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.