Interplay of oxidative sensitivity and protein glutathionylation in Darier Disease

Summary: Abstract Body: The acantholytic phenotype of Darier Disease (DD), a condition caused by heterozygous mutation of the endoplasmic reticulum calcium pump SERCA2, presents during the second decade of life and responds to environmental triggers. Delayed onset, despite lifelong SERCA2 deficiency, suggests that DD cells harbor sensitivities to age-related stressors, such as oxidative damage. The antioxidant glutathione protects certain proteins, including SERCA2, from oxidative damage through reversible glutathionylation. We find that DD keratinocytes (KCs) have an increased level of SERCA2 glutathionylation, as well global protein glutathionylation, and a partially depleted free glutathione pool compared to CTLs. This suggests that SERCA2 deficiency may cause mild oxidative stress that must be buffered at baseline. This theory is supported by our finding that, prior to any apparent adhesion defect, glutathionylation in CTL KCs increases linearly in response to treatment with the SERCA inhibitor thapsigargin. Compared to CTLs, upon the addition of exogenous stress DD KCs generate twice the reactive oxygen species, recruit desmosomal components to junctions 89% less efficiently, and show an 8-fold decrease in adhesion strength via dispase. Desmosomal proteins in DD cells collapse into a perinuclear ring, highly colocalized with the signal for glutathionylation; proximity ligation confirms significantly increased desmoplakin glutathionylation in stressed DD cells. We propose a model whereby SERCA2 heterozygosity causes mild oxidative stress that is buffered by glutathionylation. When subjected to additional external stress, free glutathione is shunted to perinuclear desmoplakin at the expense of SERCA2, rendering the calcium pump vulnerable to irreversible oxidation/inactivation. Lesional flares, then, could represent cases of total SERCA2 inhibition, perturbing intercellular adhesion via ER stress, altered cadherin trafficking, and cytoskeletal disruption. Erin McCarthy¹, Robert Harmon¹, Lisa M. Godsel¹, Russell Dahl³, Eran Cohen Barak², Roni Dodiuk-Gad², Amy Paller¹, Kathleen Green¹ 1. Northwestern University Feinberg School of Medicine, Chicago, IL, United States. 2. Technion Israel Institute of Technology, Haifa, Haifa District, Israel. 3. Neurodon LLC, Crown Point, IN, United States. Genetic Disease, Gene Regulation, Gene Therapy & Epigenetics