Proteomic assessment of sulfur mustard-induced protein adducts and other protein modifications in human epidermal keratinocytes.
Although some toxicological mechanisms of sulfur mustard (HD) have been uncovered, new knowledge will allow for advanced insight in the pathways that lead towards epidermal-dermal separation in skin. In the present investigation, we aimed to survey events that occur at the protein level in human epidermal keratinocytes (HEK) during 24 h after exposure to HD. By using radiolabeled (14)C-HD, it was found that proteins in cultured HEK are significant targets for alkylation by HD. HD-adducted proteins were visualized by two-dimensional gel electrophoresis and analyzed by mass spectrometry. Several type I and II cytokeratins, actin, stratifin (14-3-3sigma) and galectin-7 were identified. These proteins are involved in the maintenance of the cellular cytoskeleton. Their alkylation may cause changes in the cellular architecture and, in direct line with that, be determinative for the onset of vesication. Furthermore, differential proteomic analysis was applied to search for novel features of the cellular response to HD. Partial breakdown of type I cytokeratins K14, K16 and K17 as well as the emergence of new charge variants of the proteins heat shock protein 27 and ribosomal protein P0 were observed. Studies with caspase inhibitors showed that caspase-6 is probably responsible for the breakdown of type I cytokeratins in HEK. The significance of the results is discussed in terms of toxicological relevance and possible clues for therapeutic intervention.
Mol
Marijke A E
MA
TNO Defence, Security and Safety (formerly TNO Prins Maurits Laboratory), PO Box 45, Rijswijk, The Netherlands. marijke.mol@tno.nl
van den Berg
Roland M
RM
Benschop
Henk P
HP
D000478:Alkylation / Q000187:drug effects; D000595:Amino Acid Sequence; D053178:Caspase 6 / Q000378:metabolism; D061945:Caspase Inhibitors; D002478:Cells, Cultured; D002619:Chemical Warfare Agents / Q000633:toxicity*; D006801:Humans; D053547:Keratin-14 / Q000302:isolation & purification*; D015603:Keratinocytes / Q000187:drug effects* / Q000378:metabolism; D009151:Mustard Gas / Q000633:toxicity*; D040901:Proteomics / Q000379:methods*
D061945:Caspase Inhibitors; D002619:Chemical Warfare Agents; D053547:Keratin-14; C505514:CASP6 protein, human; D053178:Caspase 6; D009151:Mustard Gas