Project Summary The desmosome is a proteinaceous cell-cell adherence structure that is only found in tissues that undergo significant mechanical stress. One of its main functions is to connect the intermediate filament latticework of adjacent cells. Desmoplakin (DSP) is one of the central structural proteins within the desmosome. At least 80% of Arrhythmogenic Cardiomyopathy (AC) cases are linked to variants in genes that encode desmosomal proteins and about 5-15% of AC cases are specifically linked to variants in the gene encoding desmoplakin (dsp). dsp variants are also strongly linked to Skin Fragility Wooly Hair Syndrome (SFWHS) and Dilated Cardiomyopathy with Wooly Hair and Keratoderma (DCWHK), two rare diseases characterized by fragile skin or fragile skin plus cardiomyopathy. Our labs (PI Wright and co-PI Borzok) have previously worked together to identify and characterize a novel molecular mechanism of pathogenicity common to all three of these diseases. Namely, we found that a subset of dsp variants result in the exposure of a usually occluded calpain cleavage site in DSP, rendering the variant hypersensitive to proteolysis. This results in a loss of cellular DSP levels, creates nonfunctioning desmosomes, and manifests clinically in weakened tissue stability. We also previously discovered multiple strategies that can reverse this calpain hypersensitivity: the introduction of secondary mutations that overlay the calpain cleavage site, incubation of DSP with antibodies that occlude the calpain cleavage site, and incubation of DSP with small molecules that block access to the calpain cleavage site. While we have data showing that these strategies to manipulate DSP degradation are effective, the molecular underpinnings of the strategies have not yet been thoroughly analyzed. Here, we propose to examine the molecular mechanism of how these strategies work on the protein level (aim 1). We will also test whether these strategies are functional within the context of a cell (aim 2). These two aims will define the parameters of how we can use pharmacological methods to correct a cell adhesive deficiency.