178 Cooperation of keratin mutations and posttranslational modifications

Abstract

The skin fragility disorder Epidermolysis bullosa simplex (EBS) mostly results from KRT5 and KRT14 mutations which cause aggregation of the keratin cytoskeleton and render the skin sensitive to mild trauma, followed by recurrent blisters. The prevalence of more serious disease during warmer summer months in most EBS patients suggests that temperature-sensing mechanisms cooperating with keratin mutations contribute to this phenomenon. In order to dissect the underlying mechanism and to exploit this for a therapy approach, we analyzed several KRT5 and KRT14 mutations in a cell culture based heat stress assay. Hence, we identified a subset of KRT5 mutations forming extensive keratin networks with KRT14 at normal culture conditions but reversible aggregation of the keratin cytoskeleton upon short exposure to elevated temperature. This heat-induced aggregation of the keratin cytoskeleton subsequently led to impaired desmosomes and diminished intercellular adhesion. A phosphoproteomic approach revealed elevated Ser phosphorylation of mutant KRT5, but not KRT14, upon brief heat exposure, indicating the involvement of specific kinases in the heat-induced keratin aggregation. Currently, we are investigating kinase inhibitors to prevent the heat-induced filament collapse. Moreover, parthenolide, a plant sesquiterpene lactone that exerts anti-cancer and anti-inflammatory properties and modulates sirtuin function, was found to affect keratin organization, likely by reducing elevated Lys acetylation of mutant keratins. Most importantly, pre-treatment of a temperature-sensitive KRT5 mutant with parthenolide prevented heat-induced keratin aggregation and strengthened desmosome adhesion. Collectively, we show that keratin mutations promote select posttranslational modifications, partially in combination with elevated temperature. Our assay system and results enable the development of a rational therapy for the improvement of EBS.