Contact dermatitis: in pursuit of sensitizer's molecular targets through proteomics

Protein haptenation, i.e., the modification of proteins by small reactive chemicals, is the key step in the sensitization phase of allergic contact dermatitis (ACD). Despite the research effort in past decades, the identification of immunogenic hapten-protein complexes that trigger a relevant pathogenic immune response in ACD, as well as the haptenation reaction molecular site, and the elements of a potentially conditioning environment during each of these stages, remain poorly understood. These questions led us to employ a proteomics-based approach to identify modified proteins in the dendritic-like cell line THP-1 sensitized with fluorescein isothiocyanate (FITC), through a combination of 2D-gel electrophoresis, nano-LC and mass spectrometry. A specific set of 39 targeted proteins was identified and comprised proteins from various cellular locations and biological functions. One of FITC targets was identified as MLK, a member of the mixed-lineage kinase family known to act as a mitogen-activated protein kinase kinase kinase and to control the activity of specific mitogen-activated protein kinase pathways, namely p38 and JNK pathways. Haptenated in the vicinity of its active site, our results point to MLK being a relevant target due to a consistent non-activation at early time points of these pathways upon FITC sensitization in THP-1 cells. Moreover, FITC pre-treatment significantly decrease phospho-p38 and phospho-JNK levels induced upon exposure to a classical activator such as lipopolysaccharide or to the sensitizer 2,4-dinitrofluorobenzene. Overall, our data point to specific amino acid residues haptenation within critical proteins as the key step in the subsequent signaling pathways modulation responsible for DC activation and maturation events.