The chemical chaperone trimethylamine N-oxide ameliorates the effects of mutant keratins in cultured cells

Abstract

Conflicts of interest: none declared. Sir, Mutations in keratin genes are the cause of at least 19 distinct disorders, many of which affect the skin and its appendages.1 The clinical symptoms vary according to the expression pattern of the mutated keratin but typically involve cell fragility and hyperkeratosis. Although keratin disorders are rarely life threatening, they can be painful and debilitating and most treatments are limited to pain relief and prevention of infection. The observation that many keratin mutations are associated with cytoplasmic keratin aggregates led us to hypothesize that keratin disorders are protein‐folding disorders and that chemical chaperones, which promote normal protein folding, will reduce the cellular effects of the mutant keratins.2 To test this hypothesis we examined the ability of the chemical chaperone trimethylamine N‐oxide (TMAO) to reduce the effects of hyperosmotic stress in cells expressing mutant keratins. We first examined the effect of hyperosmotic stress on a cell line derived from a patient with epidermolysis bullosa simplex (KEB‐1; K5:Glu475Gly).3 Incubation of KEB‐1 cells with medium containing 300 mmol L−1 sorbitol for 30 min led to collapse of the keratin cytoskeleton and the formation of cytoplasmic keratin aggregates in more than 20% of the cells. In contrast, keratin aggregates formed fewer than 5% of controls cells (SVK14 cell line) following similar treatment. Pretreatment of KEB‐1 cells with TMAO reduced the number of cells with keratin aggregates to control levels (Fig. 1). A decrease in the number of keratin aggregate‐containing cells was noted after prolonged exposure (> 30 min) of KEB‐1 cells to hyperosmotic conditions. This could indicate that cells containing aggregates were lost from the culture dish or that the keratinocytes were re‐establishing their keratin cytoskeleton. To distinguish between these possibilities, KEB‐1 and SVK14 cells were incubated with sorbitol for various times and the number of cells remaining attached to the plate determined. There was a time‐dependent decrease in the number of KEB‐1 cells indicating that cells containing keratin aggregates were preferentially lost from the culture dish. No effect on cell number was observed in control cells. Pretreatment with TMAO protected KEB‐1 cells from hyperosmotic‐induced cell loss (Fig. 1).