α-Sens: The improved ARE-Nrf2-based sensitization screening assay using normalized transcriptional activity.

Two non-animal test methods, KeratinoSens™ and LuSens, have been approved by the Organization of Economic Cooperation and Development (OECD) test guidelines for evaluating the sensitization potential of chemicals, and been positioned as a method for appraising key event (KE)-2, namely, the keratinocyte response component of the Adverse Outcome Pathway (AOP) in sensitization process. However, these two methods require separate cytotoxicity tests to determine the concentrations to be tested in the main test. Therefore, we developed a simple and highly accurate KE-2 test method named α-Sens that uses the dual luciferase assay system and attempted a further application of luciferase-based determination of cell viability to calculate the normalized Antioxidant response element (ARE)-mediated transcriptional activity, named normalized ARE Activity (nAA), to evaluate the sensitizing potential of chemicals. A cell line carrying the ARE-inducible Firefly luciferase reporter gene and Thymidine kinase (TK) promoter-driven Renilla luciferase gene was established and used for the α-Sens. A total of 28 chemicals, consisting of 19 skin sensitizers and nine non-skin sensitizers were tested by this assay system. The α-Sens yielded an accuracy (%), sensitivity (%), and specificity (%) against corresponding values for local lymph node assay of 96.4 %, 95.0 %, and 100 %, respectively, and for human data of 100 % for all. The α-Sens gave clear positive results for phenyl benzoate and eugenol, chemicals for which KeratinoSens™ or LuSens yielded false-negative results, using a new parameter. Our results suggest that better prediction capacity could be achieved by using nAA as a classifier compared to other existing KE-2 test methods. In conclusion, the α-Sens is promising as a simple and highly accurate in vitro skin sensitization test method for evaluation of KE-2.

Vertical-scanning mutagenesis of amino acids in a model N-myristoylation motif reveals the major amino-terminal sequence requirements for protein N-myristoylation.

In order to determine the amino-terminal sequence requirements for protein N-myristoylation, site-directed mutagenesis of the N-terminal region was performed using tumor necrosis factor (TNF) mutants as model substrate proteins. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system using rabbit reticulocyte lysate. A TNF mutant having the sequence MGAAAAAAAA at its N-terminus was used as the starting sequence to identify elements critical for protein N-myristoylation. Sequential vertical-scanning mutagenesis of amino acids at a distinct position in this model N-terminal sequence revealed the major sequence requirements for protein N-myristoylation: the combination of amino acids at position 3 and 6 constitutes a major determinant for the susceptibility to protein N-myristoylation. When Ser was located at position 6, 11 amino acids (Gly, Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, His) were permitted at position 3 to direct efficient protein N-myristoylation. In this case, the presence of Lys at position 7 was found to affect the amino acid requirement at position 3 and Lys became permitted at this position. When Ser was not located at position 6, only 3 amino acids (Ala, Asn, Gln) were permitted at position 3 to direct efficient protein N-myristoylation. The amino acid requirements found in this study were fully consistent with the N-terminal sequence of 78 N-myristoylated proteins in which N-myristoylation was experimentally verified. These observations strongly indicate that the combination of amino acids at position 3, 6 and 7 is a major determinant for protein N-myristoylation.