; Outcome Measure; Pathologic; Pathway interactions; Patients; Phenotype; Plasma; Positioning Attribute; Predisposition; Proteins; Proteomics; Serpins; Structure; Swelling; Testing; Therapeutic; Transfection; Trypsin; Western Blotting; autosome; biophysical properties; clinically significant; disease-causing mutation; endoplasmic reticulum stress; experimental study; genetic approach; hereditary angioneurotic edema; inhibitor; misfolded protein; monocyte; mortality; mutant; novel; prevent; protein misfolding; protein protein interaction; response; trafficking

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.

遗传性血管性水肿（HAE）是由Serping1突变引起的一种常染色体显性疾病。几乎 所有HAE患者均为杂合子，具有一个正常和一个突变的Serping1副本 同一细胞中突变体和野生型C1抑制剂（C1INH）蛋白的同时表达。这 预计导致的单倍不足会导致患者具有正常C1INH水平的50％ 他们的等离子体；但是，HAE患者通常具有血浆水平的功能C1INH，介于10-之间 正常的20％。导致这种出乎意料低的功能C1INH的机制从来没有 被理解，是该应用程序的重点。我们已经表明，突变的C1INH蛋白会干扰 野生型（WT）C1INH蛋白的分泌。该应用的总体假设是突变体C1inh 对野生型C1INH产生主要负面影响，降低了功能性C1INH的水平 肿胀所需的阈值，因此负责HAE的主要负面表型。 将使用两个转染的细胞来研究这种主要负表型的机制 野生型和突变体C1INH以及HAE患者单核细胞。野生型和突变体的特定标记 C1INH蛋白将用于专门跟随两种C1INH蛋白的运输和分泌 转染的细胞。 HAE和对照单核细胞也将进行研究。 AIM 1将表征细胞内 在表达野生型和突变型C1INH蛋白的细胞中野生型C1INH的运输和处置。 我们将使用共焦和免疫电子确定这些蛋白保留在细胞中的位置 显微镜。然后，我们将使用天然凝胶确定野生型C1inh是否与突变体C1inh形成低聚物 具有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和突变体C1INH的细胞中的自噬通量，并将自噬与抑制WT相关 C1inh分泌。我们还将分析ER应力途径，包括展开的蛋白质反应 以及与ER相关的降解，对主要的负面影响。 AIM 2然后将阐明生物物理 用突变体表达的C1INH的特性有助于其对细胞内保留的敏感性 Serpin蛋白。我们将通过交换同源结构来创建嵌合C1inh和A1-AT蛋白 并定义表现出主要负面表型所需的临界序列。我们还将确定 与细胞中C1INH相互作用的蛋白质。最后，我们将使用多元分析来了解如何 这些参数中的每一个都可能有助于HAE单核细胞中功能C1INH的分泌。 到该项目结束时，预计对野生型C1inh分泌的主要负面影响 HAE将被清楚地理解。这将为随后的研究奠定了阶段 可以消除这种主要负面影响，增加野生型C1inh分泌的治疗方法， 并恢复C1INH的水平接近正常的50％，在哪个水平的患者无症状的情况下。